in castrated syngeneic hosts. .... that the AIT, grown in castrated hosts, overexpressed TGFa ..... (A) Expression of EGFR in the AIT tumor, and VPs and DLPs of.
Carcinogenesis vol.17 no.12 pp.2571-2579, 1996
Involvement of transforming growth factor a (TGFa) and epidermal growth factor receptor (EGFR) in sex hormone-induced prostatic dysplasia and the growth of an androgen-independent transplantable carcinoma of the prostate Paula J.Kaplan1, Irwin Leav2, Jessica Greenwood1, Paul W.L.Kwan2 and Shuk-mei Ho1-3 'Department of Biology, Tufts University, Medford, Massachusetts and Department of Pathology, Tufts University, Schools of Medicine and Veterinary Medicine, Boston, Massachusetts, USA 2
'To whom correspondence should be addressed
We previously reported the induction of dysplasia, a putative precursor of carcinoma, in the dorsolateral prostates (DLPs) of Noble rats by the combined administration of testosterone (T) and estradiol-Hp1 (E2) for 16 weeks. Additionally, we demonstrated growth of the AIT, a DLPderived, androgen-independent, transplantable solid tumor, in castrated syngeneic hosts. In this investigation, using Northern blot hybridization, radioimmunoassays and radioligand assays, we showed that transforming growth factor-a (TGFa) and epidermal growth factor receptor (EGFR) were expressed at close to non-detectable levels in the ventral prostates but at low, but measurable, levels in the DLPs of untreated rats. Enhanced expression of this ligand and its receptor was detected in the DLPs harboring dysplasia and marked overexpression of these molecules was noted in the AIT. In contrast, epidermal growth factor (EGF) expression was found to be constitutively expressed, at high levels, in both normal and dysplastic DLPs, but virtually absent in the AIT. Immunohistochemical data suggested that EGF, TGFa and EGFR were aprocine secretory products of the normal DLP, with TGFa and EGF localized to the supranuclear complexes and EGFR to the apical membranes of epithelial cells. Alterations in immunostaining patterns for TGFa and EGFR were exclusively detected in the dysplastic lesions in the DLPs of T + E2-treated rats. Enhanced intracytoplasmic localization for both peptides were found to accompany the loss of cell polarity in dysplastic foci. Strong intracytoplasmic immunostaining for TGFa was observed in some AIT cells whilst staining for EGFR was present in the membranes of tumor cells that formed psuedoacini. Taken together, our findings suggest that autocrine mechanisms may play an important role early in the carcinogenic process and that progression to an androgen-independent neoplastic growth may be modulated by this signaling pathway.
Introduction Gonadal steroids are essential for the development, differentiation, and maintenance of the prostate. However, cellular transformation and tumor development in the prostate generally involves a progressive loss of gonadal steroid-dependency and evolution of autonomous growth mechanisms (1,2). In this *Abbreviatlons: DLPs, dorsolateral prostates; T, testosterone; TGFa, transforming growth factor-a; EGFR, epidermal growth factor receptor, E2, estradiol-17f); EGF, epidermal growth factor, PIN, prostatic intraepithelial neoplasia; BSA, bovine serum albumin; PBS, phosphate buffered saline. © Oxford University Press
regard, it has been proposed that neoplastic transformation of prostatic epithelium may result from the inappropriate expression of a growth factor and/or its receptor, thus permitting the affected cells to overcome normal growth constraints (1,2). Escalation of unopposed growth and/or development of new autocrine pathways may then lead to the formation of a carcinoma. The epidermal growth factor (EGF*)-related growth factors and their receptor (3-5) have long been suspected to be involved in the pathogenesis of carcinoma of the prostate (1,2,6-13). EGF, transforming growth factor-a (TGFa), and their cognate receptor, EGF receptor (EGFR), are expressed in the normal prostates of humans (6-16) and rodents (17-21). Adult prostatic tissues, urine and seminal fluid contain high concentrations of EGF (14-16,22) whilst TGFa is expressed in rat prostatic epithelium during prenatal or perinatal development (23). However, only low levels of EGFR have been reported in normal rodent and human prostates (6-11,23). In rodents, EGFR has been localized to luminal epithelial cells (23), whilst in humans it has been found predominantly in basal epithelial cells (6-11). Androgens are believed to regulate both EGF and EGFR expression in rodent prostates (17-20) and receptor expression in the human gland (24,25). In vivo, these growth factors likely regulate multiple functions including growth, morphogenesis, and differentiation in the prostate and perhaps also in other organs of the male reproductive tract (14,16,20-22). In vitro, EGF and TGFa clearly serve as mitogenic factors to cultured rat and human