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Article |
Address correspondence to Lucia Languino, Dept. of Cancer Biology, University of Massachusetts Medical School, 364 Plantation St., Worcester, MA 01605. Tel.: (508) 856-1606. Fax: (508) 856-3845. email: lucia.languino{at}umassmed.edu
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Abstract |
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Key Words: laminin; prostate; Gab1; IRS-1; PI 3-kinase
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Introduction |
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The integrin cytoplasmic domains modulate several signal transduction pathways (Fornaro and Languino, 1997). The cytoplasmic domain of ß1 exists in five different spliced forms and is known in its most widely expressed form, i.e., ß1A, to modulate cell proliferation and migration as well as receptor localization (Fornaro and Languino, 1997). The ß1C integrin, an alternatively spliced variant of the ß1 subfamily that contains a unique 48amino acid sequence in its cytoplasmic domain inhibits normal and cancer cell proliferation (Fornaro and Languino, 1997). This cytoplasmic variant is expressed in nonproliferative, differentiated epithelium and is selectively down-regulated in prostate and breast carcinoma (Fornaro and Languino, 1997; Manzotti et al., 2000).
Insulin-like growth factors (IGFs) and the IGF type I receptor (IGF-IR) are important modulators of growth and differentiation, play a crucial role in the establishment and maintenance of the transformed phenotype (Baserga, 2000), and are potential targets for anticancer treatment (Surmacz, 2003). The IGF axis appears to contribute to prostate cancer progression, but the mechanisms responsible for this process have not been studied (Reiss et al., 1998). IGF-I and IGF-II bind to IGF-IR and stimulate several signaling pathways including phosphatidylinositol 3-kinase (PI 3-kinase) and MAPK (Valentinis and Baserga, 2001; LeRoith and Roberts, 2003); both pathways are also known to be activated by integrin engagement (Damsky and Ilic, 2002). IGF-I induces association of ß1 integrins and IGF-IR and increases adhesion of myeloma cells to fibronectin (FN; Tai et al., 2003). It has also been shown to increase adhesion of breast cancer cells to LN and this increase is inhibited by -IR3, an antibody to the IGF-IR (Dunn et al., 1998).
The present work unravels a novel mechanism that regulates cell adhesion to LN-1 in response to IGF without affecting cell proliferation or tumor growth and is mediated by the association of ß1 integrins with IGF-IR and with IGF-IR downstream effectors.
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Results |
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Expression of ß1C inhibits IGF-stimulated cell proliferation and tumor growth
We investigated whether ß1 integrins could interfere with PC3 cell proliferative response to IGFs. We found that both IGF-I and IGF-II increased PC3 cell proliferation in the presence of ß1A, whereas expression of ß1C prevented IGF-I or IGF-II stimulation of cell proliferation (Fig. 4 A). Based on the finding that ß1 integrin is essential for tumor growth (Bloch et al., 1997) and IGF-IR promotes transformation (Baserga, 2000), we hypothesized that ß1C would prevent tumor growth given its ability to inhibit IGF-stimulated cell proliferation. Because prostate cancer cells form tumors in an IGF-IRdependent manner when injected subcutaneously in nude mice (Burfeind et al., 1996; Reiss et al., 1998), we injected PC3 cell transfectants expressing either ß1A or ß1C and followed tumor growth. Tumor formation by PC3-ß1A cells was not affected by the presence or absence of tet in the drinking water and there were no differences in tumor formation and growth at all the examined time points (Fig. 4 B). The tumors that formed in the tet-deprived animals injected with PC3-ß1C cells were significantly smaller in size compared with those in animals given tet (Fig. 4 C). PC3-ß1C cells showed tumor formation at significantly later time points indicating that the cells were alive (not depicted). These data show that ß1C significantly reduced prostate cancer cell proliferation and tumor growth, in vivo.
