The Prostate 14: 123-132 (1989)

Binding of Epidermal Growth Factor by Human Normal, Hypertrophic, and Carcinomatous Prostate Peter Davies and Colby L. Eaton

Tenovus Institute for Cancer Research, University of Wales College of Medicine, Cardiff, United Kingdom

Saturable binding sites for radioiodinated epidermal growth factor (EGF) have been quantified in surgical specimens of benign prostate hypertrophy (BPH), histologically normal (HN) prostate, and prostate cancer. Values for EGF binding did not differ significantly between HN prostate and prostate cancer, although dedifferentiated samples tended to higher levels. These were coincident with lower comparative levels of androgen receptors. Unfractionated BPH tissue contained lower levels of EGF binding than either HN or carcinomatous prostate, but in separated epithelial cells EGF binding fell into the same range. Saturation analyses showed two affinity classes of binding in all except dedifferen- tiated tissues.

Key words: dedifferentiation, affinitive, androgen receptors

INTRODUCTION

Androgens are essential for the development, differentiation, and maintenance of the prostate gland. However, their intervention alone appears insufficient to account for all the phenomena involved in prostate epithelial cell proliferation [ 1-41. Therefore, it has been proposed that androgens are a single predominant component of a regulatory network employing an indefinite number of other coordinated growth-influencing agents [4]. Prime candidates for this latter role are the peptide growth factors.

The interaction of steroid hormones and peptide growth factors has been highlighted by studies of estrogenic mechanisms in the mammary carcinoma cell line MCF-7 [ 5 ] . Malignant cells in culture are characterized by decreased dependence on exogenous growth factors. This may be attributed to production of and response to stimulatory growth factors in an autocrine system of growth control [6] or repression of and/or insensitivity to growth inhibitory agents [7]. It is attractive to speculate on uncoupling, exploitation, or circumvention of steroid-growth factor interdependen- cies as important features of growth aberration and acquisition of hormone depen- dence.

Received for publication September 6, 1988; accepted January 5 , 1989.

Address reprint requests to Peter Davies, Tenovus Institute for Cancer Research, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XX, U.K.

0 1989 Alan R. Liss, Inc.

124 Davies and Eaton

Epidermal growth factor (EGF) is a potent mitogen for a wide variety of cells [8,9]. Levels of receptors for EGF appear to be modulated by estrogens [lo], progestins [ 111, and glucocorticoids [ 121 in respectively responsive cell types. EGF stimulates growth of normal and tumor epithelial cells from rat [13], dog [14], and human [15] prostates. Receptors for EGF have been demonstrated in rat ventral prostate [ 16,171, canine prostate epithelial cells [ 141, human prostate tumor cells in culture [ 181, and specimens of benign prostatic hypertrophy (BPH) [ 191 and human prostate carcinoma [14]. In some instances, androgens have been reported to modulate prostatic EGF receptor levels [ 16-18].

We have analyzed the specific retention of EGF by fractions of hypertrophic, carcinomatous, and histologically normal prostates to investigate whether this growth factor may feature significantly in diseased glands, and in loss of differentiation and of androgen responsiveness.

MATERIALS AND METHODS Tissue

Hypertrophic and carcinomatous prostate tissue was obtained with the cooper- ation of the surgical staff at several local hospitals, whose pathology departments provided an assessment of the tissue used for initial categorization in the laboratory. Allocation of tumor grade was based on criteria outlined by Mostofi [20], taking account of glandular differentiation and nuclear anaplasia. For this report, cancer samples were categorized according to their glandular differentiation status as well (grade I), moderately (grade 11), and poorly (grade 111) differentiated. All specimens of prostate cancer were removed by cold-punch resection or electroresection: these latter samples, and BPH samples collected by this means, were selected for assay on bases of size and condition already defined [21,22]. Infrequently, BPH tissue was obtained after retropubic prostatectomy, and on these occasions tissues classified as histologically normal (HN) were derived from peripheral areas of these samples. These tissues were chopped into approximate 3 mm3 pieces [23], which were checked histologically to confirm assessment on whole tissues.

