Cancer Biology & Therapy Tumor progression and

This article was downloaded by: [5.135.159.162] On: 08 September 2015, At: 23:32 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London, SW1P 1WG

Cancer Biology & Therapy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kcbt20

Tumor progression and metastasis from genetic to microenvironmental determinants Yves A. De Clerck, Bernard E. Weissman, Dihua Yu, Ramon Parsons, Menashe Bar-Eli, Pradip Roy-Burman, Victoria L. Seewaldt, Anne E. Cress, Lucia R. Languino, Surinder K. Batra, Careen K. Tang, Shije Sheng, Wen-Tien Chen, Srikumar P. Chellappan, Shi-Yuan Cheng, Stephan Ladisch, James B. McCarthy, Lisa M. Coussens & Michael B. Cohen Published online: 31 Dec 2006.

To cite this article: Yves A. De Clerck, Bernard E. Weissman, Dihua Yu, Ramon Parsons, Menashe Bar-Eli, Pradip Roy-Burman, Victoria L. Seewaldt, Anne E. Cress, Lucia R. Languino, Surinder K. Batra, Careen K. Tang, Shije Sheng, Wen-Tien Chen, Srikumar P. Chellappan, Shi-Yuan Cheng, Stephan Ladisch, James B. McCarthy, Lisa M. Coussens & Michael B. Cohen (2006) Tumor progression and metastasis from genetic to microenvironmental determinants, Cancer Biology & Therapy, 5:12, 1588-1599, DOI: 10.4161/cbt.5.12.3660 To link to this article: http://dx.doi.org/10.4161/cbt.5.12.3660

PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions

[Cancer Biology & Therapy 5:12, 1588-1599, December 2006]; ©2006 Landes Bioscience

Meeting Report

Tumor Progression and Metastasis from Genetic to Microenvironmental Determinants

Yves A. De Clerck1,* Bernard E. Weissman2 Dihua Yu3 Ramon Parsons4 Menashe Bar-Eli3 Pradip Roy-Burman1 Victoria L. Seewaldt5 Anne E. Cress6 Lucia R. Languino7 Surinder K. Batra8 Careen K. Tang9 Shijie Sheng10 Wen-Tien Chen11 Srikumar Chellappan12 Shi-Yuan Cheng13 Stephan Ladisch14 James B. McCarthy15 Lisa M. Coussens16 Michael B. Cohen17,*

Introduction

CE

.D

ON

OT D

IST

RIB

UT E

.

The peer review process at the NIH began in 1946 in the Division of Research Grants (DRG), which eventually became the Center for Scientific Review (CSR). Amongst the inaugural study sections in the DRG was one in Pathology, which split into Pathology A (PTH A) and Pathology B (PTH B) in the early 1960’s; PTH C briefly existed in the early 2000’s. PTH A was dissolved in the mid 1990's and PTH B was reorganized in 2003 into TPM (Tumor Progression and Metastasis), TME (Tumor Microenvironment), and CG (Cancer Genetics), with TPM continuing on with the major historical focus of PTH B: mechanisms of tumor invasion and metastasis. For much of it's time (the early 1980's until 2006), PTH B, and thereafter TPM, was run by an Executive Secretary, later a Scientific Review Administrator (SRA), named Martin L. Padarathsingh, Ph.D. Thus, many investigators whose scientific work focused on the mechanisms of tumor biology had their work reviewed by PTH B and TPM, and were in close contact with Martin during the review process. In addition, investigators were recruited, groomed, and selected to serve on PTH B and TPM by him. Martin Padarathsingh grew up in Trinidad and Tobago, and came to the US to get his bachelor’s and master’s degrees from Howard University. At Howard, he was also an All American soccer player, and his passion for soccer has endured. From George Washington University he garnered a M.Phil. and a Ph.D. Martin worked briefly in the private sector after graduation but the vast majority of his professional career was at the NIH. His contributions to PTH B and TPM, and in fact to the peer review process as a whole, cannot be underestimated. For more than two decades he was the face of PTH B and TPM. By adhering to strict ethical guidelines that came from within, and which were imparted to study section members, Martin set a standard for professionalism that will be difficult to surpass. His advocacy for the investigator, his pleas for maintaining confidentiality, and his focus on the science were aspects of Martin's leadership that had a constant presence as applications were being reviewed. To many of us, he was a mentor, a colleague, and a friend. It was in the spirit of such friendship that current and former members of PTH B and TPM organized a Workshop in his honor on May 31, 2006 before his retirement from NIH on June 30, 2006. This workshop consisted of presentations from several present and past members of PTHB and TPM study sections, and covered genetic and epigenetic, cellular, and microenvironmental determinants of tumor progression and metastasis.

IEN

1University of Southern California Keck School of Medicine; Los Angeles, California USA

3University of Texas MD Anderson Cancer Center; Houston, Texas USA

SC

2University of North Carolina Lineberger Cancer Center; Chapel Hill, North Carolina USA

BIO

4Columbia University College of Physicians and Surgeons; New York, New York USA 5Duke University Medical Center; Durham, North Carolina USA

ES

6University of Arizona Cancer Center; Tucson, Arizonia USA

7University of Massachusetts School of Medicine; Worcester, Massachusettes USA

ND

8University of Nebraska Eppley Cancer Center; Omaha, Nebraska USA

9Georgetown University Lombardi Cancer Center; Washington, DC USA

LA

10Wayne State University; Detroit, Michigan USA

11State University of New York; Stony Brook, New York USA

06

12University of South Florida H. Lee Moffitt Cancer Center; Tampa, Flordia USA 13University of Pittsburgh Cancer Institute; Pittsburgh, Pennsylvania USA

20

14George Washington University; Washington, DC USA

15University of Minnesota Cancer Center; Minneapolis, Minnesotta USA

©

Downloaded by [5.135.159.162] at 23:32 08 September 2015

A Workshop of the Tumor Progression and Metastasis NIH Study Section in Honor of Dr. Martin L. Padarathsingh, May 31, 2006, Georgetown, Washington, DC

16University of California San Francisco Cancer Center; San Francisco, California USA 17University of Iowa; Iowa City, Iowa USA

*Correspondence to: Yves A. DeClerck; Division of Hematology-Oncology, Childrens Hospital Los Angeles; 4650 Sunset Boulevard, MS #54; Los Angeles, California 90027 USA; Tel.: 323.669.2150; Fax: 323.664.9455; Email: [email protected]/Michael Cohen; Department of Pathology; University of Iowa; 200 Hawkins Drive; C670 GM; Iowa City, Iowa 52242 USA; Tel.: 319.384.9009; Fax: 319.384.9613; Email: [email protected] Original manuscript submitted: 11/14/06 Manuscript accepted: 12/06/06 Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/abstract.php?id=3660

1588

GENETIC AND EPIGENETIC DETERMINANTS OF TUMOR PROGRESSION Chromatin remodeling and epithelial mesenchymal transformation. Chromatin structure plays an important role in the regulation of gene expression. In eukaryotes, the packaging of DNA into repeating units of nucleosomes functions inherently to suppress gene transcription. Chromatin modifying and remodeling complexes have evolved to counteract nucleosome mediated repression of transcription.1 These complexes act by covalently modifying histones or by altering chromatin structure in an ATP-dependent fashion. Acetylation of histone amino terminal tails by histone acetyltransferase (HAT) complexes represents one enzymatic mechanism that leads to a “loosening” of the wrap of the DNA around the histone core. Others include histone phosphorylation, methylation, ubiquination, ADP ribosylation and glycosylation. Motor driven chromatin remodeling utilizes the energy of ATP to disrupt nucleosome structure. In humans, the SWI/SNF (SNF2), RSF (ISWI), and NURD (CHD) complexes use this mechanism.2 During the past eight years, multiple reports have demonstrated that alterations in the human SWI/SNF complexes play important roles in development of human diseases especially Cancer Biology & Therapy

2006; Vol. 5 Issue 12

Tumor Progression and Metastasis NIH Study Section


Photos of selected NIH Pathology B Study Section Meetings showing participants in 1986. The NIH Pathology B Study Section met regularly between 1946 and 2003.

