| Malignant cancer is a major refractory disease affecting human health.Due to the resistance to current traditional chemoradiotherapy and immunotherapy,quiescent cancer stem cells(CSCs)are responsible for malignant tumor formation,tumor recurrence and metastasis postoperatively.Current clinical tumor therapeutics often target most active tumor cells but fail to eradicate therapy-resistant CSCs in quiescent state.Since any quiescence-linked therapy resistance would be removed if such CSCs could be activated and the eradication of quiescent CSCs could eliminate the malignancy of tumor,strategies of tumor therapy are therefore considering the activation of quiescent CSCs for eradication or the maintenance of CSCs quiescence.Since the number of quiescent CSCs is very small and the quiescent CSCs are in extreme low cellular metabolic state,there have been few reports on malignant cancer therapy targeting quiescent CSCs.In the previous studies,we established a research model of cell quiescence and activation during the Artemia dormant embryos formation.We found that DEK,as a key regulatory factor,epigenetically regulates the activation of Artemia dormant embryos and we also elucidated the molecular mechanism of its regulation.This study lays an important foundation for the regulation of quiescent CSC activation and the application of tumor therapy targeting quiescent CSCs.In this study,we isolated and identified three types of quiescent breast CSCs from two types of mouse breast tumors(4T1 and EMT6)and one type of human breast tumor(MCF7)in cancer cell line derived xenograft(CDX)mice using assays of chemoradiotherapy resistant selection.After the removal of chemical drugs,the activated quiescent CSCs showed over 88% tumorsphere formation capacity in vitro by single cells and 100% tumor formation ability in vivo by 10 cells.By analysis of immunofluorescence and Western blot,we found that DEK was deficient in quiescent CSCs and highly expressed in activated CSCs.Experiments of DEK knockdown and rescue in a CSCs showed that DEK plays an important role in the activation of quiescent CSCs.In addition,supplementation of in vitro synthesized DEK protein to quiescent CSCs showed that exogenous DEK could enter the cells and induce the activation of quiescent CSCs.The results suggest that DEK is a key activator of CSCs.Based on the above results,we begun to study the molecular mechanism of quiescent CSC activation triggered by DEK.Firstly,transmission electron microscopy analysis and detection of related histone modification showed that the level of heterochromatin significantly decreased and the level of euchromatin significantly increased after DEK induced quiescent CSC activation.Next,we performed Ch IP-seq using specific anti-DEK antibody.The results showed that DEK binding peaks were widely distributed on all 23 human chromosomes and highly enriched near the transcription start site with 64.35% in promoter regions,and annotated DEK-bound genes were mainly involved in protein binding,intracellular signal transduction and organism development.Analysis of ATAC-seq showed that DEK induced quiescent CSC activation exhibited a higher chromatin accessibility and widespread gene active.Finally,we performed RNA-seq between quiescent and activated CSCs and revealed the upregulated genes and downregulated genes in the activation of quiescent CSCs caused by DEK.As the downstream target genes and signaling pathways regulated by DEK,we verified the molecular mechanism involved in up-regulation of MYC targets and down-regulation of p53 signaling pathway induced by DEK.These results clarify the epigenetic mechanism in DEK induced quiescent CSC activation.Based on the above results,we continued to perform a study on the elimination of tumor malignance by activating quiescent CSCs by DEK to negate their resistance to therapy and eradicating them combined with chemoradiotherapy.In vitro experiments showed that the supplementation of DEK protein could effectively activate and kill all quiescent CSCs under the condition of chemoradiotherapy.To achieve the effective delivery of DEK protein in immune-competent mice,we constructed DEK-containing exosomes with excellent biocompatibility and immune evasion.In vitro experiments showed that the supplementation of DEK-containing exosomes could also effectively activate and kill all quiescent CSCs under the condition of chemoradiotherapy.Finally,using two types of murine breast cancer model(4T1 and EMT6 cell line derived xenografts)and one type of human breast cancer model(MCF7 cell line derived xenograft),we performed the study to achieve the cure of malignant cancer by activating quiescent CSCs using DEK-containing exosomes and then eradicating them combined with chemoradiotherapy.The results showed that the tumors from 4T1-CDX mouse model were totally eradicated by radiotherapy treatment combined with DEKexosome injection with no recurrence observed within 3 months and without detectable pulmonary metastasis foci.In addition,the median survival times of the mice were significantly prolonged after the treatment.Meanwhile,we achieved recurrence-free tumor treatment in EMT6-CDX and MCF7-CDX mouse models.These results showed that the injection of DEK-containing exosomes combined with chemoradiotherapy in mice resulted in the activation and eradication of quiescent CSCs and finally achieved the cure of cancer without recurrence or metastasis.In conclusion,this study reveals DEK could activate quiescent CSCs and clarifies its molecular mechanisms of epigenetic regulation.The combination of DEKcontaining exosomes with chemoradiotherapy could activate and eradicate quiescent CSCs,eliminate the ability of malignant evolution of tumor in three mouse tumor models,and achieve complete cure of malignant tumor without recurrence or metastasis.The study lays an important theoretical foundation for the mechanism of CSC quiescence and activation regulation,and has important significance for the clinical treatment targeting quiescent CSCs. |
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