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In multiple myeloma, abnormal plasma cells accumulate and proliferate in the bone marrow. Recently, we observed that Runx2, a bone-specific transcription factor, is highly expressed in multiple myeloma cells and is a major driver of multiple myeloma progression in bone. The primary goal of the present study was to identify Runx2-targeting miRNAs that can reduce tumor growth. Expression analysis of a panel of miRNAs in multiple myeloma patient specimens, compared with healthy control specimens, revealed that metastatic multiple myeloma cells express low levels of miR-342 and miR-363 but high levels of Runx2. Reconstituting multiple myeloma cells (CAG) with miR-342 and miR-363 reduced the abundance of Runx2 and the expression of metastasis-promoting Runx2 target genes RANKL and DKK1, and suppressed Runx2 downstream signaling pathways Akt/β-catenin/survivin, which are required for multiple myeloma tumor progression. Intravenous injection of multiple myeloma cells (5TGM1), stably overexpressing miR-342 and miR-363 alone or together, into syngeneic C57Bl/KaLwRij mice resulted in a significant suppression of 5TGM1 cell growth, decreased osteoclasts and increased osteoblasts, and increased antitumor immunity in the bone marrow, compared with mice injected with 5TGM1 cells expressing a miR-Scramble control. In summary, these results demonstrate that enhanced expression of miR-342 and miR-363 in multiple myeloma cells inhibits Runx2 expression and multiple myeloma growth, decreases osteolysis, and enhances antitumor immunity. Thus, restoring the function of Runx2-targeting by miR-342 and miR-363 in multiple myeloma cells may afford a therapeutic benefit by preventing multiple myeloma progression.Implications: miR-342 and miR-363–mediated downregulation of Runx2 expression in multiple myeloma cells prevents multiple myeloma progression. Mol Cancer Res; 16(7); 1138–48. ©2018 AACR. more...

Antibodies used for Western blotting and FACS.

5TGM1 cells stably overexpressing miR-342 and miR-363 were established by lentiviral vector transduction.

PURPOSE OF REVIEW-: Precise and temporal expression of Runx2 and its regulatory transcriptional network is a key determinant for the intricate cellular and developmental processes in adult bone tissue formation. This review analyzes how microRNA functions to regulate this network, and how dysregulation results in bone disorders. RECENT FINDINGS-: Similar to other biologic processes, microRNA (miRNA/miR) regulation is undeniably indispensable to bone synthesis and maintenance. There exists a miRNA–RUNX2 network where RUNX2 regulates the transcription of miRs, or is post transcriptionally regulated by a class of miRs, forming a variety of miR-RUNX2 regulatory pathways which regulate osteogenesis. SUMMARY-: The current review provides insights to understand transcriptional–post transcriptional regulatory network governed by Runx2 and osteogenic miRs, and is based largely from in vitro and in vivo studies. When taken together, this article discusses a new regulatory layer of bone tissue specific gene expression by RUNX2 influenced via miRNA. more...

In multiple myeloma, abnormal plasma cells accumulate and proliferate in the bone marrow. Recently, we observed that Runx2, a bone-specific transcription factor, is highly expressed in multiple myeloma cells and is a major driver of multiple myeloma progression in bone. The primary goal of the present study was to identify Runx2-targeting miRNAs that can reduce tumor growth. Expression analysis of a panel of miRNAs in multiple myeloma patient specimens, compared with healthy control specimens, revealed that metastatic multiple myeloma cells express low levels of miR-342 and miR-363 but high levels of Runx2. Reconstituting multiple myeloma cells (CAG) with miR-342 and miR-363 reduced the abundance of Runx2 and the expression of metastasis-promoting Runx2 target genes RANKL and DKK1, and suppressed Runx2 downstream signaling pathways Akt/β-catenin/survivin, which are required for multiple myeloma tumor progression. Intravenous injection of multiple myeloma cells (5TGM1), stably overexpressing miR-342 and miR-363 alone or together, into syngeneic C57Bl/KaLwRij mice resulted in a significant suppression of 5TGM1 cell growth, decreased osteoclasts and increased osteoblasts, and increased antitumor immunity in the bone marrow, compared with mice injected with 5TGM1 cells expressing a miR-Scramble control. In summary, these results demonstrate that enhanced expression of miR-342 and miR-363 in multiple myeloma cells inhibits Runx2 expression and multiple myeloma growth, decreases osteolysis, and enhances antitumor immunity. Thus, restoring the function of Runx2-targeting by miR-342 and miR-363 in multiple myeloma cells may afford a therapeutic benefit by preventing multiple myeloma progression. Implications: miR-342 and miR-363–mediated downregulation of Runx2 expression in multiple myeloma cells prevents multiple myeloma progression. Mol Cancer Res; 16(7); 1138–48. ©2018 AACR. more...

