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Cancer Research 67, 2693, March 15, 2007. doi: 10.1158/0008-5472.CAN-06-3654
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Integrin {alpha}vß3 Controls Activity and Oncogenic Potential of Primed c-Src

Stephan Huveneers1,2, Iman van den Bout1, Petra Sonneveld1, Ana Sancho1, Arnoud Sonnenberg1 and Erik H.J. Danen1,2

1 Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands and 2 Division of Toxicology, Leiden Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands

Requests for reprints: Erik H.J. Danen, Division of Toxicology, Leiden Amsterdam Center for Drug Research, Leiden University, Einsteinweg 55, P.O. Box 9502, 2300 RA, Leiden, The Netherlands. Phone: 31-71-527-4486; E-mail: e.danen{at}lacdr.leidenuniv.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Increased activity of the proto-oncogene c-Src and elevated levels of integrin {alpha}vß3 are found in melanomas and multiple carcinomas. Regulation of c-Src involves "priming" through disruption of intramolecular interactions followed by "activation" through phosphorylation in the kinase domain. Interactions with overexpressed receptor tyrosine kinases or mutations in the SRC gene can induce priming of c-Src in cancer. Here, we show that {alpha}vß3 promotes activation of primed c-Src, causing enhanced phosphorylation of established Src substrates, survival, proliferation, and tumor growth. The ß3 cytoplasmic tail is required and sufficient for integrin-mediated stimulation of all these events through a mechanism that is independent of ß3 tyrosine phosphorylation. Instead, experiments using Src variants containing the v-Src Src homology 3 (SH3) domain and using mutant ß3 subunits indicate that a functional interaction of the ß3 cytoplasmic tail with the c-Src SH3 domain is required. These findings delineate a novel integrin-controlled oncogenic signaling cascade and suggest that the interaction of {alpha}vß3 with c-Src may represent a novel target for therapeutic intervention. [Cancer Res 2007;67(6):2693–700]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interactions of tumor cells with their microenvironment are important for cancer development and progression (1). Tumor cells connect with the extracellular matrix through various members of the integrin family of adhesion receptors and upon malignant transformation cells often undergo specific changes in the expression levels of integrins. High levels of integrin {alpha}vß3 correlate with growth and/or progression of melanoma (2, 3), neuroblastoma (4), and multiple different carcinomas (5–9). Moreover, individuals homozygous for the ß3L33P polymorphism that enhances the affinity of ß3 integrins have an increased risk to develop breast cancer, ovarian cancer, and melanoma (10). Despite the fact that {alpha}vß3 in the tumor vasculature has been identified as a valuable drug target, endothelial {alpha}vß3 is dispensable for tumorigenesis (11, 12). It remains unclear if and how increased levels of {alpha}vß3 on tumor cells contribute to cancer development.

Following ligand binding, integrins cluster and organize into multiprotein complexes termed cell-matrix adhesions that connect to the actin cytoskeleton through a variety of cytoskeletal linker proteins. Cell-matrix adhesions also contain various signaling intermediates, including non–receptor tyrosine kinases (non-RTK) such as focal adhesion kinase (FAK) and c-Src (13). Integrin-mediated adhesion stimulates FAK and c-Src activities, and, in turn, c-Src modulates the stability of cell-matrix adhesions through phosphorylation of several components, including integrin cytoplasmic tails (14–16). In addition, the FAK/c-Src complex is involved in the transmission of information from the extracellular matrix into the cell to regulate cellular signaling cascades in control of apoptosis and proliferation (17).

In unstimulated cells, c-Src is folded into a closed, autoinhibitory conformation. Its activation requires dephosphorylation of the COOH-terminal Tyr530 residue (amino acid numbering used in this study is for human c-Src) to disrupt intramolecular binding of this residue to the Src homology 2 (SH2) domain. A disruption of the interaction between the SH3 domain and prolines in the linker region further contributes to the formation of an unfolded or "primed" conformation. Finally, for full enzymatic activity, primed c-Src must be phosphorylated in its kinase domain at residue Tyr419 by transphosphorylation (18, 19). The oncogenic product of Rous sarcoma virus (v-Src) is constitutively activated through amino acid substitutions in the SH3 domain and the kinase domain as well as a deletion of the regulatory COOH-terminal tyrosine (18, 20). Although Rous sarcoma virus is avian specific, c-Src plays a critical role in cancer development (21, 22). Indeed, levels of c-Src activity are frequently increased in human melanoma and carcinomas of the breast, colon, and other epithelia (23–25). It is incompletely understood how c-Src activity is enhanced in tumors. Increased levels of c-Src and binding of overexpressed RTKs to the c-Src SH2 domain may enhance c-Src priming. In addition, mutations in the SRC gene stabilizing a primed conformation of c-Src through truncation of the regulatory COOH terminus have been detected in colon and endometrial cancer, although such mutations seem to be rare (26–29).

