Runx2 Transcription Factor Regulates Heparanase-Induced Bone Resorption in Multiple Myeloma

Despite advances in treatment strategies, myeloma remains incurable, and bone destruction is a major cause of morbidity in myeloma patients. We have documented in earlier studies that heparanase enzyme is preferentially expressed in myeloma cells and induces severe bone destruction in myeloma. We also discovered that heparanase increases the production of two major bone resorbing factors named Receptor Activator of NF-κB Ligand (RANKL) and Matrix Metalloproteinase 9 (MMP-9) by myeloma cells. Runx2, a member of the runt-related gene family, is a bone-specific transcription factor. Runx2 regulates osteoblast differentiation and is essential for bone tissue development. Interestingly, Runx2 also controls expression of RANKL and MMP-9 genes in osteoblasts. Recent evidence indicates that ectopic induction and overexpression of Runx2 in breast, uterine and prostate cancer cells is associated with bone-metastasis, and osteolytic bone disease in these cancers. However, very little is known about the function of Runx2 in myeloma cells. Here we report for the first time that heparanase engages the Runx2 pathway to promote expression of RANKL and MMP-9 in myeloma cells.

Molecular, biochemical, cellular and in vivo approaches were used to assess the role of Runx2 in heparanase-induced expression of RANKL and MMP-9. These included: (1) Real-time PCR and Western blot analysis to monitor Runx2 levels in CAG myeloma cells expressing high level of heparanase (HPSE-high cells) or with knockdown of endogenous heparanase (HPSE k/d cells), and the corresponding control cells. (2) Chromatin Immunoprecipitation (ChIP) assay to determine in vivo occupancy of the RANKL and MMP-9 gene promoters in myeloma cells by Runx2. (3) Zymography to determine MMP-9 activity in both human and murine myeloma cells. (4) Real-time PCR to determine changes in RANKL and MMP-9 gene expression in Runx2 knockdown MM1.S and 5TGM1 myeloma cells. (5) Assessment of tumor growth/burden and bone resorption in the 5TGM1 syngenic model of murine myeloma. 5TGM1 cells with specific knockdown of Runx2 or non-target shRNA were injected into C57BL/KaLwRij mice through the tail vein and levels of IgG2b and TRAP5b were monitored in the sera of the mice by ELISA.

We find Runx2 expression is significantly increased in CAG myeloma cells expressing a high level of heparanase and dramatically reduced in HPSE k/d cells. Increased Runx2 in HPSE-high myeloma cells results in an increase in expression of RANKL and MMP-9 mRNA as well as MMP-9 activity. In sharp contrast, knockdown of Runx2 in human myeloma MM1.S and murine myeloma 5TGM1 cells results in a significant reduction of RANKL, MMP-9 gene expression and MMP-9 activity. Thus heparanase promotes RANKL and MMP-9 expression via Runx2. Analysis of initial 1-kb sequences of RANKL and MMP-9 gene promoter in human and mice, revealed presence of multiple high affinity Runx2-binding sites. ChIP assay confirmed that Runx2 mediates induction of RANKL and MMP-9 gene transcription in the both human and murine myeloma cells by direct association with the proximal promoter of these genes. Together these results demonstrate that Runx2 is a positive regulator of RANKL and MMP-9 gene expression in myeloma cells. Finally, in the syngenic model, knockdown of Runx2 in 5TGM1 murine myeloma cells remarkably inhibits the growth of these cells in vivo and bone resorption.

Runx2 is a central regulator of heparanase induced myeloma bone disease. Our discoveries provide new insight into the mechanism of myeloma-induced bone disease and identify Runx2 as a novel target to block myeloma bone disease.

No relevant conflicts of interest to declare.