Functional Study And Molecular Characteristics Of Quiescent Cancer Stem Cells With Chemoradiotherapy Resistance In Breast Cancer Of MMTV-PyMT Mice

A small population of cancer stem cells in quiescent state are able to survive in resistant to chemoradiotherapy,which is the main reason for the incomplete eradication of tumor.They can remain quiescent after the treatment is stopped,but once they restore the cell cycle,they will then cause tumor relapse or metastasis.In our previous reports,we showed that SET domain-containing protein 4(SETD4)epigenetically regulates quiescence of diapause embryos in Artemia and cancer stem cells(CSCs)in human breast cancer cell lines by facilitating heterochromatin formation via H4K20me3 catalysis.However,Artemia is an inferior arthropod,and cell lines have left the regulation of internal environment in the body,neither of which can reflect the pathological state of cancer patients.To well simulate the complex situation of clinical breast cancer patients,and clarify the specific characteristics of quiescent CSCs,here we not only isolated quiescent CSCs using chemoradiotherapy resistance assays on tumors of MMTV-Py MT transgenic mice,but also explored their functions in chemoradiotherapy resistance and specific molecular characteristics.In this study,we found that quiescent CSCs exhibited high capacity of tumorsphere formation in vitro and tumor-initiation in vivo beyond activation.In addition,quiescent CSCs specifically expressed high levels of SETD4 but lacked Ki67.Quiescent CSCs were showed to be resistant to chemotherapy treatment in either in vivo or in vitro models.Similarly,SETD4 overexpression caused primary cancer cells to exhibit clear chemoradiotherapy resistance.Subsequent analysis of chromatin structure showed reduced degree of H3K9 ac modification,increased degree of H4K20me3 modification and a high proportion of heterochromatin in quiescent CSCs,compared with that of activated CSCs.Transcriptomic analysis revealed that quiescent CSCs were in a state of mitotic cycle arrest,low energy metabolism,and reduced cellular response to radiotherapy stimulation.Further analysis showed that the molecular processes associated with cellular quiescence included those of cell cycle,cell proliferation and Wnt/β-catenin signaling pathway.Taken together,we showed that SETD4 marks quiescent CSCs,and revealed their other molecular characteristics,which not only made up for the previous inability to label them,but also helps to explore their targets for the clinical treatment.