The localization of transforming growth factor alpha and epidermal growth factor receptor in stromal and epithelial compartments of developing human prostate and hyperplastic, dysplastic, and carcinomatous lesions

The Localization of Transforming Growth Factor Alpha and Epidermal Growth Factor Receptor in Stromal and Epithelial Compartments of Developing Human Prostate and Hyperplastic, Dysplastic, and Carcinomatous Lesions IRWIN LEAV, DVM, JOHN E. McNEAL, MD, JEFFREY ZIAR, BA, AND JOSEPH ALROY, DVM To gain insight into autocrine/paracrine mechanisms that may influence normal and abnormal growth of the human prostate, we studied the immtmohistochemical localization of transforming growth factor alpha (TGF-c0 and epidermal growth factor receptor (EGFr) in fetal, neonatal, prepnbertal, and young adult glands. Results were compared with findings in specimens of benign prostatic hyperplasia (BPH), dysplasia (prostatic intraepithelial neoplasia--PIN), and carcinoma. EGFr was strongly and exclusively expressed in fetal basal cells, whereas TGF-c~ was localized in these and secretory cells as well as in differentiating smooth muscle cells. In neonatal and prepubertal glands, EGFr continued to be found only in basal cells, whereas TGF-ct was now present in smooth muscle and infrequently in secretory cells. In the normal adult prostate, the receptor was Strictly localized in basal cells and in the lateral plasma membranes of secretory cells, whereas its ligand was exclusively expressed in smooth muscle. This pattern persisted in PBH, but both EGFr and TGF-a staining appeared to be enhanced in their respective cellular eompart-

ments. Irrespective of grade, in dysplasia diffuse-moderate EGFr and strong TGF-a staining were both present in a majority of secretory cells. Similarly, most cells in Gleason grade 3 and 4 carcinomas expressed both EGFr and TGF-~. Our findings suggest that an unregulated paracrine mode of growth attends the development of BPH, whereas malignant transformation and progression involves autocrine/paracrine mechanisms reminiscent of those found in the developing prostate. HUM PATHOL29:668-675. Copyright © 1998 by W.B. Sannders Company Key words:prostate, hyperplasia, dysplasia, carcinoma. Abbreviations:AlL androgen receptor; TGF-~, transforming growth factor alpha; EGFr, epidermal growth factor receptor; BPH, benign hyperplasia; PIN, prostate, intraepithelial neoplasia; DHT, 5-c~, dihydrotestosterone; PZ, CZ, TZ, peripheral, central, transition zones; HMC, high-molecular-weight cytokeratin; SMA, Smooth muscle actin; PCNA, proliferating cell nuclear antigen.

Epithelial-stromal interactions play a prominent role both in modulating embryonic organogenesis and in the maintenance of adult organ structure and differentiated function. However, the key interacting molecules and the nature of their reciprocal effects in these two periods are only partly understood for any organ. For example, in rodents, androgen stimulation is known to be important for prostatic organogenesis. 14 Before birth, however, the androgen receptor (AR) is only immunodetectable in the mesenchymal cells of the gland. 4 Hence, the prenatal stimulation of epithelial proliferation by androgen is not thought to be a direct effect but rather a consequence of interposed nonsteroidal signaling molecules elaborated by the mesenchymal (stromal) cells. 14 In the adult prostate of both rodents and humans, AR continues to be demonstrable in stroma, usually in occasional differentiated smooth muscle cells, but is

now also strongly expressed in the nuclei of secretory epithelium. 4-7 Accumulating evidence now indicates that epithelial proliferation is largely confined to ARnegative basal cells, 5,7,9,1° which are believed to be precursors of secretory epithelia, s,9 Accordingly, it has been proposed that in the adult gland the influence of androgen on epithelial proliferation continues to be indirect a n d depends on the elaboration of peptide growth factor signals by the AR-positive smooth muscle cells, which interact with cognate receptors in ARnegative basal cells. 1113 Along with other peptide growth factors, transforming growth factor alpha (TGF-e0 has been implicated in mediating proliferative effects of androgens in the prostate. 11,12,14This peptide has been shown to promote mitogenesis and differentiation of ectodermal, mesodermal, and endodermal cells in a variety of organs and tissues. 15 TGF-o~binds to and induces autophosphorylation of its receptor, epidermal growth factor receptor (EGFr). a5 Phosphorylation of transmembrane EGFr initiates a cascade of protein kinase activations, culminating with mitogen-activated proteins, which are translocated to the nucleus. 16,17 Autocrine/paracrine, intraepithelial, or stromal epithelial interactions between TGF-~ and its receptor have been thought to be involved in the pathogenesis of a variety of cancers, ls'19 including carcinoma of the prostate and its progression to an androgen-indepen-