neoplasia. For example, Dr. Weissman’s laboratory (University of North Carolina, Chapel Hill, NC) has shown that mutations and/or loss of the human SWI2 homolog, BRG1, occur in approximately 10% of human cancer cell lines and that loss of both BRG1 and BRM in non small cell lung cancer (NSCLC) represents a poor prognostic indicator, independent of stage.3 Focusing upon how the loss of SWI/SNF complex activity can lead to a poor outcome for NSCLC patients, they hypothesized that in the absence of the complex, increased DNA methylation might occur leading to more frequent gene silencing. Indeed, re expression of BRG1 or BRM or treatment with 5 dAzaC in doubly deficient cell lines resulted in restoration of E cadherin and CD44 expression.4 By knocking down BRG1 expression in the BRM deficient MiaPaca2 cell line using shRNA, they observed that loss of SWI/SNF complex activity resulted in a dramatic change in cellular morphology accompanied by altered actin cytoskeletal organization.5 Similar observations were made in a HeLa cell derivative D98oR. Knockdown of either BRM or BRG1 alone by stable expression of RNA did not apparently affect cellular morphology. However, dual knockdown induced a similar change in morphology to the BRG1 knockdown of the MiaPaca2 cells. Furthermore, the BRG1/BRM deficient D98oR cell line showed a significant reduction in CD44 expression. A preliminary gene expression analysis suggests that an epithelial to mesenchymal transition (EMT) may have taken place in the absence of SWI/SNF complex activity. The data thus implicate a role for the SWI/SNF complex in the regulation of cell differentiation and EMT. 14-3-3 plays a critical role in promoting multiple types of cancer. The 14-3-3 proteins are an important family of highly conserved, dimeric, 29 31kDa proteins ubiquitously expressed in all eukaryotic organisms. In humans, seven different isoforms have been identified.6 14-3-3 proteins lack endogenous enzymatic activity but instead modulate a variety of cellular processes by-binding to a broad spectrum of target proteins. 14-3-3 proteins bind primarily to phosphorylated serine/threonine motifs on their target proteins. However, some target proteins bind 14-3-3 independent of phosphorylation. To date, more than two hundred proteins are reported to interact with one or more of the 14-3-3 proteins. Through modulation of these interaction partners, 14-3-3 proteins have been implicated in regulation of a diverse number of cellular processes including cell cycle progression, mitogenic and stress signaling, apoptosis, transcription, metabolism, and cytoskeletal integrity.7 Deregulation of 14-3-3 proteins may disrupt normal cellular homeostasis and contribute to www.landesbioscience.com

the initiation and progression of cancer and other diseases. For example, the loss of 14-3-3ζ, a known tumor suppressor, is a frequent event in breast cancer. Through serendipitous observation, Dr. Yu’s laboratory (MD Anderson, Houston, TX) discovered that 14-3-3ζ is overexpressed in breast tumors and sarcomas, two tumor types with diverse origins. Although 14-3-3 proteins have been hypothesized for years to be important contributors to the oncogenic process, evidence linking 14-3-3 proteins directly to tumorigenic properties had been minimal for canonical 14-3-3 isoforms (β, γ, ε, η, τ and ζ). Her laboratory recently found that 14-3-3ζ was overexpressed in many different tumor types and that overexpression correlated with poor survival in patients. Additionally, modulating 14-3-3ζ expression altered tumorigenic properties such as survival, proliferation, and anchorage independent growth in multiple cancer cell lines suggesting that 14-3-3ζ drives malignancy through regulation of a signaling pathway/network critical for survival or growth in many cells. They have identified a novel mechanism by which 14-33ζ promotes oncogenesis, which may also contribute to immunity, obesity, diabetes, angiogenesis, neuronal survival, cardiac function, hematopoiesis, and stem cell self renewal. PTEN pathways and genetic instability in cancer. The PTEN and PI3 kinase pathway is frequently involved in tumor progression. PTEN and PI3K are enzymes that regulate the level of the second messenger phosphatidylinositol 3,4,5 triphosphate (PIP3) in opposing directions.8 Both PIK3CA (the gene that encodes the p110 catalytic subunit of PI3Ka) and PTEN (the gene that encodes the PIP3 phosphatase that removes phosphate from the 3' position of the phosphatidyl inositol ring) are mutated in human cancers leading to activation of PIK3CA or inactivation of PTEN. Dr. Parsons’ laboratory (Columbia University, New York, NY) had previously reported that in breast tumor development reduced PTEN protein expression and PIK3CA mutations occurred in invasive carcinoma.9 Alterations of both genes rarely overlapped in the same tumor and appeared to occur in different subsets of breast carcinoma, since PIK3CA mutations were usually found in tumors that were estrogen receptor positive while PTEN deficiency was found in tumors that were typically estrogen receptor negative.10 Analysis of the effects of Pten mutation in mice revealed that PTEN was essential for development soon after the embryo implants into the uterus.11 Pten heterozygous mice developed tumors in a wide variety of organs including the uterus, mammary gland, and adrenal medulla. Tumors in all organs were associated with increased levels of PI3K signaling detected via increased phosphorylation and activation of AKT and S6 kinases. Tumors frequently developed either through inactivation of the wildtype allele via a two hit mechanism or through a haploid sufficient one hit mechanism that required cooperation with other tumor suppressors or oncogene pathways.12 Tumors in the mice could be treated with rapamycin analogs that inhibit S6 kinase activation.13 Activated AKT kinase phosphorylated a wide range of substrates in

Cancer Biology & Therapy

1589



Photos of selected NIH Pathology B Study Section Meetings showing participants from the 1989 and the 1990s. The NIH Pathology B Study Section met regularly between 1946 and 2003.

the cells that stimulated tumor progression. One of these substrates was CHK1, which was critical for maintaining genomic stability in cycling cells. AKT was able to phosphorylate CHK1 and inhibit its activity by altering its cellular location and pattern of ubiquitination.14 Attenuated CHK1 function in PTEN deficient cells was likely to be playing a role in their genetic instability and poor checkpoint response to DNA damage. Analysis of the effect of PTEN on human breast tumor cell lines revealed that PTEN was able to activate the expression of insulin receptor substrate 2 (IRS2), an adaptor protein that activates PI3K.15 This suggests that cells have a feedback loop to upregulate PIP3 levels. Interestingly, examination of Pten+/tumors of the prostate showed that they were associated with increased levels of IRS2 protein and activation of AKT and S6 kinase, indicating that the feedback regulation of IRS2 was not occurring in these tumors as would have been expected. Analysis of the effects of the reversal of feedback regulation of IRS2 in tumors is an area of current active investigation by Dr. Parsons’ laboratory. Transcription factors AP 2 and CREB/ATF 1 in melanoma progression: Implications for therapy. The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP, metastatic phenotype) are not yet well defined. Dr. Bar Eli’s laboratory (MD Anderson, Houston, TX) has demonstrated that the progression of human melanoma is associated with loss of expression of the transcription factor AP 2. In metastatic melanoma cells, this loss resulted in overexpression of MCAM/MUC18 and MMP 2 and lack of c KIT expression. In addition, inactivation of AP 2 in primary cutaneous melanoma cells by dominant negative AP 2 (AP 2B) augmented their tumorigenicity in nude mice. His laboratory recently demonstrated that loss of AP 2 expression in metastatic melanoma cells resulted in overproduction of the thrombin receptor, PAR 1, which in turn contributes to the metastatic phenotype of melanoma by upregulating the expression of adhesion molecules, proteases and angiogenic factors. Using melanoma tissue microassays coupled with Laser Scanning Cytometry, they demonstrated an inverse correlation between AP 2 and PAR 1 expression in metastatic melanoma tumor specimens. Overall the data support the notion that AP 2 acts as a master regulator in the progression of human melanoma.16 Additionally, the transition of melanoma cells from RGP to VGP is associated with overexpression of the transcription factors CREB and ATF1, both of which may act as survival factors for human melanoma cells. Inactivation of CREB/ATF 1 activities in metastatic melanoma cells by dominant negative CREB or by anti ATF 1 single chain antibody fragment (ScFv), resulted in deregulation of MMP 2 and MCAM/MUC18, 1590

increased the sensitivity of melanoma cells to apoptosis, and inhibited their tumorigenicity and metastatic potential in vivo.17 In recent studies they have identified the platelet activating factor (PAF) which is secreted by cells within the tumor microenvironment as a factor involved in phosphorylation and activation of CREB/ATF 1 in melanoma cells. Since only metastatic melanoma cells express an active CREB/ATF 1, these cells may be better equipped to respond to PAF within their immediate tumor microenvironment.17 In an attempt to inhibit tumor growth and metastasis of human melanoma in vivo, Dr. Bar Eli’s laboratory developed and generated two fully human antibodies targeting MCAM/MUC18 and IL 8. Treatment of mice harboring melanoma tumors with each antibody alone resulted in an inhibition of their growth and metastatic potential.18 Testing the efficacy of these antibodies in combination with chemotherapeutic drugs is ongoing. Mouse models to study aberrant genetic determinants in cancer. There is much interest in developing mouse models that mimic the aberrant genetic determinants commonly encountered in human prostate cancer. In this regard, the application of Cre/loxP technology to inactivate relevant tumor suppressor alleles in the prostate epithelium has resulted in successful development of several mouse models of prostate cancer. Dr. Roy Burman’s laboratory (University of Southern California, Los Angeles, CA), has further modified such a Cre/lox adenocarcinoma model19 to increase its efficiency and utility. In this model, the genetic determinants of PB Cre4 transgene and Pten floxed at exon 5 phosphatase domain, were combined to a conditional luciferase reporter line. The Cre/loxP-dependent approach that inactivates Pten alleles in the prostate epithelium is used to activate the luciferase transgene in the same cells. Using this combinatorial model, it is possible to visualize prostate tumor development, its temporal growth, regression of primary tumor after


