OR2T6 Promotes Breast Cancer Progression Via Initiating Epithelial-mesenchymal Transition And MAPK/ERK Pathway

Research purposes:Breast cancer is the most common malignant tumor in Chinese women.In recent years,the incidence of breast cancer increased year by year,which has become a serious threat to women's life and health.Although the mortality rates have declined in the past decades due to advanced screening program and adjuvant treatments,the relapse rate is still high and the prognosis is poor.However,the molecular mechanism has not been fully elucidated,which limits the individualized treatment and prognostic evaluation of breast cancer patients.Therefore,it is still an important premise for prevention and control of breast cancer to explore the relevant signal molecules and clarify the mechanism of breast cancer development.ORs(Olfactory receptors),as a large superfamily of G protein-coupled receptors(GPCRs),are found to be located on olfactory neurons initially and play a key role in the process of recognition odors.Recently some researches reveal that ORs can be ectopic expressed in testis,liver,fat,adrenal gland,kidney,heart and other non-olfactory tissues and involve in the development of some tumors such as prostate cancer,colon cancer,lung cancer,melanoma,leukemia and so on.In addition,the biological functions of ORs are not consistent among malignant tumors.The same receptor even plays a "dual role" of either promoting or inhibiting cancer in different tumors.The expression of ORs in breast cancer has also been reported in a few cases.The overexpression of 34 OR genes was reported in breast tumors with the CHEK21100delC mutation.The data of a large number of breast cancer samples shows the gene amplification of some OR molecules in breast cancer tissues and cells.And the expression of.many molecules from the same olfactory receptor family,such as OR2B6,OR2W3,OR2T8,are abnormally overexpressed.This implied that ORs may be a potential biomarker of breast cancer.The previous study showed ORs was related to the abnormal DNA methylation of breast cancer cells,which may induce tamoxifen resistance in tumor cells.These findings suggested that ORs may play a role in the development of breast cancer.However,there are few reports about the effect and regulatory mechanism of ORs in the biological function of breast cancer cell.It is significant to clarify the function and mechanism of ORs for the diagnosis and treatment of breast cancerOur previous study showed a large number of differentially expressed genes with high-throughput gene expression profiling between the tumors with lymph node metastasis and non-metastatic group,and in the metastatic group,the expression of OR2T6 was significantly higher compared with the non-metastatic group.However,the role and mechanism of OR2T6 in tumors are not clear.In this study,the expression of OR2T6 mRNA in fresh breast cancer and normal breast tissue were further detected.The expression of OR2T6 protein were examined in 102 cases of paraffin embedded breast cancer and 60 cases of normal breast tissue by immunohistochemistry and the relationship among the OR2T6,prognosis and clinicopathological parameters were analyzed.In vitro experiments,we studied the effects of OR2T6 on the proliferation,apoptosis,invasion and migration of breast cancer cells and EMT by EdU,Transwell and flow cytometry.In order to explore the molecular mechanism of OR2T6 promoting breast cancer cell proliferation and inhibiting apoptosis,we preliminarily verified the regulatory effect of OR2T6 on MAPK/ERK signaling pathway by high-throughput mRNA expression microarray screening,Real-time quantitative PCR and Western blotMethods:1.The expressions of OR2T6 mRNA were detected in 41 fresh breast cancer tissues and 9 normal breast tissues by Real-time quantitative PCR2.The expressions of OR2T6 protein were detected in 102 breast cancer pareffin tissues and 60 normal breast tissues by immunohistochemistry,and the relationship between the expression of OR2T6 and clinic pathological parameters was studied.3.The effect of OR2T6 on the overall and disease-free survival was investigated by performing the Kaplan-Meier curve and the log-rank test.Cox regression analysis was applied to assess the impact of OR2T6 on the prognosis of breast cancer patients.4.The genetic alterations of OR2T6 gene in breast cancer were studied comprehensively by analyzing multiple datas in the cBioPortal cancer gene database 5.The effect of OR2T6 on cell proliferation and apoptosis was investigated by EdU and flow cytometry after transfection of pcDNA3.1-OR2T6 plasmid and siRNA into breast cancer cells.6.The effect of OR2T6 on cell invasion and migration was investigated by Transwell assay after transfection of pcDNA3.1-OR2T6 plasmid and siRNA into breast cancer cells.7.The role of OR2T6 in EMT process was investigated by Western blot analysis after transfection of pcDNA3.1-OR2T6 plasmid and siRNA for further study on cell migration and invasion8.The MCF-7 breast