Targeting p62^ZIP in Marrow Stromal Cells Is Highly Effective at Inhibiting Myeloma Cell Growth and Osteoclast Formation.

Marrow stromal cells play a critical role in osteolytic bone destruction in multiple myeloma, and promote tumor growth. In particular, adhesive interactions between myeloma cells and marrow stromal cells increase RANK ligand (RANKL), a potent inducer of osteoclast formation, IL-6 and TNF-α production by marrow stromal cells. IL-6 enhances the growth and prevents apoptosis of myeloma cells, and TNF increases production of RANKL and IL-6. Recently, a new member of NF-κB signaling pathway, p62^ZIP, has been identified. p62^ZIP plays a critical role in NF-κB activation induced by TNF-α and RANKL and is involved in multiple signaling pathways that result in enhanced IL-6 production, tumor cell survival and bone destruction. It is our hypothesis that inhibiting p62^ZIP expression will profoundly diminish myeloma growth by blocking the effects of IL-6 produced in the tumor-bone microenvironment in response to TNF-α. Therefore, we used p62^ZIP−/− mice to determine the effects of deleting p62^ZIP in stromal cells on the growth of myeloma cells. Marrow cells from p62^ZIP −/− or wild type mice were used to establish long-term Dexter-type marrow cultures to isolate marrow stromal cells. Marrow stromal cells from p62^ZIP −/− or wild type mice were cocultured for 48 h with a GFP-labeled human MM.1S myeloma cell line (MM.1S) and murine and human RANKL, IL-6 and TNF-α levels were determined in conditioned media from these cocultures using commercial ELISA assays. Cocultures of MM.1S with wild type marrow stromal cells resulted in much greater upregulation of murine IL-6 than p62−/− stromal cell coculture (IL-6 in p62−/− stromal cell cultures; 114+70 vs. WT 1900+9 pg/ml). In addition, deleting p62^ZIP in stromal cells markedly decreased the growth of tumor cells. Coculture with wild type stromal cells induce 1.4-fold greater increase in MM.1S cell growth at 72 h compared to p62−/− stromal cells. Further, addition of neutralizing antibodies to TNF-α and IL-6 to the cocultures of MM.1S cells with WT stromal cells similarly affected the growth of the MM.1S. Since TNF-alpha can increase the expression of adhesion molecules on stromal cells and tumor cells, we measured expression levels of ICAM-1 and VCAM-1 by Western blot. VCAM-1 and ICAM-1 levels on p62^ZIP−/− bone marrow stromal cells were not changed compared to WT stromal cells. We then determined the capacity of p62 −/− cells to support OCL formation by normal spleen CFU-blast. OCL formation was decreased about 50 % in cocultures containing p62−/− stromal cells treated with PTH-rp, IL-6 and TNF-α compare with WT stromal cell culture. These results show that p62 plays an important role in myeloma cell growth and OCL formation induced by cytokines that are upregulated in the marrow microenvironment in patients with myeloma.