prostatic epithelial cells (26-29). We have previously reported the induction of an atypical proliferative lesion, termed dysplasia, in the dorsolateral prostate (DLP) of Noble rats treated simultaneously with testosterone (T) and estradiol-17p (E2) for 16 weeks (34). The lesion closely resembles prostatic intraepithelial neoplasia (PIN) in the human prostate where it is considered to be a precursor to carcinoma (35). Moreover, protracted administration of T + E2 to the rats gives rise to a high incidence of carcinoma likely arising from the dysplasia seen in the DLP of the 16-week T + E2-treated animals (36). Also available to our laboratory is an androgen-independent, transplantable tumor which we have termed AIT (37). This tumor was originally derived from the DLP of an estrone-treated Noble rat (38) and was subsequently established in our laboratory via subcutaneous transplantation in castrated syngeneic males (37). The AIT expresses low levels of androgen receptor with abnormal intracellular localization pattern (37,39) and grows equally well in castrated and intact hosts (37). Using the Noble rat dysplasia model and the AIT we aimed, in the current investigation, to determine whether the TGFa/ EGFR loop was involved in the early prostatic carcinogenesis and/or growth of a late-staged prostatic neoplasm. Our results indicated that the combined sex hormone treatment enhanced the expression of TGFa and EGFR in DLPs harboring the dysplasia and new intracellular localization patterns for these 2571
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two molecules emerged in the dysplastic cells. We also found that the AIT, grown in castrated hosts, overexpressed TGFa and EGFR in a manner resembling those found in the dysplastic cells. Together, these findings suggest that from the outset autocrine/paracrine mechanisms are involved in the carcinogenic process and that progression to an androgen-independent neoplasm increasingly involves modulation by this growth factor signaling pathway. Materials and methods Animals and steroid treatment Male Noble rats were purchased from Charles River, Inc. (Wilmington, MA) at 5—6 weeks old. Animals were housed at the departmental animal facility until they reached a size of 28O-3OOg at which time they were implanted with hormone-filled Silastic capsules. Two groups of rats were used in this study. The first group was surgically implanted with two 2 cm Silastic capsules (Catalog number 602-205; 1.0 mm inner diameterX2.2 mm outer diameter, Dow-Corning Corporation, Midland, MI) filled with T (Sigma, St Louis, MO) and one I cm capsule filled with E2 (Sigma) as previously described (34). Rats were killed 16 weeks post-implantation. The second group was age matched control rats. The AIT The AIT, a transplantable, solid rat prostatic tumor is maintained in our laboratory by passage in castrated male Noble rats (37) Chronic exposure of Noble rats to estrone, a weak estrogen, induced low incidence of adenocarcinomas in the DLP of the treated animals (38). One of such induced DLP adenocarcinoma, successfully grew in syngeneic intact hosts, was named the Nb-2Pr-E line (38). Derived from the Nb-2Pr-E line was an androgenresponsive subline, referred to as Nb-2Pr-A, which we brought into our laboratory, transplanted to castrated hosts, and established an androgenindependent line, known as the AIT (37). The growth characteristics, androgen receptor status and responsiveness, and histological features of the AIT have previously been published by this laboratory (37,39). The same castration and animal care procedures, and tumor implantation, monitoring and harvesting techniques as previously described (37) were used in the present study. Tumor fragments (2-3 mm3) were transplanted subcutaneously into a castrated male host and allowed to grow to approximately 1.5 cm to 2.0 cm in diameter, at which time the animal was killed and the tumor was excised for biochemical studies. Buffers and chemicals All chemicals used were of reagent grade and were purchased from Sigma Chemical Company (St Louis, MO) or from Fisher (Pittsburgh, PA) unless otherwise indicated Restriction enzymes were from New England Biolabs (Beverly, MA). 10X Borate buffer (pH 8.0) contained 0.5 M boric acid, 50 mM sodium borate, 100 mM sodium sulfate, 10 mM ethylenediamine tetraacetic acid-disodium salt (EDTA). 1 M Phosphate buffer (pH 7.0) was 1 M dibasic sodium phosphate adjusted to pH 7.0 using phosphoric acid. 50X Denhardt's solution (pH 7 0) was made up of 1% ficoll (type 400-DL), 1% poly vinyl pyrrohdone, 1% bovine serum albumin (fraction V). 