Research On Functional Mechanism Of GABRP In Breast Cancer Stem Cells And Chemotherapy Drug Sensitivity

Objectives:Triple-negative breast cancer is a kind of breast cancer with high degree of malignancy,strong heterogeneity,and poor prognosis.Chemotherapy is currently the mainstay of systemic medical treatment for TNBC.Chemotherapy drugs can not kill tumor stem cells,leading to the accumulation of tumor stem cells that contribute to treatment resistance and relapse.Thus,tumor stem cells are one of the difficulties in the research of TNBC treatment.The elucidation of pathways or molecules that regulate BCSC will provide new therapeutic strategies for TNBC.We previously found that GABRP gene was highly expressed in TNBC.Further bioinformatics analysis revealed that high levels of GABRP are associated with poor clinical outcomes in TNBC patients.Moreover,we observed that the profile of GABRP-overexpression group enriched for genes associated with stemness using GSEA analysis.These results suggest that GABRP promotes tumor progression by modulating cancer cell stemness.Our findings reveal that GABRP is a promising strategy to inhibit tumor progression and sensitize TNBC to chemotherapeutic agents.Methods:1.The mRNA expression level of GABRP was detected by RT-PCR in human breast cancer cell lines and breast cancer stem cells.2.Knockdown of GABRP was performed by lentiviral transfection system in HCC1806 and HCC1937,and the knockdown efficiency was detected by western blot and qRT-PCR.The stable overexpression GABRP cell line was constructed in MDA-MB-231,and the overexpression efficiency was detected by western blot and qRT-PCR.3.MTS and colony information assay were performed in the stable overexpression GABRP cell line,and ALDH assay was used to detect the regulation of GABRP on cancer stem cell marker.4.The cell viability of knockdown GABRP cell line treated with chemotherapy drugs(paclitaxel,doxorubicin)was detected by MTS assay.5.Treatment by conventional chemotherapies,including PTX(1 nM),DOX(5 nM),and cisplatin(100 nM),significantly enriched the BCSCs,then the clone formation assay was performed after silencing GABRP in HCC1806.Treat GABRP knockdown cell line with paclitaxel(1 nM),CD24lowCD44high flow cytometry analysis was performed to detect the numbers of cancer stem cells.6.The mRNA expression level of key transcriptional regulators of stemness genes was detected by qRT-PCR after silencing GABRP in HCC1806.7.The mRNA expression level of the gene in BCSC self-renewal pathways(Hh,Wnt,Notch)was detected by RT-PCR after silencing GABRP in HCC1806.8.The protein expression of GABRP,EGFR,phosphorylated-ERK,and ERK were detected by western blot in paclitaxel-induced stemness-enriched cells.Results:1.GABRP gene was highly expressed in TNBC.2.GABRP was highly expressed in the breast cancer stem cell population.3.GABRP overexpression promoted the ability of self-renew and caused TNBC cells to be resistant to chemotherapy drugs.The ability of self-renew was decreased when GABRP knockdown in TNBC cells,and caused TNBC cells to be sensitive to chemotherapy drugs.4.GABRP overexpression increased the mRNA expression of key transcriptional regulators of stemness genes.5.The expression of GABRP,EGFR,and phosphorylated-ERK increases when TNBC cells exposing to low dose paclitaxel.6.GABRP knockdown decreased the mRNA expression of HHAP.SHH and IHH(Hh signaling pathway).Conclusion(s):We found that GABRP is highly expressed in BCSC.GABRP can maintain the stemness of BCSCs,and the overexpression of GABRP did not affect the proliferation ability of TNBC.Upon GABRP knockdown,the increase in colony formation ability induced by chemotherapy was significantly inhibited,and GABRP knockdown caused TNBC cells to be sensitive to chemotherapy drugs.GABRP overexpression increase the expression of stemness-related genes,including SOX2,OCT4,NANOG,ALDH1A1 and ALDH1A3.Among the key signaling pathways involved in the regulation of CSC,GABRP mainly affects the expression of related molecules in the Hh signaling pathway.