The Mechanism Of HES1/Slug Axis Regulating Stemness Properties Of Breast Cancer Stem Cells In Triple Negative Breast Cancer

Objective:Breast cancer is the most common malignant tumor in Chinese women,which is the leading cause of cancer-related death.Triple negative breast cancer(ER-,PR-,HER2-,TNBC)is the worst prognostic subtype of breast cancer due to the lack of effective therapeutic targets.Triple negative breast cancer is a highly heterogeneous tumor because it is enrichment with breast cancer stem cells(BCSCs).Breast cancer stem cells(BCSCs)are a small number of cell subpopulations with strong self-renewal ability,continuous proliferative capacity and multi-directional differentiation potential,which are also considered to be the source of the initiation,malignant process,treatment resistance and early recurrence and metastasis of breast cancer.The regulation mechanism of stemness properties of BCSCs in triple negative breast cancer remains unclear.Thus,the researches aiming to reveal specific stemness-related factors and regulation mechanism in BCSCs of triple negative breast cancer are more important theoretical and clinical significance.HES1 is a basic helix loop helix(b HLH)transcription factor,which is implicated in many biological processes,especially in maintaining undifferentiation status of stem cells.HES1is a bidirectional transcription factor participating in the progression of many malignant tumors,which is also a downstream targeted effector of many stemness-related signaling pathways.HES1 may play an important role in maintaining the self-renewal ability of stemness properties.Our previous published study has confirmed that HES1 is upregulated in breast cancers,which indicates worse prognosis and is closely related to triple negative subtype;HES1 can activate the occurrence of epithelial mesenchymal transition(EMT)and promote the migration and invasion of breast cancer cells.Up to now,there is no reports about the biological role of HES1 in triple negative breast cancer and its regulation mechanism on stemness properties of breast cancer stem cells in triple negative breast cancer.In this study,we clarified that HES1 regulated Slug(an EMT-driver)expression,and analyzed the biological role of HES1 and Slug in triple negative breast cancer clinical tissues;elucidated the mechanism of HES1/Slug axis regulating stemness properties of breast cancer stem cells,which is expected to find out the specific stemness-related factors and mechanisms in triple-negative breast cancer,providing theoretical basis for novel prognostic markers and specific therapeutic targets of triple negative breast cancer.Methods:1.Biological role of HES1 and Slug in triple negative breast cancers:The expression of HES1 were detected by immunohistochemistry in 150 cases of triple negative breast cancer tissue samples and the correlations between HES1 and clinicopathological factors and survival prognosis was analyzed.In triple negative breast cancer cell lines,Western blot and RT-PCR were used to identify HES1 regulating Slug(an EMT-driver).Then,the expression of Slug was detected by immunohistochemistry in150 cases of triple negative breast cancer tissue samples and the correlations between Slug and clinicopathological factors was analyzed.Kaplan-Meier survival analysis was used to evaluate the correlation between Slug and survival prognosis in triple negative breast cancer patients.The correlation between HES1 and Slug expression in 150 cases of triple negative breast cancer tissue samples was evaluated by Pearson correlation analysis.2.Molecular mechanism of Slug expression regulated by HES1:potential binding sites of HES1 in Slug promoter region were predicted by bioinformatics analysis.Chromatin immunoprecipitation assay(Ch IP)was used to verify that HES1 can bind to Slug promoter.Luciferase dual reporter assays was used to verify the regulation of HES1on the transcriptional activity of Slug promoter.3.In vitro experiments,HES1 promoted stemness properties of triple negative breast cancer stem cells via upregulating Slug:we constructed triple negative breast cancer cell lines with stable knockdown of HES1,knockdown of HES1 and overexpression of Slug as well as negative control.Based on these above cell lines groups,the expressions change of stemness markers SOX2,OCT4 and Nanog were detected by Western blot analysis.Sphere-formation assay and colony formation assay were used to detect the change of cell BCSCs self-renewal ability and breast cancer cells