From the Department of Pathology, Tufts University School of Medicine and Veterinary Medicine, Boston, MA; and the Department of Urology, Stanford University School of Medicine, Stanford, CA. Accepted for publication September 30, 1997. Supported by grant no. CA 15776 from the National Cancer Institute. Address correspondence and reprint requests to Irwin Leav, DVM, Department of Pathology, Tufts University School of Medicine and Veterinary Medicine, 136 Harrison Ave, Boston MA 02111. Copyright © 1998 by W.B. Saunders Company 0046-8177/98/2907-000358.00/0

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TGF-c~AND EGFrIN DEVELOPINGHUMAN PROSTATE(Leav et al) d e n t state. 11,12,2°,21 Studies o n the TGF-ot EGFr pathway in the prostate have t e n d e d to be i n c o m p l e t e , investigating only a p o r t i o n o f the biological s p e c t r u m f r o m n o r m a l e p i t h e l i u m to cancer, a n d s o m e findings have b e e n contradictory. 2°2s Moreover, these studies have n o t related the stromal a n d epithelial localization o f TGF-~ a n d its r e c e p t o r to p o p u l a t i o n s o f proliferating a n d AR-positive cells f o u n d in n o r m a l a n d a b n o r m a l prostate tissues. In the c u r r e n t study, we have used i m m u n o h i s t o c h e m i c a l staining to trace the evolution o f T G F ~ a n d its c o g n a t e r e c e p t o r in fetal a n d adult prostate a n d c o m p a r e d o u r findings with those in n o d u l a r b e n i g n prostate hyperplasia (BPH), duct-acinar dysplasia (prostatic intraepithelial neoplasia or PIN), a n d carcinoma. We have systematically related these findings to cell p o p u l a t i o n s c o n t a i n i n g proliferation markers or a n d r o g e n r e c e p t o r positivity. With these techniques, we have shown that consistent alterations o c c u r d u r i n g n o r m a l prostatic d e v e l o p m e n t a n d m a t u r a t i o n as well as in hyperplastic, dysplastic, a n d neoplastic growth. Taken together, o u r findings implicate the i n v o l v e m e n t o f TGF-ot a n d EGFr in b o t h n o r m a l a n d a b e r r a n t growth processes in the prostate.

MATERIALS AND METHODS Prostate Specimens Most specimens were obtained from 38 radical prostatectomies done at Stanford Medical Center during 1994 to 1995. Patients ranged from 54 to 71 years of age. After perfusion fixation in zinc-formalin overnight, the glands were sectioned and routinely processed for histopathological studies. Sections for study were selected by one of us (J.E.M.). Maps depicting the areas of interest in a particular section were drawn and the appropriate paraffin block sent to another of us (I.L.) for immunohistochemical studies. Approximately 30 6-pro thick, consecutive sections were cut. The first, middle, and last sections were stained with hematoxylin and eosin to confirm that the tissues to be studied corresponded to areas on the map. The intervening sections were used for immunohistochemical studies. The lesions studied included (1) 16 Gleason grade 3 to 4 carcinomas, (2) 10 examples of BPH that usually occurred as multiple nodules within a given section, and (3) 20 examples of dysplastic lesions of varying grades of severity, with eight arising in the central zone (CZ) and the remainder originating in the peripheral zone (PZ). We also studied 17 lesion-free examples of normal peripheral, central, and transition zones. In addition, archival tissues, obtained at autopsy, from the collections at the Stanford and the Tufts Departments of Pathology were also studied. They included 13 histologically normal prostates from men 21 to 33 years old, prostates from two fetuses at 29 and 34 weeks of gestation who died 7 and 1 days, respectively, after premature birth, three glands from neonates that were 3 hours, 27 hours, and 1 week of age, respectively, and two prostates from prepubertal individuals aged 11 and 12 years.