Because (a) c-Src selectively mediates signaling by ß3 integrins (30), (b) null mutations in the Src or the Itgb3 gene give rise to partially overlapping abnormalities (31, 32), and (c) increased expression or activity of {alpha}vß3 or c-Src has been associated with growth, progression, or poor prognosis of the same types of cancer (2–9, 23–25), we hypothesized that a functional interaction of {alpha}vß3 with c-Src may contribute to cancer development. In this report, we show that the activity and oncogenic potential of primed c-Src is in fact subject to a remarkably tight regulation by integrin {alpha}vß3. Our findings identify the ß3 cytoplasmic domain as a critical regulator of c-Src–mediated oncogenic signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and plasmids. The HBL100 cell line was obtained from the American Type Culture Collection (Rockville, MD). The ß1 integrin–deficient cell lines GD25 and GE11 were provided by Dr. Reinhard Fässler (Max Planck Institute, Martinsried, Germany) and have been described previously (33, 34). All cell lines were cultured in DMEM supplemented with 10% FCS, penicillin, and streptomycin. A Myc tag was added at the 3' end of the cDNA encoding c-SrcY530F (the plasmid encoding mouse-chicken c-Src in which the COOH-terminal regulatory Tyr was replaced by Phe was purchased from Upstate Biotechnology, Lake Placid, NY); a hemagglutin (HA) tag was added at the 3' end of the cDNA encoding mouse c-Src (Upstate Biotechnology); and the tagged constructs were cloned into the LZRS retroviral vector. Retroviral expression plasmids encoding integrin ß1 or ß3 subunits, ß1exß3in and ß3exß1in chimeras, and those encoding the extracellular and transmembrance region of the non-signaling interleukin 2 receptor {alpha} (IL2R{alpha}) subunit alone or fused to the integrin ß1 cytoplasmic domain were described before (33, 34). To generate the LZRS-IL2Rß3 plasmid, the ß1 cytoplasmic domain in IL2Rß1 was replaced with the ß3 cytoplasmic domain. The retroviral expression plasmid encoding ts72v-Src (35) was provided by Dr. Irwin H. Gelman (Roswell Park Cancer Institute, Buffalo, NY). The LZRS retroviral construct expressing chimeric vSrc/SrcYF was generated by substituting the first 131 amino acids of SrcYF with the same region from ts72v-Src. The cDNA encoding human epidermal growth factor receptor (EGFR) was provided by Dr. Frank Furnari (Ludwig Institute for Cancer Research, La Jolla, CA) and cloned into the pMSCV retroviral expression plasmid by Sophia Bruggeman (The Netherlands Cancer Institute, Amsterdam, The Netherlands). The retroviral H-RasG12V expression plasmid (RasGV) was provided by Dr. John Collard (The Netherlands Cancer Institute). The ß3Y747A, ß3Y759A, and ß3{Delta}759 mutants were provided by Dr. Jari Ylänne (University of Oulu, Finland) and subcloned into the LZRS retroviral vector (36). All cDNAs were transfected into amphotrophic or ecotrophic packaging cells to generate virus-containing culture supernatants that were used for retroviral transduction of HBL100, GD25, and GE11 cells. Subsequently, SrcYF, c-Src, ts72v-Src, vSrc/SrcYF, or RasGV expressing clones were transduced with retroviral constructs encoding wild-type, mutant, and chimeric integrin subunits or EGFR. Positive cells were bulk sorted at least twice by fluorescence-activated cell sorting for human integrin, IL2R{alpha}, or EGFR surface expression.

