β1 Integrin Adhesion Enhances IL-6 Mediated STAT3 Signaling in Multiple Myeloma Cells: Implications for Microenvironment Influence on Tumor Cell Survival and Proliferation.

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Multiple Myeloma (MM) is a B cell malignancy characterized by the monoclonal expansion of plasma cells. Although numerous genetic alterations have been implicated in MM pathogenesis, it is widely hypothesized that the bone marrow (BM) microenvironment contributes to MM cell pathogenesis. The BM microenvironmental components, interleukin (IL)-6 and fibronectin (FN), have individually been shown to influence the proliferation and survival of MM cells; however, in vivo these effectors most likely work together. We examined signaling events, cell cycle progression, and levels of drug response in MM cells either adhered to FN via β1 integrins, stimulated with IL-6, or with the two combined. IL-6 and FN adhesion have been demonstrated to protect cells from a host of cytotoxic stimuli suggesting co-stimulation of MM cell lines with IL-6 and FN-adhesion may confer a greater protection against chemotherapeutics than either effector alone. However, MTT cytotoxicity assays demonstrate that although adhesion to FN provides significant protection against treatment with mitoxantrone or doxorubicin (p=0.0002 and p=<0.0001 respectively), the addition of IL-6 provides no further protection. These findings were corroborated by analysis of drug-mediated apoptosis using FCM by Annexin-V/7-AAD. In regards to cell cycle kinetics, our laboratory has previously demonstrated that adhesion of the 8226 MM cell line to FN mediated a p27Kip1 dependent G0/G1 cell cycle arrest. As predicted, BrdU/PI analysis of 8226 cells adhered to FN for 24 hours results in an increased number of cells in G0/G1 relative to cells maintained in suspension (p=0.0028). In contrast, when cells were adhered to FN in the presence of IL-6 no accumulation of cells in G0/G1 was observed, with levels similar to that observed in cells maintained in suspension with or without stimulation by IL-6. Our studies demonstrated that the G1/S cell cycle arrest associated with FN adhesion of MM cell lines was overcome when IL-6 was added; however, the cell adhesion mediated drug resistance (CAM-DR) was maintained in the presence of IL-6. Investigation of the biochemical signaling following concomitant exposure of MM cells to IL-6 and FN adhesion revealed a synergistic increase in STAT3 phosphorylation, nuclear translocation and DNA-binding as compared to either IL-6 or FN-adhesion alone in four MM cell lines. STAT3 phosphorylation was increased in cells adhered to FN in an IL-6 dose dependent manner. Electrophoretic mobility shift assay demonstrated a parallel 3-fold increase in STAT3/DNA complexes in cells adhered to FN relative to cells in suspension. To further characterize the receptor proximal affects of FN adhesion on IL-6 signaling we immunoprecipitated the IL-6R complex with antisera to gp130. Immunoprecipitation of gp130 revealed enhanced tyrosine phosphorylation of the gp130/Jak family complexes following stimulation FN-adhered RPMI 8226 MM cells with IL-6. Consistent with increased phosphorylation of the receptor complex, increased levels of phospho-STAT3 were identified associated with gp130 under co-stimulatory conditions relative to IL-6 or FN adhesion alone. Interestingly, immunoprecipitation with gp130 antibodies also revealed an association between STAT3 (non-phosphorylated) and gp130 in the absence of IL-6 stimulation in cells adhered to FN. These results suggest that adhesion to FN facilitates an IL-6-independent association between gp130 and STAT3, resulting in enhanced STAT3 signaling. Taken together, these data demonstrate a novel mechanism by which collaborative signaling by β1 integrin and gp130 confer an increased survival advantage to MM cells.