The Effects of Insulin-Like Growth Factors on Tumorigenesis and

0163-769X/00/$03.00/0 Endocrine Reviews 21(3): 215–244 Copyright © 2000 by The Endocrine Society Printed in U.S.A.

The Effects of Insulin-Like Growth Factors on Tumorigenesis and Neoplastic Growth HASNAIN M. KHANDWALA, IAN E. MCCUTCHEON, ALLAN FLYVBJERG, KEITH E. FRIEND

AND

Section of Endocrine Neoplasia & Hormonal Disorders (H.M.K., K.E.F.) and the Department of Neurosurgery (I.E.M.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; and Medical Department M (Diabetes and Endocrinology) (A.F.), Aarhus Kommunehospital, Aarhus, DK-8000 Denmark ABSTRACT Several decades of basic and clinical research have demonstrated that there is an association between the insulin-like growth factors (IGFs) and neoplasia. We begin with a brief discussion of the function and regulation of expression of the IGFs, their receptors and the IGF-binding proteins (IGFBPs). A number of investigational interventional strategies targeting the GH or IGFs are then reviewed. Finally, we have assembled the available scientific knowledge about this relationship for each of the major tumor types. The tumors have

been grouped together by organ system and for each of the major tumors, various key elements of the relationship between IGFs and tumor growth are discussed. Specifically these include the presence or absence of autocrine IGF-I and IGF-II production; presence or absence of IGF-I and IGF-II receptor expression; the expression and functions of the IGFBPs; in vitro and in vivo experiments involving therapeutic interventions; and available results from clinical trials evaluating the effect of GH/IGF axis down-regulation in various malignancies. (Endocrine Reviews 21: 215–244, 2000)

I. Introduction II. Overview: IGF Physiology and Gene Regulation A. IGF-I gene expression B. IGF-II gene expression C. IGF-I receptor gene expression D. IGF-II receptor gene expression III. Potential Therapeutic Agents A. GHRH antagonists B. Somatostatin analogs C. GH receptor antagonists D. IGF-I receptor antibodies and analogs of IGF-I E. IGFBPs IV. Central Nervous System Neoplasms A. Gliomas/astrocytomas B. Meningiomas V. Gastrointestinal Neoplasms A. Colon cancer B. Gastric cancer C. Pancreatic cancer D. Other (esophageal/hepatocellular) VI. Head, Neck, and Pulmonary Neoplasms A. Lung cancer (small cell/non-small cell) B. Thyroid cancer VII. Female Reproductive Neoplasms A. Breast cancer B. Ovarian cancer C. Other (endometrial, vaginal, cervical) VIII. Male Reproductive Neoplasms A. Prostate cancer B. Testicular cancer

IX. Genitourinary Neoplasms A. Renal cell carcinoma B. Bladder cancer X. Bone Neoplasms A. Osteosarcoma B. Other (chondrosarcoma/fibrosarcoma) XI. Skin Neoplasms A. Melanoma B. Basal/squamous XII. Hematological Malignancies XIII. Summary

I. Introduction

T

HE ASSOCIATION between the insulin-like growth factors (IGFs) and neoplasia has been a subject of investigation for many years. In this manuscript, we have reviewed the available scientific knowledge relevant to understanding the nature of this relationship. This information has been organized by grouping the tumors together by organ system (based upon tissue of origin). For each type of neoplasm, four key elements of the relationship between the IGFs and tumorigenesis and tumor growth will be discussed. These are as follows: 1) presence or absence of autocrine IGF-I and IGF-II production, particularly in relation to normal tissue; 2) presence or absence of IGF-I and IGF-II receptor expression; 3) effects of IGF-I and IGF-II on tumorigenesis and tumor growth; and 4) relevant clinical studies involving therapeutic interventions. When possible, the mechanisms whereby the IGFs are exerting their growth-promoting effects (e.g., decreased apoptosis, increased cell proliferation, or angiogenesis) are also discussed, as is the importance of

Address reprint requests to: Keith E. Friend, M.D., Section of Endocrine Neoplasia & Hormonal Disorders, University of Texas M.D. Anderson Cancer Center, Box 15, 1515 Holcombe Boulevard, Houston, Texas, 77030 USA.

215

216

KHANDWALA ET AL.

the binding proteins. We begin the manuscript with a brief overview of what is known about the function and regulation of expression of the IGFs and their receptors.

II. Overview: IGF Physiology and Gene Regulation

IGF-I and IGF-II share approximately 50% structural homology to insulin. The majority of circulating IGF-I and IGF-II are produced by the liver, although various tissues have the capability to synthesize these peptides locally. The hepatic synthesis of IGF-I is largely GH dependent, whereas the synthesis of IGF-II is relatively independent of GH. The GH/IGF-I axis is the primarily regulator of postnatal growth while IGF-II appears to have an important role during fetal development (1–3). The IGFs are present in the circulation in combination with high- and low-affinity binders, which form the IGF-binding protein (IGFBP) superfamily. A total of six high-affinity binding proteins have been identified, IGFBP-1 through IGFBP-6 (4 –7). Although the majority of the circulating IGFBPs are synthesized in the liver, many other organs are capable of production of IGFBPs. IGFBP-3 is the most abundant binding protein in the serum. It forms a ternary complex with IGF-I and an acid-labile subunit. The IGFBPs have higher affinities for IGFs (kd ⬃ 10⫺10 m) than do the type I IGF receptors (kd ⬃ 10⫺8 m). The IGF/IGFBP complex is acted upon by proteases at the target organ, whereby IGF is released and is available for biological actions. The IGFBPs have stimulatory and/or inhibitory actions on cell proliferation, and these effects may be dependent or independent of IGF. Chen et al. (8) in 1994 demonstrated that addition of exogenous IGFBP-2 and -3 increased IGF-I stimulated [3H]thymidine incorporation in the estrogen receptor-positive cell line, MCF-7. This was thought to be due to a conformational change in the IGFBP-3 as a result of cell surface association, leading to reduced affinity and increased bioavailability of IGF-I and -II. In addition to the high-affinity IGFBPs, several low-affinity binding proteins, termed IGFBP-related proteins (IGFBPrP), have been described. The IGFBP-rPs are cysteine rich, have structural similarities to the N terminus of the IGFBPs, and bind to IGFs with a 100-fold lower affinity compared with the IGFBPs. The IGFBP-rPs have actions that are predominantly independent of IGFs. The IGF-I receptor is a transmembrane heterotetramer consisting of two ␣- and two ␤-subunits. There is approximately 60% sequence homology between the IGF-I receptor and the insulin receptor. The IGF-I receptor, like the insulin receptor, possesses tyrosine kinase activity. The postreceptor signal transduction events include phosphorylation of insulin receptor substrate-1 (IRS-1) and activation of phosphatidylinositol-3 (PI-3) and mitogen-activated protein kinases. IGF-II and insulin also bind to the IGF-I receptor but with 2to 15- and 1,000-fold lower affinity, respectively (9). The IGF-I receptor has been shown to protect cells from apoptosis in vitro and in vivo. Resnicoff et al. (10) demonstrated that for a number of different cell lines, including a human melanoma cell line, decreasing the number of IGF-I receptors by antisense oligodeoxynucleotides was associated with decreased cell growth in vitro and in vivo. The number of apoptotic cells

Vol. 21, No. 3

was significantly increased in these cell lines as measured by various techniques. Valentinis et al. (11) in 1999 suggested that the paradoxical ability of IGF-I to cause unregulated cell growth, on one hand, or lead to terminal differentiation, on the other, may depend on the balance between various intracellular substrates. In their study using murine hemopoietic cells (32D), which lack IRS-1, exogenous IGF-I induced differentiation of these cells along the granulocytic pathway after interleukin-3 withdrawal. The IGF-I-induced differentiation in the absence of IRS-1 required the C terminus of the IGF-I receptor. Expression of IRS-1 in these cells was associated with loss of differentiation and caused unregulated growth, and the degree of inhibition of differentiation correlated with IRS-1 expression. The authors also suggested that overexpression of Shc, another substrate of IGF-I receptor, potentiated 32D cell differentiation while a dominant negative mutant of Shc partially inhibited differentiation. The IGF-II/cation-independent mannose-6-phosphate (IGFII/ Man-6-P) receptor is a monomeric receptor that binds IGF-II with a 500-fold increased affinity over IGF-I. The IGF-II receptor does not bind insulin. Most of the biological actions of IGF-II are thought to be mediated via the IGF-I receptor (12, 13). Four classes of ligands are currently known to bind to the extracytoplasmic receptor domain. They include mannose 6-phosphate-containing lysosomal enzymes, IGF-II, retinoic acid, and urokinase-type plasminogen activator receptor. The IGF-II receptor is thought to function primarily as a scavenger receptor, regulating the internalization and degradation of extracellular IGF-II, thus regulating the circulating IGF-II levels. IGF-II receptor mutant animals have been demonstrated to have increased circulating and serum levels of IGF-II along with an increased birth weight, organomegaly, and perinatal mortality. The IGF-II receptor also regulates intracellular trafficking of lysosomal enzymes including cathepsin, which serves as an IGFBP proteolytic enzyme. Recently, O’Gorman et al. (14) described enhanced tumor growth in a choriocarcinoma cell line, JEG-3, after IGF-II receptor expression was decreased by antisense IGF-II cDNA constructs. The resultant decrease in cathepsin degradation, yielding an increase in IGFBP proteolysis and bioavailable IGFs, may account for this enhanced tumor growth. Although most of the mitogenic/metabolic effects of IGF-II signaling are mediated via the IGF-I receptor, some effects, including Na⫹/H⫹ exchange and production of inositol triphosphate in renal tubular cells, stimulation of Ca⫹ influx, and DNA synthesis in BALB/c 3T3 cells, may be mediated by binding of IGF-II to its receptor (12, 13). A. IGF-I gene expression

The prepro-IGF-I gene consists of six exons in most mammalian species and is located on chromosome 12 in humans. The majority of circulating IGF-I is produced in the liver, and hepatic production is principally regulated by GH. Although the liver-specific deletion of the IGF-I gene using the Cre/ loxP recombination system reduced circulating IGF-I concentration by approximately 80% in mice, the growth rates of these transgenic animals was not significantly different when compared with the wild-type animals. This was demonstrated by Yakar et al. (15) and suggests the importance of

June, 2000

IGFs AND NEOPLASIA

extrahepatic, autocrine/paracrine production of IGF-I in growth regulation. In some tissues, IGF-I gene expression is predominantly under the control of other hormones: estradiol in the endometrium, gonadotropins in the gonadal tissue, TSH in the thyroid, etc. (16 –18). IGF-I mRNA levels have been also demonstrated to be altered by nutritional state and developmental stage. For instance, fasting reduces serum IGF-I concentrations as much as 75% and negates the effects of GH stimulation. Refeeding rapidly restores parameters to normal (19). The coding region of prepro-IGF-I is flanked by complex 5⬘- and 3⬘-untranslated regions that result in considerable heterogeneity in mature IGF-I transcripts. For instance, in the rat it has been demonstrated that there are separate start sites present in both exons 1 and 2. In exon 1, transcription can be initiated from several different sites over a several hundred base pair region. This broad range of sites exists because there are no core promoter elements such as TATA and CAAT box motifs in exon 1. TATA and CAAT box motifs elements, however, are present upstream of the cluster of start sites present in exon 2. Nevertheless, in most tissues, the majority of transcripts arise from sites in exon 1. As a result of different start sites and alternative splicing, there are a variety of different 5⬘-untranslated regions that can be present in the mature mRNA transcript. Translation of prepro-IGF-I can be initiated from codons in exons 1, 2, or 3. Alternate polyadenylation sites in the 3⬘-untranslated region of the molecule also contribute to differences in transcript size. On Northern analysis, IGF-I transcripts ranging from ⬍1 kb to approximately 7.5 kb are observed (20). B. IGF-II gene expression

The human prepro-IGF-II gene consists of nine exons and is located on chromosome 11. The first six exons are noncoding. There are four promoters present (P1– 4). There is one promoter each in the regions upstream of exons 1, 4, 5, and 6. During fetal development, IGF-II expression is much higher than in the postnatal period or in the adult. A distinct pattern of promoter use correlates with expression levels during development. In the fetus, promoters P2– 4 are active in the liver. After birth, the use of these three promoters declines and P1 becomes dominant. There is some degree of tissue-specific regulation (21). The IGF-II gene is one of the few genes known to have parental allele-specific expression. As such, it is referred to as an imprinted gene. In normal cells, IGF-II is maternally imprinted in that it is expressed only from the paternal copy of the gene. The IGF-II gene is located on chromosome 11p15.5 close to H19, a paternally imprinted gene. The imprinting process is an early event, taking place at the time of gametogenesis. Loss of IGF-II imprinting has been reported in a variety of tumors, including Wilms’ tumors, gastric adenocarcinomas, lung cancers, gliomas, hepatoblastomas, leiomyosarcomas, cervical cancer, prostate cancer, choriocarcinomas, rhabdomyosarcomas, seminomas, and ovarian carcinomas (22–24). When loss of imprinting occurs, biallelic expression of IGF-II results, ultimately leading to overexpression of this potent growth factor. The precise role of loss of IGF-II imprinting in tumorigenesis and tumor growth is unknown at this time. In some neoplasms,

217

such as Wilms’ tumor, loss of imprinting is an event that occurs early in carcinogenesis. In others, such as cervical carcinoma, it is not an early event. The precise relationship with H19, which may have tumor suppressor function, is also not completely known. In Wilms’ tumor, the loss of imprinting is often linked with a reduced expression of H19. This reciprocal relationship, however, has not been commonly detected in other tumor types, such as hepatoblastomas, gliomas, testicular tumors, and cervical cancers. C. IGF-I receptor gene expression

The highest levels of IGF-I receptor mRNA expression occur during fetal development and in the early postnatal period. Although IGF-I receptor expression is significantly down-regulated in the adult, it is present in most types of tissue. Its expression is up-regulated to some degree by fasting; up-regulation has been reported in the kidney in an experimental diabetes model (25). IGF-I decreases IGF-I receptor expression in a dose-dependent manner in FRTL-5, IM-9, and endothelial cells (26, 27). The IGF-I receptor gene promoter lacks both TATA and CAAT box motifs. The 5⬘-untranslated region is very GC-rich and contains multiple Sp1 consensus-binding sequences. Accordingly, Sp1 has been demonstrated to potently activate IGF-I receptor gene transcription (28). Transcription is initiated from a single start site. Basic fibroblastic growth factor (bFGF) has also been demonstrated to increase IGF-I receptor mRNA levels and activate the IGF-I receptor promoter. The effects of bFGF have been localized to a region of the IGF-I promoter located between nucleotides ⫺476 and ⫺188 (29). Transfection experiments have demonstrated that the tumor suppressor p53 inhibits activity of the IGF-I receptor promoter. Mutant versions of p53, frequently present in malignant states, result in increased IGF-I receptor gene activation (30). The increased level of IGF-I receptor expression present in some malignant tumors may enhance response to autocrine, paracrine, or circulating IGFs. D. IGF-II receptor gene expression

The IGF-II receptor binds IGF-II and ligands containing a mannose 6-phosphate recognition marker (lysosomal enzymes). Unlike the IGF-I receptor gene, this receptor is a large single-chain peptide that has no intrinsic tyrosine kinase activity (31). Its primary function seems to be transport of its ligands to liposomes, resulting in either their activation or degradation. The majority of IGF-II receptors are located on intracellular membranes. Most of the physiological actions of IGF-II are thought to be mediated through binding to the IGF-I receptor. However, binding of IGF-II to its receptor has been demonstrated to trigger interaction with a membranebound GTP-binding protein, Gi-2, that mediates the influx of calcium into the cell (32). The mouse IGF-II receptor gene is 93 kb in size and contains 48 exons. The gene contains a strong minimal promoter of 266 bp or less. An extended 54-bp footprint within the proximal promoter containing two E-boxes and probable binding sites for Sp1, nerve growth factor-IA (NGF-IA), and related proteins has also been identified. Deletion of the

218

KHANDWALA ET AL.

54-bp segment resulted in an 8-fold decline in promoter activity. Mutational analyses demonstrated that each E box contributed to more than half of the enhancers activity (33). III. Potential Therapeutic Agents

Currently, modulating the GH/IGF-I axis remains an experimental antineoplastic strategy. Nevertheless, a number of different agents are available that offer potential benefits. Virtually every level of the GH/IGF-I axis, from the hypothalamic hormones to the receptors mediating response on the tumor tissues, can be targeted (Fig. 1). The major classes of therapeutic approaches are summarized in the following section. A. GHRH antagonists

The GH releasing property of the hGHRH molecule resides in the GHRH (1–29) sequence. Various hydrophilic or hydrophobic amino acid substitutions within that sequence, in an attempt to stabilize and enhance the amphiphilic ␣helical character of the molecule, have led to the synthesis of GHRH analogs with agonist/antagonistic activity. The GHRH antagonists inhibit in vitro GHRH-stimulated GH release in a superfused rat pituitary system and inhibit basal

Vol. 21, No. 3

and hGHRH-stimulated GH release in vivo. By decreasing pituitary GH release, hepatic IGF-I production is also reduced. Varga et al. (34), in a study evaluating 22 antagonists of GHRH, demonstrated that some agents potently inhibited in vivo hGHRH-induced GH release up to at least 60 min after administration. The GHRH antagonists have also been shown to inhibit autocrine/paracrine production of IGF-I/IGF-II by acting directly on the tumor tissues. In a study by Csernus et al. in 1999, the GHRH antagonists, MZ-4 –71 and MZ-5–156, were shown to inhibit autocrine production of IGF-II, reduce IGF-II mRNA expression, and decrease cell proliferation in a number of cell lines including the breast cancer cell lines MDA-MB-468 and ZR-75–1, prostate cancer cell lines PC-3 and DU-145, and the pancreatic cancer cell lines MiaPaCa-2, Capan-2, and SW-1990 (35). Because IGF-II is a potent mitogen for a variety of cell lines, and overexpression of IGF-II mRNA has been demonstrated for some tumors, reduction of IGF-II by GHRH antagonists could be of potential therapeutic use in the management of such tumors. Thus, the GHRH antagonists could inhibit tumor growth through direct or indirect pathways. The indirect mechanism would be secondary to a reduction in pituitary GH release and subsequent hepatic IGF-I production, leading to a decrease in circulating, “endocrine” IGF-I. The GHRH antagonists may also directly inhibit intratumor IGF-I and/or IGF-II mRNA expression and decrease the autocrine/paracrine production of these growth factors. B. Somatostatin analogs

FIG. 1. Sites of action of various pharmacological agents available to modify GH/IGF-I axis. 1, GH-releasing hormone (GHRH) antagonists; 2, somatostatin analogs; 3, GH receptor antagonists; 4, IGF-I receptor antibody; 5, IGF-I/II mRNA antisense vector strategies; and 6, IGFBPs.

Somatostatin is a peptide synthesized as part of a large precursor molecule that is cleaved and processed to yield several mature products, two of them being somatostatin-14 and somatostatin-28, the latter a congener of somatostatin extended at the N terminus. Somatostatin acts as a neurotransmitter in the central nervous system, regulates the release of GH and TSH, and has a regulatory role in the gastrointestinal system and endocrine and exocrine pancreas. The actions of somatostatin are mediated through somatostatin receptors, five of which have been characterized. These membrane receptors are present in multiple organ systems. The receptors are linked to adenylate cyclase through a coupling mechanism involving guanine nucleotide-binding (G) protein. As the half-life of natural somatostatin is very short, longer acting analogs have been developed, octreotide being the first such analog to come into clinical use. Other analogs include octreotide-LAR, lanreotide, and vapreotide. Somatostatin analogs can be expected to reduce circulating IGF-I levels by 30 –50% in nonacromegaly patients (36, 37). The antineoplastic activity of the somatostatin analogs could result from a reduction of GH and IGF-I secretion, which would, in turn, have an inhibitory effect on cell proliferation. As the somatostatin analogs also inhibit secretion of many gastrointestinal hormones, including gastrin, cholecystokinin (CCK), secretin, and bombesin, which have been implicated as mitogens for a variety of neoplastic cells, modulation of these gastrointestinal hormones may also be responsible for the antineoplastic activity of somatostatin an-

June, 2000

IGFs AND NEOPLASIA

219

alogs. Furthermore, a more “direct” inhibitory effect of these compounds on cell proliferation has been demonstrated. Somatostatin analogs have been shown to stimulate tyrosine phosphatase activity in certain cell lines, an effect that antagonizes the mitogenic effect of growth factors acting on tyrosine kinase receptors such as epidermal growth factor (EGF), bFGF, and IGF-I (38, 39). This direct mode of action could likely account for the antiproliferative effect of somatostatin analogs observed in vitro. C. GH receptor antagonists

Pegvisomant (B2036-PEG) is a recombinant protein that is structurally similar to natural human GH with the exception that it contains 9 amino acid substitutions. It binds to the GH receptor but does not initiate signal transduction, thereby functioning as a competitive antagonist with natural GH (Fig. 2). Although the half-life of natural GH is only approximately 20 min, pegvisomant has several covalently attached polyethylene glycol molecules that significantly prolong its half-life (⬃100 h), allowing it to be administered subcutaneously on a once-daily basis. Only a limited number of studies have been performed with pegvisomant in respect to analyzing effects on tumor growth. Our laboratories, however, have demonstrated that it is possible to achieve at least a 75% reduction in circulating IGF-I concentrations when pegvisomant is administered to immunocompromised mice (Fig. 3). D. IGF-I receptor antibodies and analogs of IGF-I

The IGF-I receptor plays a critical role in the establishment and maintenance of the transformed phenotype. Kaleko et al. (40) in 1990 demonstrated that NIH 3T3 cells overexpressing IGF-I receptor formed tumors when injected in nude mice while nontransformed cells did not. Sell et al. in 1993 analyzed the effect of IGF-I receptor expression on mice embry-

FIG. 2. Pegvisomant functions as a competitive antagonist at the GH receptor. Normal GH has two binding sites, each of which must bind to a separate GH receptor to initiate signal transduction (left part of inset). Pegvisomant functions as a competitive antagonist because binding site 1 is functional and binding site 2 is not (right part of inset). Because pegvisomant is present in molar excess when compared with normal GH, it occupies the majority of available binding sites, resulting in a dose-dependent decrease in GH-stimulated IGF-I production by the liver.

FIG. 3. Dose response to the competitive GH receptor antagonist, pegvisomant. Serum IGF-I concentration in mice after 1 week of treatment with vehicle or 50 –500 mg/kg/day of pegvisomant.

onic fibroblasts transfected with Simian virus 40 large tumor antigen (41). Although the growth of the wild-type fibroblasts was stimulated more than 2-fold by SV 40 Tag, the transfection had no significant effect on cell growth on the cells lacking the IGF-I receptor. Furthermore, the wild-type cells were overtly transformed whereas the IGF-I receptordeficient cells continued to be contact inhibited. When the IGF-I receptor-deficient cells were transfected with an expression vector for human IGF-I receptor cDNA, growth equaled the wild-type cells. Resnicoff et al. in 1995 demonstrated that rat C6 glioblastoma, human and mouse melanoma, and rat rhabdomyosarcoma cells expressing the antisense IGF-I receptor vector to IGF-I receptor mRNA, which reduced IGF-I receptor expression by up to 70%, were associated with significantly decreased growth in vitro and in vivo compared with those with the sense expression vectors (10). Using several different techniques to examine apoptosis, they reported that a decrease in the number of IGF-I receptors caused the tumor cells to undergo increased apoptosis in vitro and in vivo. Liu et al. (42) in 1998, using an antisense expression vector to the IGF-I receptor in the mouse neuroblastoma cell line, demonstrated reduced proliferation in vitro and an increase in apoptotic cells. The transfected cells were unable to form tumors when allografted into any of the A/J mice. In addition, antisense plasmid injection into mice bearing established tumors caused complete tumor regression in half of the animals. Similar results were not obtained in studies using the immunocompromised scid mice, suggesting a possible immune mediated mechanism of IGF-I receptor down-regulation on tumorigenesis. The expression of dominant negative mutants of the IGF-I receptor have also been demonstrated to have antitumor effects in vitro and in vivo. Reiss et al. (43) transfected human colon, lung, prostate, kidney, and ovarian cancer cell lines with plasmids expressing the dominant negative mutant of the IGF-I receptor, 486/STOP, which has a frameshift mutation resulting in a stop codon at residue 486. They demonstrated a ⬎75% inhibition in colony formation in soft agar in all transfected clones. The in vivo tumorigenicity of the cells expressing the negative mutant of the IGF-I receptor was also significantly decreased when compared with the wild-type cells. Furthermore, when the transfected cells were coin-

220

KHANDWALA ET AL.

jected with a tumor forming cell line, there was significant inhibition of tumor growth, which suggested that the dominant negative mutant has a bystander effect. A monoclonal antibody to the IGF-I receptor (␣IR3) was developed in 1983 (44). This antibody binds to the ligandbinding domain on the IGF-I receptor and inhibits IGF-Imediated effects. This antibody acts as a competitive inhibitor and does not have a direct cytotoxic effect as its inhibitory effect can be overcome by increasing IGF-I concentration. Because IGF-II and insulin also bind to the IGF-I receptor, the use of this antibody has also been shown to inhibit cell growth in response to these growth factors. IGF-I peptide analogs that inhibit IGF-I receptor autophosphorylation have been developed (45, 46). Blakesley et al. (47) in 1996 reported a significant reduction in tumor growth in mice injected with fibroblasts which expressed mutations at the tyrosine residue of the carboxy terminus of the IGF-I receptor. E. IGFBPs

As reviewed previously, the IGFs circulate in combination with six high-affinity binding proteins, IGFBP 1– 6. The mammalian IGFBPs contain three distinct domains of roughly equivalent sizes: the N-terminal, the midregion, and the Cterminal. The N- and C-terminal domains are conserved, and the midregion is highly variable. The human IGFBPs share approximately 36% similarity. The C-terminal domain is responsible primarily for high-affinity binding, whereas the N-terminal and perhaps the midregion are involved in lowaffinity binding. The high-affinity IGFBPs modulate IGF bioavailability by undergoing proteolysis and generating fragments with reduced or no affinity for the IGFs. In the case of human IGFBP-1, posttranslational phosphorylation has been demonstrated to enhance IGF binding (7). In addition to modulating IGF bioavailability and thus IGF-dependent functions, the IGFBPs also have functions that are unrelated to IGFs and thus are IGF independent. The C-terminal domain and the midregion seem to be involved in IGF-independent actions. In addition to the IGFBPs, the IGFBP-rPs can have actions that are independent of IGFs on cell growth. Recombinant forms of these proteins, therefore, are potential therapeutic tools.

