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Multiple myeloma (MM) cells reside in the bone marrow microenvironment and form complicated interactions with nonneoplastic, resident stromal cells. We previously found that aggressive MM cells shift osteoblast progenitors toward adipogenesis. In addition, adipocytes are among the most common cell types in the adult skeleton; both mature adipocytes and preadipocytes serve as endocrine cells that secrete a number of soluble molecules into the microenvironment. Therefore, we used a combination of in vivo and in vitro methods to test the hypothesis that an increase in adipocyte lineage cells feeds back to promote MM progression. The results of this study revealed that bone marrow from patients with MM indeed contains increased preadipocytes and significantly larger mature adipocytes than normal bone marrow. We also found that preadipocytes and mature adipocytes secrete many molecules important for supporting MM cells in the bone marrow and directly recruit MM cells through both monocyte chemotactic protein-1 and stromal cell-derived factor-1a. Co-culture experiments found that preadipocytes activate Wnt signaling and decrease cleaved caspase-3, whereas mature adipocytes activate ERK signaling in MM cells. Furthermore, mature adipocyte conditioned medium promotes MM growth, whereas co-culture with preadipocytes results in enhanced MM cell chemotaxis in vitro and increased tumor growth in bone in vivo. Combined, these data reveal the importance of preadipocytes and mature adipocytes on MM progression and represent a unique target in the bone marrow microenvironment. (Am J Pathol 2016, 186: 3054e3063; http://dx

A major cause of morbidity in patients with multiple myeloma is the development and progression of bone disease. Myeloma bone disease is characterized by rampant osteolysis in the presence of absent or diminished bone formation. Heparanase, an enzyme that acts both at the cell-surface and within the extracellular matrix to degrade polymeric heparan sulfate chains, is upregulated in a variety of human cancers including multiple myeloma. We and others have shown that heparanase enhances osteoclastogenesis and bone loss. However, increased osteolysis is only one element of the spectrum of myeloma bone disease. In the present study, we hypothesized that heparanase would also affect mesenchymal cells in the bone microenvironment and investigated the effect of heparanase on the differentiation of osteoblast/stromal lineage cells. Using a combination of molecular, biochemical, cellular and in vivo approaches, we demonstrated that heparanase significantly inhibited osteoblast differentiation and mineralization, and reduced bone formation in vivo. In addition, heparanase also shifts the differentiation potential of osteoblast progenitors from osteoblastogenesis to adipogenesis. Mechanistically, this shift in cell fate is due, at least in part, to heparanase-enhanced production and secretion of the Wnt signaling pathway inhibitor DKK1 by both osteoblast progenitors and myeloma cells. Collectively, these data provide important new insights into the role of heparanase in all aspects of myeloma bone disease and strongly support the use of heparanase inhibitors in the treatment of multiple myeloma.

• Myeloma cell-derived Runx2 promotes myeloma progression.• High levels of Runx2 expression are associated with a high-risk myeloma population.The progression of multiple myeloma (MM) is governed by a network of molecular signals, the majority of which remain to be identified. Recent studies suggest that Runt-related transcription factor 2 (Runx2), a well-known bone-specific transcription factor, is also expressed in solid tumors, where expression promotes both bone metastasis and osteolysis. However, the function of Runx2 in MM remains unknown. The current study demonstrated that (1) Runx2 expression in primary human MM cells is significantly greater than in plasma cells from healthy donors and patients with monoclonal gammopathy of undetermined significance; (2) high levels of Runx2 expression in MM cells are associated with a high-risk population of MM patients; and (3) overexpression of Runx2 in MM cells enhanced tumor growth and disease progression in vivo. Additional studies demonstrated that MM cell-derived Runx2 promotes tumor progression through a mechanism involving the upregulation of Akt/b-catenin/Survivin signaling and enhanced expression of multiple metastatic genes/proteins, as well as the induction of a bone-resident cell-like phenotype in MM cells. Thus, Runx2 expression supports the aggressive phenotype of MM and is correlated with poor prognosis. These data implicate Runx2 expression as a major regulator of MM progression in bone and myeloma bone disease. (Blood. 2015;125(23):3598-3608) IntroductionMultiple myeloma (MM) is a largely incurable B-cell malignancy characterized by the clonal expansion of malignant plasma cells in the bone marrow.1-3 A hallmark of MM is the predominant localization in the bone marrow and the propensity for progression from primary bone sites to new bone sites in both local and distant bones. 2,4 Bone disease occurs in ;90% of patients with MM 5 and is the main cause of patient mortality, however, the cellular mechanisms driving MM progression in bone remain largely undefined.Runt-related transcription factor 2 (Runx2), a member of the runtrelated gene family, is a bone-specific transcription factor 6,7 considered to be the master regulator of osteoblastogenesis and bone formation. [6][7][8][9] Accumulating evidence has demonstrated that various solid tumors, such as breast and prostate cancers, also express Runx2 [10][11][12][13][14][15][16][17] and that Runx2 expression is significantly correlated with the development of bone metastasis and subsequent osteolysis. [10][11][12][13][14][15][16][17][18][19] Despite the evidence in solid tumors, the role of Runx2 in MM remains unclear. In this study, the regulatory roles and mechanisms of Runx2 in the promotion of MM growth, survival, and progression in bone were elucidated. Materials and methods Cell lines and cell cultureMouse myeloma 5TGM1 cells were a gift from Dr Claire M. Edwards (University of Oxford, Oxford, United Kingdom). Human myeloma MM.1R cells were purchased from American Type Culture Collection. All...

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