Int. J. Cancer: 77, 923–927 (1998) r 1998 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer INVOLVEMENT OF Shc IN THE SIGNALING RESPONSE OF HUMAN PROSTATE TUMOR CELL LINES TO EPIDERMAL GROWTH FACTOR John GRESHAM1,4, Patricia MARGIOTTA1,4, Ann J. PALAD1,4, KENNETH D. SOMERS1,4, PETER F. BLACKMORE3,4, GEORGE L. WRIGHT, JR.1,2,4, Paul F. SCHELLHAMMER2,4 and WILLIAM J. WASILENKO1,4* 1Department of Microbiology and Immunology, Eastern Virginia Medical School, Norfolk, VA, USA 2Department of Urology, Eastern Virginia Medical School, Norfolk, VA, USA 3Department of Pharmacology, Eastern Virginia Medical School, Norfolk, VA, USA 4Virginia Prostate Center, Eastern Virginia Medical School and the Sentara Cancer Institute, Norfolk, VA, USA Autocrine growth factors for the epidermal growth factor receptor (EGFR) have been identified in prostate tumors, implicating a role for EGFR in the progression of prostate cancer. To investigate early signaling mechanisms used by the EGFR in prostate tumor cells, we have characterized the involvement of the Shc (src homology 2/x-collagen related) adapter protein in EGFR signaling in several human prostate tumor cell lines. In androgen-responsive lymph node-prostate cancer (LNCaP) cells and androgen-insensitive PC3, DU145 and PPC-1 cells, Shc was identified as one of the most prominent phosphotyrosine proteins to be elevated in response to EGF. Equivalent levels of the 46- and 52-kDa Shc isoforms were detected in all of the tumor cell lines tested. However, levels of the 66-kDa isoform were variable among the cell lines. In all of the tumor cell lines, EGF caused an association between Shc and Grb2, another adapter protein linked to cellular ras activation. Additionally, several phosphotyrosine proteins, including a 115–120-kDa protein in EGF-treated LNCaP cells, co-associated with Shc. The profile of these Shcassociating proteins, however, differed among the tumor cell lines. Our results indicate that Shc is a common downstream element of EGFR signaling in prostate tumor cells and suggest multiple functions for Shc in prostate tumorigenesis. Int. J. Cancer 77:923–927, 1998. r 1998 Wiley-Liss, Inc. Prostate cancer is one of the most frequently diagnosed cancers in American males (Wingo et al., 1995). Although the mechanisms involved in prostate tumorigenesis are unclear, there is growing evidence to suggest that a variety of prostate-derived autocrine/paracrine growth factors contribute to the progression of prostate cancer (Culig et al., 1996; Ware, 1993). Understanding how these growth factors generate proliferative signals in prostate tumor cells would therefore aid our understanding of prostate tumorigenesis and could help identify novel targets for the treatment of prostate cancer. Epidermal growth factor (EGF) and transforming growth factor a (TGFa) are two autocrine/paracrine growth factors that have been identified in prostate tumors and tumor cell lines (reviewed by Culig et al., 1996; Connolly and Rose, 1990; Morris and Dodd, 1990; Scher, et al., 1995 ). Both of these growth factors bind to and activate the epidermal growth factor receptor (EGFR), which is a transmembrane tyrosine kinase that is often co-expressed with its activating ligands in several human carcinomas (Aaronson, 1991), including prostate carcinomas (Scher et al., 1995). The importance of the EGFR in prostate tumorigenesis has been demonstrated by studies showing that inhibitors of the EGFR kinase (Jones et al., 1997; Kondapaka and Reddy, 1996) or anti-EGFR antibodies (Fong et al., 1992) are able to suppress the proliferation of prostate tumor cell lines in vitro and to inhibit the growth of prostate tumor cells as xenografts in mice (Prewett et al., 1996). Activation of the EGFR has also been found to promote the chemomigration of PC3, TSU-pr1 and DU145 prostate tumor cell lines in vitro (Jarrad et al., 1994; Rajan et al., 1996; Xie et al., 1995). The above findings