Vol. 21, No. 3

1991 demonstrated the presence of IGF-I in cystic fluid aspirated from 5/5 astrocytomas or glioblastomas (range 3.4 – 178 ␮g/liter). Corresponding normal values were 3 ␮g/liter in cerebrospinal fluid and 388 ␮g/liter in serum. In a follow-up study, however, primary cultures from 12 human glioma surgical specimens did not demonstrate the presence of IGF-I in the conditioned media (51). Sandberg-Nordqvist et al. (52) in 1993 demonstrated IGF-I immunoreactivity in 6/9 gliomas and suggested that there appeared to be a positive correlation between IGF-I immunoreactivity and the histopathological grade of the tumor. In a study of 39 astrocytomas (World Health Organization grades II-IV), Hirano et al. (53) in 1999 demonstrated that there is indeed a positive correlation between IGF-I immunoreactivity and histopathological grade (Table 1). They also determined that there was a positive correlation between IGF-I and cell proliferation rates by examining the relationship between IGF-I and Ki-67 (MIB-1) labeling indices. Additional findings of note include the observation that in the high-grade tumors, IGF-I immunoreactivity was greatest in perivascular areas. Proliferating microvessels exhibited more intense staining than nonproliferative vessels. Reactive astrocytes at the margins of tumor infiltration also demonstrated high levels of IGF-I expression. IGF-I expression has also been demonstrated to be important in immune modulation in some syngeneic animal models. Rat C6 glioma cells express IGF-I and, when implanted into BDX rats, the animals develop rapidly growing tumors (54). IGF-I antisense transfected cells lose tumorigenicity. A CD8⫹ glioma-specific immune response is present in the tumors with the antisense expression vector and absent in those with IGF-I expression. The findings indicate that IGF-I expression may facilitate tumor growth in this animal model by allowing it to escape immune detection. 2. IGF-II expression. Sandberg et al. (48) in 1988 demonstrated that IGF-II mRNA is abundantly expressed in fetal brain but only minimally detectable in normal adult brain. Northern analysis identified approximately a 5- to 50-fold increase in IGF-II mRNA levels in 4/4 human glial tumors as compared with fetal brain. Glick et al. (50) in 1991 identified IGF-II in cyst fluid aspirated intraoperatively in 3/5 glial tumors. The TABLE 1. IGF-I immunoreactivity and IGF-I receptor expression according to tumor grade by World Health Organization (WHO) classification

IV. Central Nervous System Neoplasms A.Glioblastomas/astrocytomas

1. IGF-I expression. Sandberg et al. (48) in 1988 demonstrated that IGF-I mRNA was present in both adult and fetal human brain tissue using slot blot and Northern analysis. Within the adult brain, the following rank order of IGF-I expression was observed: pons ⬎ cerebellum ⬎ cerebral cortex ⬎ thalamus. In comparison, the concentrations measured in three human gliomas and one anaplastic astrocytoma were approximately 1.1- to 4.0-fold greater than the highest levels observed in normal brain tissue. Antoniades et al. (49) in 1992 confirmed the presence of IGF-I expression in 10/10 human astrocytoma specimens with in situ hybridization and immunohistochemistry. Using an IGF-I immunoassay, Glick et al. (50) in

Grade

Tissue sectiona

IGF-I staining ratiob

II III IV

19 15 29

22.6e 26.6 41.1f

Mean rank values IGF-I stainingc intensity

IGF-I receptor expressiond

29.9 28.9 35.1

22.6 28.8 37.2

[Reproduced with permission from H. Hirano et al.: Neuro-Oncology 1:109 –119, 1999 (53).] The IFG-I staining ratio (the percentage of cells within a tumor specimen demonstrating IGF-I immunoreactivity) correlated with WHO histopathological grade. The intensity of IGF-I staining, however, did not demonstrate a similar correlation. a The number of tissue sections examined. b Spearman’s rank correlation coefficient is 0.489 (P ⬍ 0.0001). c Spearman’s rank correlation coefficient is 0.143 (P ⫽ 0.2586). d Spearman’s rank correlation coefficient is 0.292 (P ⫽ 0.0216). e vs. f, P ⬍ 0.005.

June, 2000

IGFs AND NEOPLASIA

concentration in the cyst fluid in the IGF-II positive tumors ranged from 3.9 –131 ␮g/liter. IGF-II concentrations were 3.3 and 564 ␮g/liter in cerebrospinal fluid and serum, respectively. In a later study, Glick et al. (51) identified IGF-II in the conditioned media in 4/12 glioma primary cultures. Lichtor et al. (55) in 1993, using Northern blot analysis, did not identify IGF-II mRNA expression in two intermediate grade astrocytomas, three anaplastic astrocytomas, or two glioblastomas. No significant IGF-II mRNA expression was found in the two glioma cell lines, U-87 MG and T-98 G. Hultberg et al. (56) in 1993, also by Northern blot analysis, were unable to detect IGF-II mRNA expression in any of the 9 glial tumors studied. In contrast, Antoniades et al. (49) using in situ hybridization demonstrated the presence of IGF-II mRNA in 10 human glial tumors. 3. IGF receptor expression. Using [125I]-labeled ligand, Sara et al. (57) in 1986 identified IGF-I specific binding in plasma membrane preparations from both normal brain tissue and glioblastomas. No statistically significant binding differences were observed between the two groups, each of which were comprised of 6 specimens. Gammeltoft et al. (58) in 1988, also using a competitive ligand binding assay, identified IGF-I binding in 6/6 human glioma cell lines. Glick et al. (59) in 1989 also confirmed IGF-I receptor expression in human glioma specimen by [125I]-labeled IGF-I binding studies. Merrill and Edwards (60) in 1990 studied 18 glioma specimens for IGF-I receptor expression and found an increase in IGF-I binding sites in some of the gliomas. The mean number of IGF-I binding sites was 68 (20 –133) pmol/g in normal brain tissue and 195 (60 –356) pmol/g in the glioma specimens. No difference was detected in affinity characteristics. Cross-linking studies demonstrated that glial tumors expressed the same lower molecular mass (⬇ 118 kDa) ␣-subunit as is expressed in normal brain tissue. In the cell lines derived from the glioma specimens, however, a larger (⬇ 133 kDa) ␣-subunit was present. Using [125I] -labeled IGF-II binding studies, Sara et al. (57) also demonstrated an approximately 2-fold increase in IGF-II binding as compared with normal brain tissue. Gammeltoft et al. (58) found 2 to 5 times more type II IGF binding sites than type I sites. Type II sites bind IGF-II with 10 times greater affinity than IGF-I; type 1 binding sites bind IGF-I and IGF-II with approximately equal affinity. 4. IGF effects on tumor growth. The mitogenic effect of exogenous IGF-I on human glioma cell lines was studied by Merrill and Edwards (60). Using [3H]thymidine incorporation as a tumor growth marker, they reported that 9/10 glioma cell lines responded to IGF-I with increases in [3H]thymidine incorporation that ranged from 1.3 to 4 times the control value. A dose-dependent response to IGF-I was observed. The effect of IGF-I receptor expression on the in vitro and in vivo growth of glioma cell lines has been analyzed by many investigators (61– 65). Resnicoff et al. (66) in 1994 reported complete inhibition of IGF-I-stimulated growth in vitro in C6 rat glioblastoma cells incubated with antisense oligodeoxynucleotides to the IGF-I receptor mRNA. Cells stably transfected with a plasmid expressing an antisense IGF-I receptor mRNA also demonstrated near-complete in-

221

hibition of IGF-I- and IGF-II-stimulated growth. This was associated with a reduction in IGF-I receptor phosphorylation and correlated with approximately 50% reduction in IGF-I binding sites. BD-IX rats injected with C6 glioma cells stably transfected with the antisense IGF-I receptor plasmids did not develop tumors, whereas all animals injected with wild-type or sense cells developed tumors within a week. They also demonstrated inhibition of tumor formation when IGF-I receptor antisense C6 cells were injected 3 weeks before wild-type cells, whereas prior injection of sense C6 cells provided no protection against subsequent tumor development. Furthermore, complete regression of established wildtype tumors was seen within 2 weeks of injecting IGF-I receptor antisense cells in the opposite flank. 5. Summary. Many studies have demonstrated that most gliomas express the necessary receptors to respond to IGF stimulation and do so in a wide variety of in vitro and in vivo models. The recent study by Hirano et al. (53), demonstrating that there is a correlation between endogenous IGF-I expression and histopathological grade, is important confirmation of earlier studies suggesting that autocrine IGF production is of functional significance in the clinical setting. Animal studies targeting the IGF-I receptor, such as those by Resnicoff et al. (66), have been sufficiently promising that clinical trials using this strategy have been initiated. How effectively the beneficial effects observed in animals translate into patients should therefore be at least partially answered as the results of these trials become known. B. Meningiomas

1. IGF-I expression. Glick et al. (50) in 1991 measured IGF-I by RIA in the cystic fluid aspirated from two meningiomas, obtaining concentrations of 216 and 32 ng/ml, respectively. In a subsequent study, IGF-I concentrations, indicative of autocrine production, were measurable in the conditioned media of 5/12 human meningioma primary cell cultures (51). Antoniades et al. (49) detected IGF-I mRNA and IGF-I protein expression in three meningioma specimens and one control human meninges specimen. IGF-I immunoreactivity was detected in 6/12 human meningioma specimens by Lichtor et al. (55) in 1993. 2. IGF-II expression. Lichtor et al. (67) in 1991 demonstrated IGF-II mRNA using Northern blot analysis in 2/2 human meningioma specimens. Glick et al. (50) did not detect IGF-II in cyst fluid aspirated intraoperatively from two meningiomas. In a subsequent in vitro study using human meningioma cell lines, they detected IGF-II in the culture media of 6/11 tumors (51). Antoniades et al. (49) demonstrated IGF-II mRNA expression in three meningiomas and one control meningeal specimen using in situ hybridization. Hultberg et al. (56) demonstrated IGF-II mRNA in all four meningioma specimens by Northern blot analysis and in 3/4 specimens by RIA. In the IGF-II-positive tumors, the levels measured by RIA were 40, 144, and 160 ng/g. Sandberg- Nordqvist et al. (68) in 1997, studied the expression of IGF-II mRNA and IGFBP-2 mRNA. In all of the anaplastic or atypical meningiomas, there was a high ratio of IGF-II to IGFBP-2 mRNA levels. In a 5-yr follow up period of the 8 patients with a high

222

KHANDWALA ET AL.

IGF-II to IGFBP-2 ratio, 4 died from complications of the tumor, 2 had recurrence, and 2 were lost to follow-up. Of the 14 patients with a low IGF-II to IGFBP-2 ratio, no tumorassociated deaths or recurrences were observed, leading the authors to postulate that a high IGF-II to IGFBP-2 mRNA ratio is a sign of biologically aggressive behavior in meningiomas. 3. IGF receptor expression. Kurihara et al. (69) in 1989, using [125I]-labeled ligand, demonstrated the presence of highaffinity IGF-I binding sites in 8/8 of the meningioma specimens examined. Antoniades et al. (49) identified IGF-I and IGF-II receptor mRNA in 3 meningioma specimens using in situ hybridization. Lichtor et al. (55) demonstrated IGF-I receptor immunoreactivity in 4/12 meningiomas. 4. IGF effects on tumor growth. Kurihara et al. (69) demonstrated that IGF-I increased [3H]thymidine incorporation in primary meningioma cultures in a dose-dependent manner. A maximal response of approximately 350% of control was observed at an IGF-I concentration of 10⫺8 m in serum free conditions. Tsutsumi et al. (70) in 1994 also confirmed that IGF-I was a mitogen for meningiomas. [3H]thymidine incorporation was increased 200-225% over control by IGF-I in a dose-dependent manner. Friend et al. (71) in 1999 analyzed the mitogenic effect of IGF-I on primary cultures of 14 human meningioma specimens. [3H]thymidine incorporation was increased by 21, 43, and 176% of control in response to IGF-I doses of 1, 5, and 10 ␮g/liter, respectively. The GH receptor antagonist, B2036-PEG (pegvisomant), decreased [3H]thymidine incorporation in vitro. Friend et al. (72) in 1999 reported that the GH receptor antagonist, pegvisomant, decreased meningioma xenograft growth in nude mice. After 8 weeks of treatment, the tumor volume in the pegvisomant group had decreased by 38% of the initial volume, whereas the tumor volume in the vehicle-treated group increased by 23%. The serum IGF-I concentration in the pegvisomant group decreased by 20%. 5. Summary. As with gliomas, a number of studies have demonstrated that meningiomas express the necessary receptors to respond to IGF stimulation; the GH receptor also appears to be ubiquitously expressed. Accordingly, activation of these receptors, particularly the IGF-I receptor, has been demonstrated to stimulate mitogenesis. Endogenous IGF-II production by meningiomas, specifically in association with low IGFBP-2, appears to be an accurate predictor of aggressive behavior and increased risk of mortality. This observation, made by Sandberg-Nordqvist et al. (68) in 1997, is likely an illustration of the importance of autocrine IGF-II production in propagating rapid tumor growth. High levels of IGFBP-2 production would appear to ameliorate the stimulatory actions of IGF-II, suggesting that the binding protein environment is perhaps as critical a factor as the IGF production itself. In vivo attempts to influence tumor growth by modulating GH or the IGFs have been limited, until recently, by the relative lack of reliable animal models. Using a model developed by Jensen et al. (73), in which primary cultures of human tumors are xenografted into the flank of immunocompromised mice, the authors have demonstrated that the

Vol. 21, No. 3

GH receptor antagonist, pegvisomant, significantly inhibits tumor growth (72). A newly developed meningioma model, in which human tumors are implanted orthotopically, should permit more extensive analysis concerning the efficacy of targeting GH and the IGFs in these tumors (74).

V. Gastrointestinal Neoplasms A. Colon cancer

1. IGF-I expression. Tricoli et al. (75) in 1986 demonstrated the presence of IGF-I mRNA in normal colonic epithelium using Northern blot analysis. They analyzed 20 human colon cancer specimens and found that IGF-I mRNA levels were mildly elevated (3- to 5-fold) in 20% of the tumors. Culouscou et al. (76) demonstrated IGF-I production by RIA in the conditioned medium from the human colonic adenocarcinoma HT-29. 2. IGF-II expression. Tricoli et al. (75) demonstrated the presence of IGF-II mRNA by Northern blot analysis in normal colonic epithelium and colon cancer. In approximately 40% of colon cancer specimens, the IGF-II mRNA was increased 10- to 50-fold compared with the normal colonic mucosa. This increase in IGF-II mRNA levels was limited to cancers involving the rectum and rectosigmoid with 60% of rectal and 50% of rectosigmoid cancers overexpressing IGF-II. None of the tumors of cecal or sigmoid origin demonstrated enhanced IGF-II mRNA expression. Among the IGF-IIexpressing cancers, the Dukes C tumors possessed higher IGF-II levels as compared with Dukes B tumors, leading the authors to suggest that increased IGF-II mRNA expression may be a marker for aggressive distal colon cancers. Lambert et al. (77) in 1990 examined 21 surgical specimens and found that, compared with the normal colonic epithelium, 30% of colon cancer specimen had IGF-II mRNA overexpression. The amount of overexpression ranged from 2– 800 times that seen in normal tissue. No correlation between overexpression of IGF-II mRNA and the site of the tumor was observed in this study. Zarrilli et al. (78) in 1994 demonstrated the presence of IGF-II mRNA in human colon cancer cell line CaCo-2 by Northern blot analysis. IGF-II levels were high in proliferating cells and decreased more than 10-fold when the cells stopped proliferating and differentiated. IGF-II concentrations in the conditioned medium demonstrated a similar pattern in regard to proliferating and differentiated cells. Guo et al. (79) in 1995 identified IGF-II mRNA expression in six human colon cancer cell lines, HCT 116, COLO 205, COLO 320DM, LoVo, DLD-1, and HT 29. They also demonstrated autocrine production of IGF-II by a specific RRA in the conditioned medium of COLO 205 and HCT 116 cells. Kawamoto et al. (80) in 1998 performed IGF-II immunohistochemical staining on 92 colon cancer surgical specimens and 38 normal colon specimens. They found that 74% of cancer specimens stained for IGF-II compared with 11% of normal. There was a positive correlation between IGF-II staining and size, depth of tumor invasion, and proliferative cell nuclear antigen staining. IGF-II-negative tumors were also associated with a statistically significant increased

June, 2000

IGFs AND NEOPLASIA

chance of survival (Fig. 4). Kawamoto et al. (81) in 1999, using in situ hybridization and immunohistochemical staining, demonstrated that normal liver tissue at the invasive margins of metastases overexpresses IGF-II. This finding led them to suggest that hepatic IGF-II might be an important paracrine factor in propagating the growth of hepatic metastases. Freier et al. (82) in 1999, using a RNase protection assay, reported that IGF-II mRNA was increased 40-fold in six human colon cancer specimens compared with adjacent normal colonic mucosa, while protein levels were twice as abundant. 3. IGF receptor expression. Pollak et al. (83) demonstrated the presence of specific binding sites for IGF-I in colon carcinomas using competitive binding techniques in 1987. RouyerFessard et al. (84) demonstrated the presence of IGF-I receptors in normal colonic epithelium using a similar competitive binding methodology. The presence of IGF-I receptors on the human colon cancer cell lines, HCT 116 and CoLo-205, and several human colon cancer specimens was demonstrated by Guo et al. (85). Adenis et al. (86) in 1995 examined 20 human colon cancer surgical specimens and 26 normal colon specimens and observed no significant differences in terms of IGF-I receptor concentration between malignant and normal colorectal tissues. Zenilman and Graham (87), using a quantitative PCR assay, did not observe a difference in IGF-I receptor mRNA expression between malignant and adjacent normal colonic epithelium. In 1995, Guo et al. (79) reported the presence of both IGF-I and IGF-II receptors in COLO 205 cells. Freier et al. (82) in 1999, using an RNase protection assay, reported that IGF-II receptor mRNA is approximately 2.25 times more abundant in adenocarcinoma of the colon than in adjacent normal colonic epithelium. 4. IGFBPs. Michell et al. (88) in 1997 analyzed the effect of IGFBPs on [3H]thymidine incorporation in the human colon cancer cell lines, COLO205, HT29, and SW620. In fresh medium, IGF-I was more potent in stimulating DNA synthesis than its analog des(1, 3)IGF. This analog differs from IGF-I

FIG. 4. Survival curve in patient groups with colon cancer stratified according to IGF-II staining. The survival curve was significantly higher in the IGF-II-negative group than in the IGF-II-positive group. [Reproduced with permission from K. Kawamoto et al.: Oncology 55:242–248, 1998 (80) © Karger, Basel.]

223

primarily in respect to its having a much lower affinity for the IGFBPs. In the 24-h cell-conditioned media, however, IGF-I was much less potent when compared with its analog. When the conditioned medium was analyzed for the presence of binding proteins by Western blot, it was demonstrated that all the cell lines secreted IGFBP-4 and COLO205, and SW620 secreted IGFBP-2 as well. The authors suggested that the binding proteins in the culture media formed complexes with IGF-I and therefore reduced the amount of free IGF-I available, thus decreasing IGF-I-mediated DNA synthesis. As the des(1, 3)IGF-I has reduced affinity for the binding proteins, no significant change was seen in des(1, 3)IGF-I-induced DNA synthesis. Singh et al. (89) in 1994 reported that antibody to IGFBP-4 increased both basal and IGF-I-stimulated growth in the colon cancer cell line HT-29. They also demonstrated that cells transfected with an antisense vector to IGFBP-4 cDNA had a higher basal and IGF-I stimulated growth rate when compared with cells transfected with the sense or control vectors. Transfection with the sense vector to IGFBP-4 cDNA was associated with a 5- to 10-fold increase in IGFBP-4 expression; however, it was not associated with a further inhibition of basal and IGF-I stimulated cell growth. The observation that exogenous IGF-I was unable to overcome the inhibitory effect of IGFBP-4 overexpression led the authors to suggest that the effect of IGFBP-4 on inhibition of cell growth may be IGF-I independent. Macdonald et al. (90) in 1999 demonstrated that the human colon carcinoma cell line, Caco-2, which was stably transfected with an IGFBP-3 expression construct, grew more slowly in vitro than the cells transfected with a control vector. 5. IGF effects on tumor growth. The effect of IGF-I on the growth of two mouse colon cancer cell lines was studied by Koenuma et al. in 1989 (91). IGF-I, added to serum-free media at a concentration of 10 ␮g/liter, increased the growth of two variants of mouse colon adenocarcinoma 26. The growth of a highly metastatic variant (NL-17) was increased to 490% of control while a variant with lower metastatic potential (NL44) was increased to 279% of control. The number of binding sites, 1.37 ⫻ 105/cell (NL-17) and 1.26 ⫻ 105/cell (NL-44) was similar between the two cell lines. Lahm et al. (92) in 1992 studied the effects of IGF-I and IGF-II on eight human colon cancer cell lines. IGF-I and IGF-II were roughly equipotent in stimulating tumor growth; half-maximal responses were observed in the responsive cell lines at IGF concentrations ranging from 1.9 – 6.5 ␮g/liter. In a follow-up study, Lahm et al. (93) in 1994 demonstrated that a neutralizing monoclonal antibody against the human IGF-I receptor (␣IR3) decreased the growth of 7/12 cell lines. As some responsive lines expressed only IGF-II, not IGF-I, the authors concluded that autocrine IGF-II, by binding to and activating the IGF-I receptor, was propagating tumor growth. Accordingly, the growth-inhibitory effects of ␣IR3 could be overcome by adding either exogenous IGF-I or IGF-II to the culture media. Smith and Solomon (94) in 1988 studied the effect of somatostatin on the growth of human colon cancer cell lines in vivo. Nude mice bearing xenografts of the human colon cancer cell line CX1 developed significantly smaller tumors when treated with 100 –300 ␮g/kg of somatostatin 14 twice

224

KHANDWALA ET AL.

daily. In 1991, Qin et al. (95) studied the effects of the somatostatin analog RC-160 (vapreotide) on in vivo growth of the rat colon cancer cell line, DHD/K12, using a syngeneic model. In the RC-160-treated rats (100 ␮g/kg/day), the final tumor volume was approximately 36% of control. Protein and DNA concentration in the tumors of the treatment group were decreased to 70% and 69%, respectively, of the control values. The percent of bromodeoxyuridine-labeled cells in the treatment group was 35% lower than that in the control group. No similar inhibitory effect was observed on tumor growth in vitro, leading the authors to propose that indirect mechanisms, such as effects of the somatostatin analog on GH/IGF-I, CCK, or gastrin, were mediating the antitumor effects. The in vivo effect of another somatostatin analog, octreotide (SMS 201–995), was studied in mice implanted with the colon cancer cell line CT 26 by Alonso et al. (96) in 1992. Tumor volume, tumor weight, and DNA content were significantly reduced in the mice who received the somatostatin analog. Tumor growth was inhibited by 40% and was accompanied by a prolonged survival (42.5 vs. 48.5 days). The effect of vapreotide (25 ␮g/twice daily) on the growth of hepatic metastases of the human colon cancer cell lines, 320 DM and WidR, was studied by Qin et al. in 1992 (97). The mice were randomized to receive either RC-160 or vehicle after intrasplenic tumor injection. There was a decrease in the incidence (25% vs. 38%) and mean number (60 vs. 177 in 320 DM; 77 vs. 135 in WidR) of hepatic metastases and a corresponding increase in survival times (7 days vs. 20 days) in the mice receiving RC-160. When the same cell lines were implanted subcutaneously into the flank, similar beneficial effects on tumor volumes in the treatment groups were observed. In 1992 Dy et al. (98) studied the effects of octreotide (50 ␮g/kg/day) on the growth of LIM 2412 and LIM 2405 cells implanted subcutaneously in mice. Octreotide inhibited tumor growth by approximately 50%. As inhibitory effects were also observed in in vitro studies, direct effects of octreotide on the tumor, independent of the GH/IGF-I axis, were considered potential mechanisms of action. Duan et al. (99) in 1999 analyzed the effect of pegvisomant on human colon cancer cell line, COLO 205, xenografted in nude mice. After treatment for 16 days, they reported a 39% reduction in tumor volume and a 44% reduction in tumor weight in the pegvisomant group along with reductions in circulating IGF-I and IGFBP-3 levels. 6. Clinical studies. Glass et al. in 1994 measured IGF-I levels in more than 300 healthy people scheduled for a colonoscopy for occult gastrointestinal (GI) bleeding (100). No significant difference in the serum IGF-I concentrations were observed between the group with a normal colonoscopy and the group with colonic polyps or colon cancer. Ma et al. (101) reported the results of a case-control study, nested in the physicians’ health study in 1999. IGF-I and IGFBP-3 levels were measured in more than 14,000 subjects at the beginning of the study. The 193 patients who subsequently developed colon cancer, some up to 15 yr later, had significantly higher baseline IGF-I levels and lower IGFBP-3 levels than age- and weight-matched controls from the same cohort. Iftikhar et al. (102) studied the effects of the somatostatin analog octreotide on tumor kinetic measurements in 12 pa-

Vol. 21, No. 3

tients with primary rectal cancer. Four of the 12 patients who received octreotide had a lower Ki67 immunostaining in the posttreatment biopsy compared with the pretreatment biopsy. None of the six untreated control patients had a lower Ki67 index at the time of the second biopsy. In a randomized trial by Cascinu et al. (103) in 1995, 107 patients with advanced colon cancer refractory to chemotherapy were randomized to receive octreotide 200 ␮g three times per day 5 days/week or best supportive care. The patients in the octreotide group had a median survival time of 20 weeks compared with 11 weeks in the supportive care group. Although there were no objective responses, 45% of the patients randomized to octreotide had stable disease compared with 15% in the supportive care group. Goldberg et al. (104) in 1995 reported the results of an Eastern Cooperative Oncology Group randomized trial in which 260 patients with advanced colon cancer received either octreotide 150 ␮g subcutaneously three times per day or placebo/no treatment. No significant difference was observed in time to progression or survival between the two groups. In neither the 1995 Cascinu study or the Goldberg studies were serum IGF-I levels measured. Cascinu et al., however, in a follow-up study in 1997 (105), did measure IGF-I concentrations. They randomized 75 patients with newly diagnosed colon cancer to receive either octreotide, 200 ␮g subcutaneously three times per day, or placebo for 2 weeks before surgery. There was approximately a 50% reduction in circulating IGF-I levels in the octreotide-treated group (179 vs. 86). They also studied effects of octreotide treatment on tumor cell kinetics. [3H]thymidine labeling and flow cytometry were used to assess S-phase fraction in the tissue obtained from the baseline endoscopic biopsy specimen and the tissue obtained at the time of surgery. The percentage of cells in the S-phase fraction, as measured by [3H]thymidine incorporation, decreased from 18% to 3% in the octreotide group whereas no difference was observed in the placebo group. By flow cytometry, the decrease in S-phase fraction in the octreotide group was decreased from 27 to 22%. Again, no change was observed in the placebo group. 7. Summary. The increased risk of colonic polyps and colon cancer in the acromegalic population is well known. The recent epidemiological study by Ma et al. (101) suggests that even within the normal population, a high IGF-I/IGFBP-3 ratio may increase the risk of colon cancer development. Once tumors develop, the overexpression of IGF-II that occurs in a substantial subset, originally described by Tricoli et al. (75) and subsequently confirmed by Lambert et al. (77), has been clearly demonstrated by Kawamoto et al. (80) to correlate with an aggressive phenotype. Attempts to understand the utility of agents that modulate GH or the IGFs should be analyzed with respect to whether or not the tumors express high levels of IGF endogenously. Tumors without high autocrine IGF production may be the most responsive to agents that only modify circulating or paracrine IGF production. IGF-II-overexpressing tumors, which appear to be more aggressive, will likely require strategies that decrease tumor IGF production, bioavailability, or action at the receptor level.