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ß1A associates with and supports tyrosine phosphorylation of IRS-1, whereas ß1C associates with and supports tyrosine phosphorylation of Gab1 and Shp2
Upon tyrosine phosphorylation, IGF-IR becomes the docking site for SH2 domain-containing proteins (LeRoith and Roberts, 2003), such as insulin receptor substrate-1 (IRS-1). IRS-1 is known to mediate several functions, predominantly proliferation and transformation, stimulated by the IGF-IR (Surmacz, 2003). Grb2-associated binder1 (Gab1) is another downstream effector of IGF-IR, as well as of the insulin receptor, and shares functional and structural homology with IRS-1 (Winnay et al., 2000; Gu and Neel, 2003). To investigate whether the ß1 cytoplasmic domain would affect the downstream signaling proteins via direct association with ß1A or ß1C, we used CHO transfectants, where ß1A was found to be associated with the IGF-IR (Fig. 7 A, top). Our results showed that IRS-1 associates with ß1A and that ß1C associates with Gab1 (Fig. 7 B). Cell transfectants expressed similar levels of exogenous ß1 integrins (Fig. 7 A, bottom), IRS-1, or IGF-IR (not depicted).
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To explore the role of Shp2 in IGF-I mediated cell adhesion to LN-1, CHO cells expressing ß1A or ß1C were transiently transfected with either HA-tagged wt-Shp2 or FLAG-tagged Shp2 C/S, a dominant negative form of Shp2 (Zhang et al., 2002). As shown in Fig. 7 F, enhanced cell adhesion to LN-1 in the presence of IGF-I in ß1C expressing CHO cells was completely prevented by transfection of Shp2 C/S, as compared with vector alone or wt-Shp2. The expression levels of the transfected wt-Shp2 or Shp2 C/S cDNAs were analyzed in cell lysates and found comparable in ß1A and ß1C expressing cells (Fig. 7 G).
Overall, these data show that ß1A and ß1C differentially associate with IRS-1 and Gab1 and that Shp2 activation is required for cell adhesion to LN in response to IGFs.
PI 3-kinase mediates IGF-stimulated adhesion to LN-1
PI 3-kinase is a downstream effector that mediates IGF-IR as well as integrin-stimulated signaling; PI 3-kinase activation requires Shp2 in some pathways activated by receptor-tyrosine kinases such as IGF-IR and PDGF receptor, but not by others such as EGF receptor (Reiss et al., 2001; Neel et al., 2003; Tai et al., 2003). We investigated the role of PI 3-kinase in cell adhesion stimulated by IGF-II in cell transfectants expressing ß1C integrin. We transiently transfected CHO clones expressing ß1C integrin with dominant negative form of p85 (DNp85), wild-type p85 (wt-p85), or vector alone. As shown in Fig. 8 A, IGF-II stimulated cell adhesion to LN-1 in ß1C expressing cells in either vector or wt-p85 transfected cells, but cell adhesion to LN-1 was significantly inhibited upon transfection of DNp85. In our previous work, we have shown that antibody-mediated engagement of ß1 integrins activates the PI 3-kinase/Akt pathway (Fornaro et al., 2000). We found threefold higher activation of Akt, a PI 3-kinase downstream signaling molecule, in response to IGF-I or IGF-II stimulation in ß1C expressing cells as compared with ß1A expressing cells (Fig. 8 B). Our results indicate that PI 3-kinase mediates ß1C integrin effect on cell adhesion to LN-1 in response to IGF.
