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The A6B4 Integrin Can Regulate Erbb-3 Expression: Implications for A6B4 Signaling and Function Valentina Folgiero,1 Robin E

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The A6B4 Integrin Can Regulate Erbb-3 Expression: Implications for A6B4 Signaling and Function Valentina Folgiero,1 Robin E Research Article The A6B4 Integrin Can Regulate ErbB-3 Expression: Implications for A6B4 Signaling and Function Valentina Folgiero,1 Robin E. Bachelder,2 Giulia Bon,1 Ada Sacchi,1 Rita Falcioni,1 and Arthur M. Mercurio3 1Molecular Oncogenesis Laboratory, Department of Experimental Oncology, Regina Elena Cancer Institute, Rome, Italy; 2Department of Pathology, Duke University Medical Center, Durham, North Carolina; and 3Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts Abstract A series of articles published by our laboratories provided the a h The integrin A B has been shown to facilitate key functions first evidence that 6 4 contributes to the functions of carcinoma 6 4 cells and they elucidated some of the mechanisms involved. of carcinoma cells, including their ability to migrate, invade, a h and evade apoptosis. The mechanism involved seems to be a Specifically, these studies identified key roles for 6 4 in the migration, invasion, and survival of carcinoma cells (7–9). Studies profound effect of A6B4 on specific signaling pathways, especially the phosphatidylinositol 3-kinase (PI3K)/Akt path- by several other laboratories have substantiated and extended these findings (10). In fact, there is now compelling evidence that way. An intimate relationship between A6B4 and growth factor a6h4 may actually be involved in the formation of some receptors may explain this effect of A6B4 on signaling. A B carcinomas (11). It is widely assumed that the distinct signaling Previously, we showed that 6 4 and ErbB-2 can function a h properties of 6 4 underlie its involvement in these diverse synergistically to activate the PI3K/Akt pathway. Given that a h ErbB-2 can activate PI3K only when it heterodimerizes with functions of carcinoma cells. Although 6 4 has been reported to other members of the epidermal growth factor receptor activate several key signaling molecules in carcinoma cells, its family, these data imply that other receptors cooperate in this ability to activate phosphatidylinositol 3-kinase (PI3K) has process. Here, we report that A B can regulate the expression attracted the most attention, especially with regard to its influence 6 4 on function. of ErbB-3 using several different models and that the a h consequent formation of an ErbB-2/ErbB-3 heterodimer A critical, unresolved issue is the mechanism by which 6 4 A B activates PI3K (12). This mechanism is probably not a direct promotes the 6 4-dependent activation of PI3K/Akt and a h h the ability of this integrin to impede apoptosis of carcinoma activation of PI3K by 6 4 because the 4 cytoplasmic domain cells. Our data also support the hypothesis that A B can lacks a consensus sequence for binding the p85 regulatory subunit 6 4 a h regulate ErbB-3 expression at the translational level as of PI3K. Studies, to date, on the mechanism by which 6 4 evidenced by the findings that A B does not increase ErbB- activates PI3K have implicated the insulin receptor substrate 6 4 a h 3 mRNA significantly, and that this regulation is both proteins (IRS-1 and IRS-2), the compartmentalization of 6 4 into rapamycin sensitive and dependent on eukaryotic translation membrane microdomains, and its synergistic interaction with initiation factor 4E. These findings provide one mechanism to specific growth factor receptors (13–15). These mechanisms are not mutually exclusive and the ability of a6h4 to affect growth account for the activation of PI3K by A6B4 and they also provide insight into the regulation of ErbB-3 in carcinoma factor receptor signaling may encompass all of these postulated cells. [Cancer Res 2007;67(4):1645–52] mechanisms. In this direction, studies by one of our laboratories a h provided the first evidence that 6 4 may associate withErbB-2 (16), an orphan receptor of the epidermal growth factor receptor Introduction (EGFR) family, on the surface of some breast carcinoma cell lines. a h The 6 4 integrin was identified initially as an epithelial-specific Subsequent studies using a 3T3 cell model system showed that integrin, and it was thought that expression of the h subunit was a h 4 both 6 4 and ErbB-2 are required for PI3K activation and the limited to cells of epithelial origin (1). Subsequent studies in this stimulation of chemoinvasion (17). Given that ErbB-2 is thought to direction focused on the role of this integrin in epithelial biology function only when it heterodimerizes with other members of the and hemidesmosome organization (2, 3). Prior to these studies, EGFR family, these data imply that other receptors cooperate in however, Falcioni et al. (4) identified a tumor antigen associated this process. h with metastasis (TSP-180) that was shown to be identical to the 4 In the current study, we investigated the relationship