June, 2000

IGFs AND NEOPLASIA

B. Gastric cancer

1. IGF-I and IGF-II expression. Thompson et al. (106) in 1990 reported the detection of autocrine IGF-II production in the human gastric carcinoma cell line, LIM-1839. IGF-II mRNA was present and IGF-II was measurable in the conditioned medium of cells grown to confluence. No IGF-I mRNA was detectable. Chung and Antoniades (107) in 1992 demonstrated the presence of IGF-I mRNA by in situ hybridization in three human gastric cancer surgical specimens. Expression was also observed in the adjacent normal epithelium. Guo et al. (108) demonstrated IGF-II mRNA in AGS and SIIA gastric cancer cell lines in 1993 by Northern analysis. No IGF-I mRNA was detected in either cell line. 2. IGF receptor expression. Thompson et al. (106) demonstrated the presence of IGF-I and IGF-II receptors by affinity crosslinking and competitive binding studies in LIM-1839 gastric carcinoma cells. Durrant et al. (109) demonstrated the presence of IGF-I receptors by competitive binding in the gastric cancer cell lines, St16, St42, and MKN45. The number of receptors per cell were 250,190 and 310, respectively. Chung and Antoniades (107) demonstrated the presence of IGF-I receptor mRNA by in situ hybridization in human gastric cancer specimens. 3. IGF effects on tumor growth. The mitogenic effect of exogenous IGF-I and IGF-II on human gastric cancer cell line LIM-1839 was reported by Thompson et al. (106). These growth factors increased cell growth 1.6- to 2.0-fold when added to serum-free medium at 20 and 50 ng/ml. The growth-promoting effect was inhibited by ␣IR3, indicating that both IGF-I and IGF-II stimulate growth via the IGF-I receptor. Durrant et al. (109) also demonstrated that IGF-I has mitogenic effects on the St16, St42, and MKN45 gastric cancer cell lines. C. Pancreatic cancer

1. IGF-I and IGF-II expression. Ohmura et al. (110) in 1990 demonstrated the autocrine production of IGF-I in MIAPaCa 2 cells, a human pancreatic cancer line, by measuring IGF-I by RIA in conditioned culture medium. Bergmann et al. (111) did not detect IGF-I mRNA by Northern analysis in four human pancreatic cell lines, including ASPC-1, COLO 357, T3 M4, and PANC-1 cells. In 12 pancreatic surgical specimens, a 32-fold increase was present as compared with the low levels observed in the normal pancreas. 2. IGF receptor expression. Ohmura et al. (110) demonstrated I-labeled IGF-I binding in the human pancreatic cancer cell line, MIA-PaCa 29. Ishiwata et al. (112) in 1997 reported the presence of IGF-II receptor mRNA in six human pancreatic cancer cell lines by Northern analysis (ASPC-1, CAPAN-1, COLO 357, MIA-PaCa-2, PANC-1, and T3 M4). They also found that 7 of 12 pancreatic cancer surgical specimens overexpressed IGF-II receptor mRNA (5.6-fold) in comparison to normal pancreatic tissue. 125

3. IGF effects on tumor growth. Bergmann et al. (111) demonstrated that IGF-I stimulated cell growth in human pancreatic cell lines, ASPC-1 and COLO 357. For the ASPC-1 cells, the

225

one-half maximal and maximal stimulation occurred at 1.8 and 5 nm, respectively. In the COLO 357 cells, the corresponding values were 0.3 and 1.3 nm. They also observed inhibition of IGF-I stimulated growth by ␣IR3. An antisense oligonucleotide to the IGF-I receptor inhibited the growth of ASPC-1 and COLO-357 by 34% and 35%, respectively. D. Other (esophageal, hepatocellular)

a. Esophageal cancer. Oku et al. (113) in 1991 demonstrated that both IGF-I and IGF-II were potent mitogens for the human squamous esophageal cancer cell line TE-3-OS. Both IGF-I and IGF-II increased [3H]thymidine uptake by approximately 300% in a dose-dependent manner with maximal effects being observed at 100 ␮g/liter concentrations of both peptides. The anti-IGF-I receptor antibody, ␣IR3, inhibited growth stimulated by both IGF-I and IGF-II. Chen et al. (114) in 1991 demonstrated IGF-I mRNA by Northern analysis in the human esophageal cancer cell line CE48T/VGH. IGF-I increased cell growth in serum-free media about 3.5-fold, with maximal stimulation being observed at 10⫺ 9 m. [125I]IGF-I binding studies demonstrated 2.5 ⫻ 105 receptors per cell. b. Hepatocellular carcinoma. 1. IGF-I expression. Tsai et al. (115) demonstrated the presence of IGF-I mRNA by Northern analysis in 10 of 10 human hepatoma cell lines in 1988. Su et al. (116) analyzed 7 human hepatoma surgical specimen for the presence of IGF-I mRNA in 1989. Compared with nontumorous hepatic tissue from an adjacent area, the tumors expressed relatively low levels of IGF-I mRNA. Because the expression of GH receptor mRNA in the tumors was also low on a relative basis, the authors proposed that IGF-I mRNA was low as a result of reduced GH stimulation of transcription. 2. IGF-II expression. Cariani et al. (117) in 1988 observed a 40- to 100-fold increase in IGF-II mRNA expression in 9 of 40 liver cancer surgical specimens as compared with normal adult liver. They reported that many liver cancers expressed fetal transcripts (6.0 and 5.0 kb) instead of the 5.3-kb transcript present in the normal adult. Su et al. (116) in 1989 also reported that 4 of 7 human liver cancer surgical specimens had expression of IGF-II fetal transcripts (5.6 and 4.5 kb). The authors suggested the presence of the fetal transcript of IGF-II mRNA was reflective of cellular dedifferentiation. The amount of IGF-II mRNA expression in the tumors was highly variable. Lamas et al. (118) in 1991, using in situ hybridization and immunohistochemistry, demonstrated an overexpression of IGF-II mRNA and protein in malignant cells as compared with surrounding nontumorous cells. D’Errico et al. (119) in 1994 studied IGF-II immunoreactivity in liver sections from 54 patients with hepatocellular carcinoma. IGF-II immunoreactivity was detected in 60% (9 of 15) of the specimens from hepatitis B virus-positive patients and in 26% (10 of 39) of the specimens obtained from hepatitis B virus-negative patients. Rogler et al. (120) in 1994 analyzed the effects of IGF-II overexpression by constructing transgenic mice in which human prepro-IGF-II cDNA was placed under control of the major urinary protein promoter.

226

KHANDWALA ET AL.

This led to an increase of up to 30-fold in serum IGF-II concentration in the transgenic animals compared with controls. Over the course of 18 months, the transgenic mice developed an increased number of tumors in general and hepatocellular carcinoma in particular when compared with control animals. Scharf et al. (121) in 1998 using a Northern blot technique could not detect IGF-II mRNA in the human hepatoma cell line PLC. Ng et al. (122) in 1998 reported repression of the expression of the normal adult IGF-II transcript in 93% (28 of 30) of human hepatoma surgical specimens. Fetal transcripts, however, were present in 40% of the specimens. The nontumorous hepatocytes expressed the adult IGF-II mRNA transcript in 93% of the cases. The tumors from older patients were more likely to express IGF-II. Sohda et al. (123) in 1997 detected an overexpression of IGF-II mRNA by in situ hybridization and immunohistochemistry in 50% (5 of 10) of human hepatocellular carcinoma specimens. Cells that expressed IGF-II were more likely to express the Ki-67 antigen (expressed during active cellular proliferation), suggesting that IGF-II acts as an autocrine or paracrine growth factor for these tumors. 3. IGF receptor expression. Tsai et al. (115) detected the presence of IGF-I receptor mRNA in 10 of 10 human hepatoma cell lines. Scharf et al. (121) identified IGF-I and IGF-II receptor mRNA in the hepatoma cell line PLC. 4. IGF effects on tumor growth. The effects of exogenous IGF-I and IGF-II on DNA synthesis were also demonstrated by Scharf et al. (121). Both growth factors increased [3H]thymidine incorporation in the PLC cell line in a dose-dependent manner. Lin et al. (124) in 1997 observed that high levels of IGF-II were produced by the human hepatoma cell lines HuH-7 and HepG2. Antisense oligonucleotides complementary to IGF-II mRNA led to reduction in IGF-II mRNA and protein content in the cell lines. There was also a decrease in [3H]thymidine incorporation and cell-proliferative activity. The inhibitory effect on cell growth was not observed in those cell lines that did not overexpress IGF-II. VI. Head, Neck, and Pulmonary Neoplasms A. Lung cancer (small cell/non-small cell)

1. IGF-I expression. Minuto et al. (125) in 1986 measured IGF-I concentrations by RIA in tissue extracts from 10 human lung surgical specimens (7 epidermoid/3 adenocarcinoma). The IGF-I concentration in the cancerous tissue was significantly higher than the IGF-I concentration in the surrounding normal lung tissue (615 ⫾ 123 mU/g vs. 234 ⫾ 51). The same group of investigators (126) detected autocrine IGF-I production by RIA in the conditioned culture medium of the human lung cancer cell line CALU-6. Nakanishi et al. (127) in 1988 reported the synthesis of an IGF-I precursor molecule by Western blot analysis by two small cell lung cancer (SCLC) cell lines, NCI-H345 and NCI-N417. Jaques et al. (128) in 1988 detected IGF-I immunoreactivity in the cell pellets and culture media of 11 of 14 separate SCLC cell lines. Macaulay et al. (129) in 1990 demonstrated that the “classic” SCLC cell line HC12, which has neuroendocrine features, produced IGF-I. The “variant” cell line ICR-SC17, which does

Vol. 21, No. 3

not have neuroendocrine features, did not express IGF-I. Reeve et al. (130) in 1990 also measured IGF-I immunoreactivity in the conditioned media of lung cancer cell lines. IGF-I was detected in 2 of 2 “classic” SCLC cell lines (COR-L51, COR-L47) and 3 of 3 “variant” cell lines (COR-L27, COR-L24, COR-L103). No IGF-I immunoreactivity was detected in a large cell (COR-L23) or adenocarcinoma (MOR) line. 2. IGF receptor expression. Nakanishi et al. (127) reported the presence of two high-affinity specific binding sites for 125Ilabeled IGF-I [dissociation constant (Kd) 1.3 and 4.0 nm] in the human SCLC cell line, NCI-H345. Jaques et al. (128) also demonstrated high-affinity IGF-I binding sites in 14 of 14 SCLC cell lines (Kd 0.89 –5.21 nm). The maximum binding (Bmax) ranged from 131 to 1,230 fmol/mg protein. Rotsch et al. (131) in 1992 described the presence of high-affinity IGF-I binding sites in a number of SCLC and NSCLC lines. They also demonstrated the presence of IGF-I receptor mRNA by Northern blot analysis in all of the cell lines. Schardt et al. (132) in 1993 reported that 125I-labeled IGF-II binds with high affinity (70 – 80 pm) to the IGF-I receptor and with low affinity (2– 4 nm) to the IGF-II receptor using receptor assays on microsomal and plasma membranes of the SCLC lines, NCIH841 and NCI-H82. The presence of mannose-6-P enhanced the binding of [125I]IGF-II to the IGF-II receptor. Kaicer et al. (133) in 1993, using a monoclonal antibody, demonstrated the expression of IGF-I receptor in bronchial epithelial cells of normal lung and in 22 of 24 primary lung carcinomas. A total of 17 of 24 of the specimens expressed IGF-II receptor. In this study, 18 of the 24 specimens were squamous cell carcinomas. 3. IGF effects on tumor growth. Jaques et al. (128) demonstrated that IGF-I increased [3H]thymidine incorporation in 2 of 7 human SCLC cell lines. Peak responses were observed at 0.1–1.0 nm. Minuto et al. (126) reported that IGF-I administration resulted in a 52% increase in cell number in the human lung cancer cell line, CALU-6, at concentrations between 10 and 25 ␮g/liter. Nakanishi et al. (127) demonstrated an approximately 250% increase in cell growth in several SCLC cell lines by both IGF-I and IGF-II. On a molar basis, IGF-I was 10- to 100-fold more potent than IGF-II or insulin. The growth-promoting properties of both IGF-I and IGF-II were inhibited by the IGF-I receptor antibody, ␣IR3. Rotsch et al. (131) in 1992 also observed that IGF-I was a potent mitogen, stimulating growth 1.6- to 4.2-fold in a panel of SCLC cell lines and 1.1- to 2.7-fold in a panel of NSCLC cell lines. Zia et al. (134) in 1996 demonstrated that IGF-I increased the growth of the NSCLC cell line, NCI-H1299, approximately 7-fold at a concentration of 100 ng/ml. When the tumors were implanted into nude mice, ␣IR3 significantly inhibited cell growth (134). The mean tumor weights in PBS-treated animals was 8.03 ⫾ 0.35 g after 4 weeks. When ␣IR3 was injected intraperitoneally three times weekly at a concentration of 100 ␮g, the mean tumor weight decreased to 3.40 ⫾ 0.90 g. Lee et al. (135) in 1996 constructed an adenovirus expressing an antisense version of the first 300 bp of the IGF-I receptor (Ad-IGF-Ir/as). This led to approximately a 50% reduction in IGF-I receptor expression in the human lung cancer cell lines, NCI H460 and SCC5. The soft agar clono-

June, 2000

IGFs AND NEOPLASIA

genicity of the transfected NCI H460 cells was reduced by 84%. The intraperitoneal treatment of nude mice bearing established intraperitoneal NCI H460 cells resulted in prolonged survival compared with that achieved with a reporter virus. Long et al. (136) in 1998 studied the effect of IGF-I receptor overexpression on the metastatic behavior of the Lewis lung carcinoma cell line, M-27. M-27 cells were stably transfected with a plasmid vector expressing a full-length cDNA for human IGF-I receptor under the control of the SV-40 promoter. This led to a 3-fold increase in the number of IGF-I binding sites. In their in vitro studies, IGF-I was more potent in stimulating cell growth and [3H]thymidine incorporation in the cell line overexpressing IGF-I receptor as compared with the wild type. Intrasplenic injection of the IGF-I receptor-transfected cell line, but not mock-transfected cells, gave rise to multiple tumor nodules in the liver. Taylor et al. (137) in 1988 analyzed the effect of the somatostatin analog BIM-23014C (lanreotide) on in vivo growth of the human SCLC cell line NCI-H69 after xenografting into nude mice. Treatment with this compound (500 ␮g twice daily) was associated with a prolongation of the lag time for tumor appearance and a significant inhibition of tumor growth rate when compared with control. In 1990, Bodgen et al. (138) evaluated the same compound in vivo against four SCLC cell lines (NCI-H69, NCI-N417, NCI-H345, LX-1) and a NSCLC cell line (H-165). Tumors derived from all cell lines responded, albeit in varying degrees. In some instances, direct infusion around the tumor was superior to injection on the side opposite the tumor. Pinski et al. (139) in 1994 also demonstrated an inhibitory effect of a somatostatin analog RC-160 (vapreotide) on the in vivo growth of an SCLC and an NSCLC cell line in nude mice. A greater than 50% reduction in tumor size was seen with both cell lines. 4. Clinical studies. Macaulay et al. (140) in 1991 reported the results of a small clinical trial of octreotide, 250 ␮g subcutaneously three times daily, in 20 patients with SCLC. Although serum IGF-I levels were reduced to 62 ⫾ 7% of pretreatment levels, no objective antitumor activity was observed in regard to tumor bulk or serum levels of neuronspecific enolase. Cotto et al. (141) in 1994 treated 18 patients with recurrent and/or nonresponsive SCLC with the somatostatin analog, Somatuline (lanreotide), administered as a continuous infusion in doses ranging from 2–10.5 mg/day. No antitumor activity was seen after 28 days of treatment despite reductions in serum IGF-I levels by up to a mean of 36%. Marschke et al. (142) in 1999 also did not observe significant antitumor activity of 2,000 ␮g three times daily of a somatostatin analog in a phase II study involving 18 patients with extensive SCLC. The median survival was 106 days. 5. Summary. IGF receptor expression is common in both SCLC and NSCLC. In vivo studies using multiple cell lines have been encouraging. Experiments designed to block IGF action such as those by Lee et al. (135), using an adenovirus expressing an antisense IGF-I receptor vector, have demonstrated potent antitumor activity. Conversely, IGF-I receptor overexpression experiments such as those performed by Long et al. (136) indicate that increasing IGF-I receptor numbers can dramatically increase metastatic potential. The lim-

227

ited clinical studies that have been performed, however, have been disappointing. These studies, which have primarily involved the use of somatostatin analogs in the treatment of SCLC, did not demonstrate a survival benefit. Possible reasons for this include the relatively modest GH/IGF-I inhibition achieved with somatostatin analogs and their inability to affect endogenous IGF production. B. Thyroid cancer

Tode et al. (143) in 1989 demonstrated autocrine production of IGF-I by RIA in the conditioned medium of human thyroid follicular cells in primary culture. Exogenous TSH increased the secretion of IGF-I in the medium. Minuto et al. (144) in 1989 measured IGF-I by RIA in human thyroid surgical specimens. In their study, IGF-I levels were higher in nodular and cancerous thyroid tissue compared with normal thyroid tissue, suggesting that IGF-I may be involved in goiter/cancer pathogenesis. Onoda et al. (145) in 1992 demonstrated autocrine production of IGF-I by a human papillary thyroid cancer cell line. IGF-I immunoreactivity was present in the conditioned medium of the cell line, and IGF-I mRNA was detected by a PCR-based methodology. The presence of receptors for IGF-I on normal as well as malignant human thyroid epithelium was demonstrated in 1989 by Yashiro et al. (146). Using affinity cross-linking, they demonstrated an increase in [125I]IGF-I binding to human surgical thyroid cancer specimens when compared with normal or benign thyroid neoplasms. Tode et al. (143) demonstrated the presence of IGF-I receptor by binding studies on human thyroid follicular cells in primary culture. Yashiro et al. (147) in 1991 demonstrated the presence of IGF-II receptors in human thyroid surgical specimen by [125I]-IGF-II binding studies and reported an overexpression of IGF-II receptors in papillary and follicular carcinoma specimen when compared with nonmalignant thyroid tissue. Tramontano et al. (148) in 1986 demonstrated the mitogenic effect of exogenous IGF-I on rat thyroid follicular cell line, FRTL5. They showed that IGF-I had an additive effect with TSH on cell proliferation and thymidine incorporation. DNA synthesis was increased up to 9-fold with IGF-I and up to 30-fold with IGF-I and TSH together. Onoda et al. (145) demonstrated the mitogenic effect of IGF-I on a human papillary thyroid cancer cell line. IGF-I increased growth up to 200% over control. The effect of IGF-I on cell growth was inhibited by ␣IR3, the IGF-I receptor antibody. VII. Female Reproductive Neoplasms A. Breast cancer

1. IGF-I expression. Huff et al. (149) in 1986 reported the secretion of IGF-I by human breast cancer cells by measuring IGF-I by RIA in the conditioned medium of four human breast cancer cell lines. They also detected IGF-I mRNA by Northern analysis in all four cell lines. The estrogen-independent cell lines, MDA-231 and Hs578T, secreted several fold more IGF-I than the estrogen-dependent cell lines, MCF-7 and ZR-75–1. Several other investigators, however, did not find autocrine IGF-I production by breast cancer cells.

228

KHANDWALA ET AL.