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Discussion |
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The finding that the association between ß1A integrins and IGF-IR regulate cell adhesion to a basement membrane protein in response to IGF stimulation without affecting cell proliferation or tumor growth, is novel. Although it was known that integrins and growth factor receptors act synergistically and are associated, it was not known that their cross-talk determines the specificity of their activities (Yamada and Even-Ram, 2002). The relevance of this work is that the ß1AIGF-IR complex by limiting cancer cell adhesion to the basement membrane, presumably allows the tumor mass to expand and invade. A direct involvement of IGF-IR in cell adhesion to LN has been shown earlier (Dunn et al., 1998) supporting the evidence of a synergistic and overlapping activity of integrins and IGF-IR in cell adhesion. Our discovery was made possible by the serendipitous finding that ß1C, a ß1A cytoplasmic variant that does not associate with IGF-IR, increases cell adhesion to LN-1. This variant form causes disruption of the ß1AIGF-IR association via a 25amino acid domain which is uniquely found in the ß1C integrin. Several studies have demonstrated that structural differences in the intracellular domains are expected to be important determinants of the specificity of a variety of integrin-mediated events (Fornaro and Languino, 1997). Although the cytodomain was not believed to affect ligand specificity, our data prove that the integrin cytodomain affects ligand specificity in a substrate-dependent manner. We show an inhibitory effect on IGF-IR activation without a wide change in integrin ligand binding, because cell adhesion to FN is unaffected in response to ß1C expression.
Here, as shown in Fig. 10, we demonstrate that the ß1AIGF-IR complex preserves IGF-IR phosphorylation and association with IRS-1, a molecule necessary to promote stimulation of IGF-IR signaling pathways important for cell proliferation and transformation (Reiss et al., 2000). In contrast, ß1C does not associate with either IGF-IR or with IRS-1 and this results in failure to respond to a canonical IGF stimulation. The ß1C integrin inhibits IGF-stimulated tyrosine phosphorylation of IGF-IR and cell proliferation, as well as tumor growth by associating with Gab1, and increasing phosphorylation of Gab1. Gab1, a member of the IRS-1 family of adaptor proteins, is known to be phosphorylated in response to several growth factor receptors' activation, enhances cell growth and cell transformation, but has also a negative role on cell survival through interaction with the Shp2 tyrosine phosphatase (Gu and Neel, 2003; Holgado-Madruga and Wong, 2003). ß1C is likely to achieve the goal of preventing IGF-IR and IRS-1 tyrosine phosphorylation via activation of Gab1 and consequent recruitment of a Gab1 binding protein, Shp2 (Gu and Neel, 2003), to IGF-IR and IRS-1 (Myers et al., 1998). Gab1 activates PI 3-kinase, a molecule known to mediate cell adhesion by inhibiting the tyrosine phosphorylation of IRS-1 (Reiss et al., 2001). PI 3-kinase is a downstream effector that mediates IGF-IR as well as integrin-stimulated signaling; PI 3-kinase activation requires Shp2 in some pathways activated by receptor-tyrosine-kinases such as IGF-IR and PDGF receptor, but not by others such as EGF receptor (Reiss et al., 2001; Neel et al., 2003; Tai et al., 2003). Our data show that both Shp2 and PI 3-kinase mediate ß1C-Gab1 effect on cell adhesion. On the same line, a previous set of studies by Clemmons's group had shown that inhibition of Vß3 integrin binding to its ligands prevents IGF-stimulated tyrosine phosphorylation of IGF-IR and IRS-1 as well as cell proliferation to occur via Shp2 activation (Zheng and Clemmons, 1998). In contrast, this is the first work showing Gab1 recruitment to the cell surface by integrins, because Gab1 has only been shown to bind different growth factor receptors (Gu and Neel, 2003).
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Further elucidation of the role of integrinIGF-IR association as well as of their downstream signaling pathways will provide a better understanding of the mechanisms that contribute to prostate cancer progression.
