Aurora-A Promotes Tumor Radio-and Chemoresistance Through Dysregulating DNA Damage Response

Background:Poor prognosis of cancer usually results from radio-and chemoresistance after various treatments. Aurora kinase A (Aurora-A), a serine/threonine kinase associated with spindle separation and centrosome duplication in mitosis, has been identified as an important oncogene overexpressing in many malignant cancers. Recent studies show that Aurora-A can promote tumor radio-and chemoresistance, and since Aurora-A interacts with many DNA damage responsive proteins such as p53, BRCA1, and BRCA2, we presume that Aurora-A may confer cancer cell radio-and chemoresistance through dysregulation of DNA damage response.Purposes:We aimed to study the relationship between Aurora-A and DNA damage response to reveal the mechanism of cancer radio-and chemoresistance induced by Aurora-A, through which we may find novel therapeutic targets for sufficient treatment of cancer patients.Materials and methods:Part1. Determination of Aurora-A function in cell proliferation.1).Cell lines from breast cancer, pancreatic cancer and ovarian cancer, including those with either low Aurora-A expression (MCF-7, PANC-1and OVCA420), or high Aurora-A expression (MDA-MB-231, BXPC3and OVCA429), were used for overexpression or silencing of Aurora-A via retro viral transfection/infection.2).Cell growth curve and colony formation rate were tested by cell counting and soft agar colony formation assay.3).Flow cytometry was used to test cell cycle.Part2. Examination of Aurora-A function in cancer cell radio-and chemoresistance.1).Flow cytometry was used to examine cell apoptosis after radiation and cisplatin or VX680(Aurora-A inhibitor) treatment of cells. 2).MTT was used to measure cell viability or the half maximal inhibitory concentration (IC50) after cells were treated with cisplatin.Part3. Exploration of the mechanism of cancer cell radio-and chemoresistance induced by Aurora-A.Chapter1. Examination of the association of Aurora-A with DNA damage response.1).Immunofluorescence was used to detect the relationship between Aurora-A and γ H2AX.2).Immunoblotting was used to test the impact of Aurora-A on the expression of DNA damage response proteins including ATM, Chk2, ATR, Chkl, RAD51, H2AX,yH2AX, ERCC2, p53, pp53, AKT1, AKT2.Chapter2. Determination of the effect of ATM on cancer cell radio-and chemoresistance and DNA damage response.1).ATM was inhibited by its inhibitor KU-55933, and then apoptosis was examined by folw cytometry.2).Immunoblotting was used to test the effect of ATM inhibitor on DNA damage repair proteins such as pp53, Chk2, γ H2AX and RAD51.Chapter3. Discovery of the association of BRCA1/2with Aurora-A, and investigation of the effects of BRCA1/2on cancer cell radio-and chemo-therapy and DNA damage response.1).Cell lines from breast cancer, pancreatic cancer and ovarian cancer, including those with either low Aurora-A expression (MCF-7, PANC-1and OVCA420), or high Aurora-A expression (MDA-MB-231, BXPC3and OVCA429), were used to knockdown or overexpress BRCA1/2, via retro viral transfection/infection or Fugene6transfection reagent.2).Immunoblotting was used to test the association of BRCA1/2and Aurora-A.3).Cell growth curve and colony formation rate were tested by cell counting and soft agar colony formation assay.4).Flow cytometry was used to test cell cycle.5). Flow cytometry was used to examine cell apoptosis after radiation and cisplatin treatment of cells.6). Immunoblotting was used to test the effect of BRCA1/2on DNA damage responsive proteins including p53, pp53, ATM, Chk2, RAD51, yH2AX. Results:Part1.Overexpression of Aurora-A promotes cell proliferation, cell cycle and soft agar colony formation, while silencing of Aurora-A inhibits cell proliferation, cell cycle and soft agar colony formation.Part2.Introduction of Aurora-A reduces cell apoptosis induced by IR or cisplatin treatment, while silencing of Aurora-A or treatment with Aurora-A inhibitor VX680increases cell apoptosis caused by IR or cisplatin; Transfection of Aurora-A increases IC50of cells to cisplatin, while silencing of Aurora-A reduces the IC50values of cells to cisplatin.Part3.Chapter1. Aurora-A restrains the formation of yH2AX foci and dysregulates DNA damage response.Chapter2. Inhibition of ATM promotes cell apoptosis after IR or cisplatin treatment and affects the expression of DNA damage responsive proteins.Chapter3. BRCA1/2is negatively associated with Aurora-A,and overexpression of BRCA1/2inhibits cell proliferation, cell cycle progression and cell colony formation, but increases apoptosis after radio-and chemotherapy through altered expression of DNA damage responsive proteins.Conclusion:We show that Aurora-A controls cancer cell proliferation, cell cycle progression, and anchorage-independent colony formation to confer cancer cell radio-and chemoresistance to IR and cisplatin treatment through ATM-or BRCA1/2-mediated dysregulation of DNA damage response. Therefore, we suggest that DNA damage responsive molecules should be considered for the targeted therapy against cancers with overexpression of Aurora-A.