molecular-weight cytokeratin (HMC), clone 34 (Enzo Diagnostics, Syosset, NY), 1:50; smooth muscle actin (SMA), clone HHF35 (Biogenex, San Ramon, CA), 1:200; proliferating cell nuclear antigen (PCNA), clone PC-10 (Dako Corp., Carpinteria, CA), 1:10,000; KI-67, rabbit anti-human (Dako Corp.), 1:1,000. For each case, immunohistochemical staining was done on consecutive sections, Sections were deparaffinized in three washes of xylene, rehydrated in graded concentrations of ethanol, and then incubated in 0.3% hydrogen peroxide in methanol to quench endogenous peroxidase. To unmask certain tissue antigens, the following enzymatic digestions were performed: (1) for TGF~, sections were preincubated at room temperature in Saponin diluted 1:2,000 in distilled water; (2) for EGFr, sections were first exposed to a solution of 0.1% Pronase E in Tris buffer (pH 7.4) for 10 minutes at 37°C). With the exception of PCNA and AiR, immunostaining procedures included placing sections in 0.01 m o l / L citrate buffer (pH 6) and then heating them to boiling in a microwave oven at high power for two to three cycles at 5 minutes each. Sections to be stained for PCNA were microwaved only once, but it was usually necessary to subject sections to five cycles to detect AR. All sections were exposed to appropriate blocking serum, that is, horse, for monoclonal antibodies and goat serum for polyclonal antibodies. The sections were exposed overnight at 4°C to the primary antibodies. After a rinse in buffered saline, an appropriate biotinylated secondary antibody for each primary reagent was used, followed either by stepavidin-peroxidase (Vector Laboratories, Burlingame CA), and 3-3 diaminobenzidine as the chromogen (Kirkegard & Perry, Gaithersburg, MD). For EGFr, alkaline phosphatase-conjugated strepavidin with new fuchsin was used as the chromogen (Biogenex). Sections were lightly counterstained with 10% Harris hematoxylin. For all omission controls, nonimmune or premune sera of mouse or rabbit origin were substituted for the primary antibodies at appropriate dilutions. Additionally, for all cases, competition controls were done for the TGF-e~ immunostaining. In these instances, the primary antibody was preincubated with a 10-fold excess of corresponding TGF-a pepfide (Oncogene Sciences) on sections overnight at 4°C. In selected cases, where TGF-ct staining was particularly strong, such as in BPH, serial dilutions of the primary antibody (l:l,000 to 1:10,000) was also performed as an additional control and for purposes of estimating levels of expression.

Immunohistochemistry The primary antibodies and the dilutions used were as follows: TGF-c~, clone 213-4.4 (Oncogene Science, Uniondale, NY), 1:100; EGFr, clone 31G7 (Ciba Corning, Alameda, CA), 1:50; AR, clone F39.4.1 (MeDiCa, Carlsbad CA), 1:10; high-

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RESULTS Fetal, Neonatal, Prepubertal, and Young Adult Prostate Fetal and neonatal prostates were nem'ly identical histologically. Both were composed of ductal/acinar structures with small lumens b o u n d e d by a single HMC-negative luminal row and a subjacent basal cell layer abutting the stroma (Fig 1A). In some prostates, most notably those from 29-week-old fetuses, solid nests of HMC-positive cells were f o u n d at tile periphery of the gland. In the prepubertal and y o u n g adult glands, basal cells persisted as a continuous row of HMC-positive cells. As the gland lumens enlarged, they were fined by HMC-negative secretory cells, which acquired a b u n d a n t cytoplasm. T h e localization o f b o t h TGF-ot a n d EGFr in the e p i t h e l i u m a n d s t r o m a s h o w e d a consistent pattern, which evolved with age. M o d e r a t e diffuse TGF-ot staining was f o u n d in s m o o t h muscle cells o f fetal, neonatal,

