E2F3 Induces DNA Damage Repair,Stem-like Properties And Therapy Resistance In Breast Cancer

Objective: Resistance to chemotherapy and radiotherapy is a major hurdle to the treatment of human malignant tumors.Both DNA damage repair and stem-like properties contribute to chemoresistance and radioresistance.E2F3 is overexpressed in breast cancer tissues,and promotes proliferation of breast cancer cells.Higher E2F3 level is associated with shorter survival of breast cancer patients.Loss of function studies further showed that E2F3 promotes S-phage entry,DNA replication,DNA damage repair and stem-like properties.Accordingly,E2F3 knockdown sensitizes breast cancer cells to Adriamycin,Cisplatin and Olaparib as well as X-ray.FOXM1 is a downstream effector of E2F3,and E2F3 is modulated by RNA binding protein YTHDF2 dependent on m6 A methyltransferase METTL14.These data demonstrate that YTHDF2-E2F3 is a novel target to overcome chemoresistance and radioresistance in breast cancer.Methods: 1.E2F3 expression in normal mammary tissues and breast cancer tissues was investigated in Curtis and TCGA databases in Oncomine website(https://www.oncomine.org/).The association of E2F3 expression with survival of breast cancer patients was analyzed in Kaplan-Meier plotter website(http://kmplot.com/analysis/).Gene expression in human breast cancer cell lines was investigated in the Cancer Cell Line Encyclopedia(CCLE)database.Correlation analysis was performed between two genes by Pearson statistics.The heat map was generated by Graph Pad Software 8.2.Twenty copies of breast cancer samples and ten copies of adjacent normal tissues were obtained from Cancer Hospital of China Medical University with the consent of the patients.Research was approved by Institutional Research Ethics Committee of China Medical University.Immunohistochemical staining were performed to confirm the difference of E2F3 expression between tumors and adjacent tissues.3.Human breast cancer cells MDA-MB-231,MCF-7 and SK-BR-3 were transfected with E2F3 sh RNA lentiviruses.E2F3-knockdowned MDA-MB-231 cells was constructed by FOXM1-overexpression plasmids.YTHDF2 sh RNA plasmids was used to conducted MDA-MB-231 cells.The efficiency of transfection was confirmed by western blot assays and real-time PCR.4.Western blot assays was used to monitored the markers of cell cycle.The distribution of cell cycle was detected by flow cytometry.The cell viability was confirmed by CCK-8 assay.5.The stem-like properties in E2F3-knockdown MDA-MB-231 and MCF-7 cell lines were detected by Tumorisphere formation assays.The phenotypes of stem-like properties were confirmed by real-time PCR assay.The proportion of ALDH-positive cells were detected by flow cytometry.6.The markers represent the ability of DNA damage repair were detected by western blot assay.The transfected cells were treated by adriamycin or X-ray and the immunofluorescence ofγ-H2 AX of the nuclei was visualized by a laser confocal focus microscope.7.The cells were treated with Cisplatin(0,2,4,6,8,10μM),Adriamycin(0,0.2,0.4,0.6,0.8,1.0μM),Olaparib(0,20,40,60,80,100μM)or radiated by various doses of X-ray(0,4,6,8,12,16Gy)and incubated for 48 h.The absorbance was measured at 450 nm by a Microplate reader.8.The E2F3-knockdowned MDA-MB-231 cells were infected by FOXM1 overexpression plasmids.Western blot and real-time PCR assays were used to confirm the transfection efficiency.The markers standing for DNA replication and cell cycle were monitored by western blot.Flow cytometry was used to detect the distribution of cell cycle.CCK-8 assays were performed to assess the proliferation ability and sensitivity to Cisplatin,Adriamycin,Olaparib and X-ray.OE-FOXM1 cells and OE-control cells were radiated by 6MV X-ray and detected by a laser confocal focus microscope.