p62 Signaling Is Increased in Multiple Myeloma Microenvironment.

Abstract The bone microenvironment plays a critical role in promoting both tumor growth and bone destruction in myeloma (MM). Marrow stromal cells produce factors, which stimulate both the growth of MM cells and osteoclastic bone destruction and are key regulators of these processes. Marrow stromal cells produce these factors in increased amounts when they bind MM cells through adhesive interactions mediated via VCAM-1 on stromal cells and β1 integrins on the surface of MM cells. We have examined the role of sequestosome-1 (p62), a recently described member of the NF-κB signaling pathway, in this process, since it sits at the crossroads of multiple signaling pathways potentially involved in both osteoclastogenesis and MM cell growth. In previous studies, we found that stromal cells lacking p62 minimally supported the growth of MM cells or osteoclast (OCL) formation compared to wild-type p62 containing stromal cells. We have further shown that stromal cells lacking p62 produce much lower levels of TNF-α, interleukin (IL)-6, MCP-1 and RANK ligand (RANKL), factors which increase OCL formation, and express lower levels of VCAM-1. However, the mechanisms responsible for this decreased cytokine production and VCAM-1 expression in multiple myeloma patient stromal cells lacking p62 are unknown. Further, it is unknown that targeting PKCζ, a downstream enzyme activity activated by p62, will have similar effects on myeloma patient stromal cells. Our hypotheses are that inhibiting PKCζ expression will block the effects of cytokines produced in the MM-bone microenvironment in response to TNF-α, decrease VCAM-1 expression in stromal cells, and markedly diminish osteolytic bone destruction and MM growth in MM patients. To test these hypotheses, we established long-term Dexter-type marrow cultures to isolate marrow stromal cells from MM patient and normals and screened for p62 and PKCζ/λ activation in MM marrow. We measured levels of PKCζ and p62 by Western blot analysis in marrow stromal cells from 13 patients and 11 healthy controls. We found significantly elevated levels of phosho-PKCζ/λ and total PKCζ in MM patients although the levels varied greatly among the patients. We next measured the relative expression levels of VCAM-1 on marrow stromal cells by Western blot in these marrow stromal cells. Since inhibiting p62 expression should profoundly diminish osteolytic bone destruction and myeloma growth in patients, by blocking production of RANKL, MCP-1, TNF-α and IL-6 in the tumor-bone microenvironment, and the upregulation of VCAM-1 on stromal cells, we assessed the effects of blocking p62 activity in human stromal cells. p62 siRNA (40 nM) was transduced into human stromal cells using a commercial transfection substrate. We confirmed that p62 expression was decreased by at least 50% in these stromal cells by Western blot analysis using an anti-p62 antibody. Stromal cells from the p62siRNA and control siRNA treatment and non-treatment stromal cells were cultured with or without MM1s cells for 3 days in separate experiments. The levels of VCAM-1 in p62siRNA transducer stromal cells were lower than control siRNA and untreated stromal cells. In addition, IL-6 production by MM1.s was increased when cocultured with control stromal cells but not with p62siRNA treated bone marrow stromal cells. These results show that p62 plays an important role in myeloma cell growth induced by cytokines that are upregulated in the marrow microenvironment and myeloma cells in patients with myeloma. Therefore, p62 is an attractive therapeutic target for MM growth and bone destruction.