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Growth regulation of ovarian cancer cells by epidermal growth factor and transforming growth factors a and/31 Li Z h o u a n d B e n j a m i n S. L e u n g Dit'ision o f Reproductit'e Cell Biology Department O["Obstetrics and Gynecology Unit 'ersily ~/" Minnesota, Minneapolis, M N (USA) (Received 21 May 1992)

Key words: OVCAR-3; CAOV-3; EGF receptor; Receptor phosphorylation; Cell proliferation

Regulation of ovarian cancer growth is poorly understood. In this study, the effects of EGF, TGFa and TGF/31 on two ovarian canccr cell lines (OVCAR-3 and CAOV-3) were investigated. The results showed that E G F / T G F a stimulated cell growth and DNA synthesis in OVCAR-3 cells, but inhibited cell proliferation and DNA synthesis in CAOV-3 cells. TGF/31 invariably inhibited cell proliferation and DNA synthesis in both cell lines. These effects on growth factors are dose dependent. The interaction of TGF/31 and E G F / T G F a was antagonistic in OVCAR-3 cells. In contrast, E G F / T G F a and TGF/31 had an additive inhibitory effect on CAOV-3 cells. Our results demonstrated that mature and functional EGF receptors arc present in both cell lines and that they are capable of ligand binding, internalization, processing and ligand-enhanced autophosphorylation. Both high- and low-affinity binding are present in these cell lines, with CAOV-3 cells having about 2-3-fold higher total receptors than OVCAR-3 cells. These results together with those from our previous studies show that these cells express TGFa, TGF/31 and EGF receptors and that cell growth may be modulated by these growth factors in an autocrine and paracrine manner. This report presents evidence supporting the important roles of growth factors in ovarian cancer growth and provides a foundation for further study into the mechanism of growth regulation by growth factors in these cell lines.

Introduction Ovarian cancer is the most fatal gynecologic malignancy, and although a great deal of effort has been devoted to it, prognosis has not been significantly improved. This is due in part to the occult nature of the malignancy as well as the fact that ovarian cancer's mechanism for growth regulation is as yet unknown. Recent studies demonstrate that oncogenes, growth factors and their cognate receptors are involved in the genesis and proliferation of neoplasms [1,2]. For exampie, E G F has been shown to be a potent mitogen for many types of malignant and nonmalignant cells [3,4]. E G F ' s binding to its receptor activates the tyrosine kinase which in turn initiates autophosphorylation and the signal transduction mechanism involved in many cellular functions [5]. Paradoxically, when E G F receptor is overexpressed in certain cancer cell lines, E G F can inhibit cell proliferation [6,7]. T G F a , which has high structural homology to EGF, also binds to E G F receptor and exhibits EGF-like biological activities.

Correspondence to: B.S. Leung, Division of Reproductive Cell Biology, Department of Obstetrics and Gynecology, 420 Delaware St. S.E., U M H C Box 395, University of Minnesota, Minneapolis, MN, USA.

That it functions as an autocrine factor has been reported for a number of malignant cells in culture [8,9]. TGF/31 is chemically distinct from E G F and binds to its cognate membrane receptors. It is a bifunctional regulator of cell growth and a potent growth suppressor for many epithelial cancer cells [10]. Despite the large volume of literature on the effect of growth factors on the many cell types, little is known regarding their regulation of ovarian cancer cell proliferation. Bauknecht et al. [11] reported that 5 of 12 ovarian cancers were EGF-receptor positive and that a negative relation exists between E G F receptor status and the amount of EGF-like substance produced by the tumors. With an immunohistologic staining method, Gullick et al. [12] investigated the expression of E G F receptor in 10 gynecologic cancers. They found that there was always strong staining for squamous cell carcinoma of the cervix and the vulva, but weak or heterogeneous patterns in ovarian adenocarcinoma. Recently, the expression of E G F receptor in ovarian cancer was correlated to poor survival [13] and responsiveness of ovarian cancer cells to E G F in vitro [14]. To further understand the roles of growth factors in ovarian cancer cell proliferation, we investigated the effects of EGF, T G F a and TGF/31 on cell proliferation and the expression of E G F receptor of two epithelial ovarian cancer cell lines (OVCAR-3 and CAOV-3). Our

131 results give further support to the inhibitory effect of TGF/3 on epithelial cancer cells. We also demonstrated that mature and functional E G F receptors are present in both cell lines, with a relative abundance in CAOV-3. E G F or T G F a elicited opposite responses on cell proliferation in these two cell lines, their effects appearing to be related to E G F receptor levels. Studies aiming to elucidate the mechanisms by which these growth factors stimulate or inhibit cell proliferation are in progress.