2006; Vol. 5 Issue 12



Photos of selected NIH Pathology B Study Section Meetings showing participants from the 1990s. The NIH Pathology B Study Section met regularly between 1946 and 2003.

androgen deprivation, and then recurrence of androgen depletion independent cancer, all by in vivo bioluminescence imaging of the living animals. This capacity to monitor tumorigenesis in individual mice in vivo led to collection of tumors at defined stages of development. Compared to the tumors from intact animals, the post castration recurrent tumors revealed a significant increase in focal neuroendocrine differentiation, a characteristic of human prostate cancer. Work done to date with primary tumors from the Pten deletion model in comparison to the normal prostate of littermate controls has identified several genes of interest, in particular a member of the bone morphogenetic protein (BMP) family, BMP7, illustrating how the clues obtained from the models may lead to new mechanistic insights into the human disease.20 The finding that BMP7 is strikingly upregulated during the development of adenocarcinoma in the model led to the study of this secreted signaling molecule in the context of human prostate cancer cells. All prostatic cell lines tested expressed variable levels of BMP7 and at least two of each type I and II BMP receptors required for signaling. However, the biological responses to BMP7 appeared cell type specific. The benign prostatic hyperplasia cell line, BPH 1, was growth arrested at G1, epithelial mesenchymal transdifferentiation was induced in the metastatic PC 3 cells, and stress induced apoptosis was inhibited in the LNCaP cell line and more remarkably in its bone metastatic variant C4 2B line. In all cases, BMP7 induced Smad phosphorylation in a dosedependent manner. Using the C4 2B cell line, a relationship between BMP/Smad signaling and survivin promoter activation and protein expression was demonstrated for the first time in the protection www.landesbioscience.com

from stress induced apoptosis. In addition, JNK activation was identified as another Smad independent pathway that contributed to the pro survival effect of BMP7. These effects were induced whether BMP7 was provided exogenously or produced endogenously by the cells, implicating its influence in all autocrine, paracrine and ECM contexts. Consistent with these in vitro findings of BMP7, correlative results of Smad activation, survivin expression, and JNK activation were also demonstrated in the progression of prostate cancer in the conditional Pten deletion mouse model, in which the evidence of BMP7 overexpression was first obtained. Together, the data clearly point to the importance and validity of the models for the continued scrutiny of the mechanisms of prostate tumorigenesis and progression. In addition, the ability to follow tumor growth, regression and recurrence in individual animals by bioluminescence imaging, as illustrated by the conditional Pten deletion model, should serve well for preclinical intervention studies. Identifying epigenetic markers of short term breast cancer risk. Recent studies suggest that breast cancer incidence may be substantially reduced in high risk women by tamoxifen treatment and/or prophylactic mastectomy. Although these reports are encouraging, current prevention strategies are expensive and can be associated with significant side effects. Biomarkers are needed to accurately predict short term breast cancer risk so that women who are most likely to benefit from preventive therapy can be identified, and response to chemoprevention can be accurately assessed. Observations in Dr. Seewaldt's laboratory (Duke University, Durham, NC) as well as by other investigators suggest that the retinoic acid receptor β2 (RARβ2) is a key regulator of mammary gland homeostasis.21 Epigenetic changes, mainly DNA methylation and modification of histones, are now recognized as playing a critical role in cancer initiation. Loss of RARβ2 function in mammary epithelial cells is hypothesized to be the result of both genetic and epigenetic events. Loss of heterozygosity (LOH) at the RARβ2 loci is frequently observed in invasive breast cancers and is thought to be a late mechanism for loss of RARβ2 expression. In contrast, RARβ2 promoter hypermethylation has been observed in dysplastic mammary epithelial cells and is thought to be an important early mechanism for loss of RARβ2 expression.22 To examine this aspect in patients at risk of breast cancer, Dr. Seewaldt’s group enrolled a cohort of 189 high risk women who underwent serial Random Periareolar Fine Needle Aspiration (RPFNA) assessment. RPFNA cytology was evaluated by a single dedicated cytopathologist, thus allowing for precise and reproducible identification of early cytological changes. RPFNA was readily accepted by high risk


1591



Photos of selected NIH Pathology B Study Section Meetings showing participants from the 1990s and 2000s. The NIH Pathology B Study Section met regularly between 1946 and 2003.

women and 24% of the cohort was African American. Importantly, more than 90% of women who underwent initial RPFNA were willing to undergo subsequent RPFNA, thus allowing serial cytological monitoring in these women. RPFNA provided sufficient DNA to test more than 30 genes for promoter methylation. The initial RPFNA specimens obtained from 178 high risk women on study entry were tested for promoter hypermethylation of the RARβ2 promoter at the M3 retinoic and responding element (RARE) and M4 AP 1 sites, as well as three other genes that play an important role in mammary carcinogenesis: BRCA1, tumor ERα (ESR1) and p16. This analysis revealed that RARβ2 (M3 + M4) promoter hypermethylation correlated with cytological atypia (p < 0.001). Hypermethylation of p16 was also primarily observed in high grade atypia. There was no association between BRCA1 promoter hypermethylation and cytology. Altogether the data show that RARβ2 is an important regulator of normal mammary gland homeostasis and that hypermethylation of the RARβ2 promoter at the M3 and M4 site occurs during early mammary carcinogenesis (hyperplasia). Furthermore, combined hypermethylation occurs in more than 75% of atypical samples obtained from high risk women.23

CELLULAR DETERMINANTS OF TUMOR PROGRESSION α6 integrin cleavage by uPA in prostate cancer invasion and metastasis. A major step in cancer progression is the acquisition of the invasive and metastatic phenotype and integrins are involved in this process. Previous work from Dr. Cress’ laboratory (University of Arizona, Tucson, AZ) reported a cleaved form of integrin α6 (integrin α6p) that existed in invasive human prostate cancer tissue and was absent in normal prostate tissue. This integrin has now been found in other epithelial cancer cells such as pancreatic, ovarian and breast cancer cell lines. The α6p integrin is missing over half the extracellular segment of the protein including the β propeller domain associated with ligand-binding.24 Cleavage of α6 to generate α6p can be produced by the proteolytic action of urokinase type plasminogen activator (uPA) in a plasmin independent manner.25 A putative uPA cleavage site with a consensus Q589EPSSRRR596 was found in the α6 integrin sequence. Further, the sequencing data confirmed that both the heavy and light chains of the α6p variant contained identical portions of the full length α6 integrin and that α6p contained at least amino acid, R596. This information coupled with the knowledge that the protease that cleaves the α6 integrin is uPA, led them to mutate different Arg residues upstream from R596. Site directed mutagenesis experiments identified amino acid residues R594 and R595, located in the “stalk” region of integrin α6, as essential for cleavage. The cleavage site is located on the extracellular region of 1592

the protein between the β barrel domain and the thigh domain. Inducible over expression of the cleavable, wild-type form of integrin α6 (PC3N α6 WT) or the non cleavable form of integrin α6 (PC3N α6 RR) was accomplished using prostate cancer (PC3N) cells, a variant of the PC3 prostate carcinoma cell line. The cells expressing the mutant form of the receptor (PC3N α6 RR) were defective in migration on laminin 1 in vitro. PC3N α6 WT and PC3N α6 RR cells were also implanted into immunodeficient mice. The initial growth of the tumors was independent of the integrin status whereas the ability of tumors to regrow following therapeutic and clinically relevant doses of ionizing radiation was markedly affected. These data taken together suggest that the uPA mediated cell surface cleavage of the α6 integrin extracellular domain is involved in tumor cell migration on laminin 1 and can influence tumor repopulation in vivo. Further understanding of the factors regulating integrin cleavage holds promise for either modulating or detecting early tumor cell migration. αvβ6 Integrin: a link between inflammation and prostate cancer. Prostate cancer cell functions are regulated by elaborated signaling pathways activated by extracellular stimuli. The integrin family of cell surface receptors is known to activate intracellular pathways via interactions with specific ligands in the tumor microenvironment.26,27 Among integrins, αvβ6 appears to be a key player in mediating cancer progression given its ability to mediate signals originating in the tumor microenvironment via activation of latent TGF (transforming growth factor) β. A growing trend of data shows that expression of the αvβ6 integrin, not normally found in healthy adults, is associated with neoplastic and metastatic phenotypes in colorectal, lung, oral and ovarian cancer. In addition, this epithelial specific integrin is up regulated in wound repair and is induced by inflammatory cytokines. Dr. Languino’s laboratory (University of Massachusetts, Worcester, MA) has investigated αvβ6 expression in human prostate cancer tissue specimens and demonstrated that it is up regulated in inflammatory sites where leukocyte infiltration is observed. In addition, they show that the αvβ6 integrin, while not expressed in normal


