Abstract 3081: Crosstalk between Runx2 regulatory network and PI3K/Akt signaling pathway in breast cancer cells

Abstract

Abstract The Runt-related transcription factor Runx2 is essential for skeletal development but is also aberrantly expressed in several cancers (e.g., breast, prostate and osteosarcoma) where it promotes cell migration, invasive and growth properties of cancer cells. The phosphatidylinositol 3′ kinase (PI3K)/Akt signaling pathway is linked to Runx2-dependent skeletogenesis, however, the mechanisms of Runx2 role in PI3K-Akt signaling in breast cancers, and its implication in cancer progression remains under investigated. To mechanistically determine the function of Runx2 in epidermal growth factor (EGF)-induced PI3K/Akt signaling and concomitantly ensuing downstream signaling events, we modulated Runx2 expression levels in immortalized normal MCF-10A and invasive MDA-MB-231 cells via lentiviral vector-mediated gene delivery. The Runx2 over-expression in EGF-stimulated MCF-10A cells increased the cellular response towards PI3K inhibitor LY294002 as indicated by a decline in Akt (Serine 473) phosphorylation (pAkt) levels. This effect could be restored by Runx2 suppression indicating the specificity of Runx2 function in Akt regulation. In MDA-MB-231 cells, Runx2 over-expression and, surprisingly, suppression profoundly inhibited pAkt suggesting that a threshold level of Runx2 is required for phosphorylation of Akt. The Runx2-mediated decline in pAkt was completely reversed by the Runx2-DNA binding mutant over-expression indicating that Runx2 DNA binding domain is essential for Akt inhibition. To further dissect the mechanism of Runx2-mediated regulation of pAkt levels in MDA-MB-231 cells, we examined the protein levels of phosphatases and kinases associated with PI3K/Akt signaling pathway. These analyses revealed that PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1) levels were directly responsive to Runx2. Chromatin immunoprecipitation studies showed Runx2 recruitment on the proximal promoter of PHLPP1 suggesting that Runx2 mediates its effect on Akt activity, in part by regulating PHLPP1 expression. Furthermore, Runx2 overexpression increased nuclear translocation of a forkhead box transcription factor FoxO1, a downstream molecule of PI3K/Akt signaling indicating an indirect Runx2 affect on FoxO1 transcriptional activity in MDA-MB-231 cells as examined by western blot analysis and immunofluorescence studies. Together, our results identified clinically relevant and novel mechanisms of Runx2 regulatory network in PI3K/Akt signaling pathway that could have important consequences in targeting breast cancer-associated cell survival and invasive properties. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3081. doi:1538-7445.AM2012-3081