indicate that EGFR is involved in the generation of important biochemical signals for prostate tumor cell growth and possibly plays a role in prostate tumor metastasis. In many cells, stimulation of EGFR promotes the rapid recruitment and binding of several effector enzymes and adapter proteins to specific autophosphorylation sites on the activated EGFR (Schlessinger, 1993). This sets off a myriad of cellular responses, including the stimulation of mitotic signal transduction pathways such as the ras-MAP kinase and the STAT pathway (Schlessinger, 1993). The Shc adapter protein is one particularly important signaling protein recruited to and phosphorylated by EGFR (Rozakis-Adcock et al., 1992). One distinct property of Shc proteins is their ability to act as a physical bridge between several receptor tyrosine kinases, such as EGFR, and Grb2–Sos complexes, which activate the ras signaling pathway in many cells (Buday and Downward,1993; Rozakis-Adcock et al., 1992). Shc proteins also contain src homology 2 (SH2) and phosphotyrosine binding (PTB) domains that selectively bind to phosphotyrosine sites on a variety of cellular proteins (Bonfini et al., 1996). These features identify Shc as an important convergence point involved in the relay of regulatory signals from the cell surface to the nucleus. Growth factor signaling mechanisms in prostate tumor cells are poorly understood. To address this question, we have examined the EGFR-mediated production of phosphotyrosine proteins in androgenresponsive vs. androgen-independent human prostate tumor cell lines. Our results indicate that Shc is one of the prominent tyrosine phosphoproteins appearing in EGF-treated prostate tumor cells. Interestingly, differences were observed in the types of proteins found to associate with Shc in the tumor cell lines tested, suggesting that Shc may exert multiple functions in prostate tumorigenesis. MATERIAL AND METHODS Cell lines Androgen-sensitive lymph node-prostate cancer (LNCaP) and androgen-independent PC3 and DU145 human prostate tumor cell lines were obtained from the ATCC (Rockville, MD). The androgenindependent human PPC-1 prostate tumor cell line is a variant of PC3 and was obtained from Dr. A. Brothman (Salt Lake City, UT). All cell lines were grown in RPMI-1640 medium supplemented with L-glutamine and 5–10% fetal bovine serum (FBS) (GIBCO, Grand Island, NY) and penicillin-streptomycin (50 µg/ml). Cell lysis, immunoprecipitation and Western blotting Prior to cell lysis, cell cultures were preincubated in either growth medium containing 0.5% FBS (2 hr) or growth medium containing 0.1% bovine serum albumin (BSA) (Boehringer Mannheim, Indianapolis, IN) for 18–24 hr. Following treatment with EGF (50 ng/ml) (Sigma, St. Louis, MO), cells were rinsed with phosphate-buffered saline (PBS), then lysed in a modified HNTG buffer consisting of 50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100 and 50 µg/ml leupeptin, 1 mM ethylenediamine tetraacetic acid (EDTA), 2% aprotinin, 1 mM sodium orthovanadate and 10 µg/ml APMSF (Boehringer Mannheim). Cells were scraped and triturated in the cold lysis buffer, Grant sponsors: CAPCuRE; American Cancer Society. *Correspondence to: Department of Microbiology and Immunology, Eastern Virginia Medical School, Norfolk, VA 23507, USA. Fax: (757)624– 2255. E-mail: [email protected] Received 29 January 1998 GRESHAM ET AL. 924 allowed to solubilize for 30 min (4°C) then centrifuged at 30,000 g for 30 min. Protein in the resulting supernants was quantitated using the BCA method (Pierce, Rockford, IL) and aliquots of these supernants were stored at 280°C prior to use in all analyses. In experiments involving vanadate pretreatment, cells were serumstarved for 2 hr then incubated with catalase-treated pervanadate for 15 min. The pervanadate was prepared by reacting equal volumes of 0.1M H2O2 and 0.1 M orthovanadate for 15 min at room temperature (Evans et al., 1994). To