β1 Integrin Activation Via FN Adhesion Enhances IL-6 Response in Myeloma Cells.

Many environmental factors are known to affect the progression and survival of Multiple Myeloma (MM) cells. These factors include soluble determinants such as cytokines, chemokines and growth factors and physical determinants such as MM cell adhesion to fibronectin (FN) and bone marrow stromal cells. Although these determinants likely work in concert in the in vivo MM microenvironment, they have typically been analyzed individually. Only recently has the idea of crosstalk between different environmental factors been studied and appreciated. In this study we examined signaling events, cell cycle progression, and levels of drug resistance in myeloma cells adhered to FN alone via β1 integrins, stimulated with IL-6 alone, or the two combined. We found that the combination of FN adhesion and IL-6 markedly enhanced myeloma cell Stat3 signaling when compared to either condition alone. Western blot analysis demonstrated that the phosphorylation status of Stat3 was significantly increased by combining IL-6 with FN adhesion as compared to either IL-6 or FN-adhesion alone. Furthermore, Stat3 phosphorylation was sustained with the combination of IL-6 and FN adhesion (up to 6 hours) compared to either treatment alone. A similar effect on Stat3 signaling was observed in U266, H929 and MM1.S myeloma cells. EMSA analysis also demonstrated a synergistic increase in Stat3 nuclear translocation and DNA binding with the combination of IL-6 and FN adhesion, inducing a 3.0-fold increase in protein-DNA complex formation when compared to IL-6 alone and a 10.2-fold increase over FN adhesion alone. Previously, our lab has shown that these factors individually influence cell survival. In this cell line model, the combination of IL-6 and FN adhesion did not confer increased protection to mitoxantrone (a classic topoII inhibitor) beyond that mediated by FN adhesion alone as quantitated by MTT analysis and Annexin V/7-AAD staining (Table 1). We previously demonstrated that IL-6 and FN have differing effects on MM cell proliferation; IL-6 promotes cell cycle progression, whereas FN adhesion facilitates a G0/G1 cell cycle arrest. In the present study, cell cycle analysis (using BrdU/PI) illustrated that the combination of IL-6 and FN adhesion reversed the arrest mediated by FN (Table 1). Together, these data show synergistic effects of IL-6 and FN adhesion on Stat3 signaling that potentially confers a more malignant phenotype. Drug resistance bestowed by adhesion to FN is maintained when myeloma cells are exposed to IL-6, and cells are able to proliferate and overcome the adhesion- mediated cell cycle arrest. Importantly, these results demonstrate that components of the bone marrow microenvironment act synergistically to influence myeloma cell survival and proliferation and understanding how these elements interact provides a new target for myeloma treatment.

Table 1