Materials and Methods

Materials. Mouse E G F was purchased from Sigma (St. Louis, MO). Recombinant human T G F a was purchased from Chemicon International (Los Angeles, CA). TGF/31, purified from human platelets, was a gift of C. Heldin, Uppsala, Sweden. OVCAR-3 and CAOV-3 human ovarian cancer cells were purchased from American Type Culture Collection (Rockville, MD). Cell growth determination. Cells were grown as monolayer culture at 37°C in a humidified atmosphere of 95% air and 5% CO 2 in their respective media, RPMI-1640 for OVCAR-3 cells and D M E M for CAOV-3 cells, supplemented with 2 mM L-glutamine, 10 mM Hepes, 100 units/ml penicillin, 100 #xg/ml streptomycin and 10% fetal bovine serum (FBS). For the cell proliferation study, cells were grown in 24-well Costar plates and partially synchronized at the early Gl-phase by cultivation in serum-free medium for 48 h [15]. Synchronized cells were incubated for various periods in fresh medium containing 1% FBS and different concentrations of growth factors. Cell count was done with a Coulter counter. At the end of incubation, 0.5 /xCi of [3H]thymidine in a volume of 20 /zl was added to each well. Cells were incubated for an additional hour and fixed with 5% ice-cold trichloroacetic acid. After extensive washing with ice-cold distilled water, immobilized cells were solubilized in 0.5 ml 1 M NaOH, neutralized with 1 M HC1, the solution then being mixed with 5 ml Budget-Solve scintillation fluid for the determination of incorporated radioactivity by a scintillation counter (Beckman LS 2800). Experiments were performed in triplicate. [125I]EGF binding. Near-confluent monolayer cells in 12-well tissue culture plates were washed twice with phosphate buffered saline (PBS) and [125I]EGF added ( 1 5 8 / x C i / # g ; ICN Biomedicals, Irvine, CA) in binding medium ( D M E M supplemented with 20 mM Hepes, 0.1% BSA). At the end of incubation, unbound [125I]EGF was removed and ceils were washed three times with ice-cold binding medium. Cell-bound radioactivity, solubilized with 0.5 M NaOH, was measured by a Beckman Gamma 4000 Counter. The binding affinity and capacity of E G F receptors were deter-

mined by binding various concentrations of labeled ligand at 22°C for 2 h [16] and analyzing them by Scatchard's method. Nonspecific binding was determined in the presence of 200-fold unlabeled EGF. To monitor degradation of ligand-receptor complex, a pulse-chase experiment was conducted. Following incubation with [125I]EGF at 37°C for 2 h, cells were washed to remove unbound labeled ligand and reincubated with fresh medium containing 200 n g / m l unlabeled EGF. Cell-free medium from the second incubation was lyophilized and resuspended in a small volume of distilled water. Radioactive material secreted into the medium was analyzed by Sephadex G-25 column chromatography. Cross-linking of EGF to receptors. Cells were incubated in monolayers with [J25I]EGF at 22°C for 2 h in the presence or absence of unlabeled E G F and E G F was cross-linked to its receptor with 0.25 mM disuccinimidyl suberate (DSS) [17]. The detergent-soluble fractions were subjected to 7.5% SDS-PAGE. The gels were dried and exposed to Kodak XAR-5 X-ray film at - 70oc. Biosynthesis of EGFR protein. Ceils (105) were seeded into 6-well plates in media supplemented with 10% FBS. The next day monolayers were washed with

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T i m e (h) Fig. 1. Time-course of growth factor effects on thymidine incorporation for O V C A R - 3 (A) and CAOV-3 (B) cells. After synchronization, cells were incubated in 1% FBS medium in the presence of T G F a 5 n g / m l (©), TGF/3 2.5 n g / m l ([3) or absence (o) of growth factors for various periods. At the end of each period, a 1-h pulse with [3H]thymidine was performed and mean values of close triplicates were shown.

