Role of Runx2 in transcriptional regulation of MMP9 in bone metastatic cancer cells and the control of cell invasion

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Matrix metalloproteinase-9 (MMP9) is a key regulator in the development of the growth plate during endochondral bone formation. The transcription factor runt-related gene 2, (RUNX2, CBFA1/AML3) is essential for maturation of the growth plate and bone formation. Up regulation of MMP9 expression has been implicated in invasion and metastasis of breast and prostate tumors, and Runx2 is ectopically expressed in breast (MDA-MB-231) and prostate (PC3) cancer cells that metastasize to bone. By cDNA expression array, we identified MMP9 as a novel downstream target of Runx2 using total RNA from wild type and Runx2^{ΔC /ΔC} mutant mouse. MMP9 expression, like MMP13 but not MMP2, is nearly depleted in Runx2 deficient mice. In vivo analysis by chromatin immunoprecipitation assay and in vitro by EMSA revealed recruitment of Runx2 to the MMP9 promoter. We show by mutational analysis that the Runx2 site mediates transactivation of the MMP9 promoter activity in both osteoblastic cells (MC3T3- E1) and non-osseous (HeLa) cells. Runx2 regulation of MMP9 was examined relative to the invasive properties of tumor cells. We find that over-expression of Runx2 by adenovirus delivery in both primary and bone metastatic cancer cell lines significantly increases endogenous levels of MMP9. Furthermore, knockdown of Runx2 by RNA interference results in decreased MMP9 expression. Importantly, we have demonstrated using a cell migration assay that Runx2 regulated MMP9 levels are functionally related to the invasion properties of cancer cells. In conclusion, our findings demonstrate the role of Runx2 in transcriptional regulation of MMP9 and that modulation of MMP9 expression by Runx2 influences cell migration .We suggest that Runx2 is a primary initiator for expression of genes that contribute to the metastatic properties of cancer cells and their activity in the bone environment.