In 1989, Yee et al. (150), using a ribonuclease protection assay, were not able to detect IGF-I mRNA in any of the 11 breast cancer cell lines they examined. They detected IGF-I mRNA in 12 of 20 breast cancer surgical specimens, but in situ hybridization studies demonstrated that expression was limited to the stromal elements. No IGF-I expression was observed in either normal or malignant epithelial cells. Paik (151) in 1992 studied the expression of IGF-I in 10 human surgical breast cancer specimen using in situ hybridization. IGF-I mRNA was detected only in the stromal cells adjacent to normal and benign breast tissue. No IGF-I mRNA expression was seen in the stromal cells associated with the tumor cells. IGF-I mRNA was not detected in either the normal or malignant epithelial cells. In 1998, Gebauer et al. (152) examined the MCF-7 breast cancer cell line, 6 primary breast cancer cultures, and breast cancer surgical specimen from 11 patients using a PCR-based methodology. None of the primary cultures or the MCF-7 cancer cell line expressed IGF-I mRNA. Expression was seen in 6 of 11 of the surgical specimens. The expression of IGFs in some of the surgical specimens, but not in the cancer cell lines, again suggested that in the breast, IGF-I is predominantly of stromal origin. 2. IGF-II expression. In 1989, Osborne et al. (153) studied six breast cancer cell lines (BT20, ZR75–1, MDA-231, MDA-330, T47D, and MCF-7L) for endogenous expression of IGF-II. Only two cell lines, TD47D and MCF-7L, were shown to have mRNA for IGF-II by Northern blot and RNase protection assay and to have significant secretion of IGF-II in the culture medium as measured by RIA and RRA. Paik (151) studied the IGF-II expression of 10 human surgical breast cancer specimen using in situ hybridization. He demonstrated that IGF-II mRNA expression was present in the stromal elements surrounding both normal and malignant breast epithelium. In one case of carcinoma in situ, IGF-II mRNA expression was detected in the malignant epithelium. Gebauer et al. (152) detected IGF-II mRNA in 1 of 7 breast cancer cell lines and in 7 of 11 surgical specimens, leading the authors to conclude that IGF-II, like IGF-I, is produced predominantly in the stromal tissue. 3. IGF receptor expression. Furlanetto et al. (154) in 1984 observed the presence of high-affinity type I IGF binding sites in four human breast cancer cell lines (MCF-7, MDA-231, T47-D, and HBL-100). Myal et al. (155) demonstrated similar high-affinity binding in T47D cells the same year. Pollak et al. (156) and De Leon et al. (157) demonstrated high-affinity binding in a variety of breast cancer cell lines in 1988. Peyrat and Bonneterre (158) demonstrated the presence of IGF-I receptors by competitive binding and cross-linking techniques in 5 breast cancer cell lines (BT-20, MCF-7, T-47D, HBL 100, and MDA-MB-231) in 1992. They also demonstrated IGF-I receptor mRNA by Northern analysis in each of the cell lines. The concentration of IGF-I receptor was higher in the estrogen-dependent cell lines than in the estrogen-independent cell lines. There was also a positive correlation seen between the estrogen, progesterone, and PRL receptors and IGF-I receptor expression. They also demonstrated the presence of IGF-I receptor and IGF-I receptor mRNA in human breast cancer biopsy specimens. IGF-I receptor levels in the

Vol. 21, No. 3

breast cancer biopsy specimen were higher. Gebauer et al. (152) demonstrated the presence of IGF-II receptor and IGF-II receptor mRNA in the MCF-7 breast cancer cell line, 6 primary breast cancer cell cultures from metastatic breast cancer patients, and breast cancer surgical specimens from 11 patients. 4. IGFBPs. Clemmons et al. (159) in 1990 measured IGFBP immunoreactivity in the conditioned media of breast cancer cell lines and observed that the estrogen receptor-positive lines secreted predominantly IGFBP-2 while the estrogen receptor-negative lines secreted IGFBP-1 and 3. Pratt and Pollak (160) in 1993 studied the effect of estrogen and the antiestrogens, tamoxifen and ICI 182,780, on cell growth and IGFBP expression in the estrogen-dependent cell line, MCF-7. The antiestrogens caused an increase in IGFBP-3 and a decrease in IGFBP-4 secretion, as measured by Western blot in the culture media of the cell lines, whereas treatment with estrogens had the opposite effect. Furthermore, the antiestrogen ICI 182,780 inhibited IGF-I-stimulated MCF-7 cell proliferation. The authors suggested that the antiestrogenmediated increase in IGFBP-3 was the cause of decreased cell growth. The same authors (161) in 1994 reported that exogenous IGFBP-3 inhibited estrogen-mediated cell proliferation and [3H]thymidine incorporation in MCF-7 cell line. Huynh et al. (162) in 1996 also confirmed the negative and positive effects, respectively, of estrogens and antiestrogens on IGFBP-3 expression in MCF-7 breast cancer cell line. Exogenous recombinant IGFBP-3 inhibited basal and estrogenstimulated growth. The antiestrogen, ICI 182,780, along with increasing IGFBP-3 concentration, also decreased basal and estrogen-stimulated growth. When antisense oligodeoxynucleotides to IGFBP-3 were added, the inhibitory effect of the antiestrogens on cell proliferation was attenuated, leading the authors to suggest that a part of the antiproliferative effect of antiestrogens in breast cancer cells may be due to the increase in IGFBP-3 production. Oh et al. (163) in 1993 demonstrated the secretion of IGFBP-3 in the cultured medium of the estrogen-independent breast cancer line Hs578T using Western blot and immunoprecipitation studies. This particular cell line does not respond to the mitogenic effect of either IGF-I or IGF-II. The presence of IGFBP-3 receptors was also demonstrated by affinity cross-linking studies. Exogenous IGFBP-3 was demonstrated to cause significant decrease in cell proliferation and [3H]thymidine incorporation in serum-free conditions. The inhibitory effect of IGFBP-3 on cell growth was suggested to be IGF independent as IGFs are not mitogenic for this cell line. Furthermore, when exogenous IGF-I or IGF-II was added to the medium, there was an attenuation of IGFBP-3 inhibitory effects on cell growth. However, when analogs of the IGFs with reduced affinity to IGFBP-3 were added, there was no reduction in the inhibitory effect of IGFBP-3. The authors suggested that the IGFs formed complexes with IGFBP-3 and hence made IGFBP-3 unavailable to exert its growth-inhibitory effects whereas the analog, because of its reduced affinity for IGFBP-3, left more free IGFBP-3 to exert its effects. Nickerson et al. (164) in 1997, however, suggested that the IGF-dependent effects of IGFBP-3 are more important in

June, 2000

IGFs AND NEOPLASIA

regulating cell growth. They studied the effects of IGFBP-3 and the antiestrogen ICI 182,780 on apoptosis (measured by cell death enzyme-linked immunosorbent assay) and DNA synthesis (by [3H]thymidine incorporation) in the estrogenresponsive cell line MCF-7. Both ICI 182,780 and IGFBP-3 caused a greater than 3-fold increase in apoptosis along with significant decreases in [3H]thymidine incorporation. They also demonstrated that the antiestrogen increased IGFBP-3 production and the inhibitory effect on cell growth of the antiestrogen was mediated, at least in part, by IGFBP-3. Exogenous IGF-I, in the presence of the antiestrogen and IGFBP-3, increased apoptotic rates whereas the analog of IGF-I with reduced affinity to IGFBP-3, potentially making more “free’ IGF-I available to act on the IGF-I receptor, decreased apoptotic rates. Martin et al. (165) in 1995 analyzed the production of IGFBP-3 and -6 in the estrogen-dependent cell line MCF-7. They reported a significant increase in the secretion of IGFBP-3 and -6 in the conditioned medium after exposure of the cell line to retinoic acid, (Bu)2cAMP, and forskolin (a stimulator of adenylate cyclase). They also demonstrated that MCF-7 cells, after incubation with retinoic acid and forskolin, had an attenuated response to IGF-I-stimulated [3H]thymidine incorporation. They suggested that the 6- and 12-fold increase in IGFBP-3 and -6 production, respectively, after exposure to retinoic acid and forskolin, decreased the bioavailability of IGF-I. Estradiol had differential effects on IGFBP-3 and -6 production in MCF-7 cell line with estradiol decreasing IGFBP-3 production and increasing IGFBP-6 production. Gucev et al. (166) in 1996 reported a 40% reduction in cell proliferation by retinoic acid and TGF-␤2 in the estrogen-independent breast cancer cell line MDA-MB231. They observed a 3-fold increase in IGFBP-3 production and a 2-fold increase in IGFBP-3 mRNA expression in the cell lines induced by retinoic acid and TGF-␤2. Using antisense oligodeoxynucleotides to IGFBP-3, which decreased retinoic acid and TGF-␤2-stimulated IGFBP-3 production by 90%, they demonstrated up to 40% reduction in retinoic acid and TGF-␤2 inhibitory effects on cell proliferation, indicating that a part of the growth-inhibitory effects of retinoic acid and TGF-␤2 are mediated via IGFBP-3. Gill et al. (167) in 1997 reported that exogenous IGFBP-3 decreased cell proliferation in the basal state and in response to ceramide in the estrogenindependent, IGF-I-independent breast cancer cell line Hs578T. The IGFBP-3-induced increase in metabolically inactive cells was demonstrated to be the result of an increase in apoptotic rates as measured by flow cytometry. They also reported that after the addition of exogenous IGFBP-3 in Hs578T cell media, a number of IGFBP-3 fragments were seen in addition to the intact IGFBP-3 after 24 and 48 h, suggesting that the exogenous IGFBP-3 may be proteolytically cleaved by the Hs578T cells. Yee et al. (168) in 1994 reported the effects of endogenous IGFBP-1 expression on IGF-I-mediated IGF-I receptor phosphorylation. MCF-7 cells were stably transfected with an IGFBP-1 vector, and IGFBP-1 expression was confirmed in the cells by RNase protection assay and by analyzing IGFBP-1 in the culture medium. In the transfected cells, exogenous IGF-I was unable to cause IGF-I receptor phosphorylation, whereas the analog of IGF-I with reduced affinity for IGFBP-1 caused IGF-I receptor phosphorylation. The authors

229

suggested that IGFBP-1, either exogenous or endogenously produced, binds with IGF-I, thus inhibiting ligand/receptor interaction and subsequent cell growth. Van den Berg et al. (169) in 1997 analyzed the effect of the polyethylene glycolconjugated IGFBP-1 compounds, WT-BP-1 and PEG-BP-1, on the in vitro and in vivo growth of the breast cancer cell lines, MCF-7, MDA-MB 231, and MDA-MB-435A. The colony growth of MCF-7 and MDA-MB-435A cells was significantly inhibited by both the IGFBP-1 compounds, whereas growth of MDA-MB-231 was stimulated by WT-BP-1. In the in vivo studies, PEG-BP-1 decreased tumor volume in mice bearing MDA-MB-231 but not in MCF-7-bearing mice. The in vitro effects on MDA-MB-231 tumor growth were hypothesized to occur because of effects on host IGF production. 5. IGF effects on tumor growth. Furlanetto et al. (154) studied the effect of IGF-I on DNA synthesis on breast cancer cell lines. In each of the four cancer cell lines studied (MCF-7, T47D, MDA-MB-231, and HBL-100), IGF-I stimulated DNA synthesis as measured by [3H]thymidine incorporation. The concentration of IGF-I required for half-maximal stimulation varied from a minimum of 0.03 nm in the MCF-7 cells to a maximum of 0.6 nm in the T47-D cells. The effects of the monoclonal antibody ␣IR3, which blocks IGF binding to the IGF-I receptor, were studied by Rohlik et al. (170) in 1987. Using the estrogen-responsive cell line, MCF-7, they demonstrated that ␣IR3 decreased cell growth both in the presence and absence of exogenous estrogen. The cells were propagated in 5% calf serum supplemented with 5 ng/ml of insulin. Karey and Sirbasku (171) in 1988 demonstrated that both IGF-I and IGF-II stimulated cell growth in MCF-7 and T47-D cells. IGF-I was the most potent growth factor of the panel analyzed (EGF, aFGF, bFGF, TGF␣, TGF␤, PDGF, IGFII, insulin). Arteaga and Osborne (172) described the mitogenic effect of exogenous IGF-I and IGF-II on a panel of estrogen receptor-positive (MCF-7, ZR75–1, T47D) and estrogen receptornegative (MDA 231, HS578T) cell lines in 1989. Both growth factors increased in vitro DNA synthesis in all cell lines studied. They also demonstrated that ␣IR3 inhibited IGF-I- and IGF-II-stimulated growth, suggesting that both these growth factors act via the IGF-I receptor. ␣IR3, however, did not abolish estrogen-stimulated growth in the estrogen receptorpositive cell lines. In a separate study published in 1989, Arteaga et al. (173) demonstrated that exogenous IGF-I increased the proliferation of MCF-7 and MDA-231 cells, and this increase in proliferation was inhibited in a dose-dependent manner by the addition of ␣IR3. In serum-free medium, ␣IR3 was able to inhibit growth only after exogenous administration of IGF-I, suggesting no autocrine IGF production. Osborne et al. (153) demonstrated a mitogenic effect of IGF-II in 5 of 5 of the breast cancer cell lines examined. The addition of ␣IR3 inhibited the IGF-II-mediated increase in DNA synthesis. Stewart et al. (174) in 1990 observed that in MCF-7 cells, IGF-I and IGF-II increased cell proliferation only in the presence of estradiol. They also demonstrated that estradiol induced an increase in IGF-I receptor mRNA (6.5fold) and in [125I]IGF-I binding (7.0-fold) in the MCF-7 cell line. In an estrogen receptor-negative cell line, MDA MB-231,

230

KHANDWALA ET AL.

IGF-I stimulated growth of the cell line in a manner that was unaffected by the presence or absence of estrogen in the medium. De Leon et al. (175) in 1992 studied the in vitro effects of IGF-I and IGF-II and their receptor antibodies on growth of the MCF-7 breast cancer cell line. Both growth factors stimulated cell proliferation (3- to 5-fold). When ␣IR3 was added, it inhibited IGF-I induced cell growth approximately 50%. It did not inhibit IGF-II-induced cell growth. Two different IGF-II receptor antibodies and an insulin receptor antibody also failed to significantly block IGF-II-stimulated growth. Arteaga and Osborne (172) in 1989 studied the in vivo effect of ␣IR3 on growth of breast cancer cell lines xenografted into nude mice. Three-week-old athymic mice were inoculated with either MCF-7 or MDA-231 cells. There was a greater than 80% reduction in tumor volume at 35 days in those animals that received 500 ␮g of the antibody via intraperitoneal injection on a twice weekly basis. This inhibitory effect was not observed with the MCF-7 xenografts. Weckbecker et al. (176) in 1994 reported that treatment with the somatostatin analog, octreotide, potentiated the antineoplastic activity of tamoxifen and ovariectomy in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinomas. Yang et al. (177) in 1996 xenografted MCF-7 cells in control mice and scid mice homozygous for the lit mutation. This missense mutation leads to loss of function of the GH-releasing hormone receptor and causes marked reductions of GH and IGF-I concentrations. The tumors in the lit/lit mice were approximately 50% smaller than the tumors implanted in the control mice. Huynh and Pollak (178) in 1994 reported that the inhibitory effect of tamoxifen on IGF-I was potentiated by the somatostatin analog octreotide. Combined therapy decreased serum IGF-I concentrations in rats to 49 ⫾ 10% of control values and hepatic IGF-I gene expression to 12 ⫾ 9% of control. 6. Clinical studies. Peyrat et al. (179) in 1993 demonstrated that preoperative circulating IGF-I levels in 47 patients with breast cancer were elevated when compared with controls (152 ␮g/liter vs. 115 ␮g/liter) and suggested that a positive correlation between IGF-I concentrations and breast cancer risk might exist. A positive correlation between serum IGF-I levels and the risk of premenopausal breast cancer was suggested by the study of Hankinson et al. (180) in 1998. As a nested case control study within the nurses’ health study cohort (⬎30,000 women), baseline IGF-I levels were measured in women who developed breast cancer (397 women) and case controls (620 women). The relative risk for premenopausal breast cancer was 4.58 (top tertile vs. bottom tertile) with a P value of 0.02. The relative risk for developing postmenopausal breast cancer was 2.33 with a P value of 0.08. Pollak et al. (181) in 1990 reported that breast cancer patients treated with tamoxifen for 3 months had lower IGF-I concentrations compared with a placebo-treated group (1.4 U/ml vs. 0.9 U/ml). The authors suggested that part of the antitumor effect of tamoxifen in breast cancer may be due to reduction in circulating IGF-I concentrations. The effect of octreotide in 14 patients with advanced breast cancer was reported by Vennin et al. (182) in 1989. At a dose of 100 ␮g sc twice/day there was no objective response seen in any of

Vol. 21, No. 3

the patients. In 1995, Di Leo et al. (183) reported the results of lanreotide administration (30 mg ip every 2 weeks) in 10 patients with breast cancer (predominantly estrogen receptor-positive patients). No significant responses were observed. In the analysis of serum IGF-I levels, however, lanreotide treatment did not significantly decrease circulating IGF-I concentrations. Canobbio et al. (184) in 1995 studied tamoxifen treatment combined with the somatostatin analog Somatuline. A total of 33 postmenopausal patients with breast cancer were treated. Half of the patients achieved at least a partial response. Although the IGF-I levels declined significantly in the cohort, there was no significant difference in the IGF-I levels between those that responded and those that did not. O’Byrne et al. (185) in 1999 administered the somatostatin analog RC-160 by continuous subcutaneous infusion to 14 patients with estrogen receptor-positive breast cancer. No objective responses were observed despite decreases in circulating IGF-I levels of almost 50%. In a randomized trial by Ingle et al. (186) in 1999, 135 postmenopausal women with predominantly estrogen receptor-positive breast cancer were randomized to receive tamoxifen alone or a combination of tamoxifen and octreotide (150 ␮g sc three times per day). There was no statistically significant difference in time to progression between the tamoxifen only group (14.2 months) and the combination therapy (10.3 months). The serum IGF-I concentrations were decreased 17% in the tamoxifen group and 40% in the combination group. 7. Summary. Early studies by Yee et al. (150), subsequently confirmed by others, indicate that autocrine IGF-I production by breast tumors, if it does occur, is a relatively rare phenomenon. Autocrine IGF-II production occurs in only a minority of cases. The stromal elements in the breast tissue may secrete both IGF-I and IGF-II and stimulate growth of the breast epithelial cells in a paracrine fashion (Fig. 5). IGF-I receptor expression, however, is ubiquitous or nearly ubiquitous, and activation of this receptor has been demonstrated by many investigators to be a potent stimulus for growth. Numerous animal models have demonstrated the utility of targeting the IGFs or the IGF-I receptor. The administration of binding proteins or agents that modify tumor binding protein production also would appear to be a promising therapeutic strategy. It is worth noting again that at least some of the antitumor actions of the binding proteins appear to be mediated by mechanisms that are independent of their ability to modulate IGF bioavailability. As with most other tumor types, clinical trials to date have been limited to small numbers of patients receiving somatostatin analogs. Larger studies, as well as studies employing agents targeting IGF or

FIG. 5. IGF expression in malignant breast tissue. IGF-I and IGF-II produced by the stromal cells may stimulate growth of the malignant epithelial cells. Some malignant epithelial cells may also secrete IGF-II in an autocrine fashion.

June, 2000

IGFs AND NEOPLASIA

binding protein action in ways other than the somatostatin analogs, are needed to make more definitive statements about the potential clinical utility of this type of approach. B. Ovarian cancer

1. IGF expression. Yee et al. (187) in 1991 demonstrated mRNA for IGF-I in 3 of 10 human ovarian cell lines and in 7 of 7 primary and metastatic ovarian cancer surgical specimens by RNase protection assay. The cell lines and surgical specimens predominantly expressed an IGF-I mRNA transcript with an alternate first exon. Resnicoff et al. (188) in 1993 reported the presence of IGF-I in conditioned media of OVCAR-3 and CaOV-3 cell lines. In 1994 Karasik et al. (189) reported that IGF-I levels in the fluid aspirated at surgery from malignant ovarian cysts was significantly higher than benign cysts, suggesting that there was autocrine production of IGF-I by the ovarian cancers. Conover et al. (190) in 1998 demonstrated IGF-I mRNA by RNase protection assay in 3 of 5 ovarian epithelial cancer cell cultures while IGF-II mRNA was detected in one cell line. 2. IGF receptor expression. Yee et al. (187) identified IGF-I receptor mRNA in 10 of 10 human ovarian cell lines and in 7 of 7 surgical specimens by RNase protection assay. Beck et al. (191) in 1994 demonstrated IGF-I receptor using a RIA in all primary and metastatic ovarian cancer surgical specimens examined. 3. IGFBPs. Yee et al. (187) demonstrated the presence of IGFBP-3 mRNA by Northern blot in the ovarian cancer cell lines, OVCAR-3, CaOV-4, and SK-OV-3. The first two cell lines also expressed IGFBP-2 mRNA. The IGFBP-2 and -3 peptides were also demonstrated by Western blot in the conditioned medium of OVCAR-3. Krywicki et al. (192) in 1993 analyzed the effect of estrogen and on IGFBP expression in the estrogen-responsive ovarian cancer cell line PE04. Estrogen decreased the expression of IGFBP-3 mRNA in the cells and also decreased IGFBP-3 peptide levels in the conditioned medium. IGFBP-5 mRNA levels were increased after estrogen treatment. They did not observe a significant estrogen-induced modulation of the other IGFBPs. Karasik et al. (189) in 1994 reported that IGFBP-2 levels were significantly higher in cyst fluid from invasive malignant than from benign epithelial ovarian neoplasms. Kanety et al. (193) in 1996 reported that IGFBP-2 levels were significantly higher in the protein extracts prepared from malignant ovarian surgical specimen compared with benign ovarian tumors. They also observed an increase in IGFBP-2 mRNA expression, from 2- to 30-fold, in the malignant ovarian tumors and found an association between IGFBP-2 mRNA expression and invasiveness of the tumor. Flyvbjerg et al. (194) in 1997 reported that serum IGFBP-2 levels in patients with malignant ovarian tumors were 253% (RIA) and 105% (WLB) of serum IGFBP-2 levels in control patients and in patients with benign ovarian disease. They also observed a positive correlation between serum IGFBP-2 levels and the tumor marker, cancer antigen 125 (CA 125). The serum IGF-I and IGFBP-3 levels were lower, and IGFBP-3 proteolytic activity higher, in patients with malignant ovarian tumors compared

231

with the other groups. The serum IGF-I, IGFBP-3, and IGFBP-3 proteolytic activity did not correlate with CA 125. 4. IGF effects on tumor growth. Yee et al. (187) demonstrated that exogenous IGF-I (5 nm) increased cell growth in the ovarian cancer cell line OVCAR-3 in a manner equivalent to 10% FCS. Resnicoff et al. (188) reported that IGF-I (10 ng/ml) increased OVCAR-3 and CaOV-3 cell growth approximately 50%. Antisense oligodeoxynucleotides to IGF-I receptor RNA inhibited cell growth in serum-free medium as well as in the presence of exogenous IGF-I. In 1998, Muller et al. (195) demonstrated that an antisense oligodeoxynucleotide targeted against the translation initiation site of the IGF-I receptor mRNA was associated with a decrease in IGF-I receptor mRNA and protein. These antisense oligonucleotides resulted in inhibition of basal and IGF-I stimulated growth of the human ovarian cancer cell line NIH:OVCAR-3. Coppola et al. (196) in 1999 transfected rabbit ovarian mesothelial cells with the human IGF-I receptor gene and observed increased basal (⬃8-fold) and IGF-I stimulated (⬃20-fold) growth compared with nontransfected cells. The addition of antisense oligonucleotides against the IGF-I receptor mRNA decreased basal and IGF-I-stimulated growth significantly in the transfected cells. When compared with the nontransfected cells, the transfected clone (OMIR) demonstrated fewer apoptotic cells and reduced expression of Fas-R, a cell membrane protein implicated in the apoptosis signaling pathway. The OMIR clone was also demonstrated to be tumorigenic in vivo. C. Other (endometrial, vaginal, cervical)

a. Endometrial carcinoma. 1. IGF expression. Klienman et al. (197) in 1993 demonstrated, using RIA, the presence of IGF-II but not IGF-I in the conditioned medium of Ishikawa cells, a human endometrial cancer cell line. Hana and Murphy (198) in 1994 demonstrated the presence of IGF-I and IGF-II mRNA using a PCRbased methodology in Ishikawa cells. Northern analysis was not sensitive enough to detect the IGF messages. Estradiol (10⫺7 m) increased IGF-I mRNA 366 ⫾ 20%. At the same concentration, 4-hydroxy tamoxifen increased IGF-I mRNA levels 257 ⫾ 35% above control. Neither estradiol nor 4- hydroxy tamoxifen had any effect on IGF-II mRNA expression. Klienman et al. (199) in 1996 reported that in the Ishikawa cell line, tamoxifen (10⫺6 m) decreased membrane-bound IGFBPs (3-fold) and IGFBP-3 mRNA (3.5-fold), thus likely increasing IGF availability. Tamoxifen did not affect the number or affinity of IGF-I receptors, although it was associated with increased IGF-I-stimulated phosphorylation of the IGF-I receptor. Reynolds et al. (200) also studied the autocrine production of IGF-I in four human endometrial adenocarcinoma cell lines, Ishikawa, HEC, KLE, and RL95–2. IGF-I mRNA was detected by a PCR-based methodology in all four cell lines. IGF-I was also demonstrated by RIA in conditioned medium of all four cell lines; the basal cell proliferation in serum-free medium was inhibited by the IGF-I receptor antibody ␣IR3 in 3 of 4 cell lines, further pointing to the importance of an autocrine role of IGF-I. Elkas et al. (201) in 1998 also demonstrated IGF-I expression in normal and malignant

232

KHANDWALA ET AL.

human endometrial specimen by immunohistochemistry. They also reported an increase in IGF-I (7- to 20-fold) and a decrease in IGFBP-1 expression (32–36%) in benign endometrial specimen from patients treated with tamoxifen compared with proliferative or secretory endometrium from untreated patients. 2. IGF receptor expression. Talavera et al. (202) in 1990 demonstrated the presence of IGF-I receptors by [125I]IGF-I binding studies on human endometrial biopsy specimens. They also reported a slight increase in the number of IGF-I receptors on malignant endometrial tissue when compared with the normal nonneoplastic endometrium. 3. IGFBPs. Rutanen et al. (203) in 1994 analyzed the expression of IGFBP 1, 2, 4, 5, and 6 mRNA by RT-PCR in 20 human endometrial cancer surgical specimens and normal endometrium. IGFBP-1 mRNA expression was either undetectable or as low as in the proliferative phase of the endometrium. IGFBP-1 mRNA expression increased in the late secretory phase and early pregnancy decidua. IGFBP-2, -4, and -5 mRNA expression was detectable in all cancer specimens, and their expression was not different compared with that in the cycling endometrium. They observed a cyclic variation in the expression of IGFBP-6 mRNA in the normal endometrium, and the mean level of IGFBP-6 mRNA in the endometrial cancer specimen was similar to that at midcycle. Kleinman et al. (204) in 1995 reported the down-regulation of IGFBP-3 mRNA and peptide levels in the estrogen-responsive Ishikawa endometrial cancer cell line after treatment with estrogen. Estrogen and IGF-I were synergistic in causing cell proliferation, and the decrease in IGFBP-3 could potentially make more IGF-I bioavailable. 4. IGF effects on tumor growth. Pearl et al. (205) in 1993 analyzed the mitogenic effect of IGF-I (100 ␮g/liter) and IGF-II (100 ␮g/liter) on human endometrial adenocarcinoma cell lines, HEC1-A and KLE. They reported a 2.1- to 2.7-fold increase in cell proliferation in the two cell lines. Similar increases in DNA synthesis, as measured by [3H]thymidine incorporation, were induced by both growth factors. b. Cervical cancer. Steller et al. (206) in 1996 reported the presence of IGF-II mRNA but not IGF-I mRNA in human cervical cancer cell lines using a PCR-based methodology. They also demonstrated the presence of IGF-I receptor by [125I]IGF-I binding studies and reported that compared with normal ectocervical cells, IGF-I receptor was overexpressed in cervical cancer cells. Hembree et al. (207) in 1994 reported that IGF-I increased cell growth in the human cervical cancer cell line ECE16 –1. EGF decreased IGFBP-3 concentrations in the culture medium and enhanced the growth response to IGF-I. VIII. Male Reproductive Neoplasms A. Prostate cancer