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and young adult prostates, but fibroblasts were always negative (Fig 1B and Table 1). In fetal prostates, moderate staining for TGF-~ was also present in both basal and secretory cells of immature glands (Fig 1B and Table 1). Faint staining of occasional secretory cells was also found in neonatal and young adult glands, but basal cells were always negative. EGFr was exclusively present in basal cells of the normal prostate at all ages. Strong immunostaining for the receptor was seen diffusely in the immature glands of fetal prostates (Fig ]C, 1D, Table 1). HMC localization, on consecutive sections, confirmed that all EGFrpositive cells were basal cells. These cells were selectively oriented along the perimeters of acini and ducts (Fig 1D), or, in fetal prostates, they were occasionally found in solid nests at the periphery of developing glands (Fig 1C). This pattern persisted in the prostates of young adults, although there was diminution in the intensity of EGFr staining (Table 1). In the young adult prostate, the lateral plasma membranes of secretory cells also often showed a thin border of EGFr positivity, whereas

the cell cytoplasm and luminal plasma membrane were consistently negative. PCNA and Ki-67-immunostained cells were abundant in fetal and neonatal epithelium (Fig 2). Comparison between consecutive sections showed that positivity for both proliferative markers were almost exclusively localized in basal cells. This selective localization persisted into the adult prostate and was similar in PZ, central zone CZ, and transition zone (TZ). Compared with fetal and neonatal prostates, the frequency of proliferative basal cells was markedly reduced in both prepubertal and young adult glands. Androgen receptor (AR) immunostaining was consistently absent in all cellular constituents of fetal, neonatal, and prepubertal prostates. However, the epididymal epithelia were AR-positive in all of the neonatal cases (Fig 3). This finding made it unlikely that the absence of staining in the corresponding prostates from the same individuals was artefactual. With the exception of three older archival specimens, AR staining was prominent in young adult prostates. In agreement with past reports, 5"7 AR was characteristically localized in

FIGURE 1. (A) A section of the prostate from a fetus at 29 weeks of gestation that has been immunostained for high-molecularweight cytokeratin (HMC). Note the distinct-immunopositive basal ceil layer. In some areas, HMC-positive cells were found in solid nests, but with lumen formation these cells became localized along the basement membrane. (Harris hematoxylin counterstain, original magnification x420.) (B) TGF-e-immunostained sections of the prostate of a fetus at 29 weeks of gestation. Most of the epithelial cells in both the solid nests and in glands are moderately positive, as are the smooth muscle cells, which have undergone differentiation. (Harris hematoxylin counterstain, original magnification x500.) (C, D) EGFr-immunostained section of the fetal prostate. (C) This section, immunostained for EGFr, was from a fetal prostate at 29 weeks of gestation. The ceils in these solid nests are strongly positive. These cells were all positively stained for HMC in consecutive sections. (Harris hematoxylin counterstain, original magnification x420.) (D) A primitive acinus in the prostate of a fetus at 34 weeks of gestation. Note the localized EGFr-positive cells, which were identified as basal cells by cytokeratin staining of consecutive sections. Luminal cells, which were HMC negative, are unstained for EGFr. (Harris hematoxylin counterstain, original magnification x420.)

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TGF-~ AND EGFr IN DEVELOPING HUMAN PROSTATE(Leav et al)

TABLE 1.

TGF-~ a n d EGFr Immunostaining Patterns

Stroma Fibrous

Fetal-neonate Prepub-youngadult BPH Dysplasia Carcinoma

Epithelium Smooth Muscle

Basal

Secretory

TGF-~

EGFr

TGF~

EGFr

TGF-~

EGFr

TGF-~

EGFr

0 0 0 0 0

0 0 0 0 0

++ ++ +++ + +/v + +/v

0 0 0 0 0

+ 0 0 + +/b --

+++ ++ +++ + + +/b --

+ +/v +/v +++ +++

0 +/a +++/a + +/v + +/v

Abbreviations and symbols: a, staining restricted to lateral plasma membrane; b, in mild to moderate dysplasia; +++, strong; ++, moderate; +, weak;v, variable per case or area; 0, not present; --, does not apply. secretory cells of adult glands, but no immunostaining for the receptor was present in any basal cells. In the stroma, PCNA/Ki-67-immunostained nuclei were seen in occasional cells of fetal and y o u n g adult glands. The identity of positive cells was always found, by SMA immunostaining of consecutive sections, to be smooth muscle. A n d r o g e n receptor immunostaining was also seen in occasional smooth muscle nuclei of y o u n g adult glands. Benign Prostatic Hyperplasia

The localization of HMC, EGFr, and TGF-o~ immunostaining, as well as PCNA, Ki 67, and AR, was remarkably similar for all cases of BPH we studied and resembled the distribution pattern seen in normal adult glands. In most specimens, discontinuities or gaps occurred in the layer of basal cells within hyperplastic acini when c o m p a r e d with n o r m a l glands adjacent to the nodular lesions (Fig 4). The possibility that the gaps in the layer of basal cells c o r r e s p o n d e d to stretches of attenuated cytoplasm was discounted because these areas were negative by HMC immunostaining of consecutive sections. HMC-negative cells f o u n d along the basement membrane, in basal cell gaps, had r o u n d vesicular nuclei and a b u n d a n t cytoplasm; they were histologically

