CBX4 Exhibits Oncogenic Activities In Breast Cancer Via Notchl Signaling

Background:Breast cancer is one of the most common malignant tumors that seriously endanger women’s health worldwide.The morbidity of breast cancer in North America and Europe develop countries is the highest in female malignant tumors[1].Meanwhile,breast cancer is the most common cause of cancer-related death among women in China[2,3].Although the screening of breast cancer has been carried out in developed countries,its incidence is still increasing year by year,and it has a tendency to become younger.Breast cancer is highly invasive with poor prognosis.Its mechanism of breast cancer has not been fully defined.Previous studies have showed that he polycomb group of protein family(PcG)plays the role of oncogenes or tumor suppressor genes in tumors.Among them,CBX4 has been reported to be involved in the regulation of malignant progression of various tumors,but its role and clinical significance in breast cancer is unclear.Therefore,the study of the role of CBX4 in breast cancer has important significance for further understanding the biological characteristics of breast cancer,as well as clinical diagnosis and treatment.Materials and Methods:1.Selcetion of clinical tissue specimens179 cases breast cancer paraffin-embedded specimens:A cohort of 179paraffin-embedded samples,along with the clinicopathological information,was collected from patients received surgical resection between January 2000 and December 2010.All the tissue samples were fixed with formaldehyde and embedded in paraffin.None of the breast cancer patients had received adjuvant therapies before surgery.The use of tissues for this study has been approved by the Institute Research Medical Ethics Committees of The First Affiliated Hospital of Nanchang University.30 cases breast cancer fresh specimens:We collected 30 fresh tissue specimens of breast cancer and their corresponding adjacent tissues.The mRNA and protein expression levels of tumor-associated molecules were detected.Tissue specimens were excised and immediately placed in a-80℃refrigerator for long-term storage.2.Clinical pathological parameter analysis and statistical analysisExpression of CBX4 in paraffin tissues were determined using HE staining and Immunohistochemistry(IHC).The staining results were interpreted and recorded by two independent pathologists.T test was performed at comparion of two sample count data.Wilcoxon paired test was conducted for comparion CBX4 expression between fresh tumor specimens and adjacent tissues,х~2 test for correlation analysis between CBX4 expression and clinical data.Kaplan-Meier method was used to analyze the survival of patients with high and low CBX4 expression,and log-rank was used to test the difference in survival rate between the two groups.Multivariate COX regression analysis was carried out to determined independent influence factors affecting the overall survival of breast cancer patients.3.In vivo and in vitro biological assaysUsing qPCR and Western blotting,the mRNA and protein expression of CBX4,miR-137 and Notch1 signaling pathways in clinical breast cancer tissue samples and cell lines were examined.MTT,EdU,and Transwell assay were used to detect the ability of cells to proliferate and migrate.Dual-Luciferase Reporter Assay was applied to determine the transcriptional activity of target gene promoter or 3’UTR region.In vivo,after subcutaneous tumor formation in nude mice,the growth/volume of tumors were measured and counted.CBX4 expression was detected via IHC.Results:CBX4 expression was increased in breast cancer,compared with the non-tumor tissues.High CBX4 expression was closely correlated with tumor metastasis,advanced stage and poor overall survival in a cohort of 179 patients with breast cancer.In vitro studies demonstrated that CBX4 overexpression enhanced,whereas,CBX4 knockdown inhibited cell growth and migration.Mechanistically,in a PRC1-dependent manner,CBX4 inhibited the promoter activity of miR-137 and suppressed its expression.miR-137 decreased the expression of Notch1,Jag1 and Hey2 via targeting their 3’-UTRs.The suppression of Notch1 by siRNA or overexpression of miR-137 markedly attenuated CBX4-promoted phenotypes.Conclusinon:Collectively,these findings indicate that CBX4 promotes breast cancer via miR-137-mediated Notch1 signaling.Our data,therefore,suggest that CBX4 serve as a prognostic biomarker and that targeting CBX4/miR-137 axis may provide therapeutic potent in the treatment of breast cancer.