1. IGF-I expression. Cohen et al. (208) in 1991 did not detect IGF-I in the conditioned medium of normal prostate epithelial cells in primary culture. Kaicer et al. (209) in 1991 detected an IGF-like molecule in the conditioned medium of the human prostate cancer cell line PC-3. Pietrzkowski et al. (210)

Vol. 21, No. 3

in 1993, using a RIA, detected IGF-I in the conditioned medium of three prostate cancer cell lines, LNCa.FGC, PC-3, and DU-45. Conversely, Iwamura et al. (211) in 1993 did not detect IGF-I in the conditioned media by the human prostate cancer cell lines PC-3, DU-45, and LNCaP. Connolly and Rose (212) in 1994 did not detect IGF-I in the conditioned medium of the human prostate cancer cell line DU145. Angelloz-Nicoud and Binoux (213) in 1995 also did not detect autocrine IGF-I production by PC-3 cells. Kaplan et al. (214) in 1999 analyzed IGF-I expression in prostatic tissue in the transgenic adenocarcimona of mouse prostate (TRAMP) model. TRAMP mice develop prostatic intraepithelial neoplasms and/or well differentiated prostate cancer by 10 –12 weeks. All TRAMP mice ultimately develop metastatic adenocarcinoma. Using RT-PCR, they reported an increase in IGF-I mRNA expression that correlated with cancer progression, with IGF-I mRNA from the metastatic lesions being ⬎250% of 12-week-old TRAMP mice. This relationship, however, was not observed in androgen-independent prostate cancer. They also observed that serum IGF-I levels were precociously elevated in TRAMP mice compared with the nontransgenic animals. 2. IGF-II expression. Cohen et al. (208) did not detect IGF-II in the conditioned medium of normal prostate epithelial cells in primary culture. Connolly and Rose (212) did not detect IGF-II in the conditioned medium of DU145 cells. AngellozNicoud and Binoux (213) observed that PC-3 cells are capable of autocrine IGF-II production. They also demonstrated that the IGF-I receptor antibody, ␣IR3, inhibited cell growth, suggesting that the IGF-II was important in propagating growth and that its effects were mediated via the IGF-I receptor. Tennant et al. (215) in 1996, using in situ hybridization, observed that IGF-II mRNA was 30% more abundant in prostate adenocarcinoma than in benign epithelial cells. Lamharzi et al. (216) in 1998 detected IGF-II mRNA but not IGF-I mRNA in DU-145 cells using a PCR-based methodology. Using similar methodology, Csernus et al. (35) in 1999 demonstrated IGF-II mRNA in PC-3 cells. They also reported that treatment of PC-3 cells in culture with the GHRH antagonists, MZ-4 –71 or MZ-5–156, at concentrations of 3 ␮m, decreased autocrine IGF-II mRNA to 70 and 77% of control, respectively. [3H]thymidine incorporation was also decreased by both agents. Kaplan et al. (214) observed a 75–95% reduction in IGF-II mRNA expression in the prostate of the TRAMP mice compared with the nontransgenic mice. 3. IGF receptor expression. The presence of type IGF-I receptors on normal prostate epithelial cells was demonstrated by Cohen et al. (208) using [125I]-IGF-I binding studies. No IGF-II receptors were detected. High-affinity IGF-I binding sites with dissociation constants between 0.23– 0.39 nm were detected on PC-3, DU-45, and LNCaP cells by Iwamura et al. (211) in 1993. Pietrzkowski et al. (210) identified IGF-I receptor mRNA in three human prostate cancer cell lines. In two, PC-3 and DU-45 cells, IGF-I receptor mRNA was overexpressed. Kaplan et al. (214) observed a significant reduction in IGF-I and IGF-II receptor mRNA expression in tumors from castrated TRAMP mice and in metastatic lesions when

June, 2000

IGFs AND NEOPLASIA

compared with nontransgenic mice. The authors suggested that loss of IGF-I receptor expression may be associated with the loss of differentiation and increased tumorigenicity. 4. IGFBPs. Figueroa et al. (217) in 1995 reported that recombinant IGFBP-1 inhibited basal and IGF-II stimulated growth of the androgen-independent prostate cancer cell line, DU145. This inhibitory effect of IGFBP-1 was reversed by adding an analog of IGF-I that does not bind to the IGFBPs. The authors suggested that the IGFBP-1 inhibited cell growth by binding to and thus making unavailable the autocrine growth factor(s). Damon et al. (218) in 1998 analyzed the effects of IGFBP-4 overexpression in the human prostate cancer cell line, M12 (218). IGFBP-4 mRNA and peptide levels were increased in the transfected cells. The cell line overexpressing the IGFBP-4 proliferated slower than the control cells both in the absence and presence of exogenous IGF-II. Cells overexpressing IGFBP-4 also demonstrated slower anchorage-independent growth compared with control cells. This inhibitory effect, however, was not seen after treatment of the cells with an IGF-I analog without affinity for the IGFBPs, suggesting that IGFBP-4 overexpression inhibited cell growth via an IGF-dependent mechanism. When the cell lines were implanted in nude mice, there was an initial transient lower take rate in the mice implanted with the IGFBP-4 overexpressing cell line compared to the control cell line. This inhibitory effect, however, was lost after 10 weeks, and both the transfected and the control cell lines demonstrated similar growth. Nickerson et al. (219) in 1999 analyzed the effect of castration-induced tumor regression and IGFBP-5 mRNA expression in the androgen-dependent Shionogi prostate carcinoma. They reported a 90% reduction in Shionogi tumors 10 days post castration, which was accompanied with up to a 120-fold increase in IGFBP-5 mRNA expression. The authors suggested a functional role of IGFBP-5 in androgen deprivation-mediated apoptosis in androgen-dependent prostate cancer. 5. IGF effects on tumor growth. Pietrzkowski et al. (210) reported that IGF-I is a potent mitogen for the prostate cancer cell lines, LNCaP, PC-3, and DU-145. The proliferation of each cell line in serum-free medium was inhibited by an antisense oligodeoxynucleotide complementary to IGF-I receptor mRNA. Peptide analogs of IGF-I that compete with IGF-I for binding to its receptor also inhibited growth. Iwamura et al. (211) observed that IGF-I administration stimulated DNA synthesis in the androgen-independent cell lines, PC-3 and DU-145. The growthpromoting effects of IGF-I were not dependent upon the presence of dihydrotestosterone (DHT) in the medium. In the androgen-dependent cell line, LNCaP, IGF-I was able to stimulate DNA synthesis only in the presence of DHT. Schally and Redding (220) in 1987 analyzed the effects of a LH-releasing hormone (LHRH) agonist and the somatostatin analog RC-121 (5 ␮g/day), alone and in combination, on rats bearing the androgen-dependent Dunning R-3327H rat prostate adenocarcinomas. Although the LHRH agonist was more effective than RC-121, the combined treatment was superior to either treatment alone. The combination treatment reduced tumor weight to 16% and tumor volume to

233

23% of control. In the rats that received RC-121 treatment alone, there was a 58% reduction in tumor weight and a 55% reduction in volume compared with control. Murphy et al. (221) in 1987 observed that rats bearing R-3327 prostate cancers treated with the somatostatin analog, DC-13–167, developed tumors that were 41% smaller than vehicle-treated controls. Yano et al. (222) in 1992, also using Dunning R-3327H prostate tumors, observed that there was a synergistic effect when the somatostatin analog RC-160 (vapreotide) was administered in combination with a LHRH antagonist. Pinski et al. (223) in 1993 studied the effects of the somatostatin analog RC-160 (vapreotide), alone or in combination with the bombesin/gastrin-releasing peptide antagonist (RC-3095), on growth of the androgen-independent prostate cancer cell line PC-3 implanted into nude mice. Treatment with either agent resulted in approximately a 40% reduction in tumor weight and volume, but no additive benefit was observed when the agents were administered together. The inhibitory effects on tumor growth were lost when treatment was initiated with the tumors measuring 90 mm3 rather than 10 mm3. Pinski et al. (224) in 1993 also reported approximately a 50% reduction in tumor volume and weight with RC-160 (vapreotide) treatment (100 ␮g/day sc) of nude mice bearing the androgen-independent DU-145 human prostate cancer. Burfeind et al. (225) in 1996 stably transfected PA-III cells (rat prostate adenocarcinoma) with an IGF-I receptor antisense construct. As a result, IGF-I mRNA levels were reduced several fold. Mice injected with IGF-I receptor antisensetransfected PA-III cells developed tumors that were approximately 90% smaller than controls. Jungwirth et al. (226) in 1997 reported the effects of treatment with the GHRH antagonist, MZ-4 –71, on tumor growth in nude mice implanted with the human cell lines, DU-145 and PC-3 (40 ␮g/day), and the rat prostate cancer cell line, Dunning R-3327 AT-1 (100 ␮g/day). Treated groups had significantly smaller tumors as follows: DU-145 (81% of control), PC-3 (70% of control), and Dunning R-3327 AT-1 (44% of control). Tumor IGF-I and IGF-II levels, which were 146 ⫾ 25 and 190 ⫾ 32 pg/100 ␮g protein in the control group, respectively, decreased to undetectable levels in the treatment group. The antitumor effects of MZ-4 –71 were also studied by Lamharzi et al. (216) in 1998 using DU-145 xenografts. A 71% decrease in tumor volume was observed in the GHRH-treated mice. A 77% reduction in IGF-II protein concentrations and a 58% reduction in tumor IGF-II mRNA was also seen. Although serum IGF-I concentrations were decreased by 21%, there were no significant differences observed in terms of tumor content of IGF-I. 6. Clinical studies. Mantzoros et al. (227) in 1997 reported that serum IGF-I levels in 52 patients with histologically confirmed prostate cancer were significantly higher than ageand weight-matched normal controls, 160.3 vs. 124.4 ␮g/ liter. Chan et al. (228) in 1998 reported the results of a nested case-control study within the Physicians Health Study. Of the 14,916 participants, the serum IGF-I concentrations of the 152 patients who developed prostate cancer were significantly higher than a group of matched controls (269.4 vs. 248.9 ␮g/liter). A correlation between serum IGF-I levels and

234

KHANDWALA ET AL.

prostate cancer was also suggested by Wolk et al. (229) in 1998. The IGF-I mean concentration in 210 newly diagnosed, untreated prostate cancer patients was 158.4 ␮g/liter compared with 147.4 ␮g/liter in matched controls. Parmar et al. (230) performed an open-label clinical trial of the somatostatin analog, BIM 32014 (lanreotide), in 25 patients with progressive metastatic prostate cancer. Two patients achieved a partial remission, one of which was maintained at 30 months. Figg et al. (231), in a Phase I dose escalation study of the somatostatin analog Somatuline, did not find any objective response after 28 days of treatment in 25 patients with metastatic prostate cancer. Circulating IGF-I concentrations were reduced by 39% from baseline values. Maulard et al. (232) in 1995 reported the effects of lanreotide 30 mg ip injections administered weekly for 3 months in patients with hormone-refractory prostate cancer. Prostatespecific antigen levels decreased at least 50% in 20% of the patients. A significant improvement in bone pain (35% of patients) and performance status (40% of patients) was also reported. In one patient, the bone scan normalized. 7. Summary. The case-control study by Chan et al. (228), demonstrating that men with elevated IGF-I concentrations are at increased risk for developing prostate cancer, indicates that IGF may have a role in tumorigenesis or, at least, is a marker of early, subclinical disease. Interestingly, the recently developed TRAMP model of prostate cancer is also associated with an early rise in serum IGF concentrations. The IGF-I receptor appears to be abundantly expressed in most prostate cancer cell lines, but some studies have suggested that levels are significantly reduced as the disease becomes androgen-independent. Although IGF-II autocrine production by some prostate cell lines is significantly elevated, the recent study by Csernus et al. (35) demonstrating that GHRH antagonists markedly decrease autocrine IGF-II production in PC-3 cells is an important finding. It indicates that IGF overexpression, which provides some tumor cells with a critical growth advantage, is a phenomenon that can be down-regulated. This could have significant clinical ramifications. Additionally, several IGFBPs have been demonstrated to have marked antitumor effects on prostate cancer viability and growth rates, indicating that recombinant binding proteins or agents that modify endogenous production may be of significant clinical utility as well. B. Testicular cancer

Biddle et al. (233) in 1988 demonstrated the presence of IGF-I and IGF-II receptors by binding studies on human teratoma cell line, Tera-2. Weima et al. (234) in 1989 also demonstrated IGF-I receptor on human embryonal carcinoma cell line Tera 2 by [125I]IGF-I binding studies and affinity cross-linking. Biddle et al. (233) demonstrated that both IGF-I and IGF-II increased [3H]thymidine incorporation and cell proliferation in the Tera-2 cells. IX. Genitourinary Neoplasms A. Renal cell carcinoma

Jungwirth et al. (235) in 1997 reported the presence of high-affinity binding sites on the human renal adenocarci-

Vol. 21, No. 3

noma cell line Caki-I. They also demonstrated that IGF-I increased [3H] thymidine incorporation in vitro by 66% at 10 ␮g/liter and 101% at 20 ␮g/liter. IGF-II increased [3H] thymidine incorporation by 209% at 50 ␮g/liter and 302% at 90 ␮g/liter. The GH-RH antagonist, MZ-4 –71, decreased in vitro cell growth 59% when administered at a concentration of 42.3 mg/liter. In in vivo studies in which Caki-I cells were xenografted into nude mice, MZ-4 –71, administered twice daily as a sc injection of 20 ␮g, decreased final tumor volume and weight to 10.4 and 14.6% of control, respectively. Tumor doubling time increased from 7.7 ⫾ 0.6 to 59.9 ⫾ 29 days. Serum IGF-I concentrations decreased 127.7 ⫾ 10.7 to 94.8 ⫾ 7.9 ␮g/liter, but no change was observed in serum IGF-II concentrations. Intratumor IGF-I concentrations were decreased by 50%, and intratumor IGF-II concentrations were decreased by 47%. B. Bladder cancer

Dunn et al. (236) in 1997 studied the effects of diet restriction (DR) on the effect of a bladder carcinogen, p-cresidine, in heterozygous p53-deficient mice. In their previous study, 40% DR reduced serum IGF-I concentration by 50% (237). The mice were initially fed an ad libitum (AL) diet for 15 weeks, after which they were divided into 3 groups of 10 as follows: one group continued the AL diet, one group was placed on a 20% DR diet, and the third group was on a 20% DR diet but received IGF-I (IGF-I/DR) via a miniosmotic pump to restore the serum IGF-I concentration to match the AL group. All three groups received p-cresidine in their diet for 15 weeks until randomization. When the mice were killed, 4 of 10 mice in the AL group, 6 of 10 in the IGF-I/DR, and 2 of 10 in the DR group had developed transitional cell carcinoma (TCC) of the bladder. A total of 3 of 10 tumors in the IGF-I/DR group, 1 of 10 in the AL group, and 0 of 10 in the DR group were multiple. Furthermore, the tumors in the AL and IGF-I/DR groups tended to be of a higher histopathological grade than the DR group. Serum IGF-I concentrations were reduced 26% in the DR group compared with the AL group. By measuring BrdUrd labeling as a marker for cell proliferation, the IGF-I/DR and the AL groups had a 6-fold increase in cell proliferation compared with the AL group. The mice in the IGF-I/DR and the AL group had a 10-fold reduction in apoptosis compared with the DR group. These findings indicated that the differences in the cell proliferation and apoptosis rates between the groups could be explained primarily by the modest differences in the serum IGF-I concentrations.

X. Bone Neoplasms A. Osteosarcoma

1. IGF expression. Raile et al. (238) in 1994 demonstrated IGF-II mRNA in human osteosarcoma cell line U-2 OS using solution hybridization. IGF-II immunoreactivity was also detected in the conditioned medium of U-2 OS cells grown to confluency (1–2 ␮g/liter after 24 h). Burrow et al. (239) in 1998, using a PCR-based methodology, demonstrated IGF-I

June, 2000

IGFs AND NEOPLASIA

mRNA and IGF-II mRNA expression in the majority of surgical specimens obtained from patients with osteosarcomas. 2. IGF-receptor expression. In 1990 Pollak et al. (240) demonstrated the presence of IGF-I receptors on membranes prepared from MG-63 immortalized human osteosarcoma cells and primary human osteosarcoma cells by competitive binding assays and affinity-labeling experiments. Scatchard analysis revealed that the MG-63 cells and primary osteosarcoma cells had Kd values of 2.9 and 2.5 nm, respectively. The calculated binding capacity was 0.17 pmol/mg for the primary cells and 0.21 pmol/mg for the MG-63 cells. Raile et al. (238) demonstrated IGF-I and IGF-II receptors by affinity cross-linking and identified their respective mRNAs by Northern analysis in U-2 OS cells. A total of 4.5 ⫻ 104 and 10.0 ⫻ 104 binding sites were observed for IGF-I and IGF-II, respectively. The corresponding apparent affinity constants were 0.7 ⫻ 10⫺9 and 11.2 ⫻ 10⫺9 m. Kappel et al. (241) in 1994 demonstrated IGF-I receptors in five osteosarcoma cell lines (G292, HOS, MG-63, SaOS, U-2) using a ligand-binding assay. 3. IGF effects on tumor growth. Pollak et al. (240) demonstrated a dose-dependent proliferative effect of IGF-I on MG-63 osteosarcoma cells, an effect that was blocked by the anti-IGF-I receptor antibody, ␣IR3. IGF-I was effective at concentrations as low as 10⫺10 m. Raile et al. (238) also demonstrated an in vitro mitogenic effect of exogenous IGF-I and IGF-II on the human osteosarcoma cell line U-2 OS and suggested that both act via the IGF-I receptor as ␣IR3 inhibited the stimulatory effect of both IGFs. Kappel et al. (241) demonstrated that the in vitro survival of some osteosarcoma cell lines is dependent upon the addition of IGF-I to serum-free media. They also observed that the mitogenic effect of exogenous IGF-I in some cell lines was blocked by ␣IR3 or antisense oligonucleotides complementary to the IGF-I receptor. In 1992 Pollak et al. (242) reported on the results of xenografting the human osteosarcoma cell line MGH-OGS into hypophysectomized mice. The mean serum IGF-I concentration in the control mice was 195 ⫾ 8 ␮g/liter; this was reduced to 29 ⫾ 6 ␮g/liter in the hypophysectomized animals. The tumors in the hypophysectomized animals grew significantly slower and developed significantly fewer lung metastases than the tumors in the control animals (mean number of metastases, 16 vs. ⬍1). Pinski et al. in 1995 analyzed the effects of the GH-releasing hormone antagonist MZ-4 –71 on the treatment of nude mice bearing tumors from the human osteosarcoma cell lines MNNG/HOS and SKES-1 (243). MZ-4 –71 was administered via osmotic pump at a dose of 40 ␮g/day or via sc injection, 25 ␮g/twice daily. The treated mice, either with osmotic pump or subcutaneous injection, had a significantly lower tumor weight and volume. Serum IGF-I levels were decreased by 62 (MNNG/ HOS) and 42% (SK-ES-1) below control in the subcutaneous injection experiments. MZ-4 –71, at a concentration of 10⫺5 m, decreased thymidine incorporation by 28% in SK-ES-1 cells and 85% in MNNG/HOS cells. This finding indicated that there was a direct, pituitary-independent effect of MZ-4 –71 on cell growth. Pinski et al. (244) in 1996 reported that somatostatin analog, RC-160 (vapreotide), has antitumor effects in vitro and in vivo on the growth of human osteosar-

235

coma cell lines, SK-ES-1 and MNNG/HOS. In the in vitro studies, RC-160 was able to inhibit cell growth in the somatostatin receptor-positive cell line MNNG/HOS, but not in the somatostatin receptor negative line SK-ES-1. This suggested that the antiproliferative effect of the somatostatin receptor analogs might be related to the presence or absence of membrane somatostatin receptors. However, in their in vivo study, both cell line-derived tumors were inhibited by RC-160 (100 ␮g administered subcutaneously on a daily basis), highlighting the probable importance of indirect GH/ IGF-I effects. B. Other (chondrosarcoma/fibrosarcoma)

a. Chondrosarcoma. Foley et al. (245) in 1982 demonstrated the presence of high-affinity [125I]IGF-I and [125I]IGF-II binding on Swarm rat chondrosarcoma chondrocytes. IGF-I and IGF-II were able to stimulate glycosaminoglycan synthesis as evidenced by increased [35S]sulfate incorporation into macromolecules from the medium and cell matrix of the chondrosarcoma chondrocytes. Takigawa et al. (246) in 1997 demonstrated the presence of IGF-I and IGF-II mRNA by Northern analysis in a human chondrosarcoma-derived chondrocyte cell line HCS-2/8. Conditioned medium from their cell culture also demonstrated the presence of IGF-I and IGF-II by RIA. IGF-I and IGF-II receptor mRNA was also detected using Northern analysis. Seong et al. (247) in 1994 also demonstrated a mitogenic effect of IGF-I on DNA and glycosaminoglycan synthesis as measured by [3H]thymidine incorporation and [35S]sulfate incorporation, respectively, in rat chondrosarcoma chondrocytes in culture. The stimulatory effects of IGF-I on [3H]thymidine incorporation and [35S]sulfate incorporation were inhibited by 29 and 25%, respectively, by ␣IR3. b. Fibrosarcoma. Butler et al. in 1998 analyzed the effect of IGF-I expression and development of fibrosarcomas in mice (248). Tumors arising from NIH3T3 lines expressing different numbers of the IGF-I receptor were studied. The NWTc43 line expressed 1.9 ⫻ 105 receptors/cell while the pNeo1 line expressed 1.6 ⫻ 104. Nude mice transplanted with the NWTc43 cells and treated with an IGF-I infusion (4 or 10 mg/kg administered subcutaneously daily) developed palpable tumors more quickly than those animals treated with vehicle or IGF-I concentrations ⱕ 1 mg/kg/day. Once palpable, the tumors grew more quickly with the higher IGF-I doses. Identically treated animals with the pNeo1 grafts did not demonstrate the same stimulatory response to IGF-I administration. XI. Skin Neoplasms A. Melanoma

Rodeck et al. (249) in 1987 observed that cell lines derived from metastatic (5 of 6) but not primary (0 of 5) melanoma lesions grew in serum-free media, suggesting that the metastatic melanoma lines produced growth factor(s) endogenously. They also demonstrated the presence of IGF-I receptors by cross-linking studies with [125I]-IGF-I. Exogenous IGF-I was shown to be a potent stimulator of cell growth, and this effect was inhibited by ␣IR3. Furlanetto et al. (250) in 1993

236

KHANDWALA ET AL.

demonstrated that IGF-I was a mitogen for two human melanoma cell lines in vitro, WM 373 and WM 852, whereas it did not have an effect on two other human melanoma lines, WM 239-A and WM 266 – 4. ␣IR3 inhibited IGF-I stimulated cell growth in vitro in the responsive cell lines. In in vivo studies, ␣IR3 (500 ␮g ip twice weekly) significantly inhibited tumor growth in the mice bearing WM 373 and WM 852 xenografts but had no effect on mice implanted with the other cell lines. The effect of IGF-I on melanoma cell cultures was also analyzed by Resnicoff et al. in 1994 (251). They observed that the FO-1 human melanoma cell line, after being stably transfected with a plasmid expressing an antisense RNA to IGF-I receptor, expressed 70% fewer IGF-I receptors (49 ⫻ 104 receptors/cell vs. 14.7 ⫻ 104 receptors per cell). When 107 cells were implanted into nude mice, the latent period for tumor development was 28 days compared with 4 days for those animals with tumors transfected with an expression vector for the sense RNA or not transfected at all. Pretreatment with antisense oligonucleotides to the IGF-I receptor had a similar inhibitory effect on tumorigenesis. B. Basal/squamous

Neely et al. (252) in 1991 demonstrated the presence of IGF-I receptors by affinity cross-linking on normal adult human keratinocytes in culture and on a skin-derived squamous cell carcinoma cell line, SCL-1. At the maximum dose of 100 ␮g/liter, both IGF-I and IGF-II administration resulted in a 2.3-fold increase in normal keratinocyte cell number. In SCL-1 cells, IGF-I and IGF-II administration at 333 ␮g/liter both resulted in a 4.7-fold increase in [3H]-thymidine incorporation. Bol et al. (253) in 1997 developed transgenic mice in which expression of a human IGF-I cDNA was targeted to the interfollicular epidermis using a human keratin 1 promoter construct (HK1). The transgenic mice (HK1.IGF-I) showed evidence of epidermal hyperplasia, and 60% developed papillomas by 20 weeks in response to 12-O-tetradecanoylphorbol-13-acetate, whereas none of the nontransgenic mice developed tumors. Wilker et al. (254) in 1999 determined that HK1.IGF-I mice were more sensitive to a wide variety of tumor promoters such as chrysarobin, okadaic acid, and benzoyl peroxide when compared with nontransgenic mice. XII. Hematological Malignancies