between integrin subunit (5). This work provided the first indication that a h and ErbB signaling more rigorously, especially in the context a h 6 4 6 4 may be linked to tumor spread. Subsequently, other studies of PI3K activation. Specifically, we focused on ErbB-3, which lacks a h revealed that expression of 6 4 persists in some aggressive intrinsic protein tyrosine kinase activity but possesses in its carcinomas and that its expression may be linked to the behavior cytoplasmic tail six consensus binding sites for the SH2 domain of of these tumors (6). the p85 regulatory subunit of PI3K (18). Among the EGFR family, the ErbB-2/ErbB-3 heterodimer is the strongest stimulator of the PI3K activity (19) and the overexpression of both proteins Requests for reprints: Rita Falcioni, Molecular Oncogenesis Laboratory, promotes breast carcinoma proliferation and survival (20, 21). Our Department of Experimental Oncology, Regina Elena Cancer Institute, Via delle data reveal that a6h4 can regulate the expression of ErbB-3 at the Messi d’Oro 156, 00158 Rome, Italy. Phone: 39-06-5266-2535; Fax: 39-06-5266-2505; level of protein translation, resulting in a significant induction of E-mail: [email protected]. I2007 American Association for Cancer Research. ErbB-2/ErbB-3 heterodimerization and consequent activation of doi:10.1158/0008-5472.CAN-06-2980 PI3K. These findings provide one mechanism to account for the www.aacrjournals.org 1645 Cancer Res 2007; 67: (4). February 15, 2007 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Research a h activation of PI3K by 6 4 and they also provide insight into the phenylmethylsulfonyl fluoride (PMSF), 5 mmol/L Na3VO4, 50 mmol/L regulation of ErbB-3 in carcinoma cells. protease inhibitors (Sigma-Aldrich, Milan, Italy)]. Recombinant human heregulin h1 (HRG-h1; 10 ng/mL; R&D Systems, Milan, Italy) was added to the cells that had been serum deprived for 24 h. After 20 min of stimulation, Materials and Methods the cells were washed with cold PBS and extracted in NP40 buffer [1% Cell lines. The human breast carcinoma cell lines MDA-MB-435 and NP40, 10% glycerol, 137 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.4), MCF7 were obtained from the American Type Culture Collection 50 mmol/L NaF, 1 mmol/L PMSF, 5 mmol/L Na3VO4, protease inhibitors]. (Manassas, VA) and maintained in RPMI medium supplemented with To induce ErbB-2/ErbB-3 heterodimerization, the cells were stimulated 10% FCS (Invitrogen, Milan, Italy). The generation of MDA-MB-435 withHRG- h1 for 10 min before lysis. MDA-MB-435/mock and MDA-MB- h  6 subclones that express the a6h4 integrin and NIH 3T3 cells that express 435/ 4 cells were plated at a concentration of 1.5 10 per 100-mm dish. ErbB-2 and the a6h4 integrin has previously been described (12, 17). The The next day, the medium was replaced with serum-free medium in the generation of MCF7 scrambled short hairpin RNA (scr-shRNA) and MCF7/ presence of rapamycin 50 nmol/L (Calbiochem) for 30 and 60 min. Then, h4 shRNA cell subclones that endogenously express a6h4 integrin or are the cells were washed in PBS and extracted in radioimmunoprecipitation depleted for the expression of a6h4 integrin has previously been described assay buffer [50 mmol/L Tris-HCl (pH 8), 150 mmol/L NaCl, 1% NP40, 0.1% (22). The rat bladder epithelial cell line 804G was kindly donated by deoxycholate, 0.1% SDS, 1 mmol/L PMSF, 5 mmol/L Na3VO4, 50 mmol/L Dr. G. Meneguzzi (Faculty of Medicine, Institut National de la Sante´etdela protease inhibitors]. h h Recherche Me´dicaleU634, Nice, France). Apoptosis assay. The MDA-MD 435/ 4/scr, MDA-MB-435/ 4/siErbB-3, Reagents. The h4 antibodies (439-9B and 450-11A), the a6 antibody MCF7/scr, and MCF7/siErbB-3 cells were serum starved for 24 h. For (135-13C), and the ErbB-2 antibody (W6/100) were prepared as described Annexin V staining, 1  106 cells were washed with PBS, centrifuged, and (16, 17). The antibody W6/100 was used for immunoprecipitation experi- resuspended in 100 AL of Annexin V-FITC labeling solution (Boehringer, ments. The ErbB-2 antibodies (clone 3B5 and Ab-1) were purchased from Milan, Italy) in the presence of propidium iodide and incubated for 15 min. BD PharMingen and Oncogene (Milan, Italy) and used in Western blot After washing, the cells were incubated in SA-FLUOS solution for 20 min experiments. The immunoprecipitation experiments were also done with at 4jC, centrifuged, washed, and analyzed by fluorescence-activated cell the anti–ErbB-2 (clone 9G6) from Oncogene. The rabbit ErbB-3 antibody sorting (FACS). (C17) was obtained from Santa Cruz Biotechnology (Milan, Italy). The Flow cytometry. To assess ErbB-3 surface expression, cells were eukaryotic translation initiation factor 4E (eIF-4E), 4E-BP1, Akt, and harvested using citrate saline buffer (0.134 mol/L KCl, 0.015 mol/L Na phospho-Akt(Ser473) antibodies and the signal transducer and activator of citrate) and washed twice with cold PBS containing 0.002% EDTA and  6 j transcription 1 (Stat-1) antibody were purchased from Cell Signaling 10 mmol/L NaN3.
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