132 PBS, then incubated in 2% methionine and 2% FBSDMEM containing 50 /~Ci/ml of [-~SS]methionine at 37°C for 16 h. At the end of incubation, cells were scraped off, washed and lysed and cell lysates subjected to immunoprecipitation with protein A-Sepharose-linked anti-EGFR monoclonal antibody at 4°C for 2 h. Beads were washed and boiled and the bound radioactivities eluted with SDS-PAGE sample buffer, separated on 7.5% SDS-PAGE and visualized by fluorography. Autophosphorylation of membrane preparations and immuno-precipitation. Autophosphorylation of membrane preparations was conducted as described previously [18]. At the end of the reaction with [7-32p]ATP (4500 Ci/mmol; ICN Biomedicals, Costa Mesa, CA), in the presence or absence of EGF, membrane suspensions were immunoprecipitated by interaction at 4°C for 2 h with monoclonal antibody R1 (Amersham, IL), which is specific to the extracellular domain of the EGF receptor and immobilized on protein A-Sepharose beads. After extensive washing of the beads, antibody-bound protein was eluted by electrophoresis sample buffer and subjected to 7.5% SDS-PAGE and autoradiography.

Results

Effect of EGk, TGFa and TGF[31 on thymidine incorporation The effects of growth factors on cell proliferation were examined by thymidine incorporation. Cells were partially synchronized at Gl-phase following a 48 h period of serum deprivation. Following release, cells at G 1-phase readily traversed to S-phase as determined by [3H]thymidine incorporation into DNA (Fig. 1). Doublet peaks of radioactivity were seen in both cell lines after incubation in serum supplemented medium. The first peak represents the DNA synthetic phase (S-phase), reaching maximal incorporation at about 30 h for both OVCAR-3 and CAOV-3 cells. It is uncertain whether the second peak, which has maximal incorporation at 45 h, is derived from another S-phase population of newly committed cells. The addition of TGFa (5 ng/ml) stimulated higher [-~H]thymidine incorporation in both peaks of OVCAR-3 cells (Fig. I A), but inhibited DNA synthesis in CAOV-3 cells (Fig. 1B). Treatment of ceils with TGF/31 invariably inhibited DNA synthesis in both cell lines. Thus, TGFa or TGF/31 appears to enhance or suppress the progres-

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Fig. 2. Dose-response of TGF~, TGFI31 and combinations of T G F ~ I and TGF~ on thymidine incorporation for OVCAR-3 (A, C) and CAOV-3 (B, D) cells. Synchronized cells were incubated in fresh medium containing 1% FBS and various concentrations of T G F a in the presence or absence of 0.5 n g / m l TGF¢I1 for A and B and different concentrations of TGF~ll for C and D. [3tt]thymidine pulse-labeling was performed after 26-30 h incubation with TGFc~ (open bar) and TGFc~ and TGFI31 (cross-hatched bar). Values shown are means + S.D. of triplicates.

133 sion of Gl-phase cells to S-phase without altering the duration of Gl-phase and S phase. Partially synchronized cells were tested for their dose dependence on growth factors by incubation with various concentrations of growth factors for 26-30 h, followed by a 1-h pulse with [3H]thymidine. As shown in Fig. 2, the stimulatory effect of T G F a (0.2-5 ng/ml) in OVCAR-3 cells and the inhibitory effect in CAOV-3 cells were dose-dependent. The interaction of T G F a and TGF/31 was antagonistic in OVCAR-3 cells. As little as 0.5 n g /m l of TGF/31 completely counteracted the effect of a 10-fold excess of T G F a (Fig. 2A). In CAOV-3 cells, there was an additive inhibitory effect between T G F a and TGF/31 (Fig. 2B). EGF exhibited the same effects as T G F a in both of these cell lines (data not shown). The inhibitory effect of TGF/31 (0.1-2.5 ng/ml) in both cell lines also exhibited a dose-response relationship (Figs. 2C and 2D). The effects of EGF and TGFfl on cell proliferation for OVCAR and for CAOV-3 (results not shown) under various manipulations of serum and growth factor supplementation were consistent with results on thymidine incorporation.