40X TAE buffer contained 40 mM Tris base, 20 mM sodium acetate, 1 mM EDTA adjusted to pH 7.2 with glacial acetic acid (J.T.Baker, Inc., Phillipsburg, NJ). Northern blot preparation Total RNA was prepared by a modified single step method (40) using RNAzol B (Tel-test, Inc., Friendswood, TX). Tissue samples were homogenized in RNAzol B (2 ml per 100 mg tissue) using three 30 s bursts with a Tissuemizer (Tekmar, Cincinnati. OH). The homogenate was then extracted with chloroform twice. The RNA was precipitated with isopropanol. The precipitated RNA was washed twice with 75% ethanol and dissolved in formamide for storage (41). The concentrations of the RNA samples were determined with UV spectrophotometry. Aliquots of 10 (ig of total RNA from each sample was size-fractionated through a 0.8% agarose (electrophoresis grade, Gibco-BRL, Gaithersburg, MD), 1 M formaldehyde gel in 1 X borate buffer, transferred to a Nytran membrane (Schleicher and Schuell, Keene, NH) by a downward alkaline capillary transfer method (42), and UV crosslinked onto the membrane using a UV Stratalinker 1800 (Stratagene, LaJolla, CA). Complete transfer of RNA from gel to membrane was usually achieved in 4-6 h. Hybridization with specific cDNA probes Membranes were prehybridized for 2-3 h in 0.5 M phosphate buffer, 1% sodium dodecyl sulfate, 10X Denhardt's solution. I mM EDTA. 100 ».g/ml low molecular weight salmon testis DNA (pH 7.0) for at 60-62°C. Membranes were then hybridized with 32P-labeled cDNA probes for -16 h at 62°C in a
2572
buffer similar to the prehybndization buffer with the exclusion of the low molecular weight salmon testis DNA. Complementary DNA (20—40 ng) was routinely radiolabeled with deoxycytidine 5' triphosphate a 32P (sp. act. 3000 Ci/mmol, Dupont-New England Nuclear, Boston, MA) using the RandomPrimed DNA Labeling Kit (Boehnnger Mannheim, Indianapolis, IN) and purified by passage through a Sephadex G50 column (Pharmacia, Piscataway, NJ). The sp. act. of each probe was ~1X1O9 c.p.mVjig of cDNA and the entire reaction mixture was used in the hybridization incubation (10—15 ml). All membranes were washed in 40 mM phosphate buffer, 1% SDS, and 1 mM EDTA twice at room temperature for 10 min each and then once at the hybridization temperature for 15 mm. Membranes were washed longer at this temperature as needed. A final wash was carried out at room temperature in 0 2 M phosphate buffer. Membranes were then exposed to Fuji X-ray film at -70°C with intensifying screens for autoradiography. Following each hybridization, membranes were stripped in boiling DEPC treated H2O for 15 min and reused for hybridization with a different cDNA. To verify equal loading of RNA in each lane, membranes were re-probed with a 30-mer oligonucleotide complementary to part of the 18S rRNA at 50°C. The 30-mer ohgonucleotide [5'd(CGGCATGTATTAGC-TCTAGAATTACCACAG)3'] (43) was synthesized by Genosys Biotechnologies, Inc. (The Woodlands, TX). It was radiolabeled with adenosine tnphosphate y 32P (sp. act. 6000 Ci/mmol) by the end labeling method (44) using polynucleotide kinase (New England Biolabs, Beverly, MA) and purified by passage through a Sephadex G50 column. Hybridization to the labeled 18S rRNA probe was always the last hybridization carried out on any membrane. Northern hybridization with the rat EGFR cDNA probe revealed three EGFR transcripts of sizes 9.5, 6 5 and 5.0 kb whereas hybridization analyses with TGFa cDNA and EGF cDNA demonstrated the existence of single transcripts sized 4.5 kb and 4.8 kb, respectively. The rat EGFR cDNA, RER #10, homologous to part of the coding and 3' non-coding sequences of the receptor (George Stancel, personal communication), was a gift from Dr George Stancel at the University of Texas Health Science Center at Houston (45). The rat TGFa cDNA, prTGFOr2, which includes the coding region (46) was a gift from Dr David Lee at the Lineberger Cancer Research Center at the University of North Carolina at Chapel Hill. The EGF cDNA which includes the coding region (47) was a gift from Dr Donna Dorow at the Peter MacCallum Cancer Institute in Victoria, Australia. Hybridization signals were quantitated by densitometric scanning and normalized with respect to the corresponding 18S rRNA signal to correct for loading variations. Normalized signal intensities of samples obtained from the DLPs of untreated rats were designated as controls and arbitrarily assigned a value of 1. Signal intensities of samples obtained from the VPs of control and treated animals, DLPs of T + E2-treated rats, and AIT specimens were compared with a contiguous DLP control sample within the same Northern blot and expressed as folds of the control value (set as 1) to obtain relative mRNA levels. The prominent 9.5 kb EGFR transcript signal was used for quantitation of this message. Radioimmunoassays Samples of fresh prostatic tissues or frozen AIT tissues were homogenized in ice-cold 0.1 M acetic acid, 10 (ig/ml leupeptin, and 10 |ig/ml antipain using a buffer to tissue ratio of 10 ml per g wet weight with a Tissuemizer (Tekmar). The homogenate was centrifuged at 100 000 g for 30 min at 4°C. The supernatant, containing soluble protein, was lyophilized and resuspended in one tenth of the original volume using the appropriate buffer supplied with each radioimmunoassay kit. The soluble protein extract was analyzed separately for TGFa and EGF contents with commercially available radioimmunoassays (RIAs) according to manufacturer's instructions. Both the TGFa (Peninsula Laboratories, Belmont, CA) and EGF (Biomedical Technologies Inc., Stoughton, MA) RIA kits were specific for the rat ligands and have negligible cross-reactivity according to the company literature. Crude cell membrane preparation The membrane extraction method and binding assay used was a modification of the method described by Traish and Wotiz (17). The extraction buffer (pH 7.4) contained 20 mM PIPES, 0.25 M sucrose, 1 mM EDTA, I mM EGTA and 10 mM monothioglycerol. 