2006; Vol. 5 Issue 12




human prostate, is found in human prostatic intraepithelial neoplasia (PIN) and proliferative inflammatory atrophy (PIA) lesions, as well as in prostatic adenocarcinoma. To investigate whether αvβ6 expression would contribute to prostate cancer progression, her laboratory generated stable transfectants expressing either the αvβ6 integrin or the αvβ3 integrin using LNCaP prostate cancer cells. αvβ6 but not αvβ3 expression in LNCaP cells resulted in increased tumor volume in SCID mice. Analysis of the mechanism by which αvβ6 increases tumor growth revealed that it regulates androgen receptor transcriptional activity, thereby regulating proliferation and survival pathways. The αvβ6 effect on androgen receptor activity results in up regulation of an anti apoptotic molecule, survivin, known to promote prostate cancer progression and radio/chemo resistance. Thus, a model can be formulated in which the αvβ6 integrin, given its ability to contribute to an inflammatory phenotype and to stimulate tumor growth, provides a molecular link between inflammation and prostate cancer. These findings show, for the first time, that integrins modulate androgen receptor activity and suggest a novel integrin mediated mechanism of prostate cancer progression. Role of mucins in tumor progression and metastasis. Mucins (MUC) are large multifunctional glycoproteins whose primary functions are to protect and lubricate the surface of epithelial tissues in lining ducts and lumens of secretory organs. Mucins are also involved in more complex biological processes, such as epithelial cell renewal and differentiation, cell signaling, and cell adhesion. Under normal physiological conditions, the production of mucins is intrinsically maintained by a set of coordinated regulatory mechanisms leading to a well defined pattern of tissue, time and developmental state specific www.landesbioscience.com

expression. Mucin homeostasis, however, is altered by the action of external and internal environmental factors that affect cellular integrity. This results in a change in cell behavior that often culminates into a variety of pathological conditions. Twenty human MUC genes have been identified so far.28 In an extensive study that examined the expression of various MUC genes, Dr. Batra’s laboratory (University of Nebraska, Omaha, NE) has demonstrated the specific and differential expression of MUC4 in pancreatic adenocarcinomas as compared to the normal pancreas or chronic pancreatitis tissues. There is a strong association between MUC4 expression and progressively increasing pancreatic intraepithelial neoplasia (PanINs) and the overexpression of MUC4 in tumors is associated with poor prognosis in patients with pancreatic cancer. The full length coding sequence of MUC4 (approximately 28 kB) was established from human pancreatic tumor cDNA libraries and the complete organization of the MUC4 gene with 25 exons/introns over 100 kB is known. The deduced full length coding sequence shows a leader peptide, a serine and threonine rich non tandem repeat region, a central large tandem repeat domain containing 16 amino acid repetitive units, regions rich in potential N glycosylation sites, two cysteine rich domains, a putative GDPH proteolytic cleavage site, three EGF like domains, a hydrophobic transmembrane domain, and a short cytoplasmic tail. Due to its large size, MUC4 is predicted to exceed 2 μm in size on the cell surface. MUC4 protein is comprised of two subunits of which MUC4α is an extracellular mucin type glycoprotein subunit, and MUC4β is the growth factor like transmembrane subunit. Additionally, MUC4 also contains one NIDO (Nidogen like) and one AMOP (Adhesion associated domain in MUC4 and Other Proteins) domains at the carboxy terminus of MUC4α. Using anti sense and/or RNAi approaches, a direct association of MUC4 mucin with the metastatic pancreatic cancer phenotype was established and provided experimental evidence for a functional role of MUC4 in altered growth and invasive properties of tumor cells29. A decrease in motility and increase in adhesive properties was observed in pancreatic tumor cells with down regulated expression of MUC4. Taken together, the structure of MUC4, its aberrant expression and regulation by host microenvironment, and its role in the tumorigenicity and metastasis of pancreatic cancer cells support that MUC4 plays an important role in tumor stromal interactions and contributes significantly to disease progression. EGFRvIII in breast carcinoma. Elevated levels of epithelial growth factor receptor (EGFR) have been detected in a variety of human cancers. Several reports have demonstrated that the Type III


1593




EGF receptor deletion mutant (EGFRvIII) is frequently detected in various human cancers, including breast cancer. Dr. Tang’s laboratory (Georgetown University, Washington, DC) demonstrated that 38% (274/721) of primary invasive breast cancers and 54% (6/11) of metastatic lymph nodes, express EGFRvIII by immunohistochemical analysis with two monoclonal antibodies (DH8.3/1b 18 and 4–5 H). Furthermore, in positive samples, the normal mammary gland exhibited negative staining for EGFRvIII, while the tumor cells were positive.30 They observed that the frequency of EGFRvIII expression correlated with breast cancer progression. Furthermore, EGFRvIII was constitutively activated and frequently co-expressed with wild-type EGFR (wt EGFR) in breast carcinomas. Overexpression of EGFRvIII was capable of transforming a non-tumorigenic, IL 3-dependent murine hematopoietic cell line (32D cells) into an IL 3 independent malignant phenotype in vitro and in vivo. Co expression of EGFRvIII with ErbB 2 in 32D cells further enhanced tumorigenesis in vivo, and expression of EGFRvIII in ER positive breast cancer cells (MDA MB 361) induced an estrogen-dependent, tamoxifen resistant phenotype in vivo and enhanced the invasiveness of breast cancer cells in vitro. Using the 32D cell model system to comprehensively dissect mitogenic effect mediated by EGFRvIII, they monitored the EGFRvIII mediated signaling pathways by “switching” between an IL 3-dependent pathway (‘off ’) while growing the cells in the presence of IL 3, and a EGFRvIII mediated IL3 independent pathways (‘on’), while growing the cells in the absence of IL 3. This “switch” mimics EGFRvIII facilitated “on” and “off ” signaling, and these signaling pathways were found to be directly associated with the EGFRvIII mediated mitogenic and biological effects. They observed that although treatment with EGF led to a robust and concomitant stimulation in many signaling pathways in the 32D/EGFR cells, this stimulation was transient and reduced within 24 hours. In contrast, the 32D/EGFRvIII cells displayed a sustained activation in many signaling pathways. The lifecycle of the wild-type EGFR is regulated by its internalization and degradation, which is mediated by members of the Cbl protein family. The most intriguing observation is that EGFRvIII exhibits a defect in its degradation pathway. c Cbl failed to associate with EGFRvIII and EGFRvIII was devoid of ubiquitination and degradation. Furthermore, Tyr1045 of EGFRvIII was hypophosphorylated, despite the constitutive activation of EGFRvIII. These results suggest that hypophosphorylation of Tyr1045 likely allows EGFRvIII to escape from c Cbl induced ubiquitination and degradation.31 These results suggest that the cyto1594

plasmic domain of EGFRvIII adopts a different conformation than that of the wt EGFR. This configuration of EGFRvIII may be a key determinant of this receptor’s potency. Maspin, is an epithelial derived stress regulator in tumor progression: A paradigm shift. Maspin is a 42 kDa epithelial specific serine protease inhibitor (serpin) that is expressed in several types of human cancer including breast, prostate and lung carcinomas.32 Experimental evidence consistently showed that maspin suppresses tumor growth, invasion and metastasis, induces tumor redifferentiation, and enhances tumor cell sensitivity to apoptosis. Although maspin deviates significantly from both inhibitory and non inhibitory serpins, it has become clear that the biochemical behavior of maspin significantly deviates from that of classical inhibitory serpins, i.e., it does not readily inhibit a serine protease, does not form a 1:1 suicidal complex with any serine protease, and does not have the capacity to become interactive by insertion of the reactive site loop (RSL) into a β pleated sheet. Recent studies in Dr. Sheng’s laboratory (Wayne State University, Detroit, MI) suggest that extracellular and cell surface associated maspin may prove to be a novel and efficient quencher of the signaling activities of the cell surface associated uPA/uPAR (uPA receptor) complex before pro uPA is proteolytically activated.33 Particularly pertinent to its inhibitory effect on ECM degradation, cell detachment from mature focal adhesion contacts, tumor cell motility and invasion, prostate tumor/bone interaction


2006; Vol. 5 Issue 12



Participants in the workshop: First row from left to right: Surinder Batra, Careen Tang, Martin Padarathsingh, Yves DeClerck, Lucia Languino and Menashe Bar Eli Second row from left to right: Dihua Yu (standing), Harriet Isom, Shi Yuan Cheng, Ramon Parsons, Stephan Ladisch (standing), Mary Zutter (standing) and Nalini Chandar (standing) Third row from left to right: Wen Tien Chen, James McCarthy, Bernard Weissman, Srikumar Chellappan, Sharon Stack, Michael Cohen, Anne Cress, Pradip RoyBurman, Lisa Coussens, Shijie Sheng and Kishore K. Wary.

