Targeting p62 in Marrow Stromal Cells Is Effective at Inhibiting Myeloma Cell Growth.
Authors: Kurihara Noriyoshi , Hiruma Y. , Windle J. , Hong C.S. , Shin J. , Roodman David 
Published: April 2020

Abstract
Release of growth factors from bone by osteoclastic bone destruction and by tumor-stromal cell interactions play critical roles in promoting myeloma cell growth. In particular, expression of the cytokines RANK ligand (RANKL), a potent inducer of osteoclast (OCL) formation, IL-6, tumor necrosis factor-α (TNF-α) and MCP-1 are upregulated in the bone microenvironment in response to myeloma (MM) cells as well as by adhesive interactions between myeloma cells and marrow stromal cells through VCAM-1 on stromal cells. Therefore, agents that can both inhibit OCL formation and block the effects of myeloma stromal cell interactions should have a major impact on both bone destruction and tumor growth. p62 plays a critical role in NF-κB activation induced by TNF-α, RANKL and IL-1 and is involved in multiple signaling pathways that result in enhanced tumor cell survival and bone destruction. It is our hypothesis that inhibiting p62 expression will 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. Therefore, we used p62 −/− mice to determine the effects of deleting p62 in stromal cells on the growth of myeloma cells. Marrow cells from p62 −/− or wild type mice were used to establish long-term Dexter-type marrow cultures to isolate marrow stromal cells. IL-6 and TNF-α production by p62 −/− stromal cells was decreased compared to WT stromal cells. To determine the effects of the lack of p62 on MM cell growth, GFP-labeled MM.1S cells were co-cultured with p62 −/− stromal cells. Growth of MM.1S cells was decreased by 70% in cocultures of p62 −/− mice, and IL-6 and TNF-α levels were not increased in cocultures of tumor cells with p62 −/− stromal cells. Next, we measured the relative expression levels of VCAM-1 on marrow stromal cells by Western blot in cocultures of human myeloma cells with marrow stromal cells. Stromal cells from the p62 −/− or wild type were cultured with and without MM.1S cells for 3 days in separate experiments. The levels of VCAM-1 in p62 −/− stromal cells were lower than p62 +/− stromal cells. In addition, VCAM-1 levels on p62 −/− bone marrow stromal cells were decreased compared to p62 +/− stromal cells when cocultured with MM cells. The addition of 25 ng/ml mouse TNF-α to p62 −/− stromal cells cocultured with MM.1S cells resulted in enhanced MM.1S growth and VCAM-1 production to similar levels as seen with p62 +/− stromal cells cocultured with MM.1S cells. We then determined the capacity of p62 −/− stromal cells to increase MCP-1 production, a chemoattract for myeloma cells, when they were cocultured with human myeloma cells for 48 hours. The conditioned media were collected after 48 hours of culture. Wild type stromal cells produced increased levels of MCP-1 when cocultured with MM.1S cells. MCP-1 levels in p62 −/− stromal cell conditioned media were decreased compared to wild type stromal cell cultures, regardless of whether MM.1S cells were present in the culture (MCP-1 in p62 +/− stromal cell culture, 980± 70pg/ml vs. p62 −/− 380± 10 pg/ml). These results show that p62 plays an important role in myeloma cell growth through regulation of production of cytokines that are upregulated in the marrow microenvironment in response to myeloma, and suggest that p62 is a novel target for treating myeloma.