0 5 mM PMSF, 2 |ig/ml aprotinin and 5 |lM leupeptin were added to this buffer just prior to use. Tissue was minced on ice in 10 ml extraction buffer per gram tissue and then homogenized on ice using a Tissuemizer (Tekmar). The homogenate was centrifuged at 800 g for 10 min at 4°C, and the subsequent pellet was resuspended in 5 ml buffer per gram tissue (original wet weight), rehomogenized, and recentrifuged. The supernatant from the second extraction was added to the first. The pooled supernatant of the low speed spins were centrifuged at 10 000 g for 15 min at 4°C. The supernatant of the 10 000 g spin was then centrifuged at 100 000 g for 40 min at 4°C. The pellet, containing microsomes and plasma membrane fragments, was washed with buffer and recentrifuged at 100 000 g for 30 min at 4°C. The pellet from this last spin was resuspended in 20 mM PIPES, 0.15
Elevation of T G F a and E G F R In prostate tumorigenesis M NaCl, pH 7.4 and rehomogenized on ice using a glass homogenizer to obtain the crude cell membrane preparation. Protein concentration of the preparation was determined using the Pierce BCA Protein Assay (Pierce, Rockford, IL) using bovine serum albumin (BSA) as the standard. EGF binding assay Relative EGF R content in each crude membrane preparation was estimated using an [ I25 I]EGF binding assay according to the procedures modified from those described by Traish and Wotiz (17). Preliminary experiments were conducted to determine the optimal assay conditions for the binding of rat [ I25 I]EGF to rat prostatic membrane preparations. Saturation was demonstrated at 6 nM of [ l 2 i I]EGF under equilibrium binding conditions, which were achieved after 2 h of incubation at 25°C. For routine assays, aliquots of crude cell membrane preparations (30 |ig BSA equivalent) were incubated with 6 nM [125I]rat EGF (Biomedical Technologies Inc.) in assay buffer (20 mM PIPES, 1 mM EGTA, 1 mM PMSF and 0.1% BSA at pH 7.4) for 2 h at 25°C. To determine non-specific binding, parallel incubations were earned out in the presence of 30- to 100-fold molar excess rat EGF (Biomedical Technologies Inc.). Following incubation, samples were rapidly filtered under vacuum onto Whatman GF/B glass filters (VWR Scientific, Boston, MA). The filters used had been pre-washed once with 4 ml ice-cold assay buffer before application of samples. Free radioactivity was removed from filters by washing with 4 ml of assay buffer three times. Radioactivity left on each filter was determined by direct counting in a y counter. Relative EGF R content in each sample was expressed as fmol [ 12i I]EGF binding per mg protein. Immunohistochemistry For immunohistological detection of EGF R , a monoclonal antibody (clone L4451) was obtained from BioDesign International (Kennebunk, ME). According to the literature from the supplier, this antibody cross-reacts with receptors from all species tested including those found in human and rat tissues. Tissues were fixed in 10% buffered formalin, routinely processed, and embedded in paraffin. Paraffin sections, 5 |i thick, were deparaffinized through a graded series of xylen^s and alcohols and then rehydrated in water and phosphate buffered saline (PBS). The sections were then treated with 0.1% pronase (Sigma) in Tris-HCL buffer for 10 mm at room temperature. After a quick rinse in PBS the sections were treated with special blocking solutions from Histomouse-SP Kit (Zymed Labs, South San Francisco, CA). This kit was specifically designed to allow immunohistochemical studies of mouse or rat samples with mouse- or rat-denved primary antibodies. After rinsing off the blocking solutions, the primary antibody against EGF R was applied to the sections at a dilution of 1:100. Incubation was overnight at 4°C in humid chambers. After rinsing off the primary antibody, the slides were incubated with a biohnylated goat anti-mouse link antibody from the Histomouse-SP Kit for 10 min at room temperature. For positive control, cytospins of the human tumor line A431 and sections of rat liver were used. The A43I cells were fixed in cold acetone, air dried, and rehydrated in PBS. Pronase treatment for cytospins was found to be unnecessary. For