Dr. Martin L. Padarathsingh

and tumor angiogenesis, is the fact that the maspin effect on cell surface associated uPA/uPAR may regulate or be regulated by type I collagen (α2 chain Col I), an abundant ECM protein. Maspin also interacts with glutathione s transferase (GST) and intracellular maspin/GST interaction has been shown to enhance the GST activity, to increase cellular capacity to reduce oxidative stress induced generation of reactive oxygen species (ROS) and vascular endothelial growth factor (VEGF A).34 Interestingly, however, the maspin effect on GST mediated ROS detoxification was not associated with drug resistance. Instead, maspin specifically sensitizes tumor cells to drug induced apoptosis.35 The pro apoptotic effect of maspin is associated with increased cellular expression of p21 and Bax. Given the emerging evidence that nuclear maspin correlates with better prognosis of several types of cancer,36 it is possible that maspin is involved in the regulation of gene transcription. To this end, several lines of evidence support an inhibitory effect of maspin on histone deacetylase 1 (HDAC1) through direct molecular interaction.37 The maspin effect on HDAC1 also correlated with an increased sensitivity to www.landesbioscience.com

cytotoxic HDAC inhibitor. Interestingly, GST was detected in the maspin/HDAC1 complex. Furthermore, a C terminally truncated maspin mutant, which binds to HDAC1 but not GST, did not inhibit HDAC activity. HDAC1 controlled gene transcription plays a critical role in tumor progression. It is reasonable to speculate that HDAC1 needs to be tightly regulated by a proactive mechanism in a cell type specific manner. To date, no other polypeptide inhibitors of HDAC1 have been identified. Further mechanistic insights into the endogenous inhibitory effect of glutathione coupled maspin on HDAC1 may provide new leads toward future developments of specific HDAC1 targeting strategies. Cell surface proteases in tumor invasion. Among the many proteases associated with human cancer, seprase/fibroblast activation protein α and dipeptidyl peptidase IV (DPP4/CD26) have two types of EDTA resistant protease activities: dipeptidyl peptidase and 170 to 220 kDa gelatinase activities.38 Human DPP4 is ubiquitously expressed in fibroblasts, epithelial and endothelial cells, and serves multiple functions in cleaving the penultimate positioned prolyl bonds at the N terminus of a variety of physiologically important peptides in the circulation. On the other hand, seprase is transiently expressed in mesenchymal cells during embryonic development, in fibroblasts during early stages of wound healing, and in invasive cells of malignant tumors, suggesting a role of seprase and DPP4 in the tumor invasive phenotype. Dr. Chen’s laboratory (State University of New York, Stony Brook, NY) recently demonstrated that DPP4 and seprase form a novel protease complex found in human endothelial cells that are activated to migrate and invade in the ECM in vitro.39 DPP4 and seprase were found to be co expressed with the three major protease systems, matrix metalloproteinases (MMP), plasminogen activators and type II transmembrane serine proteases, at the cell surface and organized as a complex at invadopodia like protrusions. Both proteases were co localized at the surface of endothelial cells in capillaries but not in large blood vessels present in invasive breast ductal carcinoma in vivo. Importantly, monoclonal antibodies against the gelatin-binding domain of DPP4 blocked the local gelatin degradation by endothelial cells in the presence of the major metallo and serine protease systems that modified pericellular collagenous matrices, and subsequent cell migration and invasion. The results indicate that tumor invasion involves a novel protease mechanism, in which the DPP4 seprase complex at invadopodia facilitates the local degradation of the ECM and the invasion of tumor and endothelial cells into collagenous matrices.


1595



MICROENVIRONMENTAL DETERMINANTS OF TUMOR PROGRESSION Nicotine, an environmental factor affecting the tumor microenvironment. It is well established that cigarette smoking correlates with higher incidences of lung cancer as well as cancers of other organ sites. Tobacco specific carcinogens present in cigarette smoke are known to form adducts with DNA, mutating vital tumor suppressor genes as well as oncogenes initiating oncogenesis. Nicotine, the major addictive component of tobacco smoke is not carcinogenic; at the same time, recent studies have shown that nicotine can promote cell proliferation, inhibit apoptosis and promote angiogenesis.40 This raises the possibility that nicotine might be able to facilitate the growth and progression of tumors already initiated. These effects of nicotine are mediated by specific nicotinic acetylcholine receptor (nAChR) subunits that are present on non neuronal tissues. An analysis of multiple non small cell lung carcinoma cell lines performed by Dr. Chellappan’s laboratory (University of South Florida, Tampa, FL) showed the presence of various nAChR subunits in these cell lines41 and demonstrated that these cells line proliferated significantly upon stimulation with 1 μM nicotin, an amount equivalent to that present in the blood stream of many smokers.42 Attempts to elucidate the molecular mechanisms underlying nicotine mediated induction of cell proliferation showed that nicotine induced the phosphorylation of Rb, subsequent to its-binding to the Raf 1 kinase. Further, similar to growth factor stimulation, nicotine activated kinases associated with cyclins D and E; these events led to the phosphorylation of Rb and its dissociation from E2F regulated proliferative promoters, as seen by chromatin immunoprecipitation assay42. It was also found that nicotine stimulation led to the activation of the Src kinase. nAChRs do not have intrinsic tyrosine kinase activity and the activation of Src required the mediation of the scaffolding protein, β arrestin. Depletion of β arrestin 1 prevented nicotine induced activation of Src as well as cell proliferation. Experiments to assess whether nicotine could enhance the adhesive and invasive properties of non small cell lung carcinomas in Boyden chamber assays demonstrated that nicotine could effectively enhance the adhesion, invasion and migration of A549 cells. Furthermore, treatment with 1μM nicotine led to the enhanced growth of A549 cells in soft agar. Interestingly, Src kinase activity was needed for nicotine induced, but not VEGF induced, formation of angiogenic tubules in matrigel.43 Preliminary studies in Dr. Chellappan’s laboratory showed that nicotine could alter gene expression pattern in A549 cells consistent with an epithelial mesenchymal transition. Treatment of A549 cells with 1 μM nicotine for 48 hours led to the upregulation of vimentin and fibronectin while E cadherin and β catenin were downregulated. These observations support the contention that exposure to nicotine can lead to the growth and progression of tumors in a receptor-dependent manner, even in the absence of genotoxic tobacco carcinogens. Angiopoietin 2 stimulation in cancer invasion and metastasis. Angiopoietin 2 (Ang2) is an angiogenic factor that plays critical roles in angiogenesis and tumor progression. During angiogenesis, Ang2 antagonizes Ang1 activity by competitively inhibiting the-binding of Ang1 to its cognate endothelial receptor, Tie2, causing destabilization of the vasculature. Ang2 also acts in concert with VEGF to regulate vessel growth.44 In human cancers, increased expression of Ang2 in tumor cells is closely correlated to the progression, invasiveness and metastases of lung, gastric, colon and breast cancers. Dr. Cheng’s laboratory (University of Pittsburgh, Pittsburgh, PA) has investigated the role of Ang2 in glioma cell invasion using clinical 1596

primary tumor specimens, orthotopic xenograft tumors in mice, and cell culture model systems. In primary human glioma tumor biopsies, they found high levels of expression of Ang2 in the invasive areas but not in the central regions of those tumors. In the invasive regions where Ang2 was overexpressed, increased levels of MMP 2 were also apparent.45 Consonant with these features, intracranial xenografts of glioma cells engineered to express Ang2 were highly invasive into adjacent brain parenchyma compared to isogenic control tumors. In regions of the Ang2 expressing tumors that were actively invading the brain, high levels of expression of MMP 2 and increased angiogenesis were also evident. A link between these two features was apparent as stable expression of Ang2 by U87MG cells or treatment of several glioma cell lines with recombinant Ang2 in vitro caused activation of MMP 2 and acquisition of increased invasiveness. Conversely, MMP inhibitors suppressed Ang2 stimulated activation of MMP 2 and Ang2 induced cell invasion.45 Upregulation of Ang2, MMP 2, membrane type 1 MMP (MT1 MMP) and laminin 5γ2 (LN 5γ2) in tumor cells are also correlated with glioma invasion. Analyses of 57 clinical human glioma biopsies including WHO grade I to IV tumors displaying a distinct invasive edge and 39 glioma specimens that only contain the central region of the tumors showed that Ang2, MMP 2, MT1 MMP and LN 5γ2 were co overexpressed in invasive areas, but not in the central regions of the glioma tissues and that there was a significant link between the preferential expression of these molecules and invasiveness. Protein analyses of microdissected primary glioma tissue showed an up regulation and activation of MT1 MMP and LN 5γ2 at the invasive edge of the tumors, supporting this observation. Concordantly, in human U87MG glioma xenografts engineered to express Ang2, increased expression of MT1 MMP and LN 5γ2 in actively invading glioma cells, along with MMP 2 upregulation, was also evident. In cell culture, stimulation of glioma cells by overexpressing Ang2 or exposure to exogenous Ang2 promoted the expression and activation of MMP 2, MT1 MMP and LN 5γ2.46 Dr. Cheng’s laboratory has identified a novel mechanism by which Ang2 stimulates MMP 2 expression leading to glioma cell invasion. His laboratory demonstrated that Ang2 interacted with αvβ1 integrin in Tie2 deficient human glioma cells and activated focal adhesion kinase (FAK), p130Cas, extracellular signal regulated protein kinase (ERK)1/2 and JNK. It also enhanced MMP 2 expression and secretion. Signaling was attenuated by functional inhibition of αvβ1 integrins, FAK, p130Cas, ERK1/2 or JNK. Furthermore, expression of a negative regulator of FAK, FAK related non kinase (FRNK) by U87MG/Ang2 expressing glioma xenografts suppressed Ang2 induced MMP 2 expression and glioma cell infiltration in the murine brain.47 Recently, whether Ang2 plays crucial roles in promoting breast cancer metastasis was also examined. The data demonstrate that Ang2 stimulated breast cancer metastasis through the α5β1 integrin mediated signaling pathway. Immunohistochemical analysis of 185 primary human breast cancer specimens that include 97 metastatic tumors revealed a significant correlation between the expression of Ang2 and E cadherin, Snail, metastatic potential, tumor grade and lymphatic vessel invasion during breast cancer progression. In a xenograft model, overexpression of Ang2 in poorly metastatic MCF 7 breast cancer cells promoted metastasis to the lymph nodes and lung of mice. In cell culture, Ang2 promotes cell migration and invasion in Tie2 deficient breast cancer cells through the α5β1 integrin/ILK mediated pathway activating Akt, GSK 3β, Snail and E cadherin in breast cancer cells. Functional inhibition of β1, α5 integrins and ILK suppressed Ang2 activation of Akt, GSK 3β, Snail, E cadherin and breast cancer cell migration and invasion. Together,