Antibodies and other materials. Anti-human ß1 monoclonal antibodies were TS2/16, clone 18 (BD Transduction Laboratories, Lexington, KY), and K20 (Biomeda, San Francisco, CA). Anti-human ß3 monoclonal antibodies were C17 (provided by Dr. Ellen van der Schoot, Sanquin, Amsterdam, The Netherlands), 23C6 (provided by Dr. Michael Horton, University College London, United Kingdom), and SSA6 (provided by Dr. Sanford Shattil, University of California San Diego, CA). Other monoclonal antibodies were anti-c-Src (B-12; Santa Cruz Biotechnology, Santa Cruz, CA), anti-{alpha}-tubulin (B-5-1-2; Sigma, St. Louis, MO), anti-ß-actin (AC-15; Sigma), anti-EGFR (Ab-1 clone 528; Calbiochem, La Jolla, CA), anti–phosphorylated signal transducer and activator of transcription 3(Y705) [anti-p-Stat3(Y705); 3E2; Cell Signalling Technology], and anti-bromodeoxyuridine (anti-BrdUrd; Bu20a; DAKO, Carpinteria, CA). The following rabbit polyclonal antibodies were used: anti–phosphorylated Src(Y419) [anti-p-Src(Y419); Biosource, Camarillo, CA], anti-c-Src (SRC 2; Santa Cruz Biotechnology), anti-myc (A-14; Santa Cruz Biotechnology), anti-HA (GeneTex, Inc., San Antonio, TX), anti-FAK (C-20; Santa Cruz Biotechnology), anti–phosphorylated FAK(Y925) [anti-p-FAK(Y925); Biosource], anti-human ß1 (provided by Dr. Ulrike Mayer, University of Manchester, United Kingdom), anti-Stat3 (K-15; Santa Cruz Biotechnology), anti-human IL2R{alpha} (N-19; Santa Cruz Biotechnology), and polyclonal goat anti-human ß3 (N-20; Santa Cruz Biotechnology). Texas Red–conjugated phalloidin was purchased from Molecular Probes (Eugene, OR). Human plasma fibronectin was prepared as described previously (34).

Flow cytometry, immunofluorescence, and Western blot analysis. These experiments were done as described previously (34).

Immunoprecipitations. For immunoprecipitations, cells were lysed for 15 min at 4°C in lysis buffer [1% NP40, 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L sodium vanadate, 0.5 mmol/L sodium fluoride, and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO)]. Lysates were clarified by centrifugation at 14,000 rpm for 15 min at 4°C and precleared with protein A-Sepharose (Amersham Pharmacia Biotech AB, Uppsala, Sweden) for 2 h at 4°C. Proteins were immunoprecipitated o/n at 4°C with antibodies to c-Src (B-12), ß1 (K20), or ß3 (SSA6), coupled to protein A-Sepharose.

For in vitro Src kinase assays, cells were lysed for 15 min at 4°C in lysis buffer [0.5% NP40, 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 5 mmol/L MgCl2, 1 mmol/L sodium vanadate, and protease inhibitor cocktail (Sigma-Aldrich)]. Lysates were clarified by centrifugation at 14,000 rpm for 15 min at 4°C. SrcYF protein was isolated from the clarified lysates by immunoprecipitation with 5 µg anti-myc antibody for 2 h at 4°C, and immune complexes were collected with protein A-Sepharose. Kinase activity of isolated SrcYF protein was determined by use of a Src kinase assay kit (Upstate Cell Signaling Solutions, Charlottesville, VA).

Soft agar and tumorigenicity assays. For soft agar assays, six-well plates were first coated with 0.6% low melting point (LMP) agarose (Roche, Indianapolis, IN). Subsequently, 100,000 cells were suspended in culture medium containing 0.35% LMP agarose and seeded on top of the 0.6% LMP agarose layer. For tumorigenicity assays, cells were harvested, washed, and resuspended in 0.2 mL sterile PBS per injection. Female 6-week-old athymic BALB/c mice were then s.c. injected into the left and right flanks. After cell inoculation, tumor volumes were measured using calipers at the indicated times. All animal experiments were approved by the animal welfare committee of the Netherlands Cancer Institute.