Current studies offer little insight on how epigenetic remodeling of bone-specific chromatin maintains bone mass in vivo. Understanding this gap and precise mechanism is pivotal for future therapeutic innovation to prevent bone loss. Recently, we found that low bone mass is associated with decreased H3K27 acetylation (activating histone modification) of bone specific gene promoters. Here, we aim to elucidate the epigenetic mechanisms by which a miRNA cluster controls bone synthesis and homeostasis by regulating chromatin accessibility and H3K27 acetylation. In order to decipher the epigenetic axis that regulates osteogenesis, we studied a drug inducible anti-miR-23a cluster (miR-23a ClZIP) knockdown mouse model. MiR-23a cluster knockdown (heterozygous) mice developed high bone mass. These mice displayed increased expression of Runx2 and Baf45a, essential factors for skeletogenesis; and decreased expression of Ezh2, a chromatin repressor indispensable for skeletogenesis. ChIP assays using miR-23a Cl knockdown calvarial cells revealed a BAF45A-EZH2 epigenetic antagonistic mechanism that maintains bone formation. Together, our findings support that the miR-23a Cl connection with tissue-specific RUNX2-BAF45A-EZH2 function is a novel molecular epigenetic axis through which a miRNA cluster orchestrates chromatin modification to elicit major effects on osteogenesis in vivo. more...

Interleukin-6 (IL-6) plays an important role in progressive bone loss in rheumatoid arthritis (RA). However, the molecular mechanisms through which IL-6 propels RA synovial fibroblasts (RASFs) to contribute to bone loss are not fully understood. Treatment of human RASFs with IL-6 and IL-6 receptor (IL-6/IL-6R, 100 ng/ml each) alone or in combination with M-CSF (2 ng/ml) and RANKL (5 ng/ml) for 9 days resulted in the phenotypic changes to osteoclast-like features as determined by increase in TRAP staining and Dentin pit resorption area, which were comparable to M-CSF/RANKL group. IL-6/IL-6R induced a dose-dependent increase in the expression of transcription factor Ets2 and enhanced its nuclear translocation alone or combinatorially in M-CSF/RANKL-differentiated human RASFs. Chromatin immunoprecipitation (ChIP) analysis of Cathepsin K and B promoters showed Ets2 binding and transcriptional activation upon IL-6/IL-6R stimulation, which was further increased with M-CSF/RANKL. Interestingly, the untargeted proteomics analysis of the conditioned media from activated RASFs using Mass-Spectrometry (MS) technique and Trans-Proteomic Pipeline tool showed the production of 113 analytes unique to IL-6/IL-6R stimulation. Further analysis of the proteomics data using STRING database for pathway analysis showed the impact of IL-6/IL-6R on immunometabolic reprogramming of human RASFs towards osteoclast-like phenotype, which was partly mediated through Ets2. These preliminary studies identified a novel role of IL-6/IL-6R-mediated transsignaling in the metabolic reprogramming in RASFs that could potentially be targeted by inhibiting Ets2 to limit their role in bone resorption in RA. more...

MicroRNAs (miRs) and Hox transcription factors have decisive roles in postnatal bone formation and homeostasis. In silico analysis identified extensive interaction between HOXA cluster mRNA and microRNAs from the miR-23a cluster. However, Hox regulation by the miR-23a cluster during osteoblast differentiation remains undefined. We examined this regulation in preosteoblasts and in a novel miR-23a cluster knockdown mouse model. Overexpression and knockdown of the miR-23a cluster in preosteoblasts decreased and increased, respectively, the expression of the proteins HOXA5, HOXA10, and HOXA11; these proteins' mRNAs exhibited significant binding with the miR-23a cluster miRNAs, and miRNA 3′-UTR reporter assays confirmed repression. Importantly, during periods correlating with development and differentiation of bone cells, we found an inverse pattern of expression between HoxA factors and members of the miR-23a cluster. HOXA5 and HOXA11 bound to bone-specific promoters, physically interacted with transcription factor RUNX2, and regulated bone-specific genes. Depletion of HOXA5 or HOXA11 in preosteoblasts also decreased cellular differentiation. Additionally, stable overexpression of the miR-23a cluster in osteoblasts decreased the recruitment of HOXA5 and HOXA11 to osteoblast gene promoters, significantly inhibiting histone H3 acetylation. Heterozygous miR-23a cluster knockdown female mice (miR-23a Cl(WT/ZIP)) had significantly increased trabecular bone mass when compared with WT mice. Furthermore, miR-23a cluster knockdown in calvarial osteoblasts of these mice increased the recruitment of HOXA5 and HOXA11, with a substantial enrichment of promoter histone H3 acetylation. Taken together, these findings demonstrate that the miR-23a cluster is required for maintaining stage-specific HoxA factor expression during osteogenesis. more...