Abstract
Abstract Lung cancer is one of the major causes of cancer-related deaths. Several genetic and epigenetic alterations are now established for lung cancer progression. However, the regulatory mechanisms of these alterations are still unclear. Here, we show that the transcription factor Runx2 is highly expressed in lung cancer cells and downregulates a tumor suppressor bone morphogenetic protein-3B (BMP-3B/GDF10). The Runx2 transcription factor, an essential bone cell differentiation factor, is previously implicated in breast and prostate cancer progression by activating cancer related genes and promoting invasive properties. Our studies in Runx2 deficient animals demonstrated a significant increase in BMP-3B expression levels compared to wild type littermates as examined by cDNA arrays and real time PCR analysis. BMP-3B was previously identified as a silenced gene by DNA methylation in restriction landmark genomic scanning in lung cancer cells, thus suggesting an important function linked to maintaining the normal cell phenotype. Characterization of two normal lung fibroblasts (WI-38, IMR-90) and three lung cancer cells lines (A549, H720, H1299) showed an inverse relationship between Runx2 and BMP-3B expression as examined by real time PCR and western blot analysis. The ectopic expression of Runx2 in normal lung fibroblasts showed a significant downregulation of BMP-3B levels. Expression studies with point and deletion mutants of Runx2 in normal lung fibroblasts and lung cancer cells showed that the C-terminal domain of Runx2 is essential for BMP-3B downregulation. Chromatin immunoprecipitation assays in H1299 cells confirmed the mechanism of Runx2 mediated suppression of BMP-3B. Our results demonstrated Runx2 and H3-K9 specific histone methyltransferase SUV39H1 recruitment on the BMP-3B proximal promoter and concomitant increase in histone methylation (H3-K9) status by Runx2 to mediate repression of the BMP-3B gene locus. Furthermore, co-immunoprecipitation studies showed a direct interaction of Runx2 and SUV39H1 in lung cancer cells. Depletion of Runx2 in H1299 cells by stable expression of shRunx2 resulted in reduced H3-K9 methylation of the BMP-3B promoter and increased expression levels. Modulation of Runx2 levels in H1299 cells modestly altered cell growth properties as examined by MTT cell proliferation assay. Taken together, our studies identified BMP-3B as a novel Runx2 target gene and showed that Runx2 interaction with SUV39H1 down-regulates BMP-3B by affecting local chromatin histone methylation at BMP-3B regulatory regions. Our studies suggest that Runx2 not only activates cancer related genes but also plays a critical role in epigenetic silencing of tumor suppressors during cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4989. doi:10.1158/1538-7445.AM2011-4989
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