Terminal deoxynucleotidyl transferase–mediated nick-end labeling staining and BrdUrd incorporation assays. Cells (75,000) were plated in culture medium on fibronectin coated coverslips, and after 4 h, the cells were either kept in culture medium or switched to serum-free medium for 24 or 48 h. The cells were labeled with 15 µmol/L BrdUrd (Sigma) for 4 h before fixation in 2% paraformaldehyde. For BrdUrd staining, cells were permeabilized in 0.5% Triton X-100; DNA was denatured with 2 mol/L HCl and neutralized with 0.1 mol/L sodium borate; and coverslips were labeled with anti-BrdUrd antibody followed by FITC-conjugated secondary antibody. For terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) staining, cells were permeabilized in 0.1% Triton X-100 with 0.1% sodium citrate in PBS and stained with an in situ cell death detection kit (Roche). For both procedures, nuclei were visualized with TOPRO-3 (Molecular Probes), and preparations were mounted in MOWIOL 4-88 solution supplemented with DABCO (Calbiochem).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Integrin {alpha}vß3 supports primed c-Src–mediated tumor growth. A c-Src mutant in which a primed conformation is induced by the substitution of Tyr530 by Phe (SrcYF) was introduced alone or in combination with the ß3 integrin subunit in HBL100, GD25, and GE11 cells. Although SrcYF-transformed HBL100 cells were tumorigenic, tumors grew much faster when the surface expression levels of {alpha}vß3 were increased (Supplementary Fig. S1). The cooperation between {alpha}vß3 and SrcYF was even more striking in GD25 and GE11: whereas cells expressing ß1 integrins (GDSrcYFß1 and GESrcYFß1) were virtually unable to grow in soft agar or form tumors in mice, cells lacking ß1 integrins but expressing high levels of {alpha}vß3 (GDSrcYFß3 and GESrcYFß3) were highly tumorigenic (Fig. 1 ; Supplementary Fig. S2A; see ref. 34). Ectopic expression of ß1 integrins in GESrcYFß3 cells did not affect tumor growth, indicating that {alpha}vß3 supports SrcYF-mediated tumor formation, irrespective of the expression of ß1 integrins (Fig. 2A ). Moreover, when a 1:1 mixture of GESrcYFß1 and GESrcYFß3 cells was injected s.c., GESrcYFß3 cells (recognized by an antibody directed against human ß3) were readily detectable in the resulting tumors, whereas GESrcYFß1 cells (recognized by an antibody directed against human ß1) were virtually absent, indicating that {alpha}vß3 supports Src-mediated tumor formation in a cell-autonomous fashion (Fig. 2B).


Figure 1
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  		Figure 1. Integrin {alpha}vß3 supports oncogenic transformation by SrcYF. A and C, Western blot analysis of SrcYF (Myc-tag antibody), total Src (c-Src antibody), and ß-actin in lysates of GD25 (A) or GE11 cells (C) expressing the indicated constructs. Note that lysates were separated on a 10% polyacrylamide gel in (C), allowing the visualization of c-Src and the Myc-tagged SrcYF as separate bands (middle), whereas 4-20% gels were used in all other cases. B and D, colony formation and tumorigenicity of GD25 (B) or GE11 cells (D) expressing SrcYF and ß1 or ß3 integrins as indicated. Phase-contrast images of soft agar assays were taken 14 d after plating. Columns, average number of colonies larger than five cells per image of at least two independent experiments; bars, SE. The small clusters of GDSrcYFß1 cells (smaller than five cells) did not grow out with time. Points, average tumor volume of 1 x 106 injected cells (n ≥ 4 for GD25 lines, n = 11 for GE11 lines) obtained from two independent experiments; bars, SE.

 

Figure 2
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  		Figure 2. Specific cooperation of {alpha}vß3 and primed c-Src stimulates tumor growth in a cell-autonomous fashion. A, points, average tumor volume of GESrcYFß3 cells lacking (bullet) or expressing ß1 ({square}) obtained from two independent experiments (n = 8) where 1 x 106 cells were injected; bars, SE. B, columns, quantification of integrin expression on cells isolated from four tumors of a 1:1 mix of 5 x 105 GESrcYFß1 and GESrcYFß3 cells. Bars, SE. Expression of human ß1 (open columns) and human ß3 integrins (filled columns) was determined by fluorescence-activated cell sorting before injection ("input") or after one month of tumor growth ("tumors"). The fact that the percentage of GESrcYFß1 and GESrcYFß3 cells does not add up to 100% can be explained by the presence of stromal cells (lacking human ß1 and ß3 integrins). C, points, average tumor volume of GERasGVß1 ({square}) and GERasGVß3 cells (bullet) obtained from two independent experiments (n ≥ 7) where 1 x 106 cells were injected; bars, SE. D, columns, average tumor volume at 33 d after injection of GEß1 (open columns) and GEß3 cells (filled columns) containing the indicated expression constructs obtained from two independent experiments (n ≥ 12) where 1x106 cells were injected; bars, SE. *, P < 0.01, significant difference between mean values (Student's t test).

