Activation Autophagy by the Combination of a Histone Deacetylase Inhibitor and Radiation Limits BNIP3 Regulation in Triple-Negative Breast Cancer Cells and Orthotopic Mouse Model

Triple-negative breast cancer (TNBC) is defined by its lack of estrogen-receptor (ER) and progesterone-receptor (PR) expression, along with the absence of human epidermal growth factor receptor 2 (HER2) overexpression or gene amplification. Conventional hormonal or anti-HER2 targeted therapies have no favorable value for TNBC which lacks known common therapeutic targets. Targeting chromatin-remodeling pathways is a means of disrupting transcriptional regulation in malignant cells. Many evidences have implicated histone deacetylase (HDAC) enzymes in the development of cancer and as potential therapeutic targets. Systematic analysis of cancer cells found that many had higher levels of expression of HDACs than in corresponding normal tissues. Some HDAC inhibitors (HDACi) have been approved by FDA for treating cutaneous T-cell lymphoma. Furthermore, recent evidence shows that HDACi may enhance radiosensitivity through inhibiting DNA repair processes. Tumor cells treated with anticancer drugs experience the induction of type I programmed cell death (PCD), apoptosis, and type II PCD, autophagy. However, the detailed mechanisms of how HDACi increase the radiosensitivity of tumor cells remain largely unknown. Additionally, positive Bcl-2 expression has been associated with poor survival and reduced sensitivity to chemotherapy in patients with TNBC. Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) is a member of a Bcl-2 subfamily. Many studies demonstrated a pro-survival role BNIP3 was shown to provide survival advantage in cancer cells. BNIP3 mRNA was not identified in normal breast and up-regulation in breast cancer. Our preliminary data observed decreased BNIP3 protein level in cells treated with combined IR and HDACi treatment in TNBC cells. Here, we outline research goals that hope to achieve within one year. Aim 1: To investigate the synergistic anti-cancer effects of ionizing radiation (IR) combined with HDACi in human and mouse TNBC cells. Synergistic effects will be determined through calculation the combination index by CalcuSyn softwore. Aim 2: To analyze the mechanisms underlying apoptosis or autophagy induced by combined with IR and HDACi in TNBC cells. Aim 3: To investigate whether the BNIP3 signaling pathway is involved in the combined treatment-induced cell death. Aim 4: To evaluate the anticancer effects of combined treatment in orthotopic xenograft breast cancer model. Tumor volumes will be monitored by the bioluminescence IVIS Imaging System. Aim 5: To determine the expression of the BNIP3 and cell death-related proteins in orthotopic breast tumors. The expression of markers of autophagy, apoptosis and BNIP3 will be determined by immunohistochemistry and western blot analysis. We expect that targeting BNIP3 or combined with IR and HDACi may be considered as a potential therapeutic strategy for the treatment of TNBC.