remove residual peroxide, catalase was added at a final concentration of 2 µg/ml followed by incubation at room temperature for 30 min. Immunoprecipitations were performed using 400–800 µg of protein in 300–500 µl of lysis buffer. For Shc immunoprecipitations, polyclonal antibody against Shc (Upstate Biotechnology, Lake Placid, NY) was added to the cell lysates and incubated either 4 hr or overnight at 4°C with gentle rotation. Protein A beads (Repligen, Cambridge, MA) or protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA) were then added for 1 hr. The immunoprecipitates were subsequently washed in lysis buffer containing vanadate and then boiled for 5 min in sodium dodecyl sulfate (SDS) sample buffer. For Western blotting, cell lysate proteins were resolved on 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to Immobilon P membrane (Millipore, Bedford, MA), which was blocked using 3% BSA at 37°C for 1 hr. Blots were then probed for 1 hr with the designated primary antibodies (see Fig. legends). Proteins on the blots were detected using enhanced chemiluminescence (ECL) (Amersham, Arlington Heights, IL). RESULTS EGF stimulates the tyrosine phosphorylation of Shc in prostate tumor cell lines The profile of phosphotyrosine proteins induced by EGF in the androgen-responsive LNCaP prostate tumor cell line was examined in order to investigate early signaling responses related to EGFR activation in prostate tumor cells. Western blots of crude cellular lysates were probed with anti-phosphotyrosine antibodies. EGF was found to rapidly increase the level of tyrosine phosphorylation of several proteins with approximate molecular masses of 170–185, 110–120, 50–55 and 40–46 kDa (Fig. 1a). Immunoprecipitation and Western blot analysis indicated that the 170- and 180-kDa proteins were the EGFR and HER2 proteins, respectively (data not shown). Because the observed 50–55- and 40–46-kDa phosphotyrosine proteins are similar in size to Shc proteins, we immunoprecipitated Shc from these same cell lysates and probed the Shc proteins with anti-phosphotyrosine antibodies. EGF caused a rapid (30 sec) elevation in the tyrosine phosphorylation of the 46- and 52-kDa Shc isoforms. The tyrosine phosphorylation of Shc remained elevated throughout the 10-min period of treatment with EGF (Fig. 1b). The level of Shc proteins and their tyrosine phosphorylation in response to EGF was further investigated in several androgenindependent prostate tumor cell lines (DU145, PPC-1 and PC3) in addition to LNCaP cells. Comparable expression of the 46- and 52-kDa Shc proteins was found in the 4 prostate tumor cell lines. However, the relative level of the 66-kDa Shc protein was lower in PC3 and LNCaP cells as compared with the other cell lines (Fig. 2). EGF caused an elevation in the tyrosine phosphorylation of Shc in all of the tumor cell lines tested; the 52-kDa Shc isoform was the predominant form of Shc phosphorylated (Fig. 3). EGF also caused an elevation of Shc tyrosine phosphorylation in PRNS-1 cells, an immortalized human prostate epithelial cell line (data not shown). A prominent phosphotyrosine protein of approximately 115–120 kDa was routinely detected with Shc from EGF-treated LNCaP cells (Fig. 3). The association of this protein with Shc was not detectable in the other tumor cell lines under these assay conditions. Collectively, these findings demonstrate that Shc is a common intermediate in downstream signaling by the EGFR in malignant human prostate tumor cells. FIGURE 1 – Epidermal growth factor (EGF)-mediated elevation of tyrosine phosphoproteins and Shc tyrosine phosphorylation in lymph node-prostate cancer (LNCaP) cells. (a) Western blot analysis of phosphotyrosine proteins expressed in LNCaP cells treated with EGF (50 ng/ml). Cells were pre-incubated in growth medium containing 0.5% fetal bovine serum (FBS), treated with EGF for varying periods of time (0–10 min) then lysed in HNTG