2006; Vol. 5 Issue 12



these results from two distinct human cancer models underscore the significant contribution of Ang2 in cancer progression, not only by stimulating angiogenesis but also promoting invasion and metastasis, and provide potential mechanisms by which cancer cells acquire an enhanced invasive phenotype contributing to cancer invasion and metastasis. Ganglioside modulation of angiogenic signaling. Gangliosides are sialic acid containing cell surface glycosphingolipids that are shed in substantial quantities by many different tumor cells, and bind efficiently to host cells in the tumor microenvironment. They modulate growth factor receptors, such as EGF and others,48 and in vivo, ganglioside shedding can have an important permissive effect on tumor progression.49 Dr. Ladisch’s laboratory (George Washington University, Washington, DC) described a new concept, that VEGF-dependent signaling can be modified by tumor gangliosides. Gangliosides are avidly taken up by normal human umbilical cord endothelial cells (HUVEC) and lower the threshold and increase the efficiency of HUVEC response to angiogenic factors such as VEGF. At optimal VEGF concentrations, ganglioside enriched HUVEC showed a 92% increase in DNA synthesis, compared to an only 28% increase in control cells exposed to the same concentration of VEGF. VEGFR activation (autophosphorylation) and downstream signaling molecules (PKC and PI3K, MAPK) were more highly phosphorylated in ganglioside enriched cells, while-binding studies showed that GD1a enrichment of the membrane resulted in a significant increase in the number of effective receptors, without an increase in the total number of receptor proteins, supporting the concept that gangliosides shed by tumor cells and subsequently taken up by endothelial cells in the immediate tumor microenvironment could enhance VEGF-binding, resulting in a lowered VEGF requirement for responsiveness by endothelial cells in vivo. The particularly striking discovery of this work was that very low and suboptimal concentrations of VEGF, which normally do not trigger VEGF receptor activation and downstream signaling could do so when the cells had been enriched in gangliosides. Both VEGFR dimerization and autophosphorylation were enhanced by membrane ganglioside enrichment, even at the very low VEGF concentration of 0.1 ng/ml, or about 2% of the optimum concentration in vitro, which itself initiates only minimal VEGFR autophosphorylation and is non stimulatory to VEGFR signaling control, in normal HUVEC. Importantly, the unexpected activation of signal transduction pathways in ganglioside enriched HUVEC by very low concentrations of VEGF was accompanied by a magnified, rapid onset of HUVEC DNA synthesis and a marked increase in cell migration.50 These findings strongly support the concept that ganglioside enrichment modifies the growth factor responsiveness of normal cells in the tumor microenvironment along a continuum towards autonomy. This increasingly autonomous state is characterized by conversion from growth factor dependence to a state of response to almost trace amounts of growth factor concentrations. In tumors, this may be very important, because ganglioside shedding is an active process resulting in up to micromolar concentrations of tumor gangliosides, even in the peripheral circulation. The highly efficient-binding of tumor shed gangliosides to target cells and the shared biological properties of structurally diverse gangliosides underscore their impact on increasing the sensitivity of host cells to growth factors produced by tumor cells in the tumor microenvironment. Hyaluronan, a pericellular environment promoting metastasis and angiogenesis in prostate cancer. Tumor progression is accompanied by changes in the composition or integrity of the ECM. www.landesbioscience.com

Hyaluronan is a high molecular weight carbohydrate associated with the progression of several tumor types, including prostate cancer.51 Progression of prostate cancer is characterized by atypical growth, altered glandular architecture and increased invasion into the surrounding stroma, which includes lymphatic vessels and tumor associated blood vessels. Increased levels of hyaluronan are associated with the stroma of primary tumors, and retrospective studies have linked the level of stromal hyaluronan to poor prognosis in patients.52 The later stages of primary tumor progression are also associated with increased levels of hyaluronan in a percentage of parenchymal cells within the tumor. Studies on prostate cancer cell lines performed in Dr. McCarthy’s laboratory (University of Minnesota, Minneapolis, MN) have shown that highly metastatic, androgen independent cells exhibit increased synthesis of hyaluronan. The hyaluronan synthesized by these cells is both secreted into the medium and is also assembled into a pericellular matrix that can be visualized.53-55 The high level of hyaluronan synthesized by these cells is due to increased levels of two of three mammalian isozymes that synthesize hyaluronan, termed hyaluronan synthases 2 and 3 (HAS 2 and HAS 3). These enzymes are integral membrane proteins that catalyze the synthesis and extrusion of the hyaluronan polymer into the microenvironment. Poorly metastatic prostate cancer cell lines express low levels of hyaluronan and little or non detectable levels of HAS2 and 3. Tumor cell associated hyaluronan facilitates the adhesion of prostate cancer cells to bone marrow endothelial cells, consistent with a role for this polymer in mediating adhesion and extravasation within microvessels at sites of metastasis, including bone marrow.54,55 Subcutaneous tumor formation is also highly-dependent on hyaluronan, since antisense mediated inhibition of hyaluronan synthase expression inhibits tumor growth by 60–80%.56 The formation of a tumor associated vasculature is also inhibited to a similar degree, consistent with a role for hyaluronan in stimulating angiogenesis. Importantly, this inhibition of tumor growth can be completely reversed by the addition of exogenous hyaluronan, indicating that it is the product of hyaluronan synthases, rather than the isozymes per se, that is important for tumor growth. Additionally, these results indicate that tumors can utilize hyaluronan from different sources (e.g., tumor associated stroma) to grow, invade and metastasize. These results suggest an important role for hyaluronan in the progression and metastasis of prostate cancer cells. Interfering with the biological effects of hyaluronan in prostate tumors may represent a novel therapeutic strategy for prostate cancer patients with advanced disease. Inflammation and cancer. The functional relationship between inflammation and cancer is not new. In 1863, Virchow hypothesized that the origin of cancer was at sites of chronic inflammation, in part based on the observation that some classes of irritants, tissue injury and the ensuing inflammation they cause, enhance cell proliferation.57 While it is now clear that proliferation of cells alone does not cause cancer, sustained cell proliferation in an environment rich in inflammatory cells, growth factors, activated stroma and DNA damage promoting agents, certainly potentiates and/or promotes neoplastic risk.58 During acute tissue injury associated with wounding, cell proliferation is enhanced while the tissue regenerates but proliferation and inflammation typically subside after the assaulting agent is removed or the repair completed. In contrast, proliferating cells that have sustained DNA damage and/or mutagenic assault, e.g., initiated cells, will continue to proliferate in microenvironments rich in inflammatory cells and growth/survival factors that support their growth. In a sense, tumors act as wounds that fail to heal.59 Today, the causal relationship between inflammation, innate immunity and