Individual specimens of chips and solid tumors were randomly sampled and consensus histology assigned on the basis of examination of several samples from multiple fragments. This approach did not completely account for heterogeneities within samples, but provided a defined framework within which sufficient sampling for receptor determinations and accurate histology could be readily achieved and correlations made. Specimens of equivocal histology were excluded from the study. Retrospective analysis of stored sections in most cases confirmed initial categoriza- tion: for those few instances in which doubt was incurred, data has not been included or reported.

Cleansing and storage of tissue and techniques of disruption involved in its preparation for analysis have been described elsewhere [ 14,2 1-23].

Preparation of Subcellular Fractions

All procedures were carried out at 0-4°C unless otherwise stated. Tissue was homogenized (1 g/3 ml) in 20 mM HEPES, pH 7.4, containing 1 mM EDTA, 1 mM EGTA, 0.25 M sucrose, and 1 mM phenylmethylsulfonyl fluoride (PMSF). A maxi-

Human Prostate EGF Receptors 125

mum of 0.1 vol. of homogenate was retained for measurement of protein [24] and DNA [25]. Whenever quantities permitted, the remainder was divided. One portion was processed for the measurement of androgen receptors (AR) [22,23]. The other was centrifuged at 800g for 15 min and the pellet rehomogenized in the same buffer (3 ml/g tissue equivalents) and again centrifuged. The two supernatants were combined and centrifuged at 3,OOOg for 15 min. The resultant supernatant (fraction S) was centrifuged at 40,OOOg,, for 1 h. The pellet, comprising the membrane fraction, was resuspended by gentle hand-homogenization in all-glass homogenizers in 20 mM HEPES, pH 7.4, containing 0.15 M NaCl and 1 mM PMSF at 1 ml per 1 g starting material. Unless intended for immediate use, aliquots of membrane suspension were frozen at -70°C. Protein measurements [24] were carried out on 100- pl aliquots of fractions (combined 8OOg supernatants, fractions S, and membrane suspensions) after addition of 2 M NaOH (100 pl), heating at 90°C for 15 min, and dilution with water as necessary.

This process was routinely feasible with tissue from retropubic prostatectomy (some BPH and all HN tissue reported herein), but resected samples obtained from individual prostates comprised insufficient tissue for complete processing. Specimens were processed as far as possible: prostate cancer data were all derived from analyses of EGF binding in fractions S .

Measurement of EGF Binding Sites

Saturation analysis of EGF binding sites was achieved by incubation of aliquots (100 pl containing 8-24 pg protein) of fractions with ['251]EGF (specific activity 600-800 Ci/mmol; Amersham International PLC) at concentrations ranging from 0.1 nM to 10 nM in the absence and presence of a 100-fold molar excess of unlabeled EGF (Sigma) for 1 h at 25"C, in 20 mM HEPES, pH 7.4, containing 1 mM EGTA and 0.1% (w/v) bovine serum albumin. After incubation, samples were rapidly filtered under vacuum using Whatman GF/C filters and washed four times with ice-cold assay buffer; retained radioactivity was assessed in a Nuclear Enterprises Ltd. NE 1600 counter at 75% efficiency.

Saturation analyses on preparations from individual prostates could be carried out only upon tissue from retropubic prostatectomy. Similar analyses on resected samples, to be statistically significant, could be done only by combining those of rigorously indisputably similar histology, In most cases, individual resected samples were assessed by a single-point assay using a saturating concentration (15 nM) of ['251]EGF, with and without EGF (1.5 pM). Assays were done only if sufficient protein was available for at least triplicate determinations. Validation of this procedure resulted from estimations on five samples of BPH from retropubic prostatectomy yielding values from the single-point assay 98.6% 2 7.8% (SD) of those from saturation analyses. All values were calculated as fmol/mg homogenate DNA.