buffer. Proteins in cell lysates (75 µg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Tyrosine phosphoproteins were detected by immunoblotting with anti-phosphotyrosine (PY20; Transduction Labs, Lexington, KY) and visualized by enhanced chemiluminescence (ECL). (b) Shc tyrosine phosphorylation in EGF- treated LNCaP cells. Shc was immunoprecipitated from the LNCaP cell lysates (above) using polyclonal anti-Shc (Upstate Biotechnology). Samples were resolved by SDS-PAGE, transferred to PVDF membranes and probed for Shc phosphorylation using anti-phosphotyrosine (PY20; Transduction Labs). Bound antibodies were detected by ECL. FIGURE 2 – Expression of Shc proteins in human prostate tumor cell lines. Nearly confluent cell cultures of LNCaP, PC3, PPC-1 and DU145 prostate tumor cells were lysed as described in the Material and Methods section. Equivalent amounts (50 µg) of proteins from the cells were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to membranes for blotting. Shc proteins were detected on the blots using anti-Shc (Upstate Biotechnology) and enhanced chemiluminescence (ECL). SHC IN PROSTATE TUMOR CELLS 925 FIGURE 3 – Epidermal growth factor (EGF) stimulates the tyrosine phosphorylation of Shc proteins in human prostate tumor cell lines. Human prostate tumor cell lines were pre-incubated in growth medium containing 0.5% fetal bovine serum (FBS; 2 hr), treated with EGF (50 ng/ml; 2 min) and then lysed. Shc proteins were immunoprecipitated from equal amounts (500 µg) of cell lysate protein using anti-Shc polyclonal antibody (Upstate Biotechnology). The samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted and then probed for Shc tyrosine phosphorylation using anti-phosphotyrosine antibody. Phosphorylated proteins were visualized using enhanced chemiluminescence (ECL). LNCaP2 is a second set of LNCaP cell lysates demonstrating nonspecific proteins accompanying the immunoprecipitations using normal rabbit serum (NRS) and lysates from LNCaP cells. *Denotes location of p115–120 and p170–180 kDa proteins that co-associate with Shc immunoprecipitates. FIGURE 4 – Association of phosphotyrosine proteins with Shc in epidermal growth factor (EGF)-treated prostate tumor cell lines. Cell cultures were incubated overnight in growth medium containing 0.1% bovine serum albumin (BSA). The prostate tumor cell lines were then briefly pretreated (15 min) with pervanadate before the addition of EGF (50 ng/ml; 2 min) as described in the Material and Methods section. Cells were lysed and Shc proteins were immunoprecipitated from equal amounts (800 µg) of lysate protein. Immunoprecipitates were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted. Phosphotyrosine proteins that associated with Shc were visualized on the blots with anti-phosphotyrosine (RC-20; Transduction Labs) antibody and enhanced chemiluminescence (ECL) detection. NRS-L and NRS-P, specificity control immunoprecipitation using normal rabbit serum and lysates of the EGF-treated LNCaP and PC3 cells, respectively. Differential association of phosphotyrosine proteins with Shc in prostate tumor cell lines To further characterize the utilization of Shc in prostate tumor cell signaling, we compared the profiles of tyrosine phosphoproteins that associated with Shc in the different types of prostate tumor cell lines. The cells were briefly pretreated with pervanadate, a tyrosine phospha- tase inhibitor, to enhance the detection of tyrosine phosphoproteins in signaling complexes with Shc (Evans et al., 1994). Following pervanadate pretreatment, a constitutive but variable phosphorylation of Shc proteins on tyrosine was found in DU145, LNCaP and PC3 cells (Fig. 4). The predominant phosphorylated species of Shc was the 52-kDa isoform in all of the cell lines. Major differences were also observed GRESHAM ET AL. 926 among the tumor cell lines with regard to the profile of phosphotyrosine proteins that co-associated with