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Fraction Fig. 4. Elution profiles of dissociated radioactivity from [125I]EGFreceptor complex from OVCAR-3 (A) and CAOV-3 (B) cells. Following incubation with [125I]EOF at 37°C for 2 h, cells w e r e washed and reincubated in fresh medium containing 200 ng/m] unlabeled EGF for various periods. Cell-free medium from the chase phase was analyzed by a Sephadex 0-25 column from 30 rain chase (o) and 3 h chase (©).

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Time (min) Fig. 3. Time-course of E G F binding and internalization for OVCAR-3 (A) and CAOV-3 (B) cells. Cells were incubated with [J25I]EGF at 37°C. At the end of each time point, cells were washed with ice-cold binding medium and incubated for 4 min at 4°C with 0.5 M NaCI (titrated with acetic acid to pH 2.5). Radioactivity eluted by acid treatment (©) and from acid resistant reaction (o).

Internalization of EGF-receptor complex was demonstrated by a simple experiment (Fig. 3). Cellmembrane-bound [125I]EGF was eluted by acidic 0.5 M NaC1, but internalized EGF was not affected. [125I]EGF bound to both OVCAR-3 and CAOV-3 cells was internalized and processed in an identical fashion. In both cell lines, surface-bound radioactivity was maximal at 15 min of incubation with labeled ligand. The internalized radioactivity increased rapidly during the initial 2 h of incubation. In a pulse-chase experiment, it was shown that internalized EGF was processed and subsequently secreted as degradative products to the medium (Fig. 4). The radioactivity secreted into medium at 30 min of chase was eluted in the void volume, in the same region as the intact EGF. However, following a 3-h chase, two peaks were shown with the majority of the radioactivity eluted as low-molecular-weight degraded products.

Characterization of the EGF receptor The binding affinity and capacity of the EGF receptor were quantified by Scatchard analysis. As shown in

134 Fig. 5, the specific binding of [J25I]EGF revealed curvilinear plots. Indicative of two classes of E G F receptor in both cell lines is the high-affinity binding site with K d 0.19 nM and 0.35 nM and low-affinity binding site with K d 1.19 nM and 1.14 nM for OVCAR-3 and CAOV-3 cells, respectively. The total binding sites for OVCAR-3 is 136000 (42000 high and 94000 low) and for CAOV-3 is 292000 (116000 high and 176000 low) per cell. E G F receptor was further characterized by affinity labeling of [125I]EGF. Ligand-receptor complexes showed a 170 kDa radioactive band compatible with 200-fold unlabeled E G F on SDS-PAGE for both cell lines, consistent with results obtained by Western blot technique (results not shown). The mature E G F R at 170 kDa was again confirmed by [35S]methionine incorporation, as compared with A431 cells (Fig. 6A).

Autophosphorylation of the EGF receptor The ability of the E G F receptor to be autophosphorylated by its tyrosine kinase was tested on cell membrane preparations in the presence and absence of added E G F and immunoprecipitated with monoclonal antibody against E G F receptor. In the absence of

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added E G F to culture cells limited autophosphorylation was seen, as shown by a weak radioactive band at the position of E G F receptor. Addition of EGF markedly enhanced autophosphorylation in both cell lines, as shown by a much more intense radioactive band (Fig. 6B). Discussion