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Materials and methods |
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The following murine mAbs were used: to human ß1 integrin TS2/16 (American Type Culture Collection); K-20 (Immunotech); P4C10 (CHEMICON International, Inc.); 13 and clone 18 to ß1 integrin (BD Biosciences); 7D5/BF10 to ß1C integrin (Fornaro et al., 2001a); to chicken ß1 integrin W1B10 (Sigma-Aldrich); CSAT (Developmental Studies Hybridoma Bank [DSHB]); to hamster ß1 integrin 7E2 (DSHB). In addition, mAb to FLAG (Sigma-Aldrich); mAb 12CA5 to HA (American Type Culture Collection); 1C10 to vascular endothelial surface protein (Life Technologies); -IR3 to IGF-IR (Oncogene); PY20 to phosphotyrosine (Santa Cruz Biotechnology, Inc.); rat mAb to
6 GoH3 (CHEMICON International, Inc.). The following rabbit polyclonal Abs were used: to IGF-IR-ß (Santa Cruz Biotechnology, Inc.); to Gab1 and to IRS-1 (Upstate Biotechnology); to hIGF-II (PeproTech); to Shp2 (Santa Cruz Biotechnology, Inc.); to phospho-Akt (Ser 473), to Akt, to phospho-Shp2 (Tyr542; Cell Signaling); to ERK1 (Santa Cruz Biotechnology, Inc.); to LN-1 described previously (Tsiper and Yurchenco, 2002; provided by P. Yurchenco, Robert Wood Johnson Medical School, Piscataway, NJ). Non-immune Abs were ni-mIgG (Pierce Chemical Co.), ni-rIgG and mIgM (Sigma-Aldrich), and rtIgG (Cappel).
Cell lines and transfectants
Normal human PrEC cells were obtained from Clonetics and maintained as the manufacturer recommended. SV40 immortalized nontumorigenic human prostate epithelial 267B1 and pRNS-1-1 cells were grown in keratinocyte serum-free medium with 5 ng/ml human recombinant EGF and 0.05 mg/ml bovine pituitary extract (Parda et al., 1993; Peehl et al., 1997).
CHO stable cell transfectants expressing either human ß1A or ß1C integrin were cultured as described previously (Fornaro et al., 2000). PC3 stable cell transfectants expressing chimeric ß1A (clones 8 and 11) or ß1C (clones 17 and 19) integrin (chicken extracellular and human intracellular) were generated using the tet-regulated expression system and cultured as described previously (Fornaro et al., 2003). CHO-ß1A, CHO-ß1C, PC3-ß1A, and PC3-ß1C clones were cultured for 48 h in growth medium in the absence of 1 µg/ml tet to induce the expression of ß1A or ß1C integrin. R cells, mouse embryo fibroblasts obtained from IGF-IR knockout mice; and R+ cells, previously established by stably transfecting R cells with the human wild-type IGF-IR cDNA (Sell et al., 1994), provided by R. Baserga (Thomas Jefferson University, Philadelphia, PA).
Integrin surface expression was analyzed by FACS® as described previously (Fornaro et al., 2000, 2003). The pECE-ß1COM plasmid containing the ß1 integrin area of the cytoplasmic domain shared by ß1A and ß1C truncated at threonine residue 777, has been described previously (Retta et al., 1998; provided by G. Tarone, University of Torino, Torino, Italy). CHO cells were electroporated using 10 µg pECE-ß1COM along with 1 µg pFneo. G418-resistant clones were pooled and analyzed for cell surface expression of human ß1COM integrin by FACS® using TS2/16 or 12CA5, as a negative control, as described previously (Fornaro et al., 2000).
The ß1C nucleotide sequence (Languino and Ruoslahti, 1992) was used to design the RZ specific for the ß1C cytoplasmic domain. A double-stranded DNA encoding the RZ-ß1C was obtained by annealing two synthetic single-stranded oligodeoxyribonucleotides containing flanking ClaI restriction sites and spanning a 55-bp sequence. The resulting double-stranded DNA encoding the RZ-ß1C was subcloned into the ClaI site of the mammalian expression vector pBJ-1 and its sequence is as follows: 5'-cgCGGTTTACCCTGTGCAAAGCAGGAGTGCCTGAGTAGTCAGAGAGA-CAGCGGGT-3'. Lowercase letters correspond to the restriction sites, whereas the underlined sequence corresponds to ß1C regions (2420-2434 and 2437-2451) flanking the sequence encoding the catalytic domain of the RZ. The RZ activity was initially tested in in vitro cell-free assays using 32P-labeled ß1C mRNA.