A431 cell immunocytochemistry, the blocking solution used was normal goat serum (BioGenex Laboratories, San Ramon, CA) and the secondary antibody used was biotinylated goat anti-mouse antibody (BioGenex Laboratories). EGF immunohislochemistry was performed using a polyclonal rabbit antiserum against rat EGF (Biomedical Technologies Inc., BT585, Stoughton, MA). Following deparaffinization, sections were treated for 15 min with normal goat serum. The blocking serum was drained off and the primary antibody, at a dilution of 1:100, was applied to the sections. Incubation was overnight at 4°C in humid chambers. After a rinse with PBS, the sections were incubated with a secondary antibody, biotinylated goat anti-rabbit antibody (BioGenex Laboratories), for 20 min at room temperature. For positive control, paraffin sections of rat submaxillary glands were used. For TGFa immunohistochemistry, a polyclonal antiserum against rat TGFa from Peninsula Laboratories (#IHC 8040, Belmont, CA) at a dilution of 1:100 was used. The blocking solution was normal goat serum and the secondary antibody was a biotinylated goat anti-rabbit antiserum. The same deparaffinizing and incubation procedures used for EGF R immunohistochemistry were employed. For positive control, paraffin sections of rat submaxillary glands were used. In all cases, after incubations with the secondary antibodies, sections were rinsed with PBS and treated with an alkaline phosphatase-labeled streptavidin (BioGenex Laboratories) for 20 min at room temperature. Following a rinse with Tris buffer, the slides were developed with new fuschin (BioGenex Laboratories). After incubation, the substrate was rinsed off the sections and counterstained with either hematoxylin or methyl gTeen. The slides were dehydrated through a series of alcohol, cleared with xylene substitute and mounted with a xylene substitute medium (both from Shandon Laboratories, Pittsburg, PA). Negative controls consisted of sections incubated without the primary antibody.
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Fig. 1. (A) Expression of EGFR in the AIT tumor, and VPs and DLPs of untreated and T + E2-treated rats. Relative EGFR message levels were quantitated by Northern hybridizations. Message levels (mean ± SEM) are expressed relative to the DLP of untreated rats, which was arbitrarily assigned a value of 1.0. (B) Relative EGFR contents estimated as [ I]rat EGF binding sites in the AIT tumor, and VP and DLP of untreated and T + E2-treated rats. EGF binding was expressed as fmol EGF bound per mg protein (mean ± SEM) A statistical difference between the group mean of T + E2-treated animals or the AIT and that of the untreated controls at a significance level of P < 0.05 is noted on figures by *.
Statistical analyses Data points (histograms) are group mean values: n indicates the number of preparations from individual animals used to obtained group mean values. One-way analysis of variance was used to analyze where there was a significant difference among the various group means, and a multiple range test using the Tukey-B procedure was used to compare the individual group means A statistical difference between the group mean of T + E2-treated animals or the AIT and that of the untreated controls at a significance level of P < 0.05 is noted on figures by *.
Results EGFR and TGFa expression is elevated in the DLPs, but not in the VPs, of Noble rats following T + E2 treatment. Northern analyses of TGFa and EGFR transcript levels (Figures 1A and 2A) as well as RIA assay of TGFa (Figure IB) and radioligand binding estimation of EGFR (Figure 2B) contents showed marked similarity between the expression of the growth factor and its receptor in all tissues studied. Low to non-detectable levels of TGFa and EGFR mRNAs were detected in the normal VP. Treatment of rats with T + E2 did not induce any increases of these two molecules in this prostatic lobe. Tissue TFGa content and EGF binding sites showed low, but measurable, levels of TFGa (62 ± 32 pg/g 2573
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Untreated T+E Treated AIT in Castrated male
• Untreated • T+E2 Treated 82 AIT in Castrated male
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Fig. 2. (A) Expression of TGFot in the AIT tumor, and VPs and DLPs of untreated and T + E2-treated rats. Relative TGFa message levels were determined by Northern hybridization. Message levels (mean ± SEM) are expressed relative to the DLP of untreated rats which was arbitrarily assigned a value of 1.0. (B) TGFa peptide concentrations in the AIT tumor, and VP and DLP of untreated and T + E2-treated rats. Concentrations, expressed as pg TGFa per gram tissue (mean ± SEM), were determined by radioimmunoassay. A statistical difference between the group mean of T + E2-treated animals or the AIT and that of the untreated controls at a significance level of P < 0.05 is noted on figures by *.