RESULTS

In preliminary experiments using BPH tissue from retropubic prostatectomy, combined 800g supernatants contained 88.2% k 2.3% (SD, n = 5) of homogenate ['251]EGF binding sites. Of these, 76% 2 3.97% (67.1% k 2.94% of homogenate)

126 Davies and Eaton

'O1

LL 2l Fig. 1. Total (measured at a single saturating concentration) saturable (displaceable by 100-fold molar excess of unlabeled EGF) binding of ['"IJEGF in samples of histologically normal (HN) prostate, benign hypertrophied prostate (BPH), and well-differentiated (grade I) , moderately differentiated (grade II), and poorly differentiated (grade Ill) prostate carcinoma. Values are for unfractionated tissue (0 ) and for separated epithelial fractions (0).

derived from rehomogenization of the primary 800s pellet. These data do not differ significantly from published results [ 191. Nonspecific, i .e. , nondisplaceable, binding was very high in homogenates, exceeding displaceable binding 3.38 * 0.72-fold. Fractions S contained 87.5% * 3.62% of combined supernatant binding sites, i.e., 77.2% t 3.62% of homogenate sites. Resuspended membrane fractions contained 90.3% * 5.42% of fraction S sites and 69.7% +- 6.35% of homogenate sites. Binding of ['2'I]EGF could be displaced by unlabeled EGF and transforming growth factor (TGF) at 100-fold molar excess but not by insulin, insulin-like growth factor I (IGFI), luteinizing hormone (LH), follicle-stimulating hormone (FSH), or nerve growth factor (NGF) at similar concentrations.

Levels of specific ['2SI]EGF binding in fractions S derived from three samples of HN prostate, 30 samples of BPH, 17 samples of grade I prostate cancer, 16 samples of grade I1 prostate cancer, and 25 samples of grade 111 prostate cancer are shown in Figure 1. Individual values (pmol/mg DNA) for the HN samples were 4.9, 6.3, and 7.4 (mean, 6.2). Ranges of concentrations (pmol/mg DNA: mean I+_ SD in parentheses) for BPH and grades I , 11, and 111 prostate cancer, respectively, were 0.82-2.99 (2.02 5 0.53), 4.28-7.3 (5.45 * 0.84), 4.05-7.62 (5.62 * 0.85), and 2.8-8.52 (6.74 -+ I .33). Degrees of dedifferentiation, therefore, could not be distinguished on the basis of [1251]EGF retention, neither from each other nor from HN samples, with the limited amount of data available. Values for grade 111 carcinoma clustered in the higher range, but distribution ensured that median values for grades I, 11, and I11 were not dissimilar (5.79, 5.84, and 5.66 pmol/mg DNA, respectively). However, levels of ['2sI]EGF binding in BPH were significantly lower than in either HN or cancer samples. Assays on separated epithelial components [22] yielded levels of ['2'I]EGF binding similar to those of HN and cancer samples, i.e., 4.7-6.6 pmol/mg DNA, mean 5.46, median 5.65). Values for HN samples were generally slightly greater than those of the epithelial fractions of BPH from which they were derived, viz. (HN, BPH: pmol/mg DNA in every case), 6.3, 4.9; 7.4, 5.7; 4.9, 4.7.

No obvious correlation was observed between ['*'I1EGF binding and AR, due to the wide range of concentrations of both parameters (Fig. 2), although grade 111

Human Prostate EGF Receptors 127

cancer samples tended to the highest levels of [ '251]EGF binding and lowest AR concentrations.

Saturation analysis of [1251]EGF binding to membrane preparations from individual samples of HN prostate and BPH (Figs. 3 and 4) obtained at retropubic prostatectomy yielded curvilinear Scatchard [26] plots, which in both cases could be resolved into two components by the method of Rosenthal [27]. Higher affinity (line A, Figs. 3 and 4) and lower affinity (line B, Figs. 3 and 4) components of HN prostates and BPH showed similar dissociation constants (Kd), viz. HN (A), K d

Kd 3.4-4.7 nM. Single-point assays yielded the approximate sum of the two components. The ratio B:A was greater than 6 in HN prostates and - 3 in BPH.