Prostate tumor growth
PC3-ß1A and PC3-ß1C transfectants were induced to express ß1A or ß1C integrin. Cells were detached using 0.05% trypsin/0.53 mM EDTA, washed, and resuspended in RPMI. Cells (106) were inoculated subcutaneously into the flank of 68-wk-old male athymic Balb/c mice (Charles River Laboratories). Mice were given water supplemented with 5% sucrose or 5% sucrose plus 100 µg/ml tet to regulate the exogenous ß1 integrin expression. Tumor size was determined using a caliper every other day. Tumor volume was calculated using the formula (vol = 0.5236 x [width]2 x [length]) and expressed as tumor volume in cubic millimeters. 10 mice/group were used in each experiment.
Transient transfection
All transient transfections were performed using Lipofectamine 2000. R and R+ cells were transiently transfected with 2 µg pCMV-ß-galactosidase (ß-gal) and either 1030 µg pBJ-ß1A or 2030 µg pBJ-ß1C. CHO clones expressing ß1A or ß1C were transiently transfected with 2 µg ß-gal and either 20 µg pCMV-HA-wt-Shp2 or 20 µg pCMV-FLAG-Shp2 C/S (Zhang et al., 2002) cDNA, provided by B. Neel (Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA). ß1C-CHO clones were transiently transfected with 2 µg ß-gal and 20 µg of vector alone (pcDNA3), 20 µg DNp85 (pSR-
p85
) or 20 µg wt-p85 (Hara et al., 1994) cDNA, provided by R. Kalb (Yale University School of Medicine, New Haven, CT). Cells were cultured in the absence of tet in growth medium for 48 h. In a separate set of experiments, ß1A- and ß1C-CHO clones were transiently transfected with 2 µg ß-gal and either 20 µg of vector alone (pcDNA3) or 20 µg 486/STOP (pCVNIGFIRsol cDNA; provided by R. Baserga). Cells were cultured in the absence of tet in growth medium for 48 h. 267B1 cells were transiently transfected with 2 µg ß-gal and either 20 µg pBJ1 or 20 µg pBJ1-RZ-ß1C cDNA. Cells were harvested 48 h after transfection and used in adhesion assays as described below. In parallel, transfected cells were seeded on 48-well plates and stained for ß-gal expression to determine transfection efficiency as described previously (Manes et al., 2003).
Cell adhesion assay
CHO cell adhesion to LN-1 or BSA (100 µg/ml), or 10 µg/ml FN was performed as described previously (Languino et al., 1993) in the presence or absence of 100 ng/ml IGF-II. Where specified, cells were incubated with either Ab to IGF-II or, as a negative control, 0.1 µg/ml ni-rIgG for 1 h on ice.
Alternatively, cell adhesion assays of 267B1, CHO clones, R and R+ cells to BSA or LN-1 (100 µg/ml), or 10 µg/ml FN after being transiently transfected with cDNA constructs were performed by incubating cells with the coated substrates for 2 h at 37°C in the presence or in the absence of IGF-II or IGF-I (100 ng/ml). After adhesion, ß-gal staining was performed as described previously (Manes et al., 2003).
For PC3 clones and PrEC, 80,000 cells were allowed to adhere to plates coated with different substrates as indicated in each figure legend for 2 h at 37°C in the presence or in the absence of IGF-I or IGF-II (100 ng/ml), -IR3 or ni-mIgG (10 or 30 µg/ml), Ab to LN-1 or ni-rIgG (30 or 100 µg/ml), or GoH3 or rtIgG (20 µg/ml). Cells were fixed and stained with crystal violet (0.5%) and OD was measured at 630 nm (Manes et al., 2003).
Analysis of ß1 integrin association with IGF-IR, IRS-1, or Gab1
PC3 and CHO clones were induced to express ß1A or ß1C integrin. Cells were detached and stimulated with 100 ng/ml IGF-I for 10 min, washed and lysed. Proteins were immunoprecipitated by incubating with K-20 and protein A-Sepharose. Immunocomplexes were dissociated and reprecipitated with Ab to IGF-IR or ni-rIgG as described previously (Fornaro et al., 2000). Proteins were separated by SDS-PAGE and immunoblotted using Abs to ß1 integrin (clone 18), to IGF-IR-ß, to IRS-1 or to Gab1. As control, expression levels of IRS-1 or ß1 integrin or IGF-IR were analyzed using respective Abs. Immunoprecipitation of chicken ß1 integrin was performed using CSAT Ab as described previously (Marcantonio and Hynes, 1988). CHO clones expressing wild-type full-length ß1A or different truncated forms of ß1 integrin were allowed to grow to 70% confluency. The cells were lysed and association between ß1 integrin and IGF-IR was studied as described above.
