Study Of SIPA1 Regulating Breast Cancer Cell Stemness Characteristics And Its Effects On Chemotherapy

Breast cancer is the most common tumor type in female,and either the number of new cases or death cases ranks first every year.Breast cancer stem cells are one of the subpopulations that exist in breast tumor tissues to maintain self-renewal and differentiation.It has been widely reported that the existence of breast cancer stem cells is the main cause of distant metastasis,chemotherapy resistance and recurrence for breast cancer patients.In the previous studies,SIPA1(signal-induced proliferation-associated protein 1)has been found as one of members of the small G protein family.In addition to the function of inactivating the actived Rap1 protein in the cell,numbers of studies have reported that SIPA1 is involved in regulating the process of the metastasis and recurrence of lymphoma,prostate cancer,breast cancer or other malignant tumors.To make clear if SIPA1 can regulate chemotherapy resistance and recurrence for breast cancer,breast cancer patients’ clinical data with more than 5 years’ follow-up records were collected and their tissue chips were used for immunohistochemical staining and survival analysis in the thesis.The results showed that SIPA1 protein expression level was significantly positively correlated with breast cancer metastasis and recurrence.The patients with high SIPA1 protein expression behabed lower 5-year survival rate and worse prognosis.To clarify the mechanism of how SIPA1 causes treatment failure,recurrence and metastasis in breast cancer patients,breast cancer cell lines MDA-MB-231,BT-549 and MCF7 with different SIPA1 protein expression levels and their derived cell lines that knock down or overexpress SIPA1 were used for further research.After comparing the cancer stem cell characteristics of these cell lines,it was found that the higher SIPA1 protein expression in the breast cancer cells,the stronger the cells were able to form tumorspheres in salmon fibrinogen gel cuture system.And the cells with higher SIPA1 expression showed less sensitive to anthracycline chemotherapeutics.These results indicate that SIPA1 protein could enhance the stemness characteristics of breast cancer cells,and lead to worse prognosis of breast cancer patients.To explore how SIPA1 regulates breast cancer cell stemness characteristics,chromatin immunoprecipitation of SIPA1 protein was performed in MDA-MB-231 cells,and a large number of candidate genes that bind to SIPA1 were retrieved.Through cluster analysis and intersection analysis of the candidate gene,CD44 gene,one of the typical breast cancer stem cell markers,was found to interact with SIPA1.This interaction was confirmed by chromatin immunoprecipitation and identified that SIPA1 could bind to the promoter region of CD44 gene.Continuously,the luciferase reporter gene assay was implemented and the results showed that SIPA1 could enhance the transcription activity of CD44 gene.Next,by using Western blot,fluorescent immunostaining and flow cytometry,it is confirmed that high expression of SIPA1 could up-regulate not only the expression of CD44 but also the membrane CD44 molecule in breast cancer cells.In addition,the m RNA levels of stemnessrelated transcription factors OCT4,NANOG,POU5F1 and BMI1 in MDA-MB-231,BT-549 and MCF7 and their derived cell lines were detected by q RT-PCR,and the results indicated that the expression of these transcription factors were up-regulatd by SIPA1 protein.Then the detection of key molecules that regulate breast cancer stem cell signaling pathways was done and the data showed that both the m RNA level and phosphorylation level of SMAD3 in the TGF-β pathway were significantly increased by SIPA1’s regulating.In vivo tumor formation experiments in nude mice were performed and the results confirmed that SIPA1 knockdown cells,MDA-MB-231/sh-SIPA1,had less tumorigenic ability compared with MDA-MB-231 cells had.After isolating the tumor cells from the tumorigenic mice,the molecules related to that of stem cell characteristics were detected.The results showed that the m RNA levels of CD44,SMAD3 and transcription factors in MDA-MB-231/sh-SIPA1 cells were much lower than MDA-MB-231 cells.These results indicated that SIPA1 protein could enhance the stemness characteristics of breast cancer cells in vivo and in vitro by upregulating the expression of the typical breast cancer stem cell marker molecule CD44 and activating SMAD3-mediated signaling pathway.To further analyze the regulation of SMAD3-mediated signaling pathway on the drug resistance in breast cancer cells with high expression of SIPA1,the phosphorylation inhibitor of SMAD3,SIS3(specific inhibitor of SMAD3),which has been reported to inhibit the activity of TGF-β pathway,was used in the following experiments.After treating the SIPA1 high-expression MDA-MB-231,BT-549 and MCF7/SIPA1 cells with 3 μM SIS3,it was found that the tumorsphere formation ability of the cells was reduced,and the sensitivity to chemotherapy drugs was increased.The m RNA levels of the typical stemness-related transcription factors were reduced in SIS3 treated cells.Even more,when adding the SIS3 into the 5 days’ tumurspheres of MDA-MB-231 cell,the growth of the tumurspheres was observed to stop.Meanwhile,with the co-treatment of epirubicin and SIS3,MDA-MB-231 cell tumurspheres were observed to disintegrate gradually,and presented more sensitive to epirubicin treatment.These results indicated that SIS3 could reverse epirubicin resistance in the breast cancer cells with high SIPA1 expression.Taken together,this study revealed a mechanism that SIPA1 could promote and maintain the stemness characteristics of breast cancer cells by increasing the expression of CD44 and SMAD2/3.Meanwhile,blocking the phosphorylation of SMAD3 could suppress the breast cancer cell stemness and increase the sensitivity to chemotherapy in breast cancer cells expressing a high level of SIPA1.These results provide a novel strategy to improve the therapeutic effect of breast cancer.