Abstract
1,25 dihydroxy vitamin D3 (1,25D) plays multiple roles in normal and malignant cell functions, including cell differentiation and proliferation, and is a regulator of bone metastasis. However, the role of 1,25D in multiple myeloma (MM) is poorly characterized. We previously showed that TAF12, a member of the TFIID transcription complex, acts as a co-activator for vitamin D receptor (VDR) mediated transcription to increase 1,25D responsivity. Further, we reported that IL-6 can increase TAF12 levels in bone marrow stromal cells (BMSC). Since IL-6 is upregulated in the MM microenvironment, we assessed TAF12 levels in BMSC from 13 MM patients and 10 normals. TAF12 protein levels were significantly increased (25x) in MM BMSC compared to normal. Further, co-culture of BMSC from 3 normals with primary human MM cells significantly increased TAF12 levels in the normal BMSC. Similarly, when 5TGM-1 murine MM cells were inoculated intratibially into C57BL/KaLwRij mice and bone marrow cells collected 3 weeks later, TAF12 protein levels in MM exposed BMSC were significantly increased compared to PBS injected control mice. We then assessed if increased TAF12 levels persisted in normal BMSC (SAKA-T cells) after they were co-cultured with JJN3 MM cells and subsequently cultured in the absence of MM cells. We found that increased TAF12 levels persisted for at least 2 weeks after removal of MM cells, consistent with our findings that BMSC from MM patients express elevated TAF12 levels in the absence of MM cells. To determine if IL-6 contributes to the increased TAF12 levels induced by MM cells, we co-cultured SAKA-T cells with JJN3 MM cells in the presence or absence of anti-IL-6. Anti-IL-6 blocked the increase in TAF12 levels in these co-cultures.
We then tested if the increased TAF12 levels enhance the 1,25D responsivity of BMSC. Addition of 1,25D (10-10 - 10-8 M) greatly increased expression of RANKL mRNA and VCAM-1 protein in MM BMSC compared to normals. The increased RANKL and VCAM-1 in MM BMSC treated with 1,25D correlated with increased osteoclast (OCL) formation, MM cell growth and adhesion of MM cells. When human OCL precursors and MM or normal BMSC were co-cultured with 1,25D with/without osteoprotegerin (OPG), OCL numbers were significantly increased in co-cultures of MM BMSC treated with 1,25D compared to normal, and the increase was blocked by OPG. Further, co-culture of JJN3 cells with normal or MM BMSC and 1,25D for 6 hours followed by removal of non-adherent MM cells, demonstrated that the number of MM cells adherent to BMSC was significantly increased with 1,25D treatment of MM BMSC co-cultures compared to normal BMSC. In addition, when JJN3 cells were co-cultured with normal or MM BMSC and 1,25D, JJN3 cells numbers were significantly increased in co-cultures with MM BMSC compared to normal BMSC. Finally, we determined if high levels of TAF12 in MM BMSC increased their 1,25D responsivity. We knocked-down (k/d) TAF12 with a siRNA in normal and MM BMSC, and found that TAF12 k/d in MM or normal BMSC decreased the levels of VCAM 1 induced by 1,25D compared to control siRNA transduced cells. To further investigate this effect, we assessed if TAF12 levels affected the stability of VDR in the presence of 1,25D, using TAF12 heterozygous or WT mouse BMSC. VDR half-life was decreased approximately 50% in TAF12 heterozygous BMSC compared with WT.
These results show that MM cells increase expression of TAF12 in BMSC that is mediated by IL-6. BMSCs expressing high TAF12 levels have greater responsivity to 1,25D that enhances RANKL and VCAM-1 expression to induce osteoclastogenesis, MM cell growth and MM cell adhesion. These results further suggest that blocking TAF12 -VDR interactions may be a potential therapeutic target for MM bone disease.
Disclosures
Roodman: Amgen: Consultancy.
Human Regulatory T Cells Do Not Suppress the Antitumor Immunity in the Bone Marrow: A Role for Bone Marrow Stromal Cells in Neutralizing Regulatory T Cells