proliferation,respectively.The CD44high and CD24 low subpopulation change of tumorspheres were detected by flow cytometry.4.In vitro experiments,HES1 promoted migration and invasion of triple negative breast cancer cells via upregulating Slug(an EMT-driver):we constructed triple negative breast cancer cell lines with stable knockdown of HES1,knockdown of HES1 and overexpression of Slug as well as negative control.Based on these above cell lines groups,the expression change of E-cadherin and N-cadherin were detected by Western blot and the change of cancer cells migration and invasion were detected by Transwell analysis.5.In vivo experiments were performed to confirm the regulation of HES1/Slug axis promoting stemness properties of breast cancer stem cells in triple negative breast cancer using xenograft model.Results:1.HES1 and Slug were oncogenic prognostic factors in triple negative breast cancer:There was a significant correlation of HES1 expression with tumor size(p=0.0262),lymph node metastasis(p=0.0129)and advanced TNM stage(p=0.0001).High expression of HES1 can predict worse prognosis.In TNBC cell lines(MDA-MB-231 and HCC-1937),the m RNA and protein levels of Slug were the most significantly downregulated with loss of HES1 compared with other EMT-drivers(Snail,ZEB1,ZEB2,Twist).There was a significant correlation of Slug expression with tumor size(p=0.0092),lymph node metastasis(p=0.0060)and advanced TNM stage(p=0.0005).High expression of Slug can predict worse prognosis.There was a significant positive correlation between the expression of HES1 and Slug.The effect of HES1 on the survival of triple negative breast cancer is largely controlled by Slug expression.2.HES1 functioned transcriptional activator in regulating expression:Bioinformatics prediction from UCSC and JASPAR database found that Slug had three putative HES1binding elements(BEs)including BE1:5?-AAACCCAGGTGCCTA-3?;BE2:5?-ATTTGCACGCGGCCGC-3?;BE3:5?-AGAGCGTGGA-3?.The Ch IP results revealed that the binding of anti-HES1 antibody was significantly enriched in BE1 and BE3,and there was no enrichment detected with BE2 and Ig G control.Dual luciferase gene reporter assay was conducted to confirm that HES1 binding(BE1 and BE3)enhances Slug promoter transcriptional activity.3.HES1 contributed to BCSCs stemness of TNBC through Slug in vitro:In TNBC cell lines(MDA-MB-231 and HCC-1937),the levels of stemness-related proteins such as SOX2,OCT4,Nanog were downregulated with loss of HES1,but restored when Slug was overexpressed upon HES1 knockdown.HES1 knockdown remarkably reduced the capacity of tumorsphere formation and the ability of clonal formation and in TNBC cell lines(MDA-MB-231 and HCC-1937),but which were restored by upregulating Slug.BCSCs subpopulation(the ratio of CD44 high CD24 low cell fractions)were clearly decreased upon HES1 knockdown in forming tumorspheres of TNBC cell lines(MDA-MB-231 and HCC-1937),whereas also restored by Slug overexpression.4.HES1 contributed to cell migration and invasion in TNBC through Slug(an EMT-diver)in vitro:In TNBC cell lines(MDA-MB-231 and HCC-1937),the level of E-cadherin protein was upregulated and the level of N-cadherin protein was downregulated with loss of HES1,but restored when Slug was overexpressed upon HES1 knockdown.HES1knockdown remarkably reduced the abilities of cell migration and invasion in TNBC cell lines(MDA-MB-231 and HCC-1937),but which were restored by upregulating Slug.5.HES1/Slug axis regulated BCSC stemness of TNBC in vivo:transplantations of BCSC populations with limiting dilution assay(1�10~3and 1�10~4 cells per mouse)revealed that the tumor formation probability was severely reduced upon HES1 knockdown and almost rescued by Slug overexpression.HES1 down-regulation decreased BCSCs-derived tumor size and weight and the effects were restored by Slug overexpression in xenograft models with the limiting dilution of 1.0�10~5 cells per mouse.Conclusion:1.HES1 and Slug expressions are specifically high and as oncogenic prognostic factors in TNBC.2.HES1 is a novel transcriptional activator for Slug.HES1promotes Slug gene transcription via binding to Slug promoter region.3.HES1 promotes BCSCs stemness properties via targeting Slug in TNBC.4.HES1 contributes to cell migration and invasion via upregulating Slug(an EMT-driver)in TNBC.The above highlights that HES1/Slug axis might be a novel candidate for BCSCs stemness regulation in TNBC and providing new clues for identifying promising prognostic biomarkers and therapeutic targets of TNBC.