COX5A Promotes Invasion And Chemotherapy Resistance Of Estrogen-Receptor Positive Breast Cancer And Its Mechanism

Background and objective:Breast cancer is the most common cancer among women worldwide,with 70%being estrogen receptor-positive(ER+).Although ER-targeted treatment is effective in treating ER+ breast cancer,patients who are resistant to endocrine therapy are still prone to metastasis and relapse,and metastatic tumors are not sensitive to endocrine therapy.Fortunately,combined use of endocrine therapy and chemotherapy can reduce the risk of metastasis and recurrence in patients with ER+ breast cancer.Therefore,chemotherapy is of great significance for metastatic or endocrine-resistant ER+ breast cancer.However,chemotherapy resistance is still an urgent problem in the treatment of breast cancer.Therefore,elucidation of the mechanisms of metastasis and chemoresistance is helpful for individualized treatment and prognosis prediction of ER+breast cancer patients.Cytochrome C oxidase 5A subunit(COX5A)is an important subunit that regulates the assembly and activity of cytochrome C oxidase holoenzyme.The expression of COX5 A is related to the intracellular levels of adenosine triphosphate(ATP)and reactive oxygen species(ROS),which play an important role in maintaining the function of respiratory chain.It was reported that COX5 A can promote the migration and invasion of non-small cell lung cancer,and is also related to the tumorigenesis of supraglottic laryngeal squamous cell carcinoma.However,the clinical relevance of COX5 A in breast cancer is still undefined.In this project,we are going to analyze the relationship between the expression of COX5 A and the tumor stage,grade,lymph node involvement and prognosis of breast cancer patients,and aim to explore the role and mechanism of COX5 A in breast cancer proliferation,invasion and chemoresistance,and to provide a new target for breast cancer therapy.Methods:1.The relationship between the expression of COX5 A and the clinical characteristics of breast cancer patients was determined by data set analysis and immunohistochemical assay.2.The si RNAs were used to knockdown COX5 A,and p CMV/COX5 A was used to up-regulation the level of COX5 A in breast cancer cells.The m RNA and protein levels of COX5 A were detected by q RT-PCR and Western Blot,respectively.3.The effect of COX5 A on breast cancer cell proliferation,invasion and chemoresistance was evaluated by cell viability assay,wound healing assay,invasion chamber analysis.The apoptosis and cell cycle of breast cancer cells with different COX5 A levels were detected by flow cytometry.4.Real-time quantitative PCR(q RT-PCR)and Western Blot were used to detect the expression levels of AMPK/m TOR,ERα,proteins regulating apoptosis and cell cycle and EMT marker molecules in breast cancer cell lines with different levels of COX5 A.5.The fluorescence intensity of luciferase catalyzed luciferin by consuming ATP was detected to reflect the content of ATP in cells;Flow cytometry was used to detect the fluorescence intensity generated by the oxidation of DCFH-DA probe to reflect the intracellular ROS level.6.Micro RNAs that can target COX5 A and their potential binding sites were predicted through the database.Bioinformatics was used to analyze the relationship between the expression of COX5 A and miR-204 in breast cancer,and the correlation with the prognosis of patients.The targeted binding of miR-204 and COX5 A was detected by the dual luciferase reporter gene analysis.7.Mi R-204 mimic was used to up-regulate the expression of miR-204 in MCF7 and T-47 D cells,and co-transfected cells with miR-204 and p CMV/COX5 A to replenish the expression of COX5 A.The effect of miR-204 or combined with p CMV/COX5 A on breast cancer cell proliferation,invasion and chemoresistance was evaluated by cell viability assay,wound healing assay,invasion chamber analysis,and flow cytometry.Intracellular ATP and ROS levels were detected by chemiluminescence and flow cytometry after miR-204 alone or co-transfected with p CMV/COX5 A.Results:1.Relationship between COX5 A and pathological characteristics of breast cancer patientsHigher COX5 A m RNA levels were associated with higher histology grade,TNM stage,larger tumor size,lymph node involvement,and poor survival of breast cancer.Higher COX5 A m RNA expression was also found to be associated with higher histology grade and TNM stage in ER+ breast cancer.More interestingly,stratified Kaplan–Meier analysis showed that COX5 A m RNA levels were associated with poor survival in ER+ rather than ER-breast cancers.Gene collection enrichment analysis(GSEA)and immunohistochemical analysis showed that m RNA and protein levels of COX5 A were associated with lymph node metastasis in ER+ breast cancer patients.Furthermore,m RNA levels of COX5 A were inversely