Increased Signaling through p62 in the Marrow Microenvironment Increases Myeloma Cell Growth and Osteoclast Formation

Abstract

Marrow stromal cells in the bone microenvironment of multiple myeloma (MM) patients play a critical role in promoting both tumor growth and bone destruction. Binding of MM cells to marrow stromal cells, through VCAM-1 on stromal cells and α4β1 integrin on MM cells, results in increased production of TNF-α, receptor activator of NF-KB ligand (RANKL) and IL-6 by marrow stromal cells. These factors in turn increase osteoclast (OCL) formation and the growth of MM cells. Adhesive interaction between MM cells and marrow stromal cells also decrease the sensitivity of MM cells to chemotherapeutic agents. Many of the downstream effects of these adhesive interactions are mediated through the NF-KB, p38 MAPK and JNK signaling pathways. The adapter protein sequestosome-1 (p62) sits at the crossroads of these signaling pathways and mediates the effects of cytokines and other factors that activate NF-KB, p38 MAPK and JNK. However, the role that increased signaling through p62 plays in MM tumor growth and bone destruction is unknown. It is our hypothesis that increased signaling through p62 in marrow stromal cells is necessary for their capacity to increase the growth of MM cells and OCL formation in MM, and thus p62 may be an attractive therapeutic target for MM. To test these hypotheses, we established long-term Dexter-type marrow cultures to isolate marrow stromal cells from MM patients and normals and measured signaling through p62 and PKCζ activation in MM marrow. We found significantly elevated levels of phosho-PKCζ, total PKCζ and VCAM-1 in MM stromal cells. The enhanced IL-6 production resulted from increased p38 MAPK activity and the increased VCAM-1 expression was NF-KB dependent. We then examined the effects of blocking p62 activity in primary patients and normal stromal cells with p62 siRNA (10μg). We confirmed that p62 expression was decreased by at least 90% in both these stromal cells by Western blot analysis. Stromal cells expressing p62siRNA or control siRNA were cultured with or without MM1.S cells for 3 days in separate experiments. Knocking-down p62 in MM derived marrow stromal cells significantly decreased the levels of PKCζ, VCAM-1 and IL-6 in marrow stromal cells and markedly decreased stromal cell support of MM cell growth and OCL formation. Similarly, marrow stromal cells from p62−/− mice produced much lower levels of IL-6, TNF-α, VCAM-1 and RANKL and minimally supported MM cell growth and OCL formation compared to normal cells. Thus, increased signaling through p62 in marrow stromal cells in patients with MM plays an important role in the increased tumor growth and OCL formation in MM, and support p62 as an attractive therapeutic target for MM.