tissue, n = 9) and EGFR (36 ± 6 fmol binding/mg protein, n = 5) in the VP of untreated rats. Following the dual hormone treatment, EGF binding (27 ± 13 fmol binding/mg protein, n = 5) in rat VP remained unchanged, whilst TGFa levels (204 ± 71 pg/g tissue, n = 7) showed an increase. In contrast, expression of TGFa and EGFR transcripts were augmented in the DLP by treatment of rats with T + E2. Relative TGFa mRNA levels (2.5-fold) and TGFa peptide contents (406 ± 44 pg/g tissue, n = 7) in the DLP of T + E2-treated rats showed trends of increases (150% in transcript levels and 33% increase in peptide levels) when compared with those observed in the DLP of untreated animals (transcript levels = 1-fold; peptide contents =307 ± 30 pg/g tissue, n = 9). Similarly, EGFR expression exhibited trends of increases at both transcript (2-fold; a 100% increase) and EGF binding site (69 ± 7 fmol/mg protein, n = 5, a 70% increase) levels when compared with values observed in the DLPs untreated rats (transcript levels = 1-fold; binding sites = 41 ± 9 fmole/ mg protein, n = 5). EGF is expressed at high levels in the DLP where the expression appears to be constitutive and is not altered by hormones. 2574
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Fig. 3. (A) Expression of EGF in the AIT tumor, and VP and DLP of untreated and T + E2-treated rats. Relative EGF message levels were quantified by Northern hybridization. Message levels (mean ± SEM) are expressed relative to the DLP of untreated rats which was arbitrarily assigned a value of 1.0. (B) EGF peptide contents in the AIT tumor, and VP and DLP of untreated and T + E2-treated rats. Concentrations, expressed as \lg EGF per gram tissue (mean ± SEM), were determined by radioimmunoassay.
High, and almost identical, levels of EGF mRNA were detected in DLPs of T + F^-treated and untreated rats, whilst non-detectable levels of the message were observed in the VPs of both groups of rats (Figure 3A and 3B). EGF (peptide) contents in rat DLP were in the range of 4.4-4.7 (ig/g tissue and appeared to be unaffected by the hormonal treatment of the animals. These values were at least 10 000-fold higher than the aforementioned TGFa contents in the DLP (300-400 pg/g tissue). EGF (peptide) contents in the VPs were in the range of 46-47 ng/g, which were about 100-fold lower than those found in the DLPs. The AIT expresses high levels of TGFa and EGFR but negligible amounts of EGF, the predominant ligand expressed by the DLP, the tissue of origin for the tumor. The AIT tumor overexpresses TGFa (Figure 1A and IB) and EGFR (Figure 2A and 2B) at both the message and peptide levels. Approximately 10-fold more EGFR message and 4-fold more [125I]EGF binding sites (levels = 165 ± 37 fmol binding/ mg proteins, n = 3) were found in the AIT as compared with levels detected in the DLP, the tissue of origin for the tumor (P < 0.05). Similarly, 4.6-fold more TGFa mRNA and 3.7fold more TGFa peptide (levels = 1140 ± 380 pg/g tissue, n = 3) were detected in the AIT when compared with values observed in the DLPs of normal controls (P < 0.05). In
Elevation of TGFa and EGFR in prostate tumorigenesis
Fig. 4. (A) Immunohistochemical localization of TGFa in epithelial cells and luminal contents in the lateral prostate lobe of an untreated rat. Note the supranuclear location of lmmunostaining in glandular cells which likely corresponds to the golgi apparatus. 400X hematoxylin counterstain (B) Immunohistochemical localization of TGFa in an evolving dysplastic lesion. There is intense cytoplasmic lmmunostaining of TGFa in epithelial cells. Some piling up of cells is evident but for the most part their polarity is not markedly altered. Note the absence of immunostaining in the lumen. 400X hematoxylin counterstain. (C) Immunohistochemical localization of TGFa in a severely dysplastic lesion. Dysplastic cells with immunopositive cytoplasmic staining are mainly found around the residual lumen and pseudolumens in this cribriform lesion. Normal cell polarity is almost entirely lost and lumens are devoid of immunopositve secretions 400X hematoxylin counterstain. (D) Immunohistochemical localization of TGFa in the AIT. Note the intense immunopositive cytoplasmic staining for TGFa in the neoplastic cell in the center of this field. Less dramatic immunostaining is also seen in small cytoplasmic vacuoles, which represent pseudolumens, are devoid of immunostained contents but in other fields and tumors this was a prominent finding. 400X hematoxylin counterstain.