Detection of dissimilar affinity classes of ['251]EGF binding sites was not possible using individual cancer samples. Saturation analysis on combined fractions S from grade I carcinomas resulted in curvilinear Scatchard plots, again most readily resolved into two linear components, the higher affinity (line A) of Kd - 0.2 nM and

0.12-0.34 nM; BPH (A), Kd 0.17-0.46 nM; HN (B), K d 2.68-3.83 nM; BPH (B),

""1

L 6 8 10 0'

2 EGFReceptorlprnol/mg DNA)

Fig. 2. moderately differentiated (A), and poorly differentiated (A) prostate carcinoma.

Comparison of EGF binding and androgen receptor content of BPH (o), well-differentiated (*),

the lower affinity (line B) of Kd - 2.96 nM (Fig. 5) . The ratio B:A approximated 2. Similar analyses on combined fractions S from grade I1 cancer samples showed approximately two quantitatively equivalent affinity classes with similar Kd values to those above (Fig. 5b). Satisfactory resolution of EGF-binding data in fractions S from grade 111 cancer samples into two components has not been achieved.

DISCUSSION

We have confirmed the presence of receptors for EGF in BPH [ 191 and prostate carcinoma [ 141 and established their presence in histologically normal prostate (Fig. 3, inset) and their persistence during dedifferentiation of prostate cancer (Figs. 1, 2, and 5) . Unfractionated BPH may not be a suitable quantitative comparison for EGF receptor assessment in prostate cancer, as gross values for BPH binding of EGF were consistently lower than those found in cancer specimens, or in HN prostate (Fig, 1). This is in accord with theories that EGF is not a significant growth-promoting agent

128 Davies and Eaton

.

2 1 6 a [Tololl EGF h M l

20 LO 60 80 100 120 [Bound] EGF (pM)

Fig. 3. Saturation analysis of EGF binding to human histologically normal prostate membranes (inset) was carried out by incubation with ['2SI]EGF (0.1-7.5 nM) in the absence (total binding, T) and presence (nonsaturable binding, NS) of 100-fold molar excess of unlabeled EGF. Saturable binding (S: T minus NS) yielded a curvilinear Scatchard [26] plot (main figure) resolved 1271 into higher affinity and lower affinity components.

for BPH [28], and that BPH stroma lacks EGF receptor [19], as separated epithelial components of BPH showed EGF receptor levels similar to those of HN prostate (Fig. 1). In any event, values for all grades of prostate cancer did not vary significantly from those in HN prostate: if EGF receptors have a role in prostate cancer growth, it is more than likely that the absolute EGF binding capacity is less important than the coexistence of receptors for EGF and other growth-regulatory factors.

Only circumstantial correlations could be found between presence or concen- tration of EGF binding sites and of AR. The tissue heterogeneity and variability that has bedevilled AR binding assays on diseased prostate tissue undoubtedly affects EGF binding similarly. However, grade 111 samples tended to display lower AR values and higher EGF receptor values (Fig. 2) and to lack lower affinity EGF binding sites (Fig. 5). Variation in the ratio of lower and higher affinity sites was observed among HN prostate, BPH, and grades I and I1 cancer without significant variation in AR levels, so these two parameters are probably not directly linked. If it may be assumed that an alteration in the proportion of lower and higher affinity receptor sites

Human Prostate EGF Receptors 129

2w 1

20 LO 60 80 100 120 [Boundl EGF ipM1

Fig. 4. Saturation analysis of EGF binding to benign hypertrophic prostate membranes (inset) was camed out by incubation with ['2'L]EGF (0.1-10 nM) in the absence (total binding, T) and presence (nonsaturable binding, NS) of 100-fold molar excess of unlabeled EGF. Saturable binding (S: T minus NS) yielded a curvilinear Scatchard [26] plot (main figure) resolved [27] into higher affinity and lower affinity components.

in favor of the latter predisposes to a more effective ligand-receptor complex, then prostate cancer may become increasingly responsive to EGF as glandular differenti- ation is lost. Presumably, this results from dysregulation, but any relationship between androgen action and EGF action is tentative.

