BLyS Regulates Human Myeloma Cell IL-6 Expression.

Abstract Multiple myeloma (MM) is a malignant disease of plasma cells that accumulate in the bone marrow. The survival mechanisms of myeloma cells are still not fully understood. There are several cytokines that are known to support the survival and growth of myeloma cells, such as IL-6, IGF-I, TNF-a, and IL-1β. Recent evidence has also shown that the TNF family member, B lymphocyte stimulator (BLyS), is critical for normal B cell development and homeostasis, as well as for survival of malignant B cells, including MM. However, it should be noted that the precise mechanisms by which BLyS promotes the survival of MM cells remain uncertain. Our group has already reported that MM cell lines and primary myeloma cells can express BLyS and its receptors. Thus, exogenous BLyS augmented the survival of MM cells, including the IL-6 dependent MM cell line, KAS-6/1. To better study the mechanism underlying BLyS-mediated survival of myeloma cells, we sought to establish a KAS-6/1 cell line variant which was solely dependent on exogenous BLyS for growth and survival. We have previously reported that KAS-6/1 cells do not appear to express autocrine IL-6, and therefore are highly dependent on exogenous IL-6 for survival. To establish a BLyS-dependent KAS-6/1 variant, cells were cultured long-term in RPMI containing 0.5% BSA or 1% FCS in the absence of exogenous IL-6, and in the presence or absence of exogenous BLyS. KAS-6/1 cells cultured in the absence of cytokines were non-viable after two weeks of culture, whereas KAS-6/1 cells cultured with BLyS alone remained viable and proliferated, albeit at a lower rate than parental KAS-6/1 cells maintained with IL-6. We next focused on identifying the mechanisms of BLyS-dependent survival of the KAS-6/1/BLyS (KAS-6/B) variant cell line. Of interest, KAS-6/B cells placed in short term culture exhibited cytokine independent survival and proliferation and this was completely inhibited using a neutralizing IL-6 antibody. These results therefore suggested that the mechanism underlying BLyS-mediated survival and proliferation of the KAS-6/B cells is induction of an autocrine IL-6 pathway. This notion is further supported by the ability of BLyS to induce IL-6 expression at the transcriptional level, and at the protein level as revealed by an IL-6 specific ELISA. Consistent with the concept that BLyS induced autocrine IL-6 expression in the KAS-6/B cells, we also observed that STAT-3 was constitutively activated in these cells. In addition, we tested the ability of BLyS-conditioned media obtained from KAS-6/B cells to stimulate the proliferation of a different IL-6 dependent MM cell line, JMW. Whereas BLyS alone failed to stimulate JMW cell proliferation, BLyS-conditioned media obtained from the KAS-6/B cells stimulated proliferation, and in a manner that was inhibited by a neutralizing IL-6 antibody. Finally, BLyS stimulation of primary patient MM cells similarly resulted in increased IL-6 expression. IL-6 is known to play a major role in the malignant progression of MM by regulating the growth and survival of tumor cells. IL-6 levels often increase during disease progression, and it has been demonstrated that IL-6 may derive from the tumor cells themselves or from other cell types in the bone marrow. Because of this, it is important to fully understand what signals are capable of inducing IL-6 expression in this disease. In summary, our results demonstrate for the first time the ability of BLyS to maintain myeloma cell survival via the induction of an autocrine IL-6 pathway. Our results underscore the importance of IL-6 in this disease, and further demonstrate the complexity of the deregulated cytokine network in this disease.