1597



cancer is more widely accepted; however, many of the molecular and cellular mechanisms mediating this relationship remain unresolved and are the focus of the research of Dr. Coussens (University of California, San Francisco, CA). Utilizing a transgenic mouse model of multi stage squamous carcinogenesis, e.g., K14 human papilloma virus (HPV) 16 mice, her laboratory had previously shown that mast cells exert a promoting role during early epithelial carcinogenesis through their release of growth factors and specific serine and metallo proteases, e.g., MMP 9, mast cell chymase and tryptase60,61 since genetic ablation of mast cells and/or their specific proteases results in attenuated neoplastic progression in HPV16 mice.62 They have also examined the molecular and cellular parameters initiating leukocyte recruitment into premalignant tissue with the goal to understand the mechanisms regulating recruitment of infiltrating leukocytes towards premalignant epithelial lesions and to determine how leukocytes functionally contribute to neoplastic development. In HPV16 transgenic mice, combined B and T lymphocyte deficiency eliminated immune complex (IC) deposition in premalignant skin and attenuated innate immune cell infiltration, that then resulted in diminished tissue remodeling activity, failure to activate angiogenic vasculature, retention of terminal differentiation programs in skin keratinocytes and a 43% reduction in the overall incidence of squamous cell carcinoma (SCC), whereas genetic elimination of CD4+ and/or CD8+ T cells alone or in combination did not result in similar reductions of neoplastic characteristics.63 Adoptive transfer of B lymphocytes or serum isolated from HPV16 mice (but not from wild-type naïve mice) into B and T lymphocyte deficient/HPV16 mice restored IC deposition, chronic innate immune cell infiltration in pre malignant skin and reinstated parameters for full malignancy. These data indicate that B lymphocyte derived factors, possibly Ig, are essential for establishing chronic inflammatory pathways that potentiate cancer development. In support of this concept, anti tumor antibodies are known to enhance outgrowth and invasion of murine and human tumor cell xenografts through recruitment and activation of granulocytes and macrophages—important sources of VEGF64 that possess pro angiogenic bioactivities. Thus, serum proteins (presumably antibodies) produced by B lymphocytes locally or peripherally, at least in some scenarios, are crucial factors that initiate chronic inflammatory programs, that are not only essential for resolving acute tissue damage, but also essential for promoting tumor development in initiated tissues. The implications of these findings support a model in which inhibition of leukocyte recruitment into premalignant neoplastic stroma significantly alters the characteristics of neoplastic development and suggests that pharmacological intervention targeting either activation of B cells, antibody deposition or leukocyte recruitment may represent viable therapeutic anti cancer strategies.

CONCLUSION At the end of this workshop, three points became clear to all the participants. First, the breadth of the scientific topics discussed naturally reflects the complex nature of the cancer process and how malignant cells and the microenvironment contribute equally to cancer progression. Second, this breadth of knowledge also reflects on the broad expertise that members of TPM bring to the peer review process, an element that is critical in insuring that focused research applications viewed within a broader context by a panel of scientists with complementary expertise. Finally, and most importantly, the organization of such a workshop among members of a study section 1598

was seen as a critical tool in building better scientific awareness and strong collegiality among members of a study section. This was clearly what Martin Padarathsingh had in mind in promoting the organization of such workshops for members of PTHB and TPM as Executive Secretary/SRA. This workshop in Martin’s honor was thus part of his legacy to the NIH peer review system. References 1. Roberts CW, Orkin SH. The SWI/SNF complex—chromatin and cancer. Nat Rev Cancer 2004; 4:133-42. 2. Cairns BR. Chromatin remodeling complexes: strength in diversity, precision through specialization. Curr Opin Genet Dev 2005; 15:185-90. 3. Reisman DN, Sciarrotta J, Wang W, Funkhouser WK, Weissman BE. Loss of BRG1/BRM in human lung cancer cell lines and primary lung cancers: correlation with poor prognosis. Cancer Res 2003; 63:560-6. 4. Banine F, Bartlett C, Gunawardena R, Muchardt C, Yaniv M, Knudsen ES, Weissman BE, Sherman LS. SWI/SNF chromatin remodeling factors induce changes in DNA methylation to promote transcriptional activation. Cancer Res 2005; 65:3542-7. 5. Rosson GB, Bartlett C, Reed W, Weissman BE. BRG1 loss in MiaPaCa2 cells induces an altered cellular morphology and disruption in the organization of the actin cytoskeleton. J Cell Physiol 2005; 205:286-94. 6. Aitken A. 14-3-3 proteins on the MAP. Trends Biochem Sci 1995; 20:95-7. 7. Tzivion G, Gupta VS, Kaplun L, Balan V. 14-3-3 proteins as potential oncogenes. Semin Cancer Biol 2006; 16:203-13. 8. Li J, Simpson L, Takahashi M, Miliaresis C, Myers MP, Tonks N, Parsons R. The PTEN/ MMAC1 tumor suppressor induces cell death that is rescued by the AKT/protein kinase B oncogene. Cancer Res 1998; 58:5667-72. 9. Bose S, Crane A, Hibshoosh H, Mansukhani M, Sandweis L, Parsons R. Reduced expression of PTEN correlates with breast cancer progression. Hum Pathol 2002; 33:405-9. 10. Saal LH, Holm K, Maurer M, Memeo L, Su T, Wang X, Yu JS, Malmstrom PO, Mansukhani M, Enoksson J, Hibshoosh H, Borg A, Parsons R. PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma. Cancer Res 2005; 65:2554-9. 11. Podsypanina K, Ellenson LH, Nemes A, Gu JG, Tamura M, Yamada KM, Cordon Cardo C, Catoretti G, Fisher PE, Parsons R. Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems. Proc Natl Acad Sci USA 1999; 96:1563-8. 12. Kwabi Addo B, Giri D, Schmidt K, Podsypanina K, Parsons R, Greenberg N, Ittmann M. Haploinsufficiency of the Pten tumor suppressor gene promotes prostate cancer progression. Proc Natl Acad Sci USA 2001; 98:11563-8. 13. Podsypanina K, Lee RT, Politis C, Hennessy I, Crane A, Puc J, Neshat M, Wang H, Yang L, Gibbons J, Frost P, Dreisbach V, Blenis J, Gaciong Z, Fisher P, Sawyers C, Hedrick Ellenson L, Parsons R. An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten(+/ ) mice. Proc Natl Acad Sci USA 2001; 98:10320-5. 14. Puc J, Keniry M, Li HS, Pandita TK, Choudhury AD, Memeo L, Mansukhani M, Murty VVVS, Gaciong Z, Meek SEM, Piwnica Worms H, Hibshoosh H, Parsons R. Lack of PTEN sequesters CHK1 and initiates genetic instability. Cancer Cell 2005; 7:193-204. 15. Simpson L, Li J, Liaw D, Hennessy I, Oliner J, Christians F, Parsons R. PTEN expression causes feedback upregulation of insulin receptor substrate 2. Mol Cell Biol 2001; 21:3947-58. 16. Tellez C, McCarty M, Ruiz M, Bar Eli M. Loss of activator protein 2 alpha results in overexpression of protease activated receptor 1 and correlates with the malignant phenotype of human melanoma. J Biol Chem 2003; 278:46632-42. 17. Melnikova VO, Mourad Zeidan AA, Lev DC, Bar Eli M. Platelet activating factor mediates MMP 2 expression and activation via phosphorylation of cAMP response element-binding protein and contributes to melanoma metastasis. J Biol Chem 2006; 281:2911-22. 18. Mills L, Tellez C, Huang SY, Baker C, McCarty M, Green L, Gudas JM, Feng X, Bar Eli M. Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer Res 2002; 62:5106-14. 19. Wang S, Gao J, Lei Q, Rozengurt N, Pritchard C, Jiao J, Thomas GV, Li G, Roy Burman P, Nelson PS, Liu X, Wu H. Prostate specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell 2003; 4:209-21. 20. Yang S, Lim M, Pham LK, Kendall SE, Reddi AH, Altieri DC, Roy Burman P. Bone morphogenetic protein 7 protects prostate cancer cells from stress induced apoptosis via both Smad and c Jun NH2 terminal kinase pathways. Cancer Res 2006; 66:4285-90. 21. Seewaldt VL, Caldwell LE, Johnson BS, Swisshelm K, Collins SJ, Tsai S. Inhibition of retinoic acid receptor function in normal human mammary epithelial cells results in increased cellular proliferation and inhibits the formation of a polarized epithelium in vitro. Exp Cell Res 1997; 236:16-28. 22. Kopelovich L, Crowell JA, Fay JR. The epigenome as a target for cancer chemoprevention. J Natl Cancer Inst 2003; 95:1747-57. 23. Bean GR, Scott V, Yee L, Ratliff Daniel B, Troch MM, Seo P, Bowie ML, Marcom PK, Slade J, Kimler BF, Fabian CJ, Zalles CM, Broadwater G, Baker JC, Jr., Wilke LG, Seewaldt VL. Retinoic acid receptor beta2 promoter methylation in random periareolar fine needle aspiration. Cancer Epidemiol Biomarkers Prev 2005; 14:790-8. 24. Davis TL, Rabinovitz I, Futscher BW, Schnolzer M, Burger F, Liu Y, Kulesz Martin M, Cress AE. Identification of a novel structural variant of the alpha 6 integrin. J Biol Chem 2001; 276:26099-106.