In rat ventral prostate, androgens downregulate EGF binding capacity [ 16,171, whereas they promote EGF binding of LNCaP cells [ 181. Our observations lean toward the former. Interestingly, androgens appear to repress c-myc expression in rat ventral prostate gland [29], while we have reported a correlation between EGF receptor capacity and c-myc expression in prostate cancer [ 141. These coincidental events may achieve importance if it is considered that the activity of the somewhat higher levels of EGF receptor in grade I11 cancers is exacerbated by the predominance of higher affinity sites and lower AR. However, it is uncertain in these tumors whether c-myc expression is an index of proliferative ability or cellular regression [29,30].

It is currently tempting to theorize on uncoupling of androgen regulation and EGF effects in development of abnormal growth. Breakdown of androgenic repres- sion of EGF responsiveness could unleash a significant proliferative agent. However, regulation of EGF receptor by members of the steroid hormone family appear complex. Binding of EGF by responsive cells is increased by estrogens [lo], progestins [ 111, and glucocorticoids [ 12,3 I] and decreased by 1,25-dihydroxyvitamin D3 [32]. Increases [ 11,321 and decreases [32] in EGF receptor levels have been

130 Davies and Eaton

0201 la1 0201 Ibl

IBaundl EGFipMI

Fig. 5 . Combined 3 , 0 0 0 ~ supernatants (fractions S) derived from ten samples each of (a) well- differentiated (grade I), (b) moderately differentiated (grade II), and (c) poorly differentiated (grade 111) prostatc cancer were analyzed for saturable binding of ["'I]EGF as described in Materials and Methods and in the legend to Figure 4. Where found, curvilinear Scatchard [26] plots were resolved (271 into higher and lower affinity components.

associated with growth inhibition. Although estrogens increase uterine EGF receptors [ lo] by increasing the steady-state concentration of EGF receptor mRNA [33], the absence of estrogen receptor in breast cancer cell lines is associated with higher levels of functional EGF receptor protein and mRNA 1341. A simple and exclusive relationship between hormone steroids and peptide growth factors is therefore unlikely to be the case.

It would be foolhardy, then, to predict an interrelationship between androgens and EGF receptors in the prostate. Relative AR and EGF receptor values may reflect favored cellular populations during dedifferentiation that could be elucidated only by immunocytochemical or in situ hybridization techniques. Somewhat encouraging to our observations are reports that androgen sensitivity has been associated with insensitivity to exogenous EGF [35], that the androgenic milieu influences the occurrence of EGF in human prostate cancer samples [36], that the presence of EGF determined histologically correlated with metastatic capability of prostate tumors [36], and that, in mammary cells, EGF receptors correlate with metastatic potential and overall state of differentiation 1341.

A role for EGF in normal prostate growth is uncertain. Much evidence supports the idea that prostatic EGF is destined for secretion and is active elsewhere [see 371. The relationship between androgens and EGF receptors [ 16,171 suggests that EGF may be called upon in an emergency caused by androgen deprivation. Disruption of this regulation provides for autocrine growth control. However, the utilization of EGF receptors by transforming growth factors cannot be discounted, and the numerous growth factors, either produced by prostiate cancer cells, or capable of acting upon them, [38-401 point to a complex situation.

ACKNOWLEDGMENTS

We are grateful to Professor Keith Griffiths for encouragement and enthusiasm and to the Tenovus Institute for their generous financial support.

Human Prostate EGF Receptors 131

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