γ-H2 AX of the nuclei was monitored to evaluate the ability of DNA damage repair.The DNA damage repair markers BRCA1 and RAD51 were detected by western blot.The real-time PCR were performed to detect BRCA1,BRCA2 and RAD51.9.The MDA-MB-231 cells were infected with YTHDF2 sh RNA plasmids.The efficiency of transfection and the expression of E2F3 were confirmed by western blot.The RNA stability assay was used to monitor the relationship between YTHDF2 m RNA and E2F3 m RNA.The transcription regulatory relevance of YTHDF2 m RNA and E2F3 was performed by RIP assay.The infected sh YTHDF2 and sh Ctrl cells were irradiated by6 MV X-ray and the deposition of γ-H2 AX in the nuclei indicated the ability of DNA damage repair.10.MDA-MB-231 cells were transfected with METLL14 sh RNA lentiviruses.Real-time PCR and western blot assays were performed to confirm the efficiency of transfection.The expression of E2F3 in METTL14-knockdown cells were monitored by western blot assay.The E2F3 m RNA levels in METTL14-knockdown cells were monitored by RNA stability and analyzed by real-time PCR.The transcription regulatory relationship of YTHDF2 m RNA and E2F3 was performed by RIP assay in METTL14-knockdown cells.The transcription correlations between YTHDF2 and E2F3,E2F3 and FOXM1 were shown to explore the mechanism of E2F3 in Breast cancer cells.Results:1.E2F3 is a putative tumor promoter in breast cancerBoth Curtis and TCGA databases were used to investigate E2F3 m RNA expression.Breast cancer tissues express higher level of E2F3 m RNA than normal mammary tissues.Consistently,breast cancer tissues express more E2F3 protein than cancer-adjacent tissues,as shown by Immunohistochemistry staining.Higher E2F3 expression is associated with shorter overall survival(OS),recurrence-free survival(RFS),distal metastasis-free survival(DMFS)and post-progression survival(PPS)of breast cancer patients.E2F3 expression was stably knockdowned in breast cancer cell lines MDA-MB-231,MCF-7 and SK-BR-3,which express abundant E2F3(Supplementary Figure 2).As expected,E2F3 knockdown significantly inhibited cell growth.2.E2F3 promotes S-phase entry and DNA replicationTo analyze the exact role of E2F3 in the cell cycle,CCLE database was firstly investigated.It was shown that E2F3 is closely associated with Cyclin E2,Cyclin A2,CDK2 and P21.E2F3 is also associated with a series of factors related to DNA replication such as PCNA,RFC4,POLE2,GINS1 and MCM4.These in silico findings suggest that E2F3 is implicated in S-phase.Loss of function study was subsequently performed to validate this assumption.As shown by Flowcytometry,E2F3 knockdown in breast cancer cells reduced S-phase cell fraction whereas increased G1-phase cell fraction.E2F3 knockdown downregulated Cyclin E2,Cyclin A2,CDK2 and PCNA while upregulated P21.Moreover,E2F3 knockdown reduced RFC4,POLE2,GINS1 and MCM4 m RNA expression.3.E2F3 enhances DNA DSB repairTo assess whether E2F3 is involved in DNA DSB repair,breast cancer cells were treated with X-ray radiation.12 or 24 hours later,cells with E2F3 knockdown displayed moreγ-H2AX-positive foci compared to cells with control knockdown,indicating that E2F3 downregulation impaired the repair capacity to DSB.Adriamycin,which inhibits DNA topoisomerase II,was also adopted to induce DNA DSB.Similarly,E2F3-deficient cells exhibited more γ-H2 AX staining compared to control cells after treatment with Adriamycin.Mechanistically,E2F3 knockdown in breast cancer cells downregulated several factors related to HR repair of DSB including RAD51,BRCA1,BRCA2 and EXO1.4.E2F3 endows cancer cells with stem-like propertiesAnalysis of CCLE database revealed a positive correlation of E2F3 