associated with prognosis in ER+breast cancer patients receiving chemotherapy.These results suggested that COX5 A was related to poor prognosis of ER+ breast cancer.2.COX5 A enhances the growth and invasion ability as well as chemoresistance of ER+ breast cancer cells(1)We found that si COX5A-treated MCF7 and T-47 D cells but not MDA-MB-231 cells showed lower cell viability than si NC-treated cells,which indicated that knockdown of COX5 A inhibited the proliferation of ER+ breast cancer cells but not ER-breast cancer cells.No significant changes in ESR1 m RNA levels were found between si NC-and si COX5A-treated groups;however,knockdown of COX5 A downregulated the expression of ERα protein levels in MCF7 and T-47 D cells.We also studied the AMPK/m TOR pathway and found that the phosphorylation of AMPKα was activated while m TOR phosphorylation was inhibited in MCF7 cells after downregulation of COX5 A.MCF7 and T-47 D cells treated with si COX5 A were found to have a higher percentage of cells arrested at the G1 phase,but not in MDA-MB-231 cells,suggesting that ER+ but not ER-breast cancer cells underwent G1 to S phase arrest after a decrease in COX5 A expression.Knockdown of COX5 A upregulated the expression of p53 while the levels of cyclin D1,CDK4,cyclin E1,CDK2,and p-Rb decreased in ER+ breast cancer cells.Nevertheless,no difference in either apoptosis or the expression level of apoptosis-related proteins(c-PARP and Bcl-2)was found between si NC-and si COX5A-treated groups in either MCF7 or MDA-MB-231 breast cancer cells.The wound healing assay showed that COX5 A knockdown inhibited the migration ability of MCF7 cells but not MDA-MB-231 cells.Furthermore,we found a significant reduction in the number of MCF7 but not MDA-MB-231 cells passing through the invasion chamber.Knockdown of COX5 A in MCF7 cells but not in MDA-MB-231 cells was accompanied by increased E-cadherin and decreased Slug and Snail at the m RNA and protein levels.Results of CCK-8 detection experiments showed that DOX had a stronger inhibitory effect on cell viability following COX5 A knockdown in MCF7 and T-47 D cells.In addition,ATP levels in the si COX5 A group were significantly lower than those in the si NC group,whereas ROS levels in the si COX5 A group were higher than those in the si NC group.(2)Overexpression of COX5 A promoted the growth of ER+ breast cancer cells,reduced the percentage of cells arrested in G1 phase and the expression of p53,while the levels of cyclin D1,CDK4,cyclin E1,CDK2 and p-Rb were increased;In MCF7 cells,overexpression of COX5 A inhibited the activation of the AMPKα/m TOR pathway and resulted in increased ERα protein levels.The results of invasion chamber assay showed that up-regulation of COX5 A enhanced the invasion ability and expression of Slug and Snail of MCF7 cells,while the expression of E-cadherin was inhibited.Correspondingly,in MCF7 and T-47 D cells,overexpression of COX5 A attenuated the inhibitory effect of DOX on cell viability and reduced the intracellular level of ROS.3.COX5 A is the target of miR-204(1)Bioinformatics analysis showed that miR-204 can target COX5 A through incomplete pairing and binding with the 3’UTR of COX5 A m RNA.And patients with higher expression of COX5 A m RNA had poorer outcomes,while patients with higher expression of miR-204 had better outcomes.(2)The dual luciferase reporter assay showed that firefly luciferase activity was significantly decreased in the group co-transfected with wild type(WT)COX5A 3’UTR and miR-204 compared with that in the control groups in both MCF7 and T-47 D cells.After treatment with miR-204,the expression of COX5 A and ERα was lower than that in the mimic-NC-treated groups.These results suggest that miR-204 binds to the 3′UTR of COX5 A and inhibits its expression in breast cancer cells.4.Mi R-204 inhibits the function of COX5 A in breast cancerOverexpression of miR-204 inhibited the proliferation and invasion of MCF7 and T-47 D cells,reduced the level of intracellular ATP,increased the level of ROS,and enhanced the inhibitory effect of DOX on cells.Furthermore,the proliferation and invasion ability of MCF7 and T-47 D cells,the resistance to doxorubicin,the intracellular ATP and ROS levels were restored by supplementing the expression of COX5 A,which indicated that miR-204 can target to inhibit the expression and function of COX5 A.Conclusion:1.COX5 A correlates with poor outcomes and lymph node metastasis in ER+ breast cancer patients.2.COX5 A enhances the expression of ERα,the growth and invasion ability as well as chemoresistance of ER+ breast cancer cells3.Mi R-204 can inhibit the protein expression and function of COX5 A in ER+ breast cancer cells by targeting binding to the 3’UTR of COX5 A m RNA.4.COX5 A may serve as a prognostic biomarker and therapeutic target in ER+ breast cancer.