 
In contrast to SrcYF, RasGV-mediated tumor growth was not affected by the expression of {alpha}vß3, indicating that this integrin is specifically required for Src-mediated tumorigenesis (Fig. 2C). In several human cancers, overexpressed and/or activated EGFR can stimulate priming of c-Src through (in)direct interactions with its SH2 domain (19, 23). We analyzed if {alpha}vß3 can also support c-Src–mediated tumor growth under such conditions. Moderate overexpression of c-Src was not sufficient by itself to induce tumor formation even in the presence of {alpha}vß3 (Fig. 2D; Supplementary Fig. S2B). However, although tumors grew slow compared with those induced by SrcYF, {alpha}vß3 significantly increased tumor growth when c-Src and EGFR were coexpressed (Fig. 2D; Supplementary Fig. S2C).

Together, these findings indicate that {alpha}vß3 specifically cooperates with primed c-Src in a cell-autonomous fashion to stimulate the formation of tumors by fibroblasts and epithelial cells.

{alpha}vß3 supports SrcYF-mediated survival, proliferation, and tumor formation upstream of FAK and Stat3 by enhancing SrcYF activation. The activation of the transcription factor Stat3 by phosphorylation at Tyr705 is strongly enhanced in cells transformed by v-Src (37, 38). SrcYF only moderately increased Stat3 activity in the presence of ß1 integrins, whereas phosphorylation was clearly enhanced in the presence of {alpha}vß3 (Fig. 3A ). This suggested that {alpha}vß3 may enhance SrcYF activity, which, like wild-type c-Src, requires transphosphorylation of Tyr419 in its catalytic domain to acquire full enzymatic activity. Indeed, whereas expression of SrcYF led to increased levels of p-Src(Y419) in the presence of either ß1 or ß3 integrins, phosphorylation was much stronger in GESrcYFß3 than in GESrcYFß1 cells (Fig. 3B). Stimulation of the levels of p-Src(Y419) and of two known Src substrates [p-FAK(Y925) and p-Stat3(pY705)] in the presence of {alpha}vß3 was also observed when cells were cultured under conditions that may mimic the tumor environment (serum-free or nonadherent; Fig. 3C; data not shown). On the other hand, equal levels of Src activity were detected in in vitro Src kinase assays on SrcYF immunoprecipitates from GESrcYFß1 and GESrcYFß3 lysates (Fig. 3D). These data raise the possibility that in vivo activation of SrcYF is regulated by {alpha}vß3, possibly by enhanced clustering and subsequent transphosphorylation in the kinase domain. Concentration of SrcYF on the beads in the in vitro Src kinase assays might result in activation of SrcYF in a similar way.


Figure 3
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  		Figure 3. The ß3 cytoplasmic tail supports SrcYF activation and SrcYF-mediated tumor growth. A, Western blot analysis of p-Stat3(Y705), total Stat3 ({alpha} and ß isoforms), and tubulin loading control in lysates of GEß1 and GEß3 cells expressing or lacking SrcYF. Dotted lines in (A) and (B) separate different regions from a single film placed together. B, Western blot analysis of p-Src(Y419) and total Src in lysates of GEß1 and GEß3 cells expressing or lacking SrcYF. C, Western blot analysis of p-Src(Y419), total Src, p-FAK(Y925), total FAK, p-Stat3(Y705), total Stat3, and loading control in lysates of GE11 cells expressing the indicated constructs and grown in the absence of serum. Columns, mean of relative p-Src(Y419), p-FAK(Y925), and p-Stat3(Y705) levels compared with GESrcYFß1 in serum-starved cultures of GE11 cells expressing the indicated constructs obtained from at least two independent experiments; bars, SE. D, activity of SrcYF immunoprecipitated from GE11 (–), GESrcYF ß1, or GESrcYFß3 cells in an in vitro Src kinase assay. E, points, average tumor volume of GESrcYFß1ex3in (bullet) or GESrcYFß3ex1in cells ({square}) obtained from two independent experiments (n ≥ 8) where 1x106 cells were injected; bars, SE.

 
To investigate the role of the ß3 cytoplasmic tail in SrcYF activation and oncogenic potential, we expressed a chimeric integrin subunit consisting of the ß3 extracellular and transmembrane regions fused to the ß1 cytoplasmic region (ß3exß1in) in GESrcYF cells (Supplementary Fig. S2A, left). Unlike wild-type ß3, ß3exß1in did not enhance the levels of p-Src(Y419), p-Stat3(Y705), and p-FAK(Y925) (Fig. 3C). On the other hand, an inverse chimeric ß1exß3in integrin subunit, unlike wild-type ß1, strongly increased the degree of phosphorylation of Src, Stat3, and FAK (Fig. 3C; Supplementary Fig. S2A, right). Moreover, surface expression of ß1exß3in supported SrcYF-mediated tumor formation, whereas expression of ß3exß1in did not (Fig. 3E).