Shc. In DU145 cells, few proteins other than the 170-kDa EGFR band were detected following EGF treatment (Fig. 4). In contrast, EGF promoted interactions between Shc and phosphotyrosine proteins with approximate sizes of 75–80,110–120 and 170–180 kDa in LNCaP cells. Distinguishable proteins of 75–80 and 140–150 kDa were also observed to bind constitutively to Shc in the PC3 cells (Fig. 4). These findings indicate that the coupling of Shc to intracellular proteins is variable in different types of prostate tumor cell lines. Grb2 association with Shc in prostate tumor cell lines Because Grb2 is a recognized binding partner of Shc in many growth factor-treated cells (Rozakis-Adcock et al., 1992), we probed Shc immunoprecipitates from the various tumor cell lines for associated Grb2 proteins. In PC3, DU145 and LNCaP tumor cells, EGF promoted the association of Shc with Grb2 (Fig. 5). Grb2–Shc complexes were also detected in the non-treated, serum-starved DU145 cells. Shc phosphorylation in response to bFGF and endothelin in DU145 cells Endothelin and bFGF have been implicated in prostate tumor development (Culig et al., 1996; Nelson et al., 1995). To investigate whether Shc might be involved with signaling responses by other prostate tumor growth stimuli, the ability of bFGF and endothelin-1 to elevate Shc tyrosine phosphorylation in DU145 cells was determined. Both of these stimuli indeed elevated the tyrosine phosphorylation of p52 Shc in these cells (Fig. 6). DISCUSSION An active EGF/TGFa autocrine growth loop has been detected in human prostate carcinomas (Culig et al., 1996; Scher et al., 1995; Ware, 1993). It is likely, therefore, that intracellular signaling by the EGFR contributes to the development of prostate cancer. Although several phosphotyrosine proteins have been identified as components of mitogenic signaling by EGFR in many cells (Schlessinger, 1993), relatively little is known about the nature of downstream phosphotyrosine proteins important to the signaling mechanisms of EGFR in prostate tumor cells. In this study, the Shc adapter protein was found to be one of the most prominent phosphotyrosine proteins to be elevated in response to EGFR activation in human prostate tumor cells. Shc was tyrosinephosphorylated in response to EGF in the androgen-sensitive LNCaP cell line and in 3 androgen-independent prostate tumor cell lines. These findings indicate that Shc is a common downstream intermediate of EGFR signaling in prostate tumor cells with FIGURE 5 – Association of Grb2 with Shc in prostate tumor cell lines. Cells were treated with or without epidermal growth factor (EGF; 50 ng/ml), lysed and then Shc was immunoprecipitated from the lysates (500 µg protein) using anti-Shc antibody (Upstate Biotechnology). The immunoprecipitated Shc proteins were then transferred to PVDF membranes, and the proteins on the membranes were immunoblotted for Grb2 using anti-Grb2 MAb (G16720, Transduction Labs). Blotted Grb2 was detected using enhanced chemiluminescence (ECL). IgG L, light chain of the IgG used in the immunoprecipitation. FIGURE 6 – Shc tyrosine phosphorylation in DU145 cells in response to bFGF and endothelin. (a) DU145 cells were treated with bFGF (30 ng/ml, 2 min) or epidermal growth factor (EGF; 50 ng/ml, 2 min). (b) Cells were pretreated with pervanadate (15min) before the addition of endothelin-1 (100 nM). For both sets of treatments, the cells were lysed and then the Shc proteins were immunoprecipitated using anti-Shc polyclonal antibody (Upstate Biotechnology). Samples were blotted and probed for tyrosine phosphorylation using anti-phosphotyrosine antibody (RC-20; Transduction Labs). dissimilar phenotypes. Additionally, 2 other prostate tumor growth stimuli, bFGF and endothelin (Culig et al., 1996; Nelson et al., 1995), elevated Shc phosphorylation in DU145 cells. The later observation suggests that Shc is commonly involved with the downstream signaling mechanisms