2006; Vol. 5 Issue 12


Tumor Progression and Metastasis NIH Study Section 25. Demetriou MC, Pennington ME, Nagle RB, Cress AE. Extracellular alpha 6 integrin cleavage by urokinase type plasminogen activator in human prostate cancer. Exp Cell Res 2004; 294:550-8. 26. Goel HL, Fornaro M, Moro L, Teider N, Rhim JS, King M, Languino LR. Selective modulation of type 1 insulin like growth factor receptor signaling and functions by beta1 integrins. J Cell Biol 2004; 166:407-18. 27. Goel HL, Breen M, Zhang J, Das I, Aznavoorian Cheshire S, Greenberg NM, Elgavish A, Languino LR. beta1A integrin expression is required for type 1 insulin like growth factor receptor mitogenic and transforming activities and localization to focal contacts. Cancer Res 2005; 65:6692-700. 28. Moniaux N, Andrianifahanana M, Brand RE, Batra SK. Multiple roles of mucins in pancreatic cancer, a lethal and challenging malignancy. Br J Cancer 2004; 91:1633-8. 29. Singh AP, Moniaux N, Chauhan SC, Meza JL, Batra SK. Inhibition of MUC4 expression suppresses pancreatic tumor cell growth and metastasis. Cancer Res 2004; 64:622-30. 30. Ge H, Gong X, Tang CK. Evidence of high incidence of EGFRvIII expression and coexpression with EGFR in human invasive breast cancer by laser capture microdissection and immunohistochemical analysis. Int J Cancer 2002; 98:357-61. 31. Han W, Zhang T, Yu H, Foulke JG, Tang CK. Hypophosphorylation of Residue Y1045 Leads to Defective Downregulation of EGFRvIII. Cancer Biol Ther 2006; In press. 32. Sheng S. The promise and challenge toward the clinical application of maspin in cancer. Front Biosci 2004; 9:2733-45. 33. Yin S, Lockett J, Meng Y, Biliran H, Jr., Blouse GE, Li X, Reddy N, Zhao Z, Lin X, Anagli J, Cher ML, Sheng S. Maspin retards cell detachment via a novel interaction with the urokinase type plasminogen activator/urokinase type plasminogen activator receptor system. Cancer Res 2006; 66:4173 81. 34. Yin S, Li X, Meng Y, Finley RL, Jr., Sakr W, Yang H, Reddy N, Sheng S. Tumor suppressive maspin regulates cell response to oxidative stress by direct interaction with glutathione S transferase. J Biol Chem 2005; 280:34985-96. 35. Liu J, Yin S, Reddy N, Spencer C, Sheng S. Bax mediates the apoptosis sensitizing effect of maspin. Cancer Res 2004; 64:1703-11. 36. Sheng S. A role of novel serpin maspin in tumor progression: The divergence revealed through efforts to converge. J Cell Physiol 2006; 209:631-5. 37. Li X, Yin S, Meng Y, Sakr W, Sheng S. Endogenous inhibition of histone deacetylase 1 by tumor suppressive maspin. Cancer Res 2006; 66:9323-9. 38. Chen WT, Kelly T. Seprase complexes in cellular invasiveness. Cancer Metastasis Rev 2003; 22:259-69. 39. Ghersi G, Zhao Q, Salamone M, Yeh Y, Zucker S, Chen WT. The protease complex consisting of dipeptidyl peptidase IV and seprase plays a role in the migration and invasion of human endothelial cells in collagenous matrices. Cancer Res 2006; 66:4652-61. 40. Heeschen C, Jang JJ, Weis M, Pathak A, Kaji S, Hu RS, Tsao PS, Johnson FL, Cooke JP. Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nature Med 2001; 7:833-9. 41. Dasgupta P, Kinkade R, Joshi B, Decook C, Haura E, Chellappan S. Nicotine inhibits apoptosis induced by chemotherapeutic drugs by up regulating XIAP and survivin. Proc Natl Acad Sci U S A 2006; 103:6332-7. 42. Dasgupta P, Rastogi S, Pillai S, Ordonez Ercan D, Morris M, Haura E, Chellappan S. Nicotine induces cell proliferation by beta arrestin mediated activation of Src and Rb Raf 1 pathways. J Clin Invest 2006; 116:2208-17. 43. Dasgupta P, Chellappan S. Nicotine mediated cell proliferation and angiogenesis: New twists to an old story. Cell Cycle 2006; 5:2324-8. 44. Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J. Vascular specific growth factors and blood vessel formation. Nature 2000; 407:242-8. 45. Hu B, Guo P, Fang Q, Tao HQ, Wang D, Nagane M, Huang HJ, Gunji Y, Nishikawa R, Alitalo K, Cavenee WK, Cheng SY. Angiopoietin 2 induces human glioma invasion through the activation of matrix metalloprotease 2. Proc Natl Acad Sci USA 2003; 100:8904-9. 46. Guo P, Imanishi Y, Cackowski FC, Jarzynka MJ, Tao HQ, Nishikawa R, Hirose T, Hu B, Cheng SY. Up regulation of angiopoietin 2, matrix metalloprotease 2, membrane type 1 metalloprotease, and laminin 5 gamma 2 correlates with the invasiveness of human glioma. Am J Pathol 2005; 166:877-90. 47. Hu B, Jarzynka MJ, Guo P, Imanishi Y, Schlaepfer DD, Cheng SY. Angiopoietin 2 induces glioma cell invasion by stimulating matrix metalloprotease 2 expression through the alphavbeta1 integrin and focal adhesion kinase signaling pathway. Cancer Res 2006; 66:775-83. 48. Liu Y, Li R, Ladisch S. Exogenous ganglioside GD1a enhances epidermal growth factor receptor-binding and dimerization 9. J Biol Chem 2004; 279:36481-9. 49. Caldwell S, Heitger A, Shen W, Liu Y, Taylor B, Ladisch S. Mechanisms of ganglioside inhibition of APC function. J Immunol 2003; 171:1676-83. 50. Liu Y, McCarthy J, Ladisch S. Membrane ganglioside enrichment lowers the threshold for vascular endothelial cell angiogenic signaling 1. Cancer Res 2006; 66:10408-14. 51. Toole BP. Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer 2004; 4:528-39. 52. Lokeshwar VB, Rubinowicz D, Schroeder GL, Forgacs E, Minna JD, Block NL, Nadji M, Lokeshwar BL. Stromal and epithelial expression of tumor markers hyaluronic acid and HYAL1 hyaluronidase in prostate cancer. J Biol Chem 2001; 276:11922-32. 53. Simpson MA, Wilson CM, McCarthy JB. Inhibition of prostate tumor cell hyaluronan synthesis impairs subcutaneous growth and vascularization in immunocompromised mice. Am J Pathol 2002; 161:849-57.

www.landesbioscience.com

54. Simpson MA, Wilson CM, Furcht LT, Spicer AP, Oegema TR, Jr., McCarthy JB. Manipulation of hyaluronan synthase expression in prostate adenocarcinoma cells alters pericellular matrix retention and adhesion to bone marrow endothelial cells. J Biol Chem 2002; 277:10050-7. 55. Simpson MA, Reiland J, Burger SR, Furcht LT, Spicer AP, Oegema TR, Jr., McCarthy JB. Hyaluronan synthase elevation in metastatic prostate carcinoma cells correlates with hyaluronan surface retention, a prerequisite for rapid adhesion to bone marrow endothelial cells. J Biol Chem 2001; 276:17949-57. 56. Yoshida H, Enomoto H, Tagawa M, Takenaga K, Tasaki K, Nakagawara A, Ohnuma N, Takahashi H, Sakiyama S. Impaired tumorigenicity and decreased liver metastasis of murine neuroblastoma cells engineered to secrete interleukin 2 or granulocyte macrophage colony stimulating factor. Cancer Gene Ther 1998; 5:67-73. 57. Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet 2001; 357:539-45. 58. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100:57-70. 59. Dvorak HF. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med 1986; 315:1650-9. 60. Coussens LM, Raymond WW, Bergers G, Laig Webster M, Behrendtsen O, Werb Z, Caughey GH, Hanahan D. Inflammatory mast cells up regulate angiogenesis during squamous epithelial carcinogenesis. Genes Dev 1999; 13:1382-97. 61. Coussens LM, Tinkle CL, Hanahan D, Werb Z. MMP 9 supplied by bone marrow derived cells contributes to skin carcinogenesis. Cell 2000; 103:481-90. 62. van Kempen LC, Rhee JS, Dehne K, Lee J, Edwards DR, Coussens LM. Epithelial carcinogenesis: dynamic interplay between neoplastic cells and their microenvironment. Differentiation 2002; 70:610-23. 63. de Visser KE, Korets LV, Coussens LM. De novo carcinogenesis promoted by chronic inflammation is B lymphocyte-dependent. Cancer Cell 2005; 7:411-23. 64. Barbera Guillem E, Nyhus JK, Wolford CC, Friece CR, Sampsel JW. Vascular endothelial growth factor secretion by tumor infiltrating macrophages essentially supports tumor angiogenesis, and IgG immune complexes potentiate the process. Cancer Res 2002; 62:7042-9.


1599