1. IGF-receptor expression. Vetter et al. (255) in 1986 demonstrated the presence of high-affinity IGF-I and IGF-II binding in three human malignant cell lines, including a Burkitt type ALL line (X308), a Hodgkin’s disease derived cell line (L 428 KSA), and a cell line derived from an acute nonlymphocytic leukemia (X 376). The number of IGF-I receptors ranged from 4,500 –11,000 per cell while the number of IGF-II receptors ranged from 9,000 –55,000 per cell. Lee et al. (256) in 1986 analyzed the presence of IGF-I receptors on 6 T- and 12 B-lymphoblast cell lines isolated from patients with lymphoid malignancies. IGF-I receptors were observed in 5 of 6 T-lymphoblast lines and in 2 of 12 B-lymphoblast lines. Pepe et al. (257) in 1987 observed that the promyelocytic cell line HL 60 had more IGF-I and IGF-II specific binding than the

Vol. 21, No. 3

less differentiated myeloblast cell line KG1 and undifferentiated blast variants of these two cell lines (HL60bast, KG1a). Sinclair et al. (258) in 1988 demonstrated the presence of high-affinity [125I]IGF-I binding sites on human myeloid (HL60) and lymphoid (Namalwa) leukemia cells. 2. IGF effects on tumor growth. Vetter et al. (255) demonstrated that IGF-I increased [3H]thymidine incorporation in a Burkitt type ALL line (X308) and a Hodgkin’s disease-derived cell line (L 428 KSA) in a dose-dependent manner. Maximal stimulation was observed at an IGF-I concentration of 50 ␮g/liter, which resulted in values about 170% of control in both cell lines. IGF-II was less potent as 50 ␮g/liter concentrations resulted in [3H]thymidine incorporation values equal to only 130% of control in each of the two cell lines. Sinclair et al. (258) demonstrated that IGF-I increased [3H]thymidine incorporation in HL60 cells up to 250% over control (100 ␮g/liter for 48 h) but had no effect on Namalwa cells. The increase in [3H]thymidine incorporation in HL60 cells was inhibited by ␣IR3. Estrov et al. (259) in 1991 observed that IGF-I has a dose-dependent effect on HL60 and Burkitts lymphoma cell lines. IGF-I also had a mitogenic effect on freshly obtained marrow cells from 4 of 5 patients with acute lymphoblastic leukemia (ALL) of childhood and 4 of 4 with acute myeloblastic leukemia (AML). Stimulatory effects were observed at IGF-I concentrations ranging from 0.05 ␮g/liter to 0.5 ␮g/liter. Maximum stimulation for the ALL and AML specimens were 105 and 65%, respectively. Hursting et al. (237) in 1993 compared the growth of mononuclear cell leukemia (MNCL) cells in DR rats to control rats fed AL. DR reduced serum GH and IGF-I levels to 30 and 44% of the AL rats, respectively. The incidence of MNCL in DR rats was lower than in the AL rats (54% vs. 77%), and the DR rats also had an increased latency period (57 ⫾ 2 vs. 52 ⫾ 1 days), a lower histological grade of the tumor, and a lower splenic weight (10.7 ⫾ 1.3 vs. 15.3 ⫾ 1 g). In vitro, serum from DR rats induced less cell proliferation in CRNK-16 cells than serum from AL-treated rats. When the DR-treated rats were infused with rat GH (resulting in serum concentrations of 5.0 ␮g/liter), their serum then induced a similar degree of proliferation in CRNK-16 cells as observed with the serum obtained from AL animals. They also studied the effect of DR on cell proliferation in situ by using implanted diffusion chambers filled with CRNK-16 cells. The in situ proliferation index (ISPI), which is the diffusion chamber cell count, was significantly lower in the DR rats than in the AL animals, suggesting that DR-sensitive endogenous factors modulate the in situ growth of MNCL cells. When the DR rats were treated with GH (6.25 ␮g/h ⫻ 5 days) or IGF-I (10 ␮g/h ⫻ 5 days), the ISPI increased to that seen with the AL rats. XIII. Summary

In this manuscript, the role of IGFs in neoplastic processes has been reviewed. While some tumors are capable of endogenous IGF production, virtually all are responsive to stimulation by these growth factors. Nevertheless, the limited clinical studies performed to date (mostly with somatostatin analogs) have not been particularly successful. There are several likely reasons for this. The potency of somatosta-

June, 2000

IGFs AND NEOPLASIA

tin analogs is limited: currently available agents appear to have a maximum IGF-I suppression capability of about 50%, and a number of the clinical studies to date have employed doses that resulted in significantly less than maximum suppression. Additionally, many clinical studies with somatostatin analogs have been extensions of animal trials in which human tumors were xenografted into immunocompromised mice. Mature mice and rats have relatively low levels of circulating IGF-II. This is in contrast to humans in whom the circulating levels of IGF-II are actually higher than IGF-I on a molar basis. Thus, the modest decrease in IGF-I concentrations obtained by somatostatin analogs in a mouse model may have more profound effects than in the human. Conlon et al.(260), in studies involving guinea pigs (which maintain relatively high IGF-II levels into adulthood), have demonstrated that modulations in the circulating concentrations of one IGF have fewer physiological ramifications (weight gain, food intake, etc.) in guinea pigs than in mature rats (significant weight gain, increased food intake). This suggests that there is something of a reciprocal relationship between the two IGFs in certain species that ameliorate the effects of changes in one or the other of these growth factors. Finally, lowering GH and IGF-I concentrations also decreases circulating IGFBP-3 concentrations, a phenomenon that minimizes the impact on availability of bioactive IGF concentrations. GHRH antagonists such as MZ-5–156 also decrease circulating IGF-I concentrations. A reduction of approximately 23% was observed in a recently reported manuscript (35). GH receptor antagonists such as pegvisomant appear to decrease serum IGF-I concentrations more effectively than the currently available somatostatin or GHRH antagonists. Reductions of up to 75– 80% are achievable with pegvisomant and occur in a dose-dependent manner, as would be expected with this competitive antagonist compound. Because escalating doses of pegvisomant above those necessary to achieve an 80% reduction do not result in additional decreases in circulating IGF-I concentrations, it would seem that about 20% of the IGF-I in the circulation is produced in a GHindependent manner. Although the pegvisomant doseresponse studies were performed in mice, the circulating levels of IGF-I in GH-deficient patients suggest that a 70 – 80% reduction would probably be the maximum achievable in humans as well. The increased potency of the GH receptor antagonists, at least as measured by reduction in serum IGF-I concentrations, probably exists because of its mechanism of action. By blocking GH binding to its receptor, pegvisomant is essentially producing complete GH deficiency, a phenomenon that, at best, could be equaled by combining a somatostatin analog with a GHRH antagonist. One of the most interesting aspects of the GHRH antagonists is their ability to decrease intratumor IGF-II production. This is an important phenomenon because it indicates that these compounds can act to diminish a fundamental growth advantage possessed by many types of tumors. For instance, MZ-4 –71 administration to animals bearing DU145 human prostate carcinoma xenografts was associated with a decrease in intratumor IGF-II concentrations from 1,486 pg/100 ␮g of tissue to 304 pg/100 ␮g of tissue. Accordingly, a marked antitumor effect was observed in the

237

treatment group. Although the IGF-II assay used in these experiments was not species-specific, this profound decrease in IGF-II production almost certainly reflects tumor production as the serum IGF-II concentrations were not significantly different between the animals that received MZ-4 –71 and those that did not. The precise mechanism of the downregulation of tumor autocrine IGF-II production by the GHRH antagonists is unknown, but it has been observed in both in vitro and in vivo experiments, indicating that it is at least partially mediated by direct actions at the cellular level. Although less extensively studied, the GH receptor antagonist pegvisomant has been demonstrated to also reduce intratumor IGF production (99). The ability to down-regulate endogenous IGF production is likely to be a critical component, if not the most critical component, in mediating the antitumor effects of the GH and GHRH antagonist compounds. In addition to blocking an autocrine growth loop, decreasing endogenous IGF production may also make tumors more responsive to traditional agents such as cytotoxic chemotherapy or radiation. Modifying the IGFBP environment, whether through agents that modify tumor IGFBP production or simply by the exogenous administration of recombinant proteins, offers significant therapeutic opportunities. The antiestrogens, tamoxifen and ICI 182,780, are excellent examples of agents that modify tumor IGFBP production. For instance, both have been demonstrated to increase IGFBP-3 production in MCF-7 cells, an estrogen-responsive breast cancer cell line. This alteration in IGFBP-3, at least in certain instances, appears to be able to inhibit IGF-I-stimulated cell proliferation. Many examples of the ability of exogenous IGFBP administration to inhibit tumor growth were detailed in the preceding text. Because IGF-II also circulates in complex with the IGFBPs, changes in the binding protein milieu should influence both IGF-I and IGF-II actions. It is important to remember, however, that the effects of all binding proteins are not always uniformly inhibitory. Additionally, at least some of their actions appear to be mediated in a manner that is independent of their actions on IGF bioavailability. Nevertheless, the IGFPBs are an important component of the natural physiological regulation of IGF actions and, as such, are promising candidates for therapeutic intervention. Agents that block IGF action at the receptor level are also excellent therapeutic candidates. For instance, IGF-I receptor blocking antibodies, such as ␣IR3, should be capable of inhibiting the stimulatory actions of all IGF-I, not just the 75– 80% that is regulated by GH. Additionally, because most of the growth-promoting actions of IGF-II appear to be mediated by the IGF-I receptor, therapeutic strategies that block the IGF-I receptor would also inhibit the actions of IGF-II. Any direct actions of GH, however, would not be attenuated with this approach. However, as the IGFs are important to many normal physiological activities in virtually every tissue, there has to be some concern about the ramifications of blocking the actions with systemic agents such as an IGF-I receptor antibody. Therefore, this type of approach may be most useful for those tumors that clearly overexpress IGF-I receptor and are therefore disproportionately susceptible to receptor blockade. The use of gene therapy approaches, such as antisense IGF-I receptor vectors that are targeted to the

238

KHANDWALA ET AL.

tumor only (either by direct injection or the use of a tissuespecific promoter), offers the possibility of circumventing the problems of agents that would have systemic actions. They would, however, suffer from the same limitations of other gene therapy vectors, namely that it is difficult to achieve a high enough transfection efficiency to generate clinically meaningful outcomes. Advances in this arena, however, would make this approach even more attractive. In summary, a new generation of potential therapeutic agents that target the IGFs is currently under investigation. These new agents are capable of modulating the IGFs and their receptors in ways not previously possible. Endocrine, paracrine, and even autocrine production of the IGFs can be down-regulated. IGF action at the receptor level, and even receptor expression itself, can be blocked. Additionally, the action of GH can be completely blocked as well. The increased potency and alternate mechanisms of actions of these new approaches suggests that some of the promising findings observed in in vitro studies and animals models may eventually come to be realized in the clinical setting. Experimental design that builds upon the vast body of knowledge accumulated over the past several decades, much of which has been reviewed in this manuscript, will have the greatest opportunity for success.

References 1. Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34 2. LeRoith D 1997 Insulin-like growth factors. N Engl J Med 336: 633– 640 3. LeRoith D 1993 Insulin-like growth factors. Ann NY Acad Sci: 692:1–9 4. Clemmons DR 1997 Insulin-like growth factor binding proteins and their role in controlling IGF actions. Cytokine Growth Factor Rev 8:45– 62 5. Ranke MB, Elmlinger M 1997 Functional role of the insulin-like growth factor binding proteins. Horm Res 48S:9 –15 6. Baxter RC, Binoux MA, Clemmons DR, Conover CA, Drop SLS, Holly JMP, Mohan S, Oh Y, Rosenfeld R 1998 Recommendations for nomenclature of the insulin-like growth factor binding protein superfamily. Endocrinology 139:4036 7. Hwa V, Oh Y, Rosenfeld RG 1999 The insulin-like growth factorbinding protein (IGFBP) superfamily. Endocr Rev 20:761–787 8. Chen JC, Shao ZM, Sheikh MS, Hussain A, LeRoith D, Roberts Jr CT, Fontana JA 1994 Insulin-like growth factor binding protein enhancement of insulin-like growth factor (IGF) mediated DNA synthesis and IGF-I binding in a human breast carcinoma cell line. J Cell Physiol 158:69 –78 9. D’Ercole AJ 1996 Insulin-like growth factors and their receptors in growth. Endocrinol Metab Clin North Am 25:573:590 10. Resnicoff M, Abraham D, Yutanawiboonchai W, Rotman HL, Kajstura J, Rubin R, Zoltick P, Baserga R 1995 The insulin-like growth factor-I receptor protects tumor cells from apoptosis in vivo. Cancer Res 55:2463–2469 11. Valentinis B, Romano G, Peruzzi F, Morrione A, Prisco M, Soddu S, Cristofanelli B, Sacchi A, Baserga R 1999 Growth and differentiation signals by the insulin-like growth factor 1 receptor in hematopoietic cells are mediated through different pathways. J Biol Chem 274:12423–12430 12. O’Dell SD, Day IN 1998 Molecules in focus: insulin-like growth factor II (IGF-II). Int J Biochem Cell Biol 30:767–771 13. Braulke T 1999 Type-2 IGF receptor: a multi ligand binding protein. Horm Metab Res 31:242–246 14. O’Gorman DB, Costello M, Weiss J, Firth SM, Scott CD 1999 Decreased insulin-like growth factor-II/mannose-6-phosphate re-

15. 16.

17. 18.

19.

20. 21. 22. 23. 24. 25.

26.

27.

28.

29.

30.

31.

32.

33. 34.

Vol. 21, No. 3

ceptor expression enhances tumorigenicity in JEG-3 cells. Cancer Res 59:5692–5694 Yakar S, Liu JL, Stannard B, Butler A, Accili D, Sauer B, LeRoith D 1999 Normal growth and development in the absence of hepatic insulin-like growth factor-I. Proc Natl Acad Sci USA 96:7324 –7329 Murphy LJ, Friesen HG 1988 Differential effects of estrogen and growth hormone on uterine and hepatic insulin-like growth factor I gene expression in the ovariectomized hypophysectomized rat. Endocrinology 122:325–332 Penhoat A, Naville D, Jaillard C, Chatelain PG, Saez JM 1989 Hormonal regulation of insulin-like growth factor I secretion by bovine adrenal cells. J Biol Chem 264:6858 – 6862 Hofbauer LC, Rafferzeder M, Janssen OE, Gartner R 1995 Insulinlike growth factor I messenger ribonucleic acid expression in porcine thyroid follicles is regulated by thyrotropin and iodine. Eur J Endocrinol 132:605– 610 Clemmons DR, Klibanski A, Underwood LE, McArthur JW, Ridgway EC, Beitins IZ, Van Wyk JJ 1981 Reduction of immunoreactive somatomedin-C during fasting in humans. J Clin Endocrinol Metab 53:1247–1250 Adamo ML, Neuenschwander S, LeRoith D, Roberts Jr CT 1993 Structure, expression, and regulation of the IGF-I gene. Adv Exp Med Biol 343:1–11 Sussenbach JS, Rodenburg RJ, Scheper W, Holthuizen P 1993 Transcriptional and post-transcriptional regulation of the human IGF-II gene expression. Adv Exp Med Biol 343:63–71 Kim HT, Choi BH, Niikawa N, Lee TS, Chang SI 1998 Frequent loss of imprinting of the H19 and IGF-II genes in ovarian tumors. Am J Med Genet 80:391–395 Nonomura N, Miki T, Nishimura K, Kanno N, Kojima Y, Okuyama A 1997 Altered imprinting of the H19 and insulin-like growth factor II genes in testicular tumors. J Urol 157:1977–1979 Wu MS, Wang HP, Lin CC, Sheu JC, Shun CT, Lee WJ, Lin JT 1997 Loss of imprinting and overexpression of IGF2 gene in gastric adenocarcinoma. Cancer Lett 120:9 –14 Lowe Jr WL, Adamo M, Werner H, Roberts Jr CT, LeRoith D 1989 Regulation by fasting of insulin-like growth factor-I and its receptor. Effects on gene expression and binding. J Clin Invest 84: 619 – 626 Werner H, Roberts Jr CT, LeRoith D 1993 The regulation of insulin-like growth factor-I receptor gene expression by positive and negative zinc-finger transcription factors. Adv Exp Med Biol 343: 91–103 Rosenfeld RG, Hintz RL 1980 Characterization of a specific receptor for somatomedin-C (SM-C) on cultured human lymphocytes: evidence that SM-C modulates homologous receptor concentration. Endocrinology 107:1841–1848 Beitner-Johnson D, Werner H, Roberts Jr CT, LeRoith D 1995 Regulation of insulin-like growth factor-I receptor gene expression by Sp1: physical and functional interactions of Sp1 at GC boxes and at a CT element. Mol Endocrinol 9:1147–1156 Hernandez-Sanchez C, Werner H, Roberts Jr CT, Woo EJ, Hum DW, Rosenthal SM, LeRoith D 1997 Differential regulation of insulin-like growth factor-I (IGF-I) receptor gene expression by IGF-I and basic fibroblastic growth factor. J Biol Chem 272:4663– 4670 Zhang L, Kasanchi F, Zhan Q, Zhan S, Brady JN, Fornace AJ, Seth P, Helman LJ 1996 Regulation of insulin-like growth factor-II P3 promoter by P53: a potential mechanism for tumorigenesis. Cancer Res 56:1367–1373 Oshima A, Nolan CM and Kyle JW, Grubb JH, Sly WS 1988 The human cation-independent mannose-6-phosphate receptor. Cloning and sequence of the full length cDNA and expression of functional receptor in cos cells. J Biol Chem 263:2553–2562 Okamoto T, Nishimoto I, Murayama Y, Ohkuni Y, Ogata E 1990 Insulin-like growth factor-II/mannose-6-phosphate receptor is incapable of activating GTP-binding proteins in response to mannose-6-phosphate, but capable in response to insulin-like growth factor-II. Biochem Biophys Res Commun 168:1201–1210 Liu Z, Mittanck DW, Kim S, Rotwein P 1995 Control of insulin-like growth factor-II/mannose-6-phosphate receptor gene transcription by proximal promoter elements. Mol Endocrinol 9:1477–1487 Varga JL, Schally AV, Csernus VJ, Zarandi M, Halmos G, Groot

June, 2000

35.

36. 37.

38. 39.

40. 41.

42.

43.

44. 45. 46. 47.

48.

49.

50. 51. 52. 53.

IGFs AND NEOPLASIA

K, Rekasi Z 1999 Synthesis and biological evaluation of antagonists of growth hormone-releasing hormone with high and protracted in vivo activities. Proc Natl Acad Sci USA 96:692– 697 Csernus VJ, Schally AV, Kiaris H, Armatis P 1999 Inhibition of growth, production of insulin-like growth factor-II (IGF-II), and expression of IGF-II mRNA of human cancer cell lines by antagonistic analogs of growth hormone-releasing hormone in vitro. Proc Natl Acad Sci USA 96:3098 –3103 Lamberts SWJ, Van Der Lely AJ, De Herder WW, Hofland L 1996 Octreotide. New Engl J Med 334:246 –254 Pollak MN, Polychronakos C, Guyda H 1989 Somatostatin analogue SMS 201–995 reduces serum IGF-I levels in patients with neoplasms potentially dependent on IGF-I. Anticancer Res 9: 889 – 892 Pollak MN, Schally AV 1998 Mechanism of antineoplastic action of somatostatin analogs. Proc Soc Exp Biol Med 217:143–152 Buscail L, Delesque N, Esteve JP, Saint-Laurent N, Prats H, Clerc P, Robberecht P, Bell GI, Liebow C, Schally AV, Vaysse N, Susini C 1994 Stimulation of tyrosine phosphatase and inhibition of cell proliferation by somatostatin analogues: mediation by human somatostatin receptor subtypes SSRT1 and SSRT2. Proc Natl Acad Sci USA 91:2315–2319 Kaleko M, Rutter WJ, Miller AD 1990 Overexpression of human insulin-like growth factor-I receptor promotes ligand-dependent neoplastic transformation. Mol Cell Biol 10:464 – 473 Sell C, Rubini M, Rubin R, Liu JP, Efstratiadis A, Baserga R 1993 Simian virus 40 large tumor antigen is unable to transform mouse embryonic fibroblasts lacking type I insulin-like growth factor receptor. Proc Natl Acad Sci USA 90:11217–11221 Liu X, Turbyville T, Fritz A, Whitesell L 1998 Inhibition of insulinlike growth factor I receptor expression in neuroblastoma cells induces the regression of established tumors in mice. Cancer Res 58:5432–5438 Reiss K, D’Ambrosio C, Tu X, Tu C, Baserga R 1998 Inhibition of tumor growth by a dominant negative mutant of the insulin-like growth factor I receptor with a bystander effect. Clin Cancer Res 4:2647–2655 Kull Jr FC, Jacobs S, Su YF, Svoboda ME, Van Wyk JJ, Cuatrecasas P 1983 Monoclonal antibodies to receptors for insulin and somatomedin-C. J Biol Chem 258:6561– 6566 Pietrzkowski Z, Wernicke D, Porcu P, Jameson BA, Baserga R 1992 Inhibition of cellular proliferation by peptide analogues of insulin-like growth factor-I. Cancer Res 52:6447– 6451 Cascieri MA, Bayne ML 1994 Analysis of the interaction of insulinlike growth factor-I (IGF-I) analogs with the IGF-I receptor and IGF binding proteins. Horm Res 41[Suppl 2]:80 – 85 Blakesley VA, Kalebic T, Helman LJ, Stannard B, Faria TN, Roberts Jr CT, LeRoith D 1996 Tumorigenic and mitogenic capacities are reduced in transfected fibroblasts expressing mutant insulinlike growth factor-I receptors. The role of tyrosine residues 1250, 1251 and 1316 in the carboxy-terminus of the IGF-I receptor. Endocrinology 137:410 – 417 Sandberg AC, Engberg C, Lake M, von Holst H, Sara VR 1988 The expression of insulin-like growth factor-I and insulin-like growth factor-II genes in the human fetal and adult brain and in glioma. Neurosci Lett 93:114 –119 Antoniades HN, Galanopoulos T, Neville-Golden J, Maxwell M 1992 Expression of insulin-like growth factors-I and II and their receptor mRNAs in primary human astrocytomas and meningiomas: in vivo studies using in situ hybridization and immunocytochemistry. Int J Cancer 50:215–222 Glick RP, Unterman TG, Hollis R 1991 Radioimmunoassay of insulin-like growth factors in cyst fluid of central nervous system tumors. J Neurosurg 74:972–978 Glick RP, Unterman TG, Van Der Voude M, Blaydes LZ 1992 Insulin and insulin-like growth factors in central nervous system tumors. J Neurosurg 77:445– 450 Sandberg-Nordqvist AC, Stahlbom PA, Reinecke M, Collins VP, Holst HV, Sara VR 1993 Characterization of insulin-like growth factor-I in human primary brain tumors. Cancer Res 53:2475–2478 Hirano H, Lopes MBS, Laws ER, Asakura T, Goto M, Carpenter JE, Karns LR, Vandenberg SR 1999 Insulin-like growth factor-I content and pattern of expression correlates with histopathologic

54.

55. 56. 57. 58. 59. 60. 61. 62. 63.

64.

65.

66.

67. 68.

69.

70.

71. 72.

73. 74.

