Abstract 634: Suppression of Akt-mTOR pathway: a novel component of oncogene induced DNA damage response (DDR) barrier in breast.

Abstract

Abstract Introduction: Activation of the DNA damage response (DDR) is an important cellular mechanism for maintaining genomic integrity in the face of genotoxic stress. While the cellular response to genotoxic stress has been extensively studied in cancer models, less is known about the cellular response to oncogenic stress in the premalignant context. Methods: DNA damage (γ-H2AX), cell proliferation (ki-67) and apoptosis (cleaved caspase-3) were assessed by immunohistochemistry in breast tissue samples from 3 cohorts of women with: i) histologically normal breast tissue (n=50), ii) histological changes of increased risk (LCIS, ADH, ALH) (n=54) but without a personal history of breast cancer and iii) ductal carcinoma in situ (n=46). DNA damage parameters were also assessed in a panel of cell lines derived from the MCF-10A line that represents the multi-step process of breast tumorigenesis (AT1, DCIS, CA1d). Lastly, we generated stable cell clones over expressing cyclin E in the normal mammary epithelial cell line, MCF10A. DNA damage was measured by immuno fluorescence of γ-H2AX in the cell lines models. Apoptosis and autophagy were measured by staining cells with acridine orange and annexin V respectively followed by cell sorting with flow cytometry. Protein expression of AKt-mTOR pathway was assessed by western blotting. Results: Breast tissues samples from women at different risk levels for invasive breast cancer revealed that DNA damage is inversely correlated with breast cancer risk with the highest levels of damage seen in histologically normal tissues and lowest levels in DCIS (p<0.05). In contrast ki-67 was directly and significantly associated with increasing neoplastic progression (p<0.05). Using tree modeling, the combination of high γ-H2AX and low ki-67 was disproportionately associated average risk for breast cancer (p<0.05). Similarly, in the MCF10A panel (AT1, DCIS, CA1d) DNA damage is inversely correlated with breast cancer progression with highest levels seen in MCF10AT1 (> 10 foci= 19%) and lowest levels (> 10 foci= 0%) in the DCIS line and in the invasive cancer cell line CA1d (p<0.001). Overexpression of cyclin E in MCF10A cells resulted in about 4-fold increase in DNA damage and triggered the suppression of AKT-mTOR pathway with resultant cell death through autophagy. Conclusion: DNA damage is inversely associated with progression from normal mammary epithelium to carcinoma. DNA damage induced suppression of the pAKT-mTOR pathway in normal mammary epithelial cells appears to play a role in the tumorigenic barrier. Citation Format: Anjana Bhardwaj, Mei Liu, Yan Liu, Nivetha Ganesan, Daniel Rosen, Carol Etzel, Constance T. Albaracin, Qiang Hao, Ashley Gullett, Isabelle Bedrosian. Suppression of Akt-mTOR pathway: a novel component of oncogene induced DNA damage response (DDR) barrier in breast. [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 634. doi:10.1158/1538-7445.AM2013-634