Abstract 551: The requirement of Runx2-dependent Akt phosphorylation in breast cancer cell survival and metastasis.

Abstract

Abstract Breast cancer is not only most commonly diagnosed in women worldwide but its metastasis shows serious life threatening consequences. The cell survival and mitogenesis via phosphatidyl inositol 3’ kinase (PI3K) and mitogen activated protein kinase (MAPK) signaling pathways are critical for breast cancer cell survival, proliferation, metabolism and metastasis. A de-regulation of osteogenic Runt-related transcription factor-2 (Runx2) is recently implicated in mammary carcinogenesis. We found that Runx2 differentially regulates the serine/threonine kinase Akt phosphorylation (pAkt), a downstream effecter of PI3K signaling, in non-tumorigenic and invasive mammary epithelial cell lines. The decline in pAkt upon Runx2 suppression in invasive MDA-MB-231 cells was completely abrogated by PI3K inhibitor LY294002. In contrast, the MAPK inhibitor, U0126, showed partial restoration of pAkt upon Runx2 depletion suggesting that Runx2-mediated crosstalk stimulates feedback mechanism within the PI3K and MAPK signaling pathways. The expression analysis of kinases and phosphatases functional in pAkt regulation revealed that the decline in pAkt upon Runx2 knockdown in MDA-MB-231 cells is due to the reduction in PI3K p85 subunit and mammalian target of rapamycin complex-2 (mTORC2). Additionally, we find altered expression of FOXO1, a downstream molecule of PI3K/Akt signaling, due to Runx2-mediated regulation of pAkt levels in MCF-10A and MDA-MB-231 cell lines. To examine the phenotypic effects of Runx2 and PI3K/Akt pathway crosstalk, we evaluated the Runx2-depleted MDA-MB-231 cells for survival under stress conditions. These studies revealed an altered morphology coupled with increased rounding and sub-G1 population in response to glucose- and serum-deprivation upon Runx2 knockdown. The increase in sub-G1 population was restored by glucose and serum re-constitution in the culture media. An increase in Annexin V staining, decline in pAkt levels and cleavage of caspase-3 confirmed the induction of apoptotic cell death. To understand the Runx2-mediated regulation of PI3K/Akt signaling pathway in breast cancer metastasis, the control or Runx2-depleted MDA-MB-231 cells were inoculated intra-cardiacally into immunodeficient mice. The tumors at lung and bone metastatic sites were harvested, FACS-sorted and examined for pAkt levels. Interestingly, to this end, the in-vivo metastatic tumor-derived Runx2 knockdown MDA-MB-231 cells showed an increase in pAkt levels in contrast to in-vitro cultured Runx2 knockdown MDA-MB-231 cells. The data suggests that the mechanism of pAkt activation might involve a dynamic requirement of transcriptional control and feedback regulation within PI3K/Akt and MAPK signaling pathways during different phases of tumor growth in primary and metastatic microenvironment. Citation Format: Manish Tandon, Zujian Chen, Jitesh Pratap. The requirement of Runx2-dependent Akt phosphorylation in breast cancer cell survival and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 551. doi:10.1158/1538-7445.AM2013-551