Copper Enhances Genotoxic Drug Resistance Via ATOX1 Activated DNA Damage Repair

Cancer is a disease characterized by abnormal cell proliferation,uncontrolled division and high metastasis.Its occurrence and development are regulated by a variety of factors and signaling pathways,and undergo a multi-step and complex biological process.According to statistics,about one-sixth of the world’s people die of cancer,and cancer is the second largest cause of death in the world after cardio-cerebrovascular disease.At present,the common treatments methods of cancer include chemotherapy,radiotherapy,surgery,immunotherapy,endocrine therapy and gene therapy,among which chemotherapy is the most common one.However,due to its high toxicityeffects and drug resistance characteristics,more than 90% of cancer patients may eventually lose their lives due to chemotherapy resistance.In the process of life,the DNA damage inevitably occurs in body’ cells.In order to deal with these damages,cells have developed a DNA damage response system(DDR)which can be accurately regulated.The mediator of DNA damage checkpoint 1(MDC1)is a scaffold protein involved in the early stages of DDR that can directly interact with the DNA damage sensing molecule γH2AX.Loss of MDC1 may serve as a biomarker for carcinogenesis and chemotherapeutic drug sensitivity.Studies have shown that a variety of chemotherapy drugs can kill tumor cells by inducing DNA damage,thereby exerting anti-tumor effects;but at the same time,they also active DDR of tumor cells,which prevents tumor cell death and leads to chemotherapy resistance.Therefore,targeted inhibition of DNA damage repair signaling pathway has become an important way to improve the chemosensitivity of tumor cells,and revealing the regulatory mechanism of DNA damage repair signaling pathway is expected to provide new clues for improving tumor treatment effects.Copper is one of the essential trace metal elements in human body and participates in a variety of biochemical and physiological processes.Studies have shown that tumor cells have a high demand for copper,and the level of copper in tumor cells of drug-resistant patients is significantly higher than that in normal cells,indicating that there is an important relationship between copper and drug resistance.Copper molecular chaperone,antioxidant protein 1(ATOX1),plays an important role in copper homeostasis.ATOX1 as a copper-dependent transcription factor,is highly expressed in a variety of cancers tissues.Although ATOX1 plays an important role in promoting tumor growth,the function and potential molecular mechanisms of ATOX1 in chemotherapy resistance remain unclear.Therefore,this study will further explore whether Cu and its molecular chaperone ATOX1 participate in the regulation of chemotherapy-induced tumor drug resistance.First,we clarified whether copper participates in the drug resistance of chemotherapy drugs and DNA damage repair.Copper was added under the condition that cells were treated by chemotherapy drugs to cause DNA damage,and the CCK8 assay showed that adding an appropriate amount of copper could inhibit chemotherapy drug-induced cell death;Western blot and immunofluorescence showed that the level of DSB marker γH2AX was significantly reduced in chemotherapy-induced cancer cells.Meanwhile,the comet assay results showed that DNA tails were significantly shorter after copper treatment than those in the normal treatment group,indicating that copper can promote DDR.In addition,the addition of the specific chelator TM for copper ions can promote the cell death induced by chemotherapeutic drugs,and can obviously increase the sensitivity of cancer cells to chemotherapy drugs,and hinder the repair of DSB of tumor cells treated with chemotherapy drugs at the same time,indicating that high copper levels in tumor cells are directly related to tumor resistance to chemotherapeutic drugs and DNA damage repair.Secondly,we investigated the effect of copper molecular chaperone ATOX1 and CCS in tumor cell resistance to chemotherapeutics and DNA double-strand break repair.Firstly,by knocking down ATOX1 or CCS,respectively,we found that knocking down of ATOX1 led to a significant increase in chemotherapy-induced tumor cell death and increased DNA damage,however,knocking down of CCS showed no significant change.In the case of DNA damage in tumor cells,the results howed that copper supplementation reduced the γH2AX level compared with control group,while the protein expression level of γH2AX in ATOX1 knockdown group did not change significantly changed,in the CCS knockdown group and control group,copper treatment had no significant effect on γH2AX levels.Biological information analysis showed the cell lines with high expression of ATOX1 showed high resistance to chemotherapeutic drugs.Results of Western blot also indicated that the GEM-resistant pancreatic cancer cell lines showed higher ATOX1 expression.Indeed,overexpression of ATOX1 weakened the cytotoxicity of chemotherapeutic drugs in pancreatic cancer cells in vitro and in nude mouse tumor models.Comet assay showed that the overexpression of ATOX1 decreased the DNA damage of pancreatic cancer cells caused by chemotherapy drugs.Besides,the combination of ATOX inhibitor DCAC50 and GEM treatment significantly reduced the growth of xenografts tumors in nude mice and increase the apoptosis of cancer cells,indicating that copper is involved in the resistance of tumor cells to chemotherapy drugs and DNA damage repair through ATOX1.Finally,this paper investigated the regulatory mechanism of ATOX1 in DNA damage repair.RNA-seq analysis preliminarily found that ATOX1 can regulate MDC1,the key protein in DNA damage repair.After GEM,ADR or DDP treatment,ATOX1 could enter into the nucleus.Dual-luciferase assay proved that ATOX1 regulated the promoter of MDC1,and CHIP assay confirmed that ATOX1 could bind to the MDC1 promoter,suggesting that ATOX1 can transcriptionally activate the expression of MDC1,and ATOX1 exerts its function as a transcription factor in a copper-dependent manner.Knockdown or knockout of ATOX1 simultaneously decreased MDC1 expression and enhanced DNA double-strand breaks in GEM,ADR,or DDP-treated Patu 8988 T cells.In summary,this study found that copper promoted drug resistance and DNA damage repair in tumor cells.Copper promoted drug resistance and DNA damage repair in tumor cells through its molecular chaperone ATOX1 instead of CCS.ATOX1 played a role in tumor resistance and DNA damage repair by transcriptionally regulating the expression of MDC1,the key protein of DNA damage response,in a copper-dependent manner.The above findings provide a new understanding of the role of high copper levels in tumor cells,and provide a new strategy for clinical treatment of tumor drug resistance.