E G F has been widely reported as a mitogen, but its growth inhibitory activity on several cell lines has also been demonstrated, including A431 human vulvar [6], R L 9 5 - 2 endometrial [19], ovarian [13,14] and certain mammary [20] and liver cancers [21]. The results of the present study revealed opposite responses of two epithelial ovarian cancer cell lines to E G F / T G F a , which stimulated cell proliferation and DNA synthesis in OVCAR-3 cell line, but inhibited cell growth and DNA synthesis in CAOV-3 cell line. In addition to the growth stimulatory activity on fibroblasts [20], TGF/31 is a potent proliferation inhibitor for many cell types [2325]. Our results show that TGF¢31 inhibits growth and DNA synthesis in both OVCAR-3 and CAOV-3 cell lines. In OVCAR-3 cells, TGF/31 also inhibited E G F / T G F a - i n d u c e d stimulation of DNA synthesis. However, in CAOV-3 cells there was an additive effect of TGF/31 and E G F / T G F a on inhibition of DNA synthesis. These results are consistent with other findings that TGF/31 is a potent inhibitor of epithelial cells [24,25]. Accordingly, the absence of an operative TGF/31 system in cells may result in uncontrolled cell growth. Since most cells are known to produce a latent form of TGF/3 [26,27], the inability of cells to activate TGF/31 may also contribute to unbridled cell proliferation. Kinetic study revealed that E G F / T G F a and TGF/31 increase or decrease the proportion of G1phase cells that enter S-phase without altering the durations of these phases. This finding differs from estrogen modulation of some breast cancer cells where the G1 duration was also shortened [15,28]. The present results suggest that the function of these growth factors is to affect the traverse of Gl-phase cells to S phase. It is reasonable to speculate that specific metabolic and mitogenic enzymes or proteins may be induced or inhibited by growth factors as post-receptor events resulting in different responses to E G F / T G F ~ by these two ovarian cancer cell lines. We have shown by the intron-differential R N A / P C R method [29] that both OVCAR-3 and CAOV-3 cells express T G F a and TGF/31. T G F a secretion in spent media was also demonstrated for the different ovarian cancer cells (data not shown). The biological response presented here, together with the gene expression of T G F a and TGF/3, shown previously, demonstrates that these growth factors may regulate ovarian cancer cells in both an autocrine and paracrine manner.

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Fig. 6. (A): Biosynthetic labeling of EGFR. The figure shows the fluorographic pattern of A431 (Lane 1), CAOV-3 (Lane 2), and OVCAR-3 (Lane 3) precipitated by extracellular-domain-specific EGFR monoclonal antibodies. The 170 kDa band, shown by an arrow, represents mature EGFR. (B): Autopbosphorylation of the EGF receptor from membrane preparations in the absence (A and C) or presence (B and D) of added EGF (5 ng/ml). X-ray films for OVCAR-3 (A, B) and CAOV-3 (C, D) were exposed 3 days and 2 h, respectively. Molecular mass markers shown were from myosin,/3-galactosidase, phosphorylase, bovine serum albumin and ovalbumin.

Accumulated evidence has shown that E G F receptor status may have prognostic potential in human cancers [13,30,31]: T G F a , an E G F receptor-like protein encoded by the v-erb-B oncogene, is produced in many types of malignant cells [8,32] and has been found in transformed cells [33], mediating its action via E G F receptor. The present report demonstrates that OVCAR-3 and CAOV-3 ovarian cancer cells possess E G F receptor which is specifically bound to E G F and that the ligand-receptor complex is internalized and degraded. It is also shown that the E G F receptors are functional in terms of ligand binding, internalization and enhanced autophosphorylation by ligand binding. The affinity-labeling study showed that E G F receptor in these ovarian cancer cells is a 170 kDa protein, consistent with reports by others of a mature E G F receptor [5,13,34]. With direct and indirect immunocytochemical staining of E G F or E G F receptor in these cells, we also observed the presence of E G F receptor in both cell lines (Leung, B.S., unpublished data). Results from our studies strongly attest to the

presence of E G F receptor in the ovarian adenocarcinoma cells studied. In this study, it was revealed that the maximum binding sites of E G F receptor was 2-3-fold higher in CAOV-3 cells as compared with OVCAR-3 cells. The quantitative differences in E G F receptor levels in these two cell lines agree with our other studies conducted with immunocytochemical techniques and the introndifferential R N A / P C R method [29]. Relatively higher expression of E G F receptor in both RNA and protein levels for CAOV-3 cells was evident by the different techniques. Gene amplification, however, was not observed in either cell line [35]. The inhibitory effect of E G F / T G F a in CAOV-3 cells might be related to the higher level of E G F receptor, similar to observations on A431 cells [36]. It has been reported that E G F inhibited cell growth for all 14 lines of human squamous cell carcinoma tested and that the inhibitory effects are correlated with an elevated level of E G F receptor [7]. However, the finding that A43tR-1, a variant of the A431 cell line, exhibits the same number