We next investigated if increased levels of {alpha}vß3 promote survival and proliferation of SrcYF-transformed cells. After 24 and 48 h of serum deprivation, there were significantly fewer TUNEL-positive GESrcYFß3 than GESrcYFß1 cells (Fig. 4A ; Supplementary Fig. S3A; P < 0.05, Student's t test). In agreement with the role of the ß3 cytoplasmic tail in tumor formation induced by SrcYF, the sensitivity to serum starvation was suppressed in the presence of the ß1exß3in but not the ß3exß1in chimeric integrin subunit (P < 0.05). Furthermore, following serum deprivation the expression of ß3 or ß1exß3in correlated with high proliferation rates, whereas a large proportion of the cells expressing ß1 or ß3exß1in underwent cell cycle arrest (P < 0.05; Fig. 4B; Supplementary Fig. S3B).


Figure 4
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  		Figure 4. The ß3 cytoplasmic tail supports SrcYF signaling to survival and proliferation. A, TUNEL assays on GE11 cells expressing the indicated constructs under standard culture conditions and after 24 and 48 h of serum starvation. Columns, mean percentage of TUNEL-positive cells of two independent experiments; bars, SE. B, BrdUrd incorporation under conditions described for (A). Columns, mean percentage of BrdUrd-positive cells of three independent experiments; bars, SE. *, P < 0.05, significant difference between mean values (Student's t test).

 
From these findings, we conclude that (a) the primed SrcYF mutant is in fact subject to tight regulation in vivo; (b) as part of a functional integrin, the ß3 cytoplasmic tail is required and sufficient to support the activity of SrcYF under conditions that mimic the tumor environment; and (c) the ability of the ß3 cytoplasmic tail to support SrcYF activation is correlated with its ability to support SrcYF-mediated survival, proliferation, and tumor formation.

Functional, spatial, and molecular association of the ß3 cytoplasmic tail with SrcYF. Having shown that the ß3 cytoplasmic tail controls the activity and oncogenic potential of SrcYF, we asked two questions: Is SrcYF-mediated phosphorylation of the ß3 tail involved? And can the ß3 tail on its own support oncogenic signaling by primed Src? Both ß3Y747A and ß3Y759A mutants stimulated tumor growth of GESrcYF cells to the same extent as wild-type ß3, indicating that recruitment of signaling and adaptor proteins to phosphorylated tyrosine residues in the ß3 cytoplasmic tail is not required (Fig. 5A ; Supplementary Fig. S4A). On the other hand, an IL2R fusion construct containing the ß3 cytoplasmic tail did not enhance tumor growth of GESrcYF cells compared with IL2R- or IL2R-ß1, indicating that the cooperation between the ß3 cytoplasmic tail and SrcYF requires the context of a functional integrin (Fig. 5B; Supplementary Fig. S4B).


Figure 5
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  		Figure 5. Functional and spatial association of the ß3 cytoplasmic tail and SrcYF. A, points, average tumor volume of GESrcYF cells expressing integrin ß3 (bullet), ß3Y747A ({square}), or ß3Y759A ({blacksquare}) obtained from two independent experiments (n = 12) where 1 x 106 cells were injected; bars, SE. B, points, average tumor volume of GESrcYF cells expressing integrin ß3 (bullet), IL2R- ({blacktriangleup}), IL2Rß1 ({circ}), or IL2Rß3 ({square}) obtained from at least two independent experiments (n ≥ 5) where 1 x 106 cells were injected; bars, SE. C, Western blot analysis of the indicated proteins in immunoprecipitations of ß3 (top) or ß1 integrin (bottom), or in total lysates of GESrcYFß1 and GESrcYFß3 cells [whole-cell lysate (WCL)]. D, localization of SrcYF (anti-Myc antibody; green), integrin ß1, ß3, or IL2Rß3 (K20, 23C6, anti-IL2R{alpha} antibodies, respectively; Texas red) in GE11 cells expressing the indicated constructs. Arrows, colocalization. Bar, 5 µm.