cancer cell line was transfected with OR2T6 siRNA or negative control and collected for mRNA gene microarray,then the screened differentially expressed genes were submitted for GO and KEGG enrichment analysis to investigate the downstream signaling pathways regulated by OR2T6 with DAVID online tool And the results were validated by Real-time quantitative PCR and Western blot.Results:1.The expression of OR2T6 in breast cancer tissues.The results of Real-time quantitative PCR showed the transcripts of OR2T6 was significantly higher in 41 cases of fresh breast cancer tissues than in 9 cases of normal breast tissues.Immunohistochemistry analysis showed that the expression of OR2T6 protein was upregulated in 102 breast cancer paraffin samples compared with 60 normal breast samples.2.The relationship between OR2T6 and clinicopathological parameters of breast cancer.The data was assessed with Chi-square test or Fisher exact test and indicated high expression of OR2T6 was related to lymph node metastasis and higher TNM stage,not related to age,tumor size and histological grade.3.The relationship between OR2T6 and prognosis of breast cancer patients.The Kaplan-Meier curve and log-rank test were used to attest that patients with high OR2T6 expression had poorer overall survival and disease-free survival.Furthermore,univariate and multivariate Cox regression analysis suggested that OR2T6 was a significant predictor together with lymph node metastasis and TNM staging,but not an independent prognostic factor4.The genetic alterations of OR2T6 gene in breast cancer.The cBioPortal for cancer genomics provided mass data and a comprehensive analysis.In 10 breast cancer studies,the genetic variation rate of OR2T6 gene was 9.21%on average in 8023 breast cancer patients.In the cases of genetic change,734 cases had gene amplification,5 cases had mutation,and 4 cases had deletion.5.The effects of OR2T6 on the proliferation and apoptosis of breast cancer cells After transfection of OR2T6 overexpression plasmid into MCF-7 and MDA-MB-231 breast cancer cells,the proliferation ability was significantly enhanced and apoptosis was inhibited.In the breast cell lines of knocking out OR2T6 gene,the proliferation cells were decreased and apoptosis cells were increased.6.The roles of OR2T6 on the migration and invasion of breast cancer cells.After transfection of OR2T6 overexpression plasmid into MCF-7 and MDA-MB-231 breast cancer cells,the capacity of migration and invasion was enhanced.The ability was weakened with interfering the expression of OR2T67.The role of OR2T6 in the EMT process of breast cancer cells.With the increase of OR2T6 in MCF-7 and MDA-MB-231 breast cancer cells,the expression of epithelial marker E-cadherin was reduced,and the expression of mesenchymal markers N-cadherin,Vimentin and ?-catenin were increased.With knocking out OR2T6 gene in breast cancer cells,the expression of E-cadherin was enhanced and the expression of N-cadherin,Vimentin and ?-catenin were decreased.8.The regulation of OR2T6 in promoting breast cancer cell proliferation and inhibiting apoptosis.The human gene expression microarray was used to screen differentially expressed genes.The mRNA levels of 28186 genes were detected between the breast cancer cells transfected with OR2T6 siRNA and negative control Bioinformatic analysis revealed that 13,452 genes were up-regulated,while 14,734 genes were down-regulated in the siOR2T6 group compared with the control group.385 genes were identifed to be differentially expressed genes(DEGs)because the expression level changed more than 2 times.In these DEGs,213 genes were up-regulated and 172 genes were down-regulated.GO analysis showed that the DEGs were mainly enriched in regulation of biological process,cellular response to chemical stimulus and biological adhesion.KEGG analysis suggested that the DEGs were enriched in cAMP,calcium signaling pathway and tumor-related pathways including Rapl,PI3K-AKT and Ras signaling pathways.RT-qPCR was conducted in MCF-7 cells to validate the mRNA levels of H-Ras,c-Myc,ERK,and c-fos genes,which belong to the MAPK/ERK pathway,were decreased after interference with OR2T6 expression.Western blot analysis further proved that when we knocked down OR2T6 gene expression,the protein signals of the above genes were reduced significantly.These results suggest that OR2T6 may promote breast cancer cell proliferation and inhibit apoptosis by regulating MAPK/ERK signaling pathway.Conclusion:1.The expression of OR2T6 is significantly higher in breast cancer tissues than in normal breast tissues.High expression of OR2T6 is associated with lymph node metastasis and TNM staging,which suggests a poor prognosis in breast cancer patients.2.OR2T6 could promote proliferation,invasion,migration and inhibit apoptosis of breast cancer cells.3.OR2T6 may enhance the migration and invasion of breast cancer cells by promoting the EMT process.4.OR2T6 may be involved in breast cancer progression via initiating the MAPK/ERK signaling pathway.