with stem-related markers CD133 and EPCAM.E2F3 is also positively correlated with ALDH1A2 and stem-related transcription factor POU5F1.Consistent with these findings,E2F3 knockdown in breast cancer cells attenuated CD133,EPCAM,ALDH1A2 and POU5F1 expression.Furthermore,E2F3 knockdown in breast cancer cells reduced ALDH1-positive subpopulation and interfered with mammosphere formation,indicating that E2F3 promotes the expansion of breast cancer stem cells.5.E2F3 induces the resistance to chemotherapy and radiotherapyE2F3 contributes to both DNA damage repair and stem-like properties,thus the role of E2F3 in chemoresistance and radioresistance was explored.As shown by Cytotoxicity assays,E2F3 knockdown sensitized breast cancer cells to DNA-damaging drugs Cisplatin and Adriamycin.Notably,E2F3 knockdown also increased the sensitivity of breast cancer cells to Olaparib,a PARP inhibitor(PARPi)which has been given to breast cancer patients with HR deficiency.Finally,E2F3 knockdown enhanced the cytotoxic effect of radiotherapy on breast cancer cells.These findings indicate that E2F3 induces both chemoresistance and radioresistance.6.FOXM1 is a downstream effector of E2F3FOXM1 has been shown to be transcriptionally modulated by E2 F family.As expected,E2F3 knockdown in MDA-MB-231 cells downregulated FOXM1 in both m RNA and protein levels.To validate that FOXM1 mediates the effect of E2F3 on breast cancer cells,FOXM1 was overexpressed in E2F3-knockdowned MDA-MB-231 cells.FOXM1 overexpression increased cell viability,elevated S-phase cell fraction,and upregulated factors related to S-phase and DNA replication.Moreover,FOXM1 overexpression upregulated stem-related factors and promoted mammosphere formation.FOXM1 overexpression induced RAD51 and BRCA1 expression,and attenuated γ-H2AX staining after X-ray radiation.Accordingly,FOXM1 overexpression desensitized MDA-MB-231 cells to Adriamycin,Cisplatin,Olaparib and X-ray.These findings demonstrate that FOXM1 overexpression restores E2F3-associated phenotype.7.E2F3 is modulated by m6 A binding protein YTHDF2Analysis of CCLE database showed a positive correlation of E2F3 with m6 A binding protein YTHDF2,suggesting that E2F3 expression is related to m6 A modification.Indeed,YTHDF2 knockdown in MDA-MB-231 cells decreased E2F3 expression.Furthermore,YTHDF2 knockdown shortened E2F3 m RNA half-lives,indicating that YTHDF2 modulates E2F3 m RNA stability.RIP test further confirmed the binding of YTHDF2 to E2F3 m RNA.YTHDF2 knockdown enhanced γ-H2 AX staining after X-ray radiation,and sensitized MDA-MB-231 cells to Adriamycin,Cisplatin,Olaparib and X-ray.To determine that YTHDF2 modulates E2F3 dependent on m6 A modification,m6 A methyltransferase METTL14 was knockdowned in MDA-MB-231 cells.METTL14 knockdown also reduced E2F3 expression and shortened E2F3 m RNA half-lives.RIP test showed that METTL14 knockdown interfered with the binding of YTHDF2 to E2F3 m RNA.These data indicate that E2F3 m RNA stability is controlled by YTHDF2-METTL14 pair.Conclusions:1.E2F3 promotes the proliferation and the process of G1-S transition of breast cancer cells;endows cancer cells with stem-like properties;enhances the DNA damage repair as an oncogene character.2.E2F3 mediated the chemoresistance and radioresistance.3.FOXM1 also possesses stem-like properties and facilitate the proliferation,cell cycle progression and DNA damage repair of cancer cells as a downstream transcription factors of E2F3.4.YTHDF2 plays an oncogene role in breast cancer cells,such as enhanced the radioresistance and chemoresistance.YTHDF2 modulates E2F3 dependent on m6 A modification.5.The molecular mechanisms highlights YTHDF2-E2F3-FOXM1 as an effective and safe therapeutic target in breast cancer.