239

grade in diffusely infiltrating astrocytomas. Neurooncology 1: 109 – 119 Trojan J, Johnson TR, Rudin SD, Ilan J, Tykocinski ML, Ilan J 1993 Treatment and prevention of rat glioblastoma by immunogenic C6 cells expressing antisense insulin-like growth factor-I RNA. Science 259:94 –97 Lichtor T, Kurpakus MA, Gurney ME 1993 Expression of insulinlike growth factors and their receptors in human meningiomas. J Neurooncol 17:183–190 Hultberg BM, Haselbacher G, Nielson FC, Wulff BS, Gammeltoft S 1993 Gene expression of insulin-like growth factor-II in human intracranial meningioma. Cancer 72:3282–3286 Sara VR, Prisell P, Sjogren B, Persson L, Boethius J, Enberg G 1986 Enhancement of insulin-like growth factor-II receptors in glioblastomas. Cancer Lett 32:229 –234 Gammeltoft S, Ballotti R, Kowalski A, Westermark B, Van Obberghen E 1988 Expression of two types of receptors for insulin-like growth factors in human glioma. Cancer Res 48:1233–1237 Glick RP, Gettleman R, Patel K, Lakhsman R, Tsibris JCM 1989 Insulin and insulin-like growth factor-I in brain tumors: binding and in vitro effects. Neurosurgery 24:791–797 Merrill MJ, Edwards NA 1990 Insulin-like growth factor-I receptors in human glial tumors. J Clin Endocrinol Metab 71:199 –209 Burgaud JL, Resnicoff M, Baserga R 1995 Mutant IGF-I receptors as dominant negatives for growth and transformation. Biochem Biophys Res Commun 214:475– 481 Baserga R 1995 The insulin-like growth factor-I receptor: a key to tumor growth? Cancer Res 55:249 –252 Resnicoff M, Burgaud JL, Rotman HL, Abraham D, Baserga R 1995 Correlation between apoptosis, tumorigenesis, and levels of insulin-like growth factor-I receptors 1995. Cancer Res 55:3739 – 3741 Resnicoff M, Li W, Basak S, Herlyn D, Baserga R, Rubin R 1996 Inhibition of rat C6 glioblastoma tumor growth by expression of insulin-like growth factor-I receptor antisense mRNA. Cancer Immunol Immunother 42:64 – 68 D’Ambrosio C, Ferber A, Resnicoff M, Baserga R 1996 A soluble insulin-like growth factor-I receptor that induces apoptosis of tumor cells in vivo and inhibits tumorigenesis. Cancer Res 56:4013– 4020 Resnicoff M, Sell C, Rubini M, Coppola D, Ambrose D, Baserga R, Rubin R 1994 Rat glioblastoma cells expressing an antisense RNA to the insulin-like growth factor-I (IGF-I) receptor are nontumorigenic and induce regression of wild-type tumors. Cancer Res 54:2218 –2222 Lichtor T, Kurpakus MA, Gurney ME 1991 Differential expression of insulin-like growth factor-II in human meningiomas. Neurosurgery 29:405– 410 Sandberg-Nordqvist AC, Peyrard M, Pattersson H, Mathiesen T, Collins VP, Dumanski JP, Schalling M 1997 A high ratio of insulin-like growth factor-II/IGFBP-2 mRNA as a marker for anaplasia in meningiomas. Cancer Res 57:2611–2614 Kurihara M, Tokunaga Y, Tsutsumi K, Kawaguchi T, Shigematsu K, Niwa M, Mori K 1989 Characterization of insulin-like growth factor-I and epidermal growth factor receptors in meningioma. J Neurosurg 71:538 –544 Tsutsumi K, Kitagawa N, Niwa M, Himeno A, Taniyama K, Shibata S 1994 Effect of suramin on 125I-IGF-I binding to human meningiomas and on proliferation of meningioma cells. J Neurosurg 80:502–509 Friend KE, Radinski R, McCutcheon IE 1999 Growth hormone receptor expression and function in meningioma: effect of a specific receptor antagonist. J Neurosurg 91:93–99 Friend KE, Flyvbjerg A, Hill H, Li J, Bennett WF, Scarlett JA, Radinski R, McCutcheon IE 1999 The growth hormone receptor antagonist, pegvisomant, inhibits the growth of human meningiomas xenografted into immunocompromised animals. Program of the 5th International Symposium on Insulin-Like Growth Factors, Brighton, UK, 1999 (Abstract P 7) Jensen RL, Leppla D, Rokosz N, Wurster RD 1998 Matrigel augments xenograft transplantation of meningioma cells into athymic mice. Neurosurgery 42:130 –135 McCutcheon IE, Friend KE, Gerdes TM, Zhang BM, Wildrick

240

75.

76.

77. 78.

79.

80.

81.

82.

83. 84. 85. 86. 87. 88. 89.

90.

91. 92.

93.

KHANDWALA ET AL. DM, Fuller GN 2000 Intracranial injection of human meningioma cells in athymic mice: an orthotopic model for meningioma growth. J Neurosurg 92:306 –314 Tricoli JV, Rall LB, Karakousis CP, Herrera L, Petrelli NJ, Bell GI, Shows TB 1986 Enhanced levels of insulin-like growth factor messenger RNA in human colon carcinomas and liposarcomas. Cancer Res 46:6169 – 6173 Culouscou JM, Remacle-Bonnet M, Garrouste F, Marvaldi J, Pommier G 1987 Simultaneous production of insulin-like growth factor-I and epidermal growth factor competing growth factors by HT-29 human colon cancer line. Int J Cancer 40:646 – 652 Lambert S, Vivario J, Boniver J, Gol-Winkler R 1990 Abnormal expression and structural modification of the of insulin-like growth factor-II gene in human colorectal tumors. Int J Cancer 46:405– 410 Zarrilli R, Pignata S, Romano M, Gravina A, Casola S, Bruni CB, Acquaviva A 1994 Expression of insulin-like growth factor-II and IGF-I receptor during proliferation and differentiation of CaCo-2 human colon carcinoma cells. Cell Growth Diff 5:1085–1091 Guo YS, Jin GF, Townsend CM, Zhang T, Sheng HM, Beauchamp RD, Thompson JC 1995 Insulin-like growth factor-II expression in carcinoma in colon cell lines: implications for autocrine actions J Am Coll Surg 181:145–154 Kawamoto K, Onodera H, Kondo S, Kan S, Ikeuchi D, Maetani S, Imamura M 1998 Expression of insulin-like growth factor-II can predict the prognosis of human colorectal cancer patients: correlation with tumor progression, proliferative activity and survival. Oncology 55:242–248 Kawamoto K, Onodera H, Kan S, Kondo S, Imamura M 1999 Possible paracrine mechanism of insulin-like growth factor-2 in the development of liver metastasis from colorectal carcinoma. Cancer 85:18 –25 Freier S, Weiss O, Eran M, Flyvbjerg A, Dahan R, Nephesh I, Safra T, Shiloni E, Raz I 1999 Expression of the insulin-like growth factors and their receptors in adenocarcinoma of the colon. Gut 44:704 –708 Pollak MN, Perdue JF, Margolese RG, Baer K, Richard M 1987 Presence of somatomedin receptors on primary human breast and colon carcinomas. Cancer Lett 38:223–230 Rouyer-Fessard C, Gammeltoft S, Laburthe M 1990 Expression of two types of receptor for insulin-like growth factors in human colonic epithelium. Gastroenterology 98:703–707 Guo YS, Narayan S, Yallampalli C, Singh P 1992 Characterization of insulin-like growth factor-I receptors in human colon cancer Gastroenterology 102:1101–1108 Adenis A, Peyrat JP, Hecquet B, Delobelle A, Depadt G, Quandalle P, Bonneterre J, Demaille A 1995 Type I insulin-like growth factor receptors in human colorectal cancer. Eur J Cancer 31A:50 –55 Zenilman ME, Graham W 1997 Insulin-like growth factor-I mRNA in the colon is unchanged during neoplasia. Cancer Invest 15:1–7 Michell NP, Dent S, Langman MJ, Eggo MC 1997 Insulin-like growth factor binding proteins as mediators of IGF-I effects on colon cancer cell proliferation. Growth Factors 14:269 –277 Singh P, Dai B, Dhruva B, Widen SG 1994 Episomal expression of sense and antisense insulin-like growth factor (IGF) binding protein-4 complimentary DNA alters the mitogenic response of a human colon cancer cell line (HT-29) by mechanisms that are independent of and dependent upon IGF-I. Cancer Res 54:6563– 6570 Macdonald RG, Schaffer BS, Kang IJ, Hong SM, Kim EJ, Park JH 1999 Growth inhibition and differentiation of the human colon carcinoma cell line, Caco-2, by constitutive expression of insulinlike growth factor binding protein-3. J Gastroenterol Hepatol 14: 72–78 Koenuma M, Yamori T, Tsuruo T 1989 Insulin and insulin-like growth factor-I stimulate proliferation of metastatic variants of colon carcinoma. Jpn J Cancer Res 80:51–58 Lahm H, Suardet L, Laurent PL, Fischer JR, Ceyhan A, Givel JC, Odartchenko N 1992 Growth regulation and co-stimulation of human colorectal cancer cell lines by insulin-like growth factor-I, II and transforming growth factor ␣. Br J Cancer 65:341–346 Lahm H, Amstad P, Wyniger J, Yilmaz A, Fischer JR, Schreyer M, Givel JC 1994 Blockade of insulin-like growth factor-I receptor inhibits growth of human colorectal cancer cells: evidence of a functional IGF-II mediated autocrine loop. Int J Cancer 58:452– 459

Vol. 21, No. 3

94. Smith JP, Solomon TE 1988 Effects of gastrin, proglumide and somatostatin on growth of human colon cancer. Gastroenterology 95:1541–1548 95. Qin Y, Schally AV, Willems G 1991 Somatostatin analogue RC-160 inhibits the growth of transplanted colon cancer in rats. Int J Cancer 47:765–770 96. Alonso M, Galera MJ, Reyes G, Calabuig R, Vinals A, Rius X 1992 Effects of pentagastrin and of the somatostatin analogue (SMS 201–995) on growth of CT26 in vivo adenocarcinoma of the colon. Surg Gynecol Obstet 175:441– 444 97. Qin Y, Schally AV, Willems G 1992 Treatment of liver metastasis of human colon cancers in nude mice with somatostatin analogue RC-160. Int J Cancer 52:791–796 98. Dy DY, Whitehead RH, Morris DL 1992 SMS 201–995 inhibits in vitro and in vivo growth of human colon cancer. Cancer Res 52: 917–923 99. Duan H, Dagnaes-Hansen F, Rasmussen L, Friend KE, Orskov H, Bennett WF, Flyvbjerg A 1999 GH receptor antagonist treatment inhibits growth of human colorectal carcinoma, COLO205 in nude mice. Program of the 5th International Symposium on Insulin-Like Growth Factors, Brighton, UK, 1999 (Abstract P 13) 100. Glass AR, Kikendall JW, Sobin LH, Bowen PE 1994 Serum concentrations of insulin-like growth factor-I in colonic neoplasia. Acta Oncol 33:70 –71 101. Ma J, Pollak MN, Giovanucci E, Chan JM, Tao Y, Hennekens CH, Stampfer MJ 1999 Prospective study of colorectal cancer risk in men and plasma levels of insulin-like growth factor-I (IGF) and IGF binding protein-3. J Natl Cancer Inst 91:620 – 625 102. Iftikhar SY, Watson SA, Morris DL 1991 The effect of long-acting somatostatin analogue SMS 201–995 therapy on tumor kinetic measurements and serum tumor marker concentrations in primary rectal cancer. Br J Cancer 63:971–974 103. Cascinu S, Ferro ED, Catalano G 1995 A randomized trial of octreotide vs. best supportive care only in advanced gastrointestinal cancer patients refractory to chemotherapy. Br J Cancer 71: 97–101 104. Goldberg RM, Moertel CG, Wieand HS, Krook JE, Schutt AJ, Veeder MH, Mailliard JA, Dalton RJ 1995 A phase III evaluation of a somatostatin analogue (octreotide) in the treatment of patients with asymptomatic advanced colon carcinoma. Cancer 76:961–966 105. Cascinu S, Ferro ED, Grianti C, Ligi M, Ghiselli R, Foglietti G, Saba V, Lungarotti F, Catalano G 1997 Inhibition of tumor cell kinetics and serum insulin-like growth factor-I levels by octreotide in colorectal cancer patients. Gastroenterology 113:767–772 106. Thompson MA, Cox AJ, Whitehead RH, Jonas HA 1990 Autocrine regulation of human tumor cell proliferation by insulin-like growth factor-II: an in vitro model. Endocrinology 126:3033–3042 107. Chung CK, Antoniades HN 1992 Expression of c-sis platelet derived growth factor-B, insulin-like growth factor-I and transforming growth factor ␣ messenger RNA and their respective receptor messenger RNA in primary human gastric carcinomas: in vivo studies with in situ hybridization and Immunocytochemistry. Cancer Res 52:3453–3459 108. Guo YS, Beauchamp DR, Jin GF, Townsend Jr CM, Thompson JC 1993 Insulin-like growth factor binding protein modulates the growth response to insulin-like growth factor-I by human gastric cancer cells. Gastroenterology 104:1595–1604 109. Durrant LG, Watson SA, Hall A, Morris DL 1991 Co-stimulation of gastrointestinal tumor cell growth by gastrin, transforming growth factor ␣ and insulin-like growth factor-I. Br J Cancer 63: 67–70 110. Ohmura E, Okada M, Onaoda N, Kamiya Y, Murakami H, Tshushima T, Shizume K 1990 Insulin-like growth factor-I and transforming growth factor ␣ as autocrine growth factors in human pancreatic cancer cell growth. Cancer Res 50:103–107 111. Bergmann U, Funatomi H, Yokoyama M, Beger HG, Korc M 1995 Insulin-like growth factor-I overexpression in human pancreatic cancer: evidence for autocrine and paracrine roles. Cancer Res 55:2007–2011 112. Ishiwata T, Bergmann U, Kornmann M, Lopez M, Beger HG, Korc M 1997 Altered expression of insulin-like growth factor-II receptor in human pancreatic cancer. Pancreas 15:367–373 113. Oku K, Tanaka A, Yamanishi H, Nishizawa Y, Matsumoto K,

June, 2000

114.

115. 116. 117.

118.

119.

120.

121.

122. 123.

124. 125.

126.

127.

128.

129.

130. 131.

132.

IGFs AND NEOPLASIA

Shiozaki H, Mori T 1991 Effects of various growth factors on growth of a cloned human esophageal squamous cancer cell line in a protein-free medium. Anticancer Res 11:1591–1596 Chen SC, Chou CK, Wong FH, Chang C, Hu CP 1991 Overexpression of epidermal growth factor and insulin-like growth factor-I receptors and autocrine stimulation in human esophageal carcinoma cells. Cancer Res 51:1898 –1903 Tsai TF, Yauk YK, Chou CK, Ting LP, Chang C, Hu CP, Han SH, Su TS 1988 Evidence of autocrine regulation in human hepatoma cell lines. Biochem Biophys Res Commun 153:39 – 45 Su TS, Liu WY, Han SH, Jansen M, Yang-Fen TL, P’eng FK, Chou CK 1989 Transcripts of insulin-like growth factor-I and II in human hepatoma. Cancer Res 49:1773–1777 Cariani E, Lasserre C, Seurin D, Hamelin B, Kemeny F, Franco D, Czech MP, Ullrich A, Brechot C 1988 Differential expression of insulin-like growth factor mRNA in human primary liver cancers, benign liver tumors, and liver cirrhosis. Cancer Res 48:6844 – 6849 Lamas E, Bail BL, Housset C, Boucher O, Brechot C 1991 Localization of insulin-like growth factor-II and hepatitis B virus mRNA and proteins in human hepatocellular carcinomas. Lab Invest 64: 98 –104 D’Errico A, Grigioni WF, Fiorentino M, Baccarini P, Lamas E, Mitri SD, Gozzetti G, Mancini AM, Brechot C 1994 Expression of insulin-like growth factor-II in human hepatocellular carcinomas: an immunohistochemical study. Path Intl 44:131–137 Rogler CE, Yang D, Rossetti L, Donohoe J, Alt E, Chang CJ, Rosenfeld R, Neely K, Hintz R 1994 Altered body composition and increased frequency of diverse malignancies in insulin-like growth factor-II transgenic mice. J Biol Chem 269:13779 –13784 Scharf JG, Schmidt-Sandte W, Pahernick SA, Ramadori G, Braulke T, Hartmann H 1998 Characterization of insulin-like growth factor axis in a human hepatoma cell line (PLC). Carcinogenesis 19:2121–2128 Ng IO, Lee JM, Srivastava G, Ng M 1998 Expression of insulin-like growth factor-II mRNA in hepatocellular carcinoma. J Gastroenterol Hepatol 13:152–157 Sohda T, Oka Y, Iwata K, Gunn J, Kamimura S, Shijo H, Okumura M, Yun K 1997 Co-localization of insulin-like growth factor-II and the proliferation marker MIB1 in hepatocellular carcinoma cells. J Clin Pathol 50:135–137 Lin SB, Hsieh SH, Hsu HL, Lai MY, Kan LS, Au LC 1997 Antisense oligodeoxynucleotides of IGF-II selectively inhibit growth of human hepatoma cells overproducing IGF-II. J Biochem 122:717–722 Minuto F, Del Monte P, Barreca A, Fortini P, Cariola G, Catrambone G, Giordano G 1986 Evidence for an increased somatomedinC/insulin-like growth factor-I content in primary human lung tumors. Cancer Res 46:985–988 Minuto F, Del Monte P, Barreca A, Alama A, Cariola G, Giordano G 1988 Evidence for autocrine mitogenic stimulation by somatomedin-C/insulin-like growth factor-I on an established human lung cancer cell line. Cancer Res 46:3716 –3719 Nakanishi Y, Mulshine JL, Kasprzyk PG, Natale RB, Maneckjee R, Avis I, Treston AM, Gazdar AF, Minna JD, Cuttitta F 1988 Insulin-like growth factor-I can mediate autocrine proliferation of human small cell lung cancer cell lines in vitro. J Clin Invest 82: 354 –359 Jaques G, Rotsch M, Wegmann C, Worsch U, Maasberg M, Havemann K 1988 Production of immunoreactive insulin-like growth factor-I and response to exogenous IGF-I in small cell lung cancer cell lines. Exp Cell Res 176:336 –343 Macaulay VM, Everard MJ, Teale JD, Trott PA, Van Wyk JJ, Smith IE, Millar JL 1990 Autocrine function for insulin-like growth factor-I in human small cell lung cancer cell lines and fresh tumor cells. Cancer Res 50:2511–2517 Reeve JG, Payne JA, Bleehan NM 1990 Production of immunoreactive insulin-like growth factor (IGF)-I and IGF binding proteins by human lung tumors. Br J Cancer 61:727–731 Rotsch M, Maasberg M, Erbil C, Jacques G, Worsh U, Havemann K 1992 Characterization of insulin-like growth factor-I receptors and growth effects in human lung cancer cell lines. J Cancer Res Clin Oncol 118:502–508 Schardt C, Rotsch M, Erbil C, Goke R, Richter G, Havemann K

133.

134. 135.

136. 137.

138. 139.

140. 141. 142.

143.

144.

145.

146.

147.

148.

149.

150.

151.

241

1993 Characterization of insulin-like growth factor-II receptors in human small cell lung cancer cell lines. Exp Cell Res 204:22–29 Kaicer U, Schardt C, Brandscheidt D, Wollmer E, Havemann K 1993 Expression of insulin-like growth factor-I and IGF-II receptors in normal human lung and in lung cancer. J Cancer Res Clin Oncol 119:665– 668 Zia F, Jacobs S, Kull Jr F, Cuttita F, Mulshine JL, Moody TW 1996 Monoclonal antibody ␣IR3 inhibits non-small cell lung cancer growth in vitro and in vivo. J Cell Biochem Suppl 24:269 –275 Lee CT, Wu S, Gabrilovich D, Chen H, Nadaf-Rahrov S, Ciernik IF, Carbone DP 1996 Antitumor effects of an adenovirus expressing antisense insulin-like growth factor-I receptor on human lung cancer cell lines. Cancer Res 56:3038 –3041 Long L, Rubin R, Brodt P 1998 Enhanced invasion and liver colonization by lung carcinoma cells overexpressing the type I IGF receptor. Exp Cell Res 238:116 –121 Taylor JE, Bogden AE, Moreau JP, Coy DH 1988 In vitro and in vivo inhibition of human small cell lung carcinoma (NCI-H69) growth by a somatostatin analogue. Biochem Biophys Res Commun 153: 81– 86 Bogden AE, Taylor JE, Moreau JP, Coy DH, LePage DJ 1990 Response of human lung tumor xenografts with a somatostatin analogue (Somatuline). Cancer Res 50:4360 – 4365 Pinski J, Scally AV, Halmos G, Szepeshazi K, Groot K, O’Bryne K, Cali RZ 1994 Effects of somatostatin analog RC-160 and bombesin/gastrin-releasing peptide antagonists on the growth of human small-cell and non-small-cell lung carcinomas in nude mice. Br J Cancer 70:886 – 892 Macaulay VM, Smith IE, Everard MJ, Teale JD, Reubi JC, Millar JL 1991 Experimental and clinical studies with somatostatin analogue octreotide in small cell lung cancer. Br J Cancer 64:451– 456 Cotto C, Quoix E, Thomas F, Henane S, Trillet-Lenoir V 1994 Phase I study of the somatostatin analogue somatuline in refractory small-cell lung carcinoma. Ann Oncol 5:290 –291 Marschke Jr RF, Grill JP, Sloan JA, Wender DB, Levitt R, Mailliard JA, Gerstner JB, Ghosh C, Morton RF, Jett JR 1999 Phase II study of high dose somatostatin analogue in patients either previously treated or untreated who have extensive-stage small cell lung cancer. Am J Clin Oncol 22:15–17 Tode B, Serio M, Rotella CM, Galli G, Franceschelli F, Tannini A, Toccafondi R 1989 Insulin-like growth factor-I: autocrine secretion by human thyroid follicular cells in primary culture. J Clin Endocrinol Metab 69:639 – 647 Minuto F, Barreca A, Del Monte P, Cariola P, Torre GC, Giordano G 1989 Immunoreactive insulin-like growth factor (IGF)-I and IGF binding protein content in human thyroid tissue. J Clin Endocrinol Metab 68:621– 626 Onoda N, Ohmura E, Tsushima T, Ohba Y, Emoto N, Isozaki O, Sato Y, Shizume K, Demura H 1992 Autocrine role of insulin-like growth factor-I in a human thyroid cancer cell line. Eur J Cancer 28A:1904 –1909 Yashiro T, Ohba Y, Murakami H, Obara T, Tsushima T, Fujimoto Y, Shizume K, Ito K 1989 Expression of insulin-like growth factor receptors in primary human thyroid neoplasms. Acta Endocrinol (Copenh) 121:112–120 Yashiro T, Tsushima T, Murakami H, Obara T, Fujimoto Y, Shizume K, Ito K 1991 Insulin-like growth factor-II/mannose-6-phosphate receptors are increased in primary human thyroid neoplasms. Eur J Cancer 27:699 –703 Tramontano D, Cushing GW, Moses AC, Ingbar SH 1986 Insulinlike growth factor-I stimulates the growth of rat thyroid cells in culture and synergizes the stimulation of DNA synthesis induced by TSH and Graves’-IgG. Endocrinology 119:940 –942 Huff KK, Kaufman D, Gabbay KH, Spencer EM, Lippman ME, Dickson RB 1986 Secretion of an insulin-like growth factor-I related protein by human breast cancer cells. Cancer Res 46:4613– 4619 Yee D, Paik S, Lebovic GS, Marcus RR, Favoni RE, Cullen KJ, Lippman ME, Rosen N 1989 Analysis of insulin-like growth factor-I gene expression in malignancy: evidence for a paracrine role in human breast cancer. Mol Endocrinol 3:509 –517 Paik S 1992 Expression of IGF-I and IGF-II mRNA in breast tissue. Breast Cancer Res Treat 22:31–38

242

KHANDWALA ET AL.

