The Increased 1,25-(OH)₂D₃ Sensitivity of Myeloma Cells and Marrow Stromal Cells Enhances RANKL Production and Tumor Cell Growth

Abstract 3946

Vitamin D plays multiple roles in normal and malignant cell function, regulating cell differentiation and proliferation as well as bone homeostasis. Epidemiologic studies suggest that low levels of vitamin D contribute to the progression of lung cancer, breast cancer, colorectal and prostate cancer as well as lymphoma and melanoma. However, the role of vitamin D in multiple myeloma (MM) is unclear. In contrast to its growth inhibition of solid tumors, vitamin D has little anti-proliferative effects on MM cells. The physiological responses of myeloma cells to vitamin D are unknown, as are its effects on the marrow microenvironment in myeloma bone disease. Vitamin D insufficiency or deficiency has been documented in the majority of myeloma patients. Vitamin D receptor (VDR) is expressed by RPMI8226 cells, but it is unknown if this is a common finding in MM. Further, the functional consequences of VDR expression in myeloma cells are not well characterized. We reported osteoclast (OCL) precursors from patients with Paget's disease (PD) of bone were hypersensitive to 1,25-(OH)₂D₃ (1,25-D₃) and formed OCL at physiologic concentrations of 1,25-D₃ rather than the pharmacologic concentrations of 1,25-D₃ required for normal OCL formation in vitro. This enhanced sensitivity to 1,25-D₃ was due to increased expression of a novel VDR co-activator, TAF12, a member of the TFIID transcription complex. We found TAF12 expression was increased in marrow stromal cells (BMSC) by increased NFκB signaling and enhanced the capacity of BMSC to produce RANKL in response to low levels of 1,25-D₃. Because the marrow microenvironment in MM and PD has many similarities in terms of increased OCL activity and enhanced NFκB signaling, we determined if MM cells induced TAF12 expression in BMSC of MM patients and if 1,25-D₃ could enhanced RANKL production in BMSC of MM patients, even in patients with low levels of 1,25-D₃. We found that both BMSC and CD138⁺ primary myeloma cells from MM patients expressed increased TAF12 levels compared to normal BMSC and CD138⁺ bone marrow cells. Four of four human MM cell lines (MM1.S, ANBL6, JJN3 and RPMI8266) expressed VDR, TAF12 and ATF7, which potentiates TAF12-mediated gene transcription. MM1.S and JJN3 but not RPMI8266 produced increased amounts of RANKL in response to very low levels of 1,25-D₃. Further, 1,25-D₃ increased VEGF, DKK1 and α4β1 integrin expression by MM1.S, JJN3 and RPMI8266 cells and enhanced adhesive interactions between MM cells and BMSC that increase MM growth. To confirm the role of TAF12 in the increased RANKL expression by MM cells treated with 1,25-D₃, we established a stable TAF12 anti-sense JJN3 cell line (AS-TAF12-JJN3). AS-TAF12-JJN3 cells had markedly decreased RANKL production, VDR content and CYP24A1 accumulation in response to 1,25-D₃. MM1.S and JJN3 myeloma cells treated with a VDR antagonist (TEI-9647) decreased RANKL production and α4β1 integrin expression in response to low levels of 1,25-D₃. Further, 1,25-D₃ induced VCAM-1 expression on normal human BMSC. Co-culture of JJN3 cells with BMSC treated with 1,25-D₃ induced both MM cell growth and cell adhesion. In contrast, co-culture with AS-TAF12-JJN3 cells resulted in decreased cell growth and cell adhesion. Further, 1,25-D₃ treatment of mouse OCL precursors co-cultured with JJN3 cells, but not AS-TAF12-JJN3 cells, increased OCL formation. These results suggest that increased TAF12 levels in MM cells and BMSC allow low levels of 1,25-D₃ significantly to increase RANKL production by both MM cells and BMSC, and enhance adhesive interactions between MM cells and BMSC, thus increasing MM cell growth and OCL formation.