The Mechanism Of CCDC170 In Breast Cancer Cell Apoptosis And Its Clinical Significance

ObjectiveBreast cancer is one of the most common malignant tumors endangering women’s health in the world.In China,breast cancer incidence rate is also the leading cause of cancer incidence and mortality.Genome-wide association study found that multiple SNPs related to the breast cancer risk were located around CCDC170.But there is still little research on CCDC170.The purpose of this study was to investigate the clinical significance and functional mechanism of CCDC170 in breast cancer,explore whether it can affect cell apoptosis through IRE1/XBP1 pathway and participate in drug resistance,explore the clinical significance of its binding protein Hsp90β and their correlation,and further clarify the molecular mechanism of CCDC170 on breast cancer cells.MethodCluspro2.0 predicted the protein structure of CCDC170 and analyzed its binding with ERα.Microarray and bioinformatics analyses identified the genes after CCDC 170 overexpression.The correlation of CCDC 170,IRE1 and XBP1 at mRNA level was analyzed in public data.Breast invasive ductal carcinoma tissues were selected for IHC assay.The correlation between CCDC 170,IRE1α,XBP1 and clinicopathological data were analyzed.Then,they were verified in TCGA and GEO data.Annexin V-FITC,TUNEL and Western blot were used to detect the effect of CCDC 170 on cell apoptosis.IF experiment was to observe the co-localization of proteins.The effect of CCDC 170 on drug sensitivity was observed by MTT assay.The binding protein Hsp90β was identified by IP,MS,and then Co-IP verification.The role of Hsp90β on CCDC 170 stability was explored by CHX experiment.Categorical variables were compared using the Chi-square test,while continuous variables were analyzed using nonparametric tests(Kruskal-Wallis test and Mann-Whitney test),one-way analysis of variance(ANOVA)and Student’s t-test.Kaplan-Meier analyses and log-rank tests were performed to estimate OS and DFS.All hypothetical tests were two-sided,and P-values less than 0.05 were considered statistically significant in all tests.Result1.Protein structure prediction indicated that CCDC170 and ERα have binding sites.Positive correlation was found between CCDC170 and ESR1 mRNA expression in breast cancer cells and tissues.After CCDC 170 overexpression in breast cancer cells,IRE1 gene changed significantly,and the differentially expressed genes could be enriched into the apoptotic signaling pathway.2.Expression of CCDC 170,IRE1α and XBP1s in breast cancer IRE1α was positively correlated with CCDC170(r=0.233,P=0.020)and XBP1s(r=0.212,P=0.034).CCDC170 was also correlated with XBP1s(r=0.339,P=0.001)positively.CCDC170 was positively with ERα(r=0.389,P=9.90×10-5);IRE1α was positively with Her-2(r=0.293,P=0.003)and Ki-67(r=0.208,P=0.038);XBP1s was positively with ERα(r=0.286,P=0.004).The expression of CCDC170(P=0.006)and IRE1α(P=0.003)were different in different molecular types.Patients with high CCDC 170 expression had better prognosis.TCGA and GEO data analysis were consistent with protein level results.3.CCDC 170 regulated IRE1 signaling pathway:Overexpression of CCDC 170 resulted in increased expression of IRE1β and XBP1s,and vice versa.4.Overexpression of CCDC 170 increased apoptosis of breast cancer cells,and vice versa.Under endoplasmic reticulum stress in vitro,the expression of IRE1 increased,and CCDC 170 promoted cell apoptosis more significantly.5.CCDC 170 bound to HSP90β:HSP90β was identified as the binding protein of CCDC 170 by IP and mass spectrometry,then it was confirmed by Co-IP.Exploring the clinical significance of HSP90β,we found that the HSP90β level was correlated with tumor size(r=0.212,P=0.034),Grade(r=0.275,P=0.016)and PR(r=-0.234,P=0.019).Overexpression or down-regulation of CCDC170 could not change the expression of HSP90β,but knockdown of it could lead to the down-regulation of CCDC 170 expression.CCDC 170 degraded more rapidly in HSP90β knockdown group than in control group.Conclusion1.CCDC 170 was related to ERα closely,and the positive correlation between them in breast cancer cells and tissues.The clinical significance of CCDC 170 in breast cancer is worth discussing.2.IRE1 was the gene that changes significantly with CCDC170,and differentially expressed genes regulated by CCDC170 can be enriched into the apoptotic signaling pathway.CCDC170 was positively correlated with IRE1 and XBP1 at mRNA and protein levels.3.In breast cancer,the high expression level of CCDC 170 indicates a better prognosis,and the same conclusion can be obtained in TCGA and GEO data mRNA levels.4.At the cellular level,CCDC170 promotes the apoptosis of breast cancer cells,which is more obvious under endoplasmic reticulum stress.5.The binding protein HSP90β of CCDC 170 was correlated with breast tumor size,histological grade and PR expression.The degradation rate of CCDC 170 increased with the knockdown of HSP90β,suggesting that CCDC 170 may be a new customer protein of HSP90β.