The Function And Mechanisms Of Histone Methyltransferase SETDB2 To Promote Breast Cancer Stem Cell Maintenance

ObjectiveBreast cancer is the most common malignant tumor in women.Breast cancer stem cells are responsible for tumor recurrence,metastasis.The histone H3K9 methyltransferase SETDB2 is involved in cell cycle dysregulation in acute leukemia and has oncogenic roles in gastric cancer.But the function in breast cancer stem cells has not been reported.We found that SETDB2 is highly expressed in breast cancer stem cells,indicated it may have a role of regulate the maintenance of breast cancer stem cells.The aim of our study was to explore the function and regulatory mechanism of SETDB2 in breast cancer stem cells.Method:1.ALDEFLUORTM Assay was used to detect the proportion of ALDH+population in SETDB2 knockdown cells by flow cytometry;2.Mammosphere formation assay analysed the pellet-forming ability in SETDB2 knockdown and SETDB2 restoring cell lines.3.The CCK8 assay and colony formation analysed the proliferation of breast cancer cells in SETDB2 knockdown cell lines.5.Orthotopic mammary adenocarcinoma xenografts analysed the effect of SETDB2 in tumor growth and tumor initiation ability.6.Western blot analysed the influence of SETDB2 on histone H3K9 level and ?Np63? protein level.7.Co-immunoprecipitation analysis of endogenous and exogenous SETDB2 interacting with ?Np63?.8.Protein stability analysis the effect of SETDB2 on ?Np63? protein level after the treatment with CHX or MG132.Result:1.Knockdown of SETDB2 reduced the proportion of ALDH+population and weakened the mammosphere formation ability in breast cancer cell lines.2.Knockdown of SETDB2 inhibited the tumor growth capacity and delayed the tumor initiation ability in situ nude mice model.3.Cell proliferation assay showed that SETDB2 had no significant effect on the proliferation ability of breast cancer cells.4.Knocking down of SETDB2 did not affect the global H3K9 level in cell lines.5.RNA-Seq analysis found that knockdown of SETDB2 down regulated mRNA levels of hedgehog signaling pathway related genes PTCH1,GLI2 and CXCR4.Western blot showed that knocking down SETDB2 reduced the ?Np63? protein level.6.Co-IP assay showed the interaction between SETDB2 and ?Np63?.7.Protein stability assay showed that SETDB2 stabilized ?Np63? protein level and protect it from degradation.Conclusion:Histone methyltransferase SETDB2 promoted breast cancer stem cell maintenance by interaction with and stabilization of ?Np63? protein and participate in the hedgehog signaling pathway.SETDB2 has an positive regulation on breast cancer stem cell mammospheres formation,promotes tumor growth and tumorigenesis.Our study reveals a novel function of SETDB2 in cancer stem cell maintenance in breast cancer.Figure[12]table[26]reference[63]...