Role of interleukin-6 signaling via the Jak/Stat pathway in human myeloma and effects on mediators of cell survival and proliferation

Abstract

Multiple myeloma is a B-cell malignancy characterized by the latent accumulation in bone marrow of monoclonal plasma cells with a low proliferative index and an extended life span. Interleukin-6 (IL-6) is the major survival factor for myeloma tumor cells and induces signaling through the Signal Transducer and Activator of Transcription (STAT) proteins. We report that one STAT family member Stat3, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line, U266. U266 cells are shown to constitutively overexpress Bcl-XL protein when compared to the IL-6 independent, Stat3 negative, human myeloma cell line 8226. Comparison of the two cell lines reveals that U266 cells are inherently resistant to Fas-mediated apoptosis and cytotoxic drugs when compared to 8226 cells. Inhibition of the IL-6/Jak2/Stat3 pathway in U266 cells by the Jak kinase inhibitor, AG490, or dominant negative Stat3 expression construct (Stat3/3) results in downregulation of Bcl-XL expression, and enhanced sensitivity to Fas-mediated apoptosis. Treatment of myeloma patient specimens with AG490 was sufficient to inhibit Stat3 phosphorylation and activation of Stat3. In addition, AG490 downregulated the expression of Bcl-XL mRNA and protein as determined by RNase protection assay (RPA) and flow cytometry, respectively. We also found that ectopic overexpression of Bcl-XL rescued Fas-induced apoptosis in U266 cells following inhibition of the Jak2/Stat3 pathway. Furthermore, enforced overexpression of Bcl-XL increased the resistance of U266 cells to chemotherapeutic drugs. However, instead of sensitizing U266 cells to drug-induced apoptosis, the JAK inhibitor AG490 antagonized drug-induced apoptosis. The inhibition of drug-induced apoptosis by AG490 was due to inhibition of cyclin D1 expression resulting in cell cycle arrest. These studies demonstrate that IL-6 induced activation of the Jak2/Stat3 pathway controls the expression of several genes that regulate cell proliferation and survival. Two of these genes are Bcl-XL and cyclin D1. Blocking Stat3 activation enhanced sensitivity of U266 myeloma cells to Fas-mediated apoptosis but reduced the efficacy of cell-cycle dependent cytotoxic drugs.