contrast to its parent tissue, the AIT expressed minimal amounts of EGF (peptide contents = 23 ± 16 ng/g tissues, n = 3) while EGF transcripts were not detectable by Northern analysis (Figure 3A and 3B). Immunohistochemical findings demonstrate alterations of TGFa and EGFR expression patterns in DLP dysplasia and AIT cells as compared with those found in normal prostatic epithelia. The majority of immunostaining for TGFa was in the DLP. The strongest staining was observed as discrete supernuclear cytoplasmic droplets in epithelial cells and in luminal secretions of the lateral prostate (LP) (Figure 4A). The pattern of TGFa immunostaining was notably altered in the DLPs of rats treated with T + E2 for 16 weeks. Within these DLPs, TGFa staining patterns in normal acini/ducts were similar to those observed in the DLP from untreated animals. However, admixed with these morphologically normal acini were dysplastic foci which often contained numerous cells with discrete, intensely intracytoplasmic immunostaining (Figure 4B and 4C). The dysplastic cells lacked polarity and the lumens of these foci were
frequently devoid of immunopositive secretions (Figure 4B and 4C). Strong immunopositive staining for TGFa was frequently observed in cells within compact areas of the AIT and in foci where tumor cells formed pseudoacini. In these regions immunostaining was found as discrete droplets in the cytoplasmic vacuoles, or along the luminal surfaces of neoplastic glands (Figure 4D). EGFR immunostaining was generally sharply delineated along the apical plasma membrane of glandular cells in the DLPs of untreated rats (Figure 5A). Like the situation for TGFa, EGFR immunostaining was predominately localized to the LP. Immunopositive apical protrusions of the plasma membrane of LP cells were often shed into the lumens of acini where they became amorphous as they blended with luminal contents (Figure 5A). In contrast, EGFR immunostaining was occasionally localized along the plasma cell membranes of dysplastic epithelia but was more frequently found to rim cytoplasmic vacuoles (Figure 5B). In severely dysplastic lesions, where intraglandular luminal formation was a feature, immunostaining for EGFR was found along the 2575
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Fig. 5. (A) Immunohistochemical localization of epidermal growth factor receptor (EGFR) in the lateral prostate lobe of an untreated rat. Note the sharply delineated immunolocalization of EGFR along the apical/luminal plasma membrane. Immunopositive matenal is found within the lumen. In other areas apical protrusions of immunopositively stained membrane were found to be shed into lumens. It is likely that these protrusions undergo degradation and are the source of the immunostained material illustrated in the lumen of this gland. 400X hematoxylin counterstain. (B) Immunohistochemica] localization of EGFR in a severely dysplastic lesion. Numerous immunopositive cytoplasmic vacuoles are present in dysplastic cells. Positive staining is also present along the apical membrane of the residual lumen (lower left). Note the loss of epithelial cell polarity in the lesion when compared with their orientation in normal glands (i.e. Figure 5A). 400X hematoxylin counterstain. (C) Immunolocalization of EGFR in the AIT. Immunopositive staining was predominantly localized along the plasma membrane borders of pseudoglandular structures in specimens of AIT. 400X hematoxylin counterstain. (D) Omission controls for EGFR immunostains. Sections of DLP were incubated without inclusion of the pnmary antibody. The same results were obtained for negative controls for TGFa immunostaining. Note the absence of immunostaining in this specimen 250X hematoxylin counterstain.
plasma membranes of these structures (Figure 5B). Immunopositively stained apical protrusions or blebs and luminal secretions were always absent in severely dysplastic foci but occasionally encountered in more mild grade lesions. As was often the case for TGFa, immunostaining for EGFR was localized along the boarders of pseudo-acini in the AIT (Figure 5C). Intraluminal staining was not found in these structures. Immunostaining for EGF showed a similar pattern of distribution as TGFa in normal epithelial cells, i.e. mostly along the apical membranes of the epithelial cells and as aprocrine products in the lumen of the acini. Hormonal treatment caused no significant changes in terms of cellular distribution and intensity in the gland (data not shown). Positive controls, i.e. A431 cells and rat mandibular salivary glands were always immunostained for EGFR and TGFa, respectively. In contrast, immunostaining for either antigen was absent in control sections where the primary antibodies were omitted from the incubation mixture (Figure 5D). Staining patterns similar to prostatic tissues appear to exist in rat 2576
mandibular salivary gland where TGFa and EGF (data not shown) are localized in serous secretory cells and EGFR immunostaining is restricted to the borders and luminal contents of intralobular and intercalated ducts (not shown). Discussion The main objective of this study was to investigate whether enhanced and/or modified expression of EGF/TGFa and their receptor, EGFR, play a role in the development of dysplasia in our sex hormone-induced prostatic dysplasia/cancer model and if growth of a prostatic neoplasm in an androgenindependent environment is associated with increased involvement of this signal transduction pathway. Although it has long been known that androgens regulate growth of the prostate, their mitogenic actions on the prostatic epithelium are currently believed to be indirect and likely mediated via growth factors such as EGF and TGFa (1,19,24— 28). In this study, we have demonstrated the co-localization
Elevation of TGFa and EGFR in prostate tumorigenesis