13~

of EGF receptors as the original cell type but is refractory to the inhibitory effect of EGF [37] suggests that other mechanisms in addition to overexpression of EGF receptor may contribute to the growth inhibitory effect of EGF. It is plausible that mutation of EGF receptor gene, receptor aggregation, or multiple signals generated in response to EGF in different cells play important roles in the different responses. Our present study shows that mature and functional EGF receptors are present in these two cell lines and that they exhibit similar properties in EGF-binding kinetics, internalization, ligand-induced autophosphorylation and degradation. Further study is necessary to elucidate other events related to the mechanisms of E G F / T G F a - i n duced or suppressed cell proliferation. The mechanisms of growth factors in promoting and inhibiting ovarian cancers remain to be defined. Studies presently undertaken in our laboratory are aimed at developing further insight into these mechanisms. Acknowledgements The technical assistance of Laurence Stout is greatly appreciated. Results were presented at the 36th Annual Meeting of the Society For Gynecologic Investigation, March 15-18, 1989. This research has been supported in part by grant 5R01 CA 47212 from the National Cancer Institute. References l Goustin, A.S., Leof, E.B., Shipley, G.D. and Moses, H.L. (1986) Cancer Res. 46, 1015-1029. 2 Keski-Oja, J., Leof, E.B., Lyons, R.M., Coffey, R.J. and Moses, H.L. (1987) J. Cell Biochem. 33, 95-107. 3 Cohen, S. and Carpenter, G. (1975) Proc. Natl. Acad. Sci. USA 72, 1317-1321. 4 Kirkland, W.L., Yang, N.S., Jorgensen, T., Langley, C. and Fumanski, P. (1979) J. Natl. Cancer Inst. 63, 29-39. 5 Cohen, S., Carpenter, G. and King, L., Jr. (1980) J. Biol. Chem. 255, 4834-4842. 6 Barnes, D.W. (1982) J. Cell Biol. 93, 1-4. 7 Kamata, N., Chida, K., Rikimaru, K., Horikoshi, M., Enomoto, S. and Kuroki, T. (1986) Cancer Res. 46, 1648-1653. 8 Todaro, G.J., Fryling, C. and De Larco, J.E. (1980) Proc. Natl. Acad. Sci. USA 77, 5258-5262. 9 Dickson, R.B., Huff, K.K., Spencer, E.M. and Lippman, M.E. (1985) Endocrinology 118, 138 - 142. 10 Sporn M.B., Roberts, A.B., Wakefield, L.M. and Assoian, R.K. (1986) Science 233, 532-534.

11 Bauknecht, T., Kiechle, M., Bauer, (;. and Sicbcrs, .I.W. (198,5) Cancer Res. 46, 2614-2618. 12 Gullick, W.J., Marsden, J.J., Whittle, N., Ward, B., Bobrow, L. lind Waterfield, M.D. (1986) Cancer Res. 46, 285 292. 13 Berchuck, A., Rodriguez, G.C., Kamel, A., Dodge, R.K., Soper, J.T., Clarke-Pearson, D.I,. and Bast. R.C., .Jr. (1991) Am. ,I. Obstet. Gynecol. 164, 669 674. 14 Rodriguez, G.('.. Berchuck, A., Whitaker, R.S., Schlossman, 1)., Clarke-Pearson, D.L. and Bast, R.C.. Jr. (1991) Am. J. Obslet. Gynecol. 164, 745-45(). 15 Leung, B.S. and Potter, A.H. (1987) Cancer lnvesl. 5, 187-194. 16 Bennett, J.P. (1978) in Neurotransmitter Receptor Binding (Yamamura, H.I., Enna, S.J. and Kuhar, M.J.. eds.), pp. 57--