of several types of prostate tumor growth factors. Shc is known to function as a bridge between a variety of tyrosine kinases and Grb2, which is another adapter protein that binds to the ras nucleotide exchange factor Sos1 (Buday and Downward, 1993; Schlessinger, 1993). In many cells, growth factors activate the ras-MAP kinase pathway through the formation of Shc–Grb2–Sos complexes (Buday and Downward, 1993; Rozakis-Adcock et al., 1992). EGF was observed to promote the association of Shc with Grb2 in all of the tested prostate tumor cell lines, thus extending to the prostate the role of Shc as a Grb2binding partner. The Shc–Grb2 complexes identified in the cells may be involved with ras activation in response to EGF and/or serve other functions in the tumor cells. Interestingly, complexes between Shc–Grb2 were also detected in non-treated DU145 cells. Such constitutive associations between Shc–Grb2 have been identified in other cell types (Rozakis-Adcock et al., 1992) and may reflect the recruitment of Shc–Grb2 into signaling pathways that are stimulated by intracellularly active tyrosine kinases. Another key finding was the observation that EGF stimulated the association of several phosphotyrosine proteins with Shc in the prostate cell lines. In preliminary experiments with LNCaP cells, one of these proteins was identified as the 170-kDa EGFR itself, which is consistent with previously observed interactions between the EGFR and Shc in EGF-treated fibroblasts (Rozakis-Adcock et al., 1992). Additionally, a 115–120-kDa phosphotyrosine protein was routinely associated with Shc from EGF-treated LNCaP cells. However, this protein was not detected in Shc complexes from the 3 androgen-nonresponsive tumor cell lines, suggesting that signaling pathways involving the EGFR and Shc may differ between androgen-sensitive vs. androgen-insensitive prostate tumor cells. When the tumor cells were pretreated with the phosphatase inhibitor pervanadate, an even more diverse set of tyrosine phosphoproteins was found to associate with Shc in the tumor cells. These observations are consistent with findings from a growing body of studies showing that Shc has multiple functions in cells, as evidenced by studies demonstrating interactions between Shc and adaptins in PC12 cells (Okabayashi et al., 1996), cadherin adhesion molecules in A431 tumor cells (Xu et al., 1997) and the inositol phosphatase SHIP in lymphocytes (Lamkin et al., 1997). Further work is needed to determine the identity of the proteins we observed in Shc complexes from the prostate tumor cells. Since the profile of these phosphotyrosine proteins varied among the differ- SHC IN PROSTATE TUMOR CELLS ent tumor cell lines, Shc may exert multiple functions in prostate tumor cells that vary with tumor phenotype and/or stage of progression. Growth factors are believed to play important roles in the development of prostate cancer (Culig et al., 1996; Ware, 1993). In several preclinical studies, the inhibition of growth factor receptors has shown promise as an alternative approach for interfering with the growth of prostate tumor cells (Jones et al., 1997; Prewett et al., 1996). Because a diverse set of growth factors and cytokines have been implicated in prostate cancer, common convergence points in 927 signaling pathways, such as Shc, may also be good targets for anticancer treatments. Further characterization of signaling through Shc in prostate tumor cells may thus help to identify new strategies for the treatment of prostate malignancies. ACKNOWLEDGEMENTS This work was supported by CAPCuRE and an institutional American Cancer Society award to WJW. REFERENCES AARONSON, S.A., Growth factors and cancer. Science, 254, 1146–1153 (1991). BONFINI, L., MIGLIACCIO, E., PELICCI, G., LANFRANCONE, L. and PELICCI, P.G., Not all Shc’s roads lead to Ras. Trends biochem. Sci., 21, 257–261 (1996). 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