FIGURE 2. Ki-67immunostaining in the prostate of a fetus at 29 weeks of gestation. Note that the prominent labeling of cells that were HMC positive in consecutive sections. A few stromal cells are also positive. (Harris hematoxylin counterstain, original magnification ×500.) 671

indistinguishable from secretory cells (Figs 4, 5E). Positive immunostaining for PCNA and KI-67 was predominantly (>98%) localized in basal cells. In contrast, neither of the markers was detected in HMC-negative cells f o u n d along the basement membrane. Staining for the two proliferation markers was also occasionally n o t e d in stromal cells within BPH nodules. Strong staining for TGF-ot was noted in the abundant smooth muscle cells within and s u r r o u n d i n g BPH nodules (Fig 5A, Table 1). Immunostaining was, however, always absent in basal cells but was occasionally weakly (<10% of cases) present in secretory cells. In all BPH cases, preincubation of the primary TGF-ot antibody and its corresponding peptide markedly reduced or completely abolished immunostaining in sections, establishing the specificity of the reagent (Fig 5B). In strongly stained BPH nodules, serial dilutions of the primary antibody at 1:1,000, or 1:10,000 always yielded negatively stained sections. In moderately stained sections, with the primary antibody at 1:100 dilution, negativity was usually achieved at a 1:1,000 dilution. Basal cells and the lateral plasma membranes in all hyperplastic glands appeared more strongly stained for EGFr when c o m p a r e d with those in y o u n g adult prostates (Fig 5C, 5D, 5E, and Table 1). The lateral mem-

FIGURE 3. Androgen receptor localization in the head of the epididymis of a 1-week-old neonate. Note the presence of positively stained nuclei (black) in principal cells lining epididymal tubules. Androgen receptor immunostaining was not demonstrable in the prostate of this individual or in glands from fetuses, other neonates, or prepubertal individuals. (Harris hematoxylin counterstain, original magnification x420.)

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FIGURE 4. High-molecular-weight cytokeratin staining in o BPH nodule. Note the focal discontinuity of the basal cell layer in several hyperplastic glands. (Harris hematoxylin counterstain, original magnification ×300.)

branes of adjacent HMC-negative secretory cells were also strongly positive for the receptor (Fig 5E). In some instances, prominent cytoplasmic staining was present in the basilar areas of these secretory cells found along the basement membrane. As previously reported, 5,24AR staining was present in the nuclei of secretory cells and to a lesser extent in stromal cells of BPH nodules. Dysplasia and Carcinoma

The pattern of expression of the TGF-0~-EGFr pathway was markedly altered in dysplasia foci. Moderate to strong TGF-~ expression was present in dysplastic secretory cells (Fig 6A and Table 1). There often was, however, considerable variability in the intensity of immunostaining for T G F ~ and its receptor between different dysplastic ducts and acini in the same specimen. Typically, moderate EGFr immunostaining was found diffusely throughout the secretory cell cytoplasm of dysplastic ceils (Fig 6B). In addition to this apparent switch to an autocrine pattern of TGF-c~ and EGFr expression, we noted, in accord with our recent study,7 that all of the AR-positive cells and most of the prolifer-