 
We next investigated if SrcYF formed a complex with the ß3 subunit but did not detect an interaction using coimmunoprecipitation, whereas an interaction of SrcYF with endogenous FAK was readily detectable (Fig. 5C; Supplementary Fig. S4C; data not shown). To investigate if a possible weak interaction may occur, we analyzed the subcellular localization of SrcYF and ß3 integrins. Irrespective of the type of integrins expressed, SrcYF induced the formation of podosomes, adhesive structures that are characteristic for Src-transformed cells. Notably, this indicates that the activity of SrcYF in cells lacking high amounts of {alpha}vß3 is sufficient to cause morphologic but not oncogenic transformation. ß1 and ß3 integrins were partially colocalized with SrcYF in podosomes (Fig. 5D, left and middle), making it possible that the ß3 cytoplasmic tail locally interacts with SrcYF and enhances SrcYF-mediated oncogenic signaling. In line with this idea, IL2Rß3 did not colocalize with SrcYF in podosomes, which may explain its inability to support SrcYF-mediated oncogenic transformation (Fig. 5D, right).

The last four amino acids of the integrin ß3 tail have been reported to mediate binding of {alpha}IIbß3 to the SH3 domain of c-Src (16). A similar interaction may explain {alpha}vß3-mediated control of the oncogenic potential of primed c-Src. Experiments using v-Src, which contains multiple mutations in its SH3 domain (18), showed efficient phosphorylation on Tyr419 and colony outgrowth in soft agar irrespective of {alpha}vß3 expression levels (Fig. 6A and B ). However, v-Src also contains activating mutations in its kinase domain (18) that may make the interaction with the ß3 cytoplasmic domain redundant. Therefore, we generated a v-Src/SrcYF chimera in which only the NH2-terminal region including the SH3 domain was derived from v-Src. This construct failed to induce oncogenic transformation even in the presence of high levels of {alpha}vß3 (Fig. 6C; Supplementary Fig. S4D). Moreover, expression of a ß3{Delta}759 mutant that lacks the four most COOH-terminal amino acids required for binding the c-Src SH3 domain (16) failed to support SrcYF-mediated tumor formation (Fig. 6D).


Figure 6
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  		Figure 6. Cooperation between SrcYF and {alpha}vß3 involves integrin interaction with the Src SH3 domain. A, Western blot analysis of p-Src(Y419), total Src, and actin loading control in lysates of GEß1 and GEß3 cells expressing ts72v-Src at nonpermissive (NPT) and permissive temperature (PT). B, colony formation of GEts72v-Srcß1 and GEts72v-Srcß3 cells. Phase-contrast images of soft agar assays were taken 10 d after plating at permissive temperature. Columns, average number of colonies larger than five cells per image of two independent experiments; bars, SE. C, points, average tumor volume of GE11 cells expressing the indicated constructs obtained from two independent experiments (n = 10) where 1 x 106 cells were injected; bars, SE. D, points, average tumor volume of GESrcYFß3 (bullet) or GESrcYFß3{Delta}759 cells ({square}) obtained from two independent experiments (n ≥ 5) where 1 x 106 cells were injected; bars, SE.

 
Together, these data support the idea that oncogenic activity of primed Src variants containing a wild-type kinase domain depends on SH3-mediated interactions with the ß3 cytoplasmic domain.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In human melanomas and carcinomas of the breast, colon, pancreas, and other organs, the activity of c-Src is frequently increased compared with that in surrounding tissue (23–25). It is not fully understood how high levels of c-Src activity contribute to human cancer. The fact that activating mutations in the SRC gene seem to be rare argues against a role in tumor initiation. Increased c-Src activity may contribute to invasion and metastasis by promoting tumor cell scattering, migration, proteolytic activity, and anoikis resistance (39, 40). For colon cancer, increased c-Src activity may also contribute to tumor growth (41), perhaps by stimulating vascular endothelial growth factor–mediated angiogenesis (42). Our findings clearly show that elevated c-Src activity promotes tumorigenicity of immortalized cells where the p53 and Rb tumor suppressor pathways are suppressed, as is almost invariably the case in human cancer. We find that elevated c-Src activity promotes tumor growth in a cell-autonomous fashion by stimulating survival and proliferation. This may be especially important during early stages of cancer development. The contribution of elevated levels of c-Src activity to tumor growth may decrease as tumors progress and acquire additional mutations (e.g., those activating Ras).