Analysis of tyrosine phosphorylation of IGF-IR, IRS-1, or Gab1
PC3 or CHO clones were detached and trypsin was neutralized with soybean trypsin inhibitor. Cells were stimulated with 100 ng/ml IGF-I, washed, lysed, and proteins were immunoprecipitated with IGF-IR, IRS-1 or Gab1 Abs and protein A-Sepharose. Immunoprecipitates were then separated by SDS-PAGE under reducing conditions and immunoblotted with PY20, IGF-IR-ß, IRS-1 or Gab1, and visualized by ECL.
Immunoblotting
pRNS-1-1 and 267B1 cells were grown to 70% confluency, lysed, and immunoblotted with mAb to ß1C or mIgM (5 µg/ml) as described previously (Fornaro et al., 2001a). 267B1 cells transiently transfected with RZ-ß1C or vector were lysed and immunoblotted with mAb to 5 µg/ml ß1C or Ab to Akt or Ab to 0.2 µg/ml IGF-IR (Fornaro et al., 2000). CHO clones expressing ß1A or ß1C were starved overnight, stimulated with IGF-I or IGF-II for 10 min, lysed, and immunoblotted with Ab to Akt or Ab to phospho-Akt (Fornaro et al., 2000). CHO clones expressing ß1A or ß1C were transiently transfected with HA-tagged wt-Shp2 or FLAG-tagged Shp2 C/S. Cells were lysed and immunoblotted with Ab to 0.5 µg/ml HA or Ab to 0.5 µg/ml FLAG or Ab to 0.2 µg/ml ERK1.
Analysis of Gab1 association with either Shp2 or phospho-Shp2
CHO clones were induced to express ß1A or ß1C integrin. Cells were detached and stimulated with 100 ng/ml IGF-I for 10 min, washed and lysed; proteins were immunoprecipitated by incubating with Ab to 1 µg Gab1 and protein A-Sepharose (Fornaro et al., 2000). Proteins were separated by SDS-PAGE and immunoblotted with Ab to 0.2 µg/ml Gab1 or 0.2 µg/ml Shp2 or phospho-Shp2 (1:1,000 dilution).
SRB assay
Proliferation was measured using SRB assay. PC3 clones were induced to express ß1A or ß1C integrin, serum starved for 12 h, detached, and seeded on 96-well plates (7.5 x 103 cells per well) in the presence or in the absence of IGF-I or IGF-II (100 ng/ml). Cells were incubated for 72 h without a medium change. After incubation, cells were fixed with 10% TCA at 4°C for 1 h, washed five times with tap water and stained with 0.2% SRB for 15 min. The plates were then washed five times with 1% acetic acid, dried, and stained cells in each well were solubilized with 10 mM Tris base. The absorbance in each well was measured at 540 nm. SRB results were confirmed by cell counting.
Statistical analysis
Differences in cell adhesion to LN were measured using t test. Tumor volume was measured for each mouse on multiple days; to analyze the data, a mixed-effect general linear model for repeated measurements was applied. Analysis was operated using SAS (version 8.2; SAS Institute, Inc.).
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Acknowledgments |
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This work was supported by grants from National Institutes of Health, RO1 CA-89720 (to L.R. Languino) and from Army, PCRP DAMD17-98-1-8506 (to L.R. Languino) and by a Consiglio Nazionale delle Ricerche fellowship IBBE (to L. Moro; bando n.203.04.17).
Submitted: 1 March 2004
Accepted: 14 June 2004
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