ating cells were found together in the dysplastic secretory cell compartment. The basal cells in dysplasia were variably reduced in numbers, and those that remained often showed altered morphology, with a triangular nucleus wedged between adjacent secretory cells. However, they remained strongly HMC-positive and, except for the most severe dysplasia, continued to express strong EGFr immunostaining (Fig 6B and Table 1). Unexpectedly, some degree of TGF-cx immunostaining also appeared in the basal cells in mild to moderate dysplastic lesions. No significant change was noted for the intensity of T G F ~ immunostaining in the stroma around dysplastic glands. The eight examples of dysplasia originating in the central zone had histological/cytological features and HMC staining patterns that were similar to those found in peripheral zone dysplasias. However, in two of the cases, TGF-~ and EGFr were not both present together in the epithelium. AR, PCNA, and Ki-67 immunostaining patterns in the central zone dysplasias were the same as those found in peripheral zone lesions. Invasive carcinomas were entirely devoid of HMCstained malignant epithelial cells. Positive nuclear staining for AR was present in most cancer cells. Proliferation markers were markedly increased when compared with dysplasia or any benign prostatic epithelium. Strong immunostaining for TGF-~ was present in all but one carcinoma (Fig 6C). EGFr expression was diffusely weak to moderate in neoplastic cells (Fig 6D and Table 1), and only one carcinoma lacked both TGF-~ and EGFr. For all tissues, preincubation ofTGF-~ peptide with its corresponding antibody resulted in negatively stained sections. Similarly for all tissue specimens, that is, fetal, prepubertal, adult, BPH, dysplasia, and carcinoma, substitution with preimmune sera of the same immunoglobulin class as the EGFr antibody resulted in unstained sections.

DISCUSSION

The persistent expression of EGFr, localized exclusively in proliferative-competent basal cells throughout )

FIGURE IS. (A) TGF-e immunostaining in a BPH nodule. Pronounced TGF-~ immunostaining is evident in interlacing bundles of stromal cells, which were confirmed to be SMA positive in consecutive sections. (Harris hematoxylin counterstain, original magnification x320.) (B) Competitive TGF-e immunostaining of a hyperplastic nodule. After preincubation with TGF-~ peptide and antibody (see Materials and Methods), no staining was found in this lesion that had been intensely positive at 1:100 dilution of the primary antibody. (Harris hematoxyiin counterstain, original magnification x320.) (C) EGFr immunostaining in the prostate of a 23-year-old. Note the localization of the receptor in basal cells. Staining of the lateral borders of secretory cells is also evident. (Harris hematoxylin counterstain, original magnification x300.) (D, E) EGFr immunostaining in a BPH nodule. (D) Note the intense staining for the receptor in basal cells and in the lateral membranes of hyperplastic secretory cells. Compare with C. (Harris hematoxylin counterstain, original magnification x320.) (E) Intense EGFr staining is evident in basal cells and in the lateral cytoplasmic membranes of hyperplastic secretory cells. Note the absence of basal cells in the top layer of secretory cells that abut the basement membrane. (Harris hematoxylin counterstain, original magnification x500.) FIGURE 6. (A) TGF-~ immunostaining in a dysplastic lesion. Intense staining for TGF-~ is evident in this moderate-grade lesion. (Harris hematoxylin counterstain, original magnification ×500.) (B) EGFr immunostaining in dysplasia. Note the diffuse (light red) immunostaining in this moderate-grade dysplasia. Two pyramidal-shaped basal cells with intense EGFr immunostaining are evident in this lesion. (Harris hematoxylin counterstain, original magnification x500.) (C) Strong TGF-a immunostaining is evident in this Gleason grade 3 carcinoma. (Harris hematoxylin counterstain, original magnification x320.) (D) EGFr immunostaining in prostate carcinoma. Diffuse light red staining is present in the cytoplasm of most cells. In some instances, a more intense stain is found in the basilar portions of cells. Compare with (A) and (B). (Harris hematoxylin counterstain, original magnification x420.) 672

TGF-~ AND EGFr IN DEVELOPING HUMAN PROSTATE(Leav et al)

Figure 5.