The molecular mechanism responsible for the increased activity of c-Src in human cancers is incompletely understood. Overexpression of RTKs has been proposed to induce a primed conformation of c-Src by disrupting the intramolecular binding of the SH2 domain to phosphorylated Tyr530 (19, 23). In addition, mutations in the COOH-terminal region of the SRC gene that lead to a primed conformation of c-Src have been detected in a small subset of carcinomas of the colon and the endometrium (26, 27). Whatever the mechanism of priming, our findings show that the oncogenic potential of primed c-Src can be strongly enhanced by integrin {alpha}vß3. The notion that {alpha}vß3 and c-Src may cooperate in human cancer is supported by a number of reports showing that an increase in the expression of {alpha}vß3 is associated with growth and/or progression of various cancers in which c-Src activity is frequently enhanced (2–9, 23–25). The loss of {alpha}5ß1 or other ß1 integrins has also been associated with oncogenic transformation and tumor growth (43, 44). We observed that ß1-deficient SrcYF transformed cells were slightly more tumorigenic than their ß1-expressing derivatives (data not shown). However, expression of ß1 in SrcYFß3 cells did not reduce their tumorigenic capacity, indicating that {alpha}vß3 supports oncogenic signaling by primed Src, irrespective of the expression of ß1 integrins. The expression of {alpha}vß3 will be important for Src-mediated aspects of cancer development, whereas {alpha}vß3 may be dispensable for those aspects that are driven by oncogenic Ras (our findings) or other oncogenes such as c-Neu (11).

The interaction between the ß3 cytoplasmic tail and the c-Src SH3 domain has been shown by others (16), but we were unable to detect the interaction of the ß3 tail and primed c-Src by coimmunoprecipitation (possibly due to the much lower levels of expression). Nevertheless, several lines of evidence support a model in which this interaction controls the oncogenic potential of primed c-Src: (a) only those integrins and integrin chimeras that contain the ß3 cytoplasmic tail promote oncogenic signaling by primed c-Src; (b) in contrast to full-length ß3, an IL2Rß3 fusion construct fails to colocalize with primed Src in podosomes and fails to support tumor growth; (c) the YRGT motif in the ß3 cytoplasmic domain that was reported to interact with the SH3 domain of c-Src (16) is required for the functional interaction of {alpha}vß3 with primed c-Src; and (d) {alpha}vß3 cannot stimulate primed c-Src variants containing the SH3 domain of v-Src despite the fact that both SH2- and SH3-mediated autoinhibition is prevented. The ß3 subunit has a tendency to form homo-oligomers and clustering of {alpha}vß3 in the plane of the membrane may cocluster primed c-Src, leading to enhanced activation through cross-phosphorylation in the kinase domain (45, 46). Indeed, such intermolecular autophosphorylation is considered the major mechanism underlying c-Src activation (47). In addition to clustering, {alpha}vß3 may support conformational alterations in the Src protein or recruit additional proteins that contribute to oncogenic signaling. In this respect, our results using Tyr to Ala mutants argue against a role for the recruitment of signaling or adaptor proteins to the conserved NpxY/NxxY motifs in the ß3 cytoplasmic tail.

In conclusion, a functional interaction with the ß3 cytoplasmic tail augments the activity and oncogenic potential of primed c-Src. Phosphorylation of FAK and Stat3 are enhanced in the presence of {alpha}vß3, but it remains to be investigated if these or other downstream pathways underlie the synergistic effect of primed c-Src and {alpha}vß3 on survival, proliferation, and tumor growth. As overexpression of {alpha}vß3 and elevated levels of c-Src activity occur in the same types of tumors, the interaction of these two proteins may be an important event in cancer development and/or progression. Interfering with their interaction might therefore be a valuable therapeutic approach in melanomas and carcinomas of the breast, colon, and several other tissues. Moreover, a combinatorial analysis of the levels of integrin {alpha}vß3 and c-Src may be useful to predict cancer development and/or progression.


   Acknowledgments
 
Grant support: Dutch Cancer Society grant NKI 2003-2858 (S. Huveneers and E.H.J. Danen) and NKI 2001-2488 (I. van den Bout).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Sophia Bruggeman, John Collard, Reinhard Fässler, Frank Furnari, Irwin Gelman, Michael Horton, Ulrike Mayer, Sanford Shattil, Ellen van der Schoot, and Jari Ylänne for their generous gifts of reagents; Ingrid Kuikman for the generation of the LZRS-ß3{Delta}759 vector; and Anton Berns, John Collard, Ed Roos, Marc Vooijs, Bob van de Water, and members of the Sonnenberg group for critical reading of the article.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

I. van den Bout and P. Sonneveld contributed equally to this work.

Received 10/ 2/06. Revised 12/28/06. Accepted 1/19/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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