152. Gebauer G, Jager W, Lang N 1998 mRNA expression of the components of the insulin-like growth factor system in breast cancer cell lines, tissues and metastatic breast cancer cells. Anticancer Res 18:1191–1196 153. Osborne CK, Coronado EB, Kitten LJ, Arteaga CI, Fuqua SA, Ramasharma K, Marshall M, Li CH 1989 Insulin-like growth factor-II: a potential autocrine/paracrine growth factor for human breast cancer acting via the IGF-I receptor. Mol Endocrinol 3:1701– 1709 154. Furlanetto RW, DiCarlo JN 1984 Somatomedin-C receptors and growth effects in human breast cells maintained in long-term tissue culture. Cancer Res 44:2122–2128 155. Myal Y, Shiu RPC, Bhaumick B, Bala M 1984 Receptor binding and growth-promoting activity of insulin-like growth factors in human breast cancer cells (T-47D) in culture. Cancer Res 44:5486 – 5490 156. Pollak MN, Polychronakos C, Yousefi S, Richard M 1988 Characterization of insulin-like growth factor-I receptors of human breast cancer cells. Biochem Biophys Res Commun 154:326 –331 157. De Leon DD, Bakker B, Wilson DM, Hintz RL, Rosenfeld RG 1988 Demonstration of insulin-like growth factor (IGF)-I and IGF-II receptors and binding protein in human breast cancer cell lines. Biochem Biophys Res Commun 152:398 – 405 158. Peyrat JP, Bonneterre J 1992 Type I IGF receptor in human breast diseases. Breast Cancer Res Treat 22:59 – 67 159. Clemmons DR, Camacho-Hubner C, Coronado E, Osborne CK 1990 Insulin-like growth factor binding protein secretion by breast carcinoma cell lines: correlation with estrogen receptor status. Endocrinology 127:2679 – 86 160. Pratt SE, Pollak MN 1993 Estrogen and antiestrogen modulation of MCF7 breast cancer cell proliferation is associated with specific alterations in accumulation of insulin-like growth factor binding proteins in conditioned media. Cancer Res 53:5193–5198 161. Pratt SE, Pollak MN 1994 Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits estrogen-stimulated breast cancer cell proliferation. Biochem Biomed Res Commun 198:292–297 162. Huynh H, Yang X, Pollak M 1996 Estradiol and antiestrogens regulate a growth inhibitory insulin-like growth factor binding protein-3 autocrine loop in human breast cancer cells. J Biol Chem 271:1016 –1021 163. Oh Y, Muller HL, Lamson G, Rosenfeld RG 1993 Insulin-like growth factor (IGF)-independent actions of IGF-binding protein-3 in Hs578T human breast cancer cells. Cell surface binding and growth inhibition. J Biol Chem 268:14964 – 4971 164. Nickerson T, Huynh H, Pollak M 1997 Insulin-like growth factor binding protein-3 induces apoptosis in MCF-7 breast cancer cells. Biochem Biomed Res Commun 237:690 – 693 165. Martin JL, Coverley JA, Pattison ST, Baxter RC 1995 Insulin-like growth factor binding protein 3 (IGFBP-3) production by MCF-7 breast cancer cells: stimulation by retinoic acid and cyclic adenosine monophosphate and differential effects of estradiol. Endocrinology 136:1219 –1226 166. Gucev ZS, Oh Y, Kelley KM, Rosenfeld RG 1996 Insulin-like growth factor binding protein 3 mediates retinoic acid and transforming growth factor ␤2-induced growth inhibition in human breast cancer cells. Cancer Res 56:1545–1550 167. Gill ZP, Perks CM, Newcomb PV, Holly JM 1997 Insulin-like growth factor binding protein 3 (IGFBP-3) predisposes breast cancer cells to programmed cell death in a non-IGF-dependent manner. J Biol Chem 272:25602–25607 168. Yee D, Jackson JG, Kozelsky TW, Figueroa JA 1994 Insulin-like growth factor binding protein-1 expression inhibits insulin-like growth factor-I action in MCF-7 breast cancer cells. Cell Growth Diff 5:73–77 169. Van den Berg CL, Cox GN, Stroh CA, Hilsenbeck SG, Weng C-N, McDermott MJ, Pratt D, Osborne CK, Coronado-Heinsohn EB, Yee D 1997 Polyethylene glycol conjugated insulin-like growth factor binding protein-1 (IGFBP-1) inhibits growth of breast cancer in athymic mice. Eur J Cancer 33:1108 –111 170. Rohlik QT, Adams D, Kull Jr FC, Jacobs S 1987 An antibody to the receptor for insulin-like growth factor receptor-I inhibits the growth of MCF-7 cells in tissue culture. Biochem Biophys Res Commun 149:276 –281

Vol. 21, No. 3

171. Karey KP, Sirbasku DA 1988 Differential responsiveness of human breast cancer cell lines MCF-7 and T47D to growth factors and 17␤-estradiol. Cancer Res 48:4083– 4092 172. Arteaga CL, Osborne CK 1989 Growth inhibition of human breast cancer cells in vitro with an antibody against the type I somatomedin receptor. Cancer Res 49:6237– 6241 173. Arteaga CL, Kitten LJ, Coronado EB, Jacobs S, Kull Jr FC, Allred DC, Osborne CK 1989 Blockade of type I somatomedin receptor inhibits growth of human breast cancer cells in athymic mice. J Clin Invest 84:1418 –1423 174. Stewart AJ, Johnson MD, May FE, Westley BR 1990 Role of insulin-like growth factor and the type I insulin-like growth factor receptor in the estrogen-stimulated proliferation of human breast cancer cells. J Biol Chem 265:21172–21178 175. De Leon DD, Wilson DM, Powers M, Rosenfeld RG 1992 Effects of insulin-like growth factors (IGFs) and IGF receptor antibodies on the proliferation of human breast cancer cells. Growth Factors 6:327–336 176. Weckbecker G, Tolcsvai L, Stolz B, Pollak M, Bruns C 1994 Somatostatin analogue octreotide enhances the antineoplastic effects of tamoxifen and ovariectomy on 7,12-DMBA induced rat mammary carcinomas. Cancer Res 54:6334 – 6337 177. Yang XF, Beamer WG, Huynh H, Pollak M 1996 Reduced growth of human breast cancer xenografts in hosts homozygous for the lit mutation. Cancer Res 56:1509 –1511 178. Huynh H, Pollak M 1994 Enhancement of tamoxifen induced suppression of insulin-like growth factor-I gene expression and serum level by a somatostatin analogue. Biochem Biophys Res Commun 203:253–259 179. Peyrat JP, Bonneterre J, Hecquet B, Vennin P, Louchez MM, Fournier C, Lefebvre J, Demaille A 1993 Plasma insulin-like growth factor-I concentrations in human breast cancer. Eur J Cancer 29A:492– 497 180. Hankinson SE, Willett WC, Colditz GA, Hunter DJ, Michaud DS, Deroo B, Rosner B, Speizer FE, Pollak M 1998 Circulating concentrations of insulin-like growth factor-I and risk of breast cancer. Lancet 351:1393–1396 181. Pollak M, Costantino J, Polychronakos C, Blauer SA, Guyda H, Redmond C, Fisher B, Margolese R 1990 Effects of tamoxifen on serum insulin-like growth factor-I levels in stage I breast cancer patients. J Natl Cancer Inst 82:1693–1697 182. Vennin PH, Peyrat JP, Bonneterre J, Louchez MM, Harris AG, Demaille A 1989 Effect of the long acting somatostatin analogue SMS 201–995 (Sandostatin) in advanced breast cancer. Anticancer Res 9:153–156 183. Di Leo A, Ferrari L, Bajetta E, Bartoli C, Vicario G, Moglia D, Miceli R, Callegari M, Bono A 1995 Biological and clinical evaluation of lanreotide (BIM 23014), a somatostatin analogue in the treatment of advanced breast cancer. Breast Cancer Res Treat 34: 237–244 184. Canobbio L, Cannata D, Miglietta L, Boccardo F 1995 Somatuline (BIM 23014) and tamoxifen treatment of postmenopausal breast cancer patients: clinical activity and effect on insulin-like growth factor-I levels. Anticancer Res 15:2687–2690 185. O’Byrne KJ, Dobbs N, Propper DJ, Braybrooke JP, Koukourakis MI, Mitchell K, Woodhull J, Talbot DC, Schally AV, Harris AL 1999 Phase II study of RC-160 (vapreotide), an octapeptide analogue of somatostatin, in the treatment of metastatic breast cancer. Br J Cancer 79:1413–1418 186. Ingle JN, Suman VJ, Kardinal CG, Krook JE, Mailliard JA, Veeder MH, Loprinzi CL, Dalton RJ, Hartmann LC, Conover CA, Pollak MN 1999 A randomized trial of tamoxifen alone or combined with octreotide in the treatment of women with metastatic breast carcinoma. Cancer 85:1284 –1292 187. Yee D, Morales FR, Hamilton TC, Von Hoff DD 1991 Expression of insulin-like growth factor-I, its binding proteins, and its receptors in ovarian cancer. Cancer Res 51:5107–5112 188. Resnicoff M, Ambrose D, Coppola D, Rubin R 1993 Insulin-like growth factor-I and its receptor mediate the autocrine proliferation of human ovarian carcinoma cell lines. Lab Invest 69:756 –760 189. Karasik A, Menczer J, Pariente C, Kanety H 1994 Insulin-like growth factor (IGF)-I and IGF binding protein-2 are increased in

June, 2000

190.

191.

192.

193.

194.

195.

196.

197.

198. 199.

200.

201. 202. 203.

204.

205.

206.

IGFs AND NEOPLASIA

cyst fluids of epithelial ovarian cancer. J Clin Endocrinol Metab 78:271–276 Conover CA, Hartmann LC, Bradley S, Stalboerger P, Klee GG, Kalli KR, Jenkins RB 1998 Biological characterization of human epithelial ovarian carcinoma cells in primary culture: the insulinlike growth factor system. Exp Cell Res 238:439 – 449 Beck EP, Russo P, Gliozzo B, Jaeger W, Papa V, Wildt L, Pezzino V, Lang N 1994 Identification of insulin and insulin-like growth factor-I receptors in ovarian cancer tissue. Gynecol Oncol 53: 196 – 201 Krywicki RF, Figueroa JA, Jackson JG, Kozelsky TW, Shimasaki S, Von Hoff DD, Yee D 1993 Regulation of insulin-like growth factor binding proteins in ovarian cancer cells by oestrogen. Eur J Cancer 29A:2015–2019 Kanety H, Kattan M, Goldberg I, Kopolovic J, Ravia J, Menczer J, Karasik A 1996 Increased insulin-like growth factor binding protein 2 (IGFBP-2) gene expression and protein production lead to high IGFBP-2 content in malignant ovarian cyst fluid. Br J Cancer 73:1069 –1073 Flyvbjerg A, Mogensen O, Mogensen B, Nielsen OS 1997 Elevated serum insulin-like growth factor binding protein 2 (IGFBP-2) and decreased IGFBP-3 in epithelial ovarian cancer: correlation with cancer antigen 125 and tumor-associated trypsin inhibitor. J Clin Endocrinol Metab 82:2308 –2313 Muller M, Dietel M, Turzynski A, Wiechen K 1998 Antisense phophorothioate oligodeoxynucleotide down-regulation of the insulin-like growth factor-I receptor in ovarian cancer cells. Int J Cancer 77:567–571 Coppola D, Saunders B, Fu L, Mao W, Nicosia SV 1999 The insulin-like growth factor I receptor induces transformation and tumorigenicity of ovarian mesothelial cells and down-regulates their Fas-receptor expression. Cancer Res 59:3264 –3270 Klienman D, Roberts Jr CT, LeRoith D, Schally AV, Levy J, Sharoni Y 1993 Regulation of endometrial cancer cell growth by insulin-like growth factors and the leutinizing hormone-releasing hormone antagonist SB-75. Regul Pept 48:91–98 Hana V, Murphy LJ 1994 Expression of insulin-like growth factors and their binding proteins in the estrogen responsive Ishikawa human endometrial cancer cell line. Endocrinology 135:2511–2516 Kleinman D, Karas M, Danilenko D, Arbeli A, Roberts Jr CT, LeRoith D, Levy J, Sharoni Y 1996 Stimulation of endometrial cancer cell growth by tamoxifen is associated with increased insulin-like growth factor (IGF)-I induced tyrosine phosphorylation and reduction in IGF binding proteins. Endocrinology 137:1089 – 1095 Reynolds RK, Hu C, Baker VV 1998 Transforming growth factor ␣ and insulin-like growth factor-I, but not epidermal growth factor elicit autocrine stimulation of mitogenesis in endometrial cancer cell lines. Gynecol Oncol 70:202–209 Elkas J, Gray K, Howard L, Petit N, Pohl J, Armstrong A 1998 The effects of tamoxifen on endometrial insulin-like growth factor-I expression. Obstet Gynecol 91:45–50 Talavera F, Reynold RK, Roberts JA, Menon KM 1990 Insulin-like growth factor-I receptors in normal and neoplastic human endometrium. Cancer Res 50:3019 –3024 Rutanen EM, Nyman T, Lehtovirta P, Ammala M, Pekonen F 1994 Suppressed expression of insulin-like growth factor binding protein-I mRNA in the endometrium: a molecular mechanism associating endometrial cancer with its risk factors. Int J Cancer 59: 307–312 Kleinman D, Karas M, Roberts Jr CT, LeRoith D, Phillip M, Segev Y, Levy J, Sharoni Y 1995 Modulation of insulin-like growth factor-I (IGF-I) receptors and membrane associated IGF-binding proteins in endometrial cancer cells by estradiol. Endocrinology 136: 2531–2537 Pearl ML, Talavera F, Gretz III HF, Roberts JA, Menon KM 1993 Mitogenic activity of growth factors in the human endometrial adenocarcinoma cell lines HEC-1-A and KLE. Gynecol Oncol 49: 325–332 Steller MA, Delgado CH, Bartels CJ, Woodworth CD, Zou Z 1996 Overexpression of insulin-like growth factor-I receptor and autocrine stimulation in human cervical cancer cells. Cancer Res 56: 1761–1765

243

207. Hembree JR, Agarwal C, Eckert RL 1994 Epidermal growth factor suppresses insulin-like growth factor binding protein-3 levels in human papillomavirus type-16 immortalized cervical epithelial cells and thereby potentiates the effects of insulin-like growth factor-I. Cancer Res 54:3160 –3166 208. Cohen P, Peehl DM, Lamson G, Rosenfeld RG 1991 Insulin-like growth factors (IGFs), IGF receptors and IGF binding proteins in primary cultures of prostate epithelial cells. J Clin Endocrinol Metab 73:401– 407 209. Kaicer EK, Blat C, Harel L 1991 Insulin-like growth factor-I and IGFBPs: stimulatory and inhibitory factors secreted by human prostate adenocarcinoma cells. Growth Factors 4:231–237 210. Pietrzkowski Z, Mulholland G, Gomella L, Jameson BA, Wernicke D, Baserga R 1993 Inhibition of growth of prostate cancer cell lines by peptide analogues of insulin-like growth factor-I. Cancer Res 53:1102–1106 211. Iwamura M, Sluss PM, Casamento JB, Cockett AT 1993 Insulinlike growth factor-I: action and receptor characterization in human prostate cancer cell lines. Prostate 22:243–252 212. Connolly JM, Rose DR 1994 Regulation of DU 145 human prostate cancer cell proliferation by insulin-like growth factors and its interaction with the epidermal growth factor autocrine loop. Prostate 24:167–175 213. Angelloz-Nicoud P, Binoux M 1995 Autocrine regulation of cell proliferation by the insulin-like growth factor (IGF) and the IGF binding protein-3 protease system in a human prostate carcinoma cell line (PC-3). Endocrinology 136:5485–5492 214. Kaplan PJ, Mohan S, Cohen P, Foster BA, Greenberg NM 1999 The insulin-like growth factor axis and prostate cancer: lessons from the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Cancer Res 59:2203–2209 215. Tennant MK, Thrasher JB, Twomey PA, Drivdahl RH, Birnbaum RS, Plymate SR 1996 Protein and messenger RNA for the type I insulin-like growth factor (IGF) receptor is decreased and IGF-II mRNA is increased in human prostate carcinoma compared to benign prostate epithelium. J Clin Endocrinol Metab 81:3774 –3782 216. Lamharzi N, Schally AV, Koppan M, Groot K 1998 Growth hormone-releasing hormone antagonist MZ-5–156 inhibits growth of DU-145 human androgen-independent prostate carcinoma in nude mice and suppresses the levels and mRNA expression of insulinlike growth factor-II in tumors. Proc Natl Acad Sci USA 95:8864 – 8868 217. Figueroa JA, Lee AV, Jackson JG, Yee D 1995 Proliferation of cultured human prostate cancer cells is inhibited by insulin-like growth factor (IGF) binding protein-1: evidence for an IGF-II autocrine growth loop. J Clin Endocrinol Metab 80:3476 –3482 218. Damon SE, Maddison L, Ware JL, Plymate SR 1998 Overexpression of an inhibitory insulin-like growth factor binding protein (IGFBP), IGFBP-4, delays onset of prostate tumor formation. Endocrinology 139:3456 –3464 219. Nickerson T, Miyake H, Gleave ME, Pollak M 1999 Castration induced apoptosis of androgen-dependent Shionogi carcinoma is associated with increased expression of genes encoding insulin-like growth factor binding proteins. Cancer Res 59:3392–3395 220. Schally AV, Redding TW 1987 Somatostatin analogs as adjuncts to agonists of leutinizing hormone-releasing hormone in the treatment of experimental prostate cancer. Proc Natl Acad Sci USA 84:7275–7279 221. Murphy WA, Lance VA, Moreau S, Moreau JP, Coy DH 1987 Inhibition of rat prostate tumor growth by an octapeptide analog of somatostatin. Life Sci 40:2515–2522 222. Yano T, Pinski J, Szepeshazi K, Milovanovic SR, Groot K, Schally AV 1992 Effects of microcapsules of leutenizing hormone-releasing hormone antagonist SB-75 and somatostatin analog RC-160 on endocrine status and tumor growth in the Dunning R-3327H rat prostate cancer model. Prostate 20:297–310 223. Pinski J, Schally AV, Halmos G, Szepeshazi K 1993 Effect of somatostatin analog RC-160 and bombesin/gastrin releasing peptide antagonist RC-3095 on growth of PC-3 human prostate cancer xenografts in nude mice. Int J Cancer 55:963–967 224. Pinski J, Halmos G, Schally AV 1993 Somatostatin analog RC-160 and bombesin/gastrin releasing peptide antagonist RC-3095 in-

244

225.

226.

227.

228.

229.

230.

231.

232.

233.

234.

235.

236.

237.

238.

239. 240. 241. 242.

KHANDWALA ET AL. hibit the growth of androgen-independent DU-145 human prostate cancer line in nude mice. Cancer Lett 71:189 –196 Burfeind P, Chernicky CL, Rininsland F, Ilan J, Ilan J 1996 Antisense RNA to the type I insulin-like growth factor receptor suppresses tumor growth and prevents invasion by rat prostate cancer cells in vivo. Proc Natl Acad Sci USA 93:7263–7268 Jungwirth A, Schally AV, Pinski J, Halmos G, Groot K, Armatis P, Vadillo-Buenfil M 1997 Inhibition of in vivo proliferation of androgen-independent prostate cancers by an antagonist of growth hormone-releasing hormone. Br J Cancer 75:1585–1592 Mantzoros CS, Tzonou A, Signorello LB, Stampfer M, Trichopoulos D, Adami HO 1997 Insulin-like growth factor-I in relation to prostate cancer and benign prostatic hyperplasia. Br J Cancer 76:1115–1118 Chan JM, Stampfer MJ, Giovanucci E, Gann PH, Ma J, Wilkinson P, Hennekens CH, Pollak M 1998 Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study. Science 279: 563–566 Wolk A, Mantzoros CS, Anderson SO, Bergstorm R, Signorello LB, Lagiou P, Adami HO, Trichopoulos D 1998 Insulin-like growth factor-I and prostate cancer risk: a population based, casecontrol study. J Natl Cancer Inst 90:911–915 Parmar H, Charlton CD, Phillips RH, Edwards L, Bejot JL, Thomas F, Lightman SL 1992 Therapeutic response to somatostatin analogue BIM 23014 in metastatic prostate cancer. Clin Exp Metastasis 10:3–11 Figg WD, Thibault A, Cooper MR, Reid R, Headlee D, Dawson N, Kohler DR, Reed E, Sartor O 1995 A phase I study of the somatostatin analogue somatuline in patients with metatstatic hormone-refractory prostate cancer. Cancer 75:2159 –2164 Maulard C, Richaud P, Droz JP, Jessueld D, Dufour-Esquerre F, Housset M 1995 Phase I-II study of the somatostatin analogue lanreotide in hormone-refractory prostate cancer. Cancer Chemother Pharmacol 36:259 –262 Biddle C, Li CH, Schofield PN, Tate VE, Hopkins B, Engstrom W, Huskisson NS, Graham CF 1988 Insulin-like growth factors and the multiplication of Tera-2, a human teratoma-derived cell line. J Cell Sci 90:475– 484 Weima SM, Stet LH, Van Rooijen MA, Van Buul-Offers SC, Van Zoelen EJ, De Laat SW, Mummery CL 1989 Human teratocarcinoma cells express functional insulin-like growth factor-I receptors. Exp Cell Res 184:427– 439 Jungwirth A, Schally AV, Pinski J, Groot K, Armatis P, Halmos G 1997 Growth hormone-releasing hormone antagonist MZ-4 –71 inhibits in vivo proliferation of Caki-I renal adenocarcinoma. Proc Natl Acad Sci USA 94:5810 –5813 Dunn SE, Kari FW, French J, Leinenger JR, Travlos G, Wilson R Barrett JC 1997 Dietary restriction reduces insulin-like growth factor-I levels which modulates apoptosis, cell proliferation, and tumor progression in p53-deficient mice. Cancer Res 57:4667– 4672 Hursting SD, Switzer BR, French JE, Kari FW 1993 The growth hormone: insulin-like growth factor-I axis is a mediator of diet restriction-induced inhibition of mononuclear cell leukemia in Fischer rats. Cancer Res 53:2750 –2757 Raile K, Hoflich A, Kessler U, Yang Y, Pfuender M, Blum WF, Kolb H, Schwarz HP, Kiess W 1994 Human osteosarcoma (U-2 OS) cells express both insulin-like growth factor (IGF)-I receptors and IGF-II receptors and synthesize IGF-II: autocrine growth stimulation by IGF-II via the IGF receptor. J Cell Physiol 159:531–541 Burrow S, Andrulis IL, Pollak M, Bell RS 1998 Expression of insulin-like growth factor receptor, IGF-I and IGF-II in primary and metastatic osteosarcoma. J Surg Oncol 69:21–27 Pollak MN, Polychronakos C, Richard M 1990 Insulin-like growth factor-I: a potent mitogen for human osteogenic sarcoma. J Natl Cancer Inst 82:301–305 Kappel CC, Velez-Yanguas MC, Hirschfeld S, Helman LJ 1994 Human osteosarcoma cell lines are dependent on insulin-like growth factor-I for in vitro growth. Cancer Res 54:2803–2807 Pollak M, Sem AW, Richard M, Tetenes E, Bell R 1992 Inhibition of metastatic behavior of murine osteosarcoma by hypophysectomy. J Natl Cancer Inst 84:966 –971

Vol. 21, No. 3

243. Pinski J, Schally AV, Groot K, Halmos G, Szepeshazi K, Zarandi M, Armatis P 1995 Inhibition of growth of human osteosarcomas by antagonists of growth hormone-releasing hormone. J Natl Cancer Inst 87:1787–1794 244. Pinski J, Schally AV, Halmos G, Szepeshazi K, Groot K 1996 Somatostatin analog RC-160 inhibits the growth of human osteosarcomas in nude mice. Int J Cancer 65:870 – 874 245. Foley Jr TP, Nissley SP, Stevens RL, King GL, Hascall VC, Humbel RE, Short PA, Rechler MM 1982 Demonstration of receptors for insulin and insulin-like growth factors on Swarm rat chondrosarcoma chondrocytes. J Biol Chem 25:663– 669 246. Takigawa M, Okawa T, Pan HO, Aoki C, Takahashi K, Zue JD, Suzuki F, Kinoshita A 1997 Insulin-like growth factor-I and II are autocrine factors in stimulating proteoglycan synthesis, a marker of differentiated chondrocytes, acting through their respective receptors on a clonal human chondrosarcoma-derived chondrocyte cell line, HCS-2/8. Endocrinology 138:4390 – 4400 247. Seong SC, Matsumura T, Lee FY, Whelan MC, Li XQ, Trippel SB 1994 Insulin-like growth factor-I regulation of Swarm rat chondrosarcoma chondrocytes in culture. Exp Cell Res 211:238 –244 248. Butler AA, Blakesley VA, Tsokos M, Pouliki V, Wood TL, LeRoith D 1998 Stimulation of tumor growth by recombinant human insulin-like growth factor (IGF)-I is dependent on the dose and the level of IGF-I receptor expression. Cancer Res 58:3021–3027 249. Rodeck U, Herlyn M, Menssen HD, Furlanetto RW, Koprowski H 1987 Metastatic but not primary melanoma cell lines grow in vitro independently of exogenous growth factors. Int J Cancer 40: 687– 690 250. Furlanetto RW, Harwell SE, Baggs RB 1993 Effects of insulin-like growth factor receptor inhibition on human melanomas in culture and in athymic mice. Cancer Res 53:2522–2526 251. Resnicoff M, Coppolla D, Sell C, Rubin R, Ferrone S, Baserga R 1994 Growth inhibition of human melanoma cells in nude mice by antisense strategies to the type I insulin-like growth factor receptor. Cancer Res 54:4848 – 4850 252. Neely EK, Morhenn VB, Hintz RL, Wilson DM, Rosenfeld RG 1991 Insulin-like growth factors are mitogenic for human keratinocytes and a squamous cell carcinoma. J Invest Dermatol 96: 104 –110 253. Bol DK, Kiguchi K, Gimenez-Conti I, Rupp T, DiGiovanni J 1997 Overexpression of insulin-like growth factor-I induces hyperplasia, dermal abnormalities, and spontaneous tumor formation in transgenic mice. Oncogene 14:1725–1734 254. Wilker E, Bol D, Kiguchi K, Rupp T, Beltran L, DiGiovanni J 1999 Enhancement of susceptibility to diverse skin tumor promoters by activation of insulin-like growth factor-I receptor in the epidermis of transgenic mice. Mol Carcinog 25:122–131 255. Vetter U, Schlickenrieder JH, Zapf J, Hartmann W, Heit W, Hitzler H, Byrne P, Gaedicke G, Heinze E, Teller WM 1986 Human leukemic cells: receptor binding and biological effects of insulin and insulin-like growth factors. Leukemia Res 10:1201–1207 256. Lee PD, Rosenfeld RG, Hintz RL, Smith SD 1986 Characterization of insulin, insulin-like growth factor-I and II and growth hormone receptors on human leukemic lymphoblasts. J Clin Endocrinol Metab 62:28 –35 257. Pepe MG, Ginzton NH, Lee PD, Hintz RL, Greenberg PL 1987 Receptor binding and mitogenic effects of insulin and insulin-like growth factor-I and II for human myeloid leukemic cells. J Cell Physiol 133:219 –227 258. Sinclair J, McClain D, Taetle R 1988 Effects of insulin and insulinlike growth factor-I on growth of human leukemia cells in serumfree and protein-free medium. Blood 72:66 –72 259. Estrov Z, Meir R, Barak Y, Zaizov R, Zadik Z 1991 Human growth hormone and insulin-like growth factor-I enhance the proliferation of human leukemic blasts. J Clin Oncol 9:394 –399 260. Conlon MA, Tomas FM, Owens PC, Wallace JC, Howarth GS, Ballard FJ 1995 Long R-3 insulin-like growth factor-I (IGF-I) stimulates organ growth but reduces plasma IGF-I, IGF-II and IGF binding protein concentration in guinea pigs. J Endocrinol 146: 247–253