Abstract
Myeloma (MM) cells stimulate bone resorption by enhancing osteoclast (OC) formation and suppress bone formation by inhibiting osteoblast differentiation, leading to destructive bone lesions. In these lesions, OCs and stromal cells with defective osteoblast differentiation create a microenvironment suitable for myeloma cell growth and survival (a MM niche) to protect MM cells from various apoptotic insults. IL-6 and the TNF family members BAFF and APRIL have been demonstrated to be among predominant anti-apoptotic cytokines for MM cells elaborated by the bone marrow microenvironment in MM. The serine/threonine kinase Pim-2 is a novel apoptotic inhibitor which is transcriptionally up-regulated to promote survival of hematopoietic cells in response to environmental growth factors and cytokines. Up-regulation of Pim-2 expression has also been observed in various malignancies including MM. However, the roles for Pim-2 in growth and survival of MM cells are largely unknown. In the present study we therefore investigated the regulatory mechanism for Pim-2 expression in MM cells and the impact of Pim-2 on MM cell growth and survival with special reference to the interaction between MM cells and bone marrow components. Pim-2 protein is constitutively overexpressed in the absence of IL-6 in IL-6-dependent INA-6 as well as IL-6-independent RPMI8226 and U266 MM cell lines. Addition of IL-6, BAFF and TNFalpha up-regulated Pim-2 protein expression in INA-6 and RPMI8226 cells. A JAK/STAT3 inhibitor, cucurbitacin I, suppresses Pim-2 expression induced by IL-6, indicating Pim-2 as a downstream target of a JAK/STAT3 pathway. Stromal cells and OCs are regarded as a predominant cell type in MM bone marrow microenvironment to produce IL-6 and the TNF family members BAFF and APRIL, respectively. Co-cultures with stromal cells as well as OCs enhanced Pim-2 expression in INA-6 cells, suggesting up-regulation of Pim-2 in MM cells by surrounding cells in the bone marrow. In order to clarify the roles for Pim-2 in growth and survival of MM cells we next looked at the effects of Pim-2 siRNA. Suppression of Pim-2 expression by Pim-2 siRNA partly reduced the proliferation of INA-6 cells stimulated by IL-6 as well as the co-cultures with stromal cells or OCs. Pim-2 silencing also enhanced the cytotoxic effects of dexamethason on MM cells. Interestingly, further addition of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces cell death in concert with Pim-2 silencing in INA-6 cells, suggesting a cooperative roles for PI3K/Akt and Pim-2-mediated pathways in growth and survival of MM cells. Furthermore, Pim-2 silencing induced the cleavage of caspase9 but not caspase8; enforced expression of Pim-2 phosphorylated the BH3 only protein Bad; Pim-2 silencing suppressed phosphorylation of Bad by IL-6. Thus, Pim-2 appears to activate the intrinsic pathway of apoptotic machinery involving Bad phosphorylation. Taken together, our results suggest that Pim-2 is an important prosurvival mediator in MM cells, and that up-regulation of its expression in MM cells by bone marrow components may at least in part contribute to resistance to spontaneous and drug-induced apoptosis in MM cells. Therefore, Pim-2 may become a target for novel therapeutic strategies against MM.
High mobility group I (Y) proteins bind HIPK2, a serine-threonine kinase protein which inhibits cell growth