of TGFa and EGFR in the normal epithelia of VP and DLP. The levels of expression in the DLP were substantially higher than those observed in the VP. Immunolocalization of both EGF, TGFa and their receptor in mature glandular epithelial cells of the DLP suggests that they are normally secreted as aprocine products into the prostatic fluid. Thus, it does not appear that these proteins normally play a role in prostatic growth regulation but may, as has been suggested (14-16,22), function to mediate cellular differentiation in the rat gland. Increased levels of TGFa and EGFR expression were found in the DLPs of T + E2-treated rats. The levels of both mRNA and protein for the ligand and receptor in these glands, however, marginally elevated when compared with those from the DLPs of untreated rats. Results from our immunohistochemical studies demonstrated that enhanced expression of both TGFa and its receptor occurred exclusively in dysplastic lesions which comprised only a small portion of the DLP. Thus, the increased levels of both ligand and receptor may have been diluted by extracts from the predominately lesionfree DLPs. In addition to the apparent upregulation of TGFa and EGFR in dysplastic foci changes in localization for both ligand and receptor was strikingly evident when normal glandular cells and those in lesions were compared. In contrast to the apical/ luminal localization in normal cells, TGFa and EGFR appeared to lose this orientation in dysplastic epithlelium. Thus, the internal location of both ligand and receptor in dysplastic cells suggest that their function may now be redirected. This finding was particularly evident in lesions where a loss of cell polarity was severe. Similar immunohistochemical findings were present in the AIT where a 10-fold elevation in both TGFa and EGFR expression had occurred. Taken together, the immunolocalization of TGFa and EGFR, in the AIT and in dysplastic cells, may reflect the participation of these autocrine factors in a growth-promoting function. In contrast, EGF, was found to be expressed at high but comparable levels in normal and dysplastic DLPs. Immunostaining for EGF revealed that this protein was localized to the apical membranes of the epithelial cells and as aprocrine products in the lumen of the acini in both normal and dysplastic DLPs. Furthermore, unlike TGFa, no focal localization of EGF in dysplastic foci was observed in the DLPs of T + E2treated rats. Since the levels and cellular distribution pattern of this growth factor in the DLP were unchanged by the dual hormone treatment, it is unlikely that it plays a role in the development of dysplasia. Rather, like its human counterpart, it appears that rat EGF is a secretory product from the DLP epithelium and may regulate and/or support functions along the male reproductive tract (14,20,22). Of interest to note was a lack of expression of EGF in the AIT, which overexpressed TGFa when compared with its nontransformed counterparts, i.e. DLPs of untreated and T + E2treated rats. The reason for the dominance of TGFa over EGF in the tumor is unclear but a comparable phenomenon had previously been observed among several human prostatic cancer cell lines (32) LNCaP, an androgen-responsive cell line, expressed low levels of TGFa and high levels of EGF mRNA. In contrast, DU145 and PC-3, both androgen-independent lines, expressed high levels of TGFa message and low levels of EGF transcripts. It has been suggested that a switch in ligand dominance from EGF to TGFa is a key factor in the progression of neoplastic growth from hormonal dependence to hormonal independence (1,32). This postulate is in concordance
with our finding that the AIT, an androgen-independent prostate neoplasm, has apparently down regulated EGF expression and principally utilizes TGFa as the ligand for the EGFR-regulated autocrine loop. EGF and TGFa share most biological actions and bind to EGFR with comparable affinity (3-5). TGFa is, however, reported to be more potent and has a more protracted action than EGF (49,50). This may be partially explained by the reported differences in the intracellular processing of the two ligand-receptor complexes (50). Whether this is associated with a more prolonged hormone action in the AIT is a subject for future investigation. The treatment of Noble rats with either T, DHT, or E2 alone does not cause the development of dysplastic lesions (34,52). The conjoint action of T and E2 is therefore required for dysplasia induction as well as for subsequent carcinoma development (36). While the precise hormonal requirements for TGFa and EGFR upregulation in rat DLP are not known, it is plausible that it is due to the combined action of an androgen and an estrogen. In this context, castration experiments have shown that EGFR expression in the rat prostate is regulated by androgens (17,19,29). In female target tissues, estrogens upregulate TGFa and its receptor (45,53,54). A potential estrogen-responsive element has been localized to the 5'-flanking region of the human TGFa gene (55). Moreover, type II estrogen binding sites, which we reported to be uniquely expressed in rat DLP and elevated by the 16 week dual hormone treatment (56,57), may also be a key mediator for the conjoint androgen-estrogenic action. In summary, our findings suggest that enhanced expression and the possible acquisition of new growth-related functions for the TGFa/EGFR pathway may be important events in the development of prostatic dysplasia. Results from our study also indicate that this pathway may modulate androgenindependent malignant prostatic growth in this experimental model system. Acknowledgements This investigation was supported by National Cancer Institute, NIH grants CA-15776, CA-60923 and CA-62269. We thank Dr George Stancel, Dr David Lee and Dr Donna Dorow for generously providing us with the EGFR, TGFa and EGF cDNA probes, respectively. We acknowledge the Center for Reproductive Research at Tufts University School of Medicine for its technical assistance.
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