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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.
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