Figure 6. 673

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development from fetus to adult, suggests that the receptor plays an important role in the growth and development of the prostate. The EGFr ligand TGF-~x, also traced throughout development in our study, became increasingly localized in smooth muscle cells as the gland underwent maturation. Its location in smooth muscle is consistent with the concept that the ligand acts as a paracrine mediator of proliferative signals arising from stromal cells and interacting with EGFrpositive basal cells. In this context, it is of interest that in adult glands basal cells have been shown to be ARnegative, 5,7 whereas stromal cells and mature secretory cells have been reported to consistently express the receptor. 5,7,24,25 Taken together, our current findings and those of others n14 support the concept that the mitogenic interactions of TGF-ot with EGFr are mediated by androgens in the adult prostate. Results from our study of the h u m a n fetal prostate, however, show that both EGFr and TGF-oL are coexpressed in proliferative-competent epithelia at a time when All is not demonstrable in these cells or stromal constituents. The fetal, neonatal, and prepubertal glands we studied correspond to periods when either circulating levels of testosterone and prostatic androgen concentration are low or sex h o r m o n e - b i n d i n g globulin is high. 2e~31The result in either case would be to lower the exposure of the gland to the effects of androgen. 2s-31 The lack of immunodetectable AR therefore may be caused by lowered tissue androgens, because it is known that the threshold levels of these hormones are required for AR expression. 32,3~ In support of this concept, the immunodetectable AR we found in the developing epididymis may be explained by the presence of an androgen-binding protein, which is believed to locally elevate the concentration of androgens in this tissue. 26,3° Results from our current study therefore invoke the possibility that an androgen-independent, autocrine intraepithelial mechanism may modulate the growth and differentiation of prostatic epithelium during certain periods of development. Of great interest in this regard is the abrupt appearance of both TGF-a and EGFr in dysplastic epithelia. The localization of both ligand and receptor in these cells occurs in conjunction with the displacement of the bulk of the proliferative compartment from basal cells to dysplastic secretory cells. 1° In this context, our previous studies 34,3~had shown that dysplastic cells lacked several differentiation markers normally found in secretory epithelia. We had postulated that these alterations represented a maturation arrest in dysplastic secretory cells, which aberrantly retained a capacity for continued replication. It is therefore possible that an intraepithelial autocrine signaling pathway, similar to that found in HMC-positive cells of the developing prostate, is recapitulated in the dysplastic phenotype, where it influences growth. In contrast to the developing cells, however, presumably functional AR is also present in dysplastic cells. Moreover, the co-localization ofAR, TGF-~, and EGFr in dysplastic cells is identical to that found in invasive carcinoma. Taken together, these findings may indicate that androgens regulate the 674

autocrine pathway during this phase of prostatic carcinogenesis. This concept is supported by one study that reported that the mitogenic action of androgens on cultures of h u m a n prostate cancer cells appears to be regulated by a TGF-o~/EGFr autocrine pathway 14 and by a subsequent report that showed that antibodies directed against TGF-c~ and EGFr can decrease the proliferation of the androgen-dependent LNCAP prostate cancer cell lineY Initially, most prostatic carcinomas are androgenresponsive but become progressively independent of the h o r m o n e for growth. ~7 Parallelling this transition, Scher et al 2° have reported that most of the androgenindependent metastatic carcinomas they studied coexpressed TGF-a and EGFr. Taken in context, our findings and those cited suggest that an androgen-modulated intraepithelial autocrine loop is established early in the carcinogenic process, where it contributes to dysplastic and then to neoplastic growth. With progression to an androgenindependent state, this autonomous growth factor/ receptor pathway may become a dominant mechanism for continued neoplastic cell proliferation. In marked contrast to the autocrine translocation of the TGF-~/EGFr pathway that generally attends prostatic carcinogenesis, in BPH the normal paracrine relationship of the ligand to the receptor is maintained but appears to be amplified. Likewise, AR localization is also unchanged from normal, a finding consistent with past reports that indicate that mechanisms of androgen activation are functionally intact in BPH tissue. 3s Thus, in BPH, as proposed for normal prostate tissue, androgens may regulate the TGF-e~/EGFr pathway through stromal/epithelial interactions. However, by contrast with normal glands, the focal absence of EGFr-positive basal cells as well as the apparent amplification of the receptor in secretory cells may reflect altered regulation of paracrine stromal-epithelial interactions in hyperplastic lesions. The importance of these alterations in the pathogenesis of BPH is not readily apparent because most proliferative activity in lesions remains localized in residual basal cells. In summary, results from our studies indicate that a TGF-0CEGFr autocrine loop is involved in the early development of the h u m a n prostate. With further maturation, we find that the loop increasingly assumes a paracrine signaling pattern with interactions between smooth muscle and basal cell constituents. In BpH, this paracrine relationship is maintained but appears markedly enhanced. In contrast, the development and progression of prostatic carcinoma recapitulates some of the autocrine features of this growth factor-receptor pathway found in basal cells of the fetal/neonatal gland. REFERENCES 1. Cunha GR, Alarid ET, Turner T, et al: Normal and abnormal development of the male urogenital tract: Role of androgens, mesenchymal-epithelial interactions and growth factors. J Androl 13:465475, 1992 2. Cunha GR, Fujji H, Neuberger BL, et al: Epithelial-mesenchymal interactions in prostatic development: Morphological observa-

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