| 1.BackgroundAs a highly heterogeneous malignancy,breast cancer is the leading cause of female cancer-related deaths.Although the mortality rate has drastically dropped as a result of improved early diagnostic methods and therapeutic advancements,a significant portion of patients eventually suffer tumor relapse and develop drug resistance.Accumulating evidence demonstrates that breast cancer stem cells(BCSCs),a tumor subpopulation which is critical for breast cancer relapse and drug resistance,possesses indefinite potential for self-renewal that drive tumorigenesis.Therefore,searching for effective strategies available for targeting CSCs is a way to improve the treatment and prevention of tumor.The signaling protein p62(also termed SQSTM1,ZIP3,A170)contains several important functional domains,which mediate the cross-talk with a number of signaling molecules to regulate the activities of downstream effectors,such as NRF2,NF-κB and m TOR.Recent studies indicate that aberrant p62 overexpression is implicated in numerous cancer types,including breast cancer.However,the detailed mechanisms for p62-mediated breast cancer initiation and progression,especially a role of p62 in promoting breast cancer stem-like properties,are still poorly characterized.In this study,we set out to investigate the potential role of p62 in the regulation of breast cancer stem-like properties and the mechanism involved.2.Methods(1)(1)ALDH1+ sorting assay was employed to isolate breast cancer stem cells.RT-q PCR and Western blot assays were performed to compare p62 expression levels between ALDH1+ and ALDH1-populations.(2)Mammosphere formation assay was employed to enrich breast cancer stem cells.RT-q PCR and Western blot assays were performed to compare p62 expression levels between sphereroids and monolayer cells.(2)(1)ALDH1+ staining assay was employed to detect whether p62 overexpression or depletion could influence the number of ALDH1+ cells.(2)Mammosphere formation assay was performed to explore whether p62 could affect breast cancer stem-like properties in vitro.(3)(1)ALDH1+ sorting assay was employed to isolate breast cancer stem cells.RT-q PCR assay was performed to detect the expression levels of p62 and related stem genes(NANOG,SOX2,POU5F1)in sh NC and shp62 sorted cells.(2)Two group with equal amounts of 5×104 ALDH1+ cells were subcutaneously inoculated into each NOD/SCID mouse(n=6).Mice were sacrificed after 6 weeks.Tumor formation frequencies and tumor volumes were analyzed.(4)(1)AFFYMETRIX Human Primeview Gene Microarray was performed to analyze expression profiles of both sh NC and shp62 xenografts tumor tissues.(2)Compare the array data(>1.5 fold)with one stem cell gene set.(3)Important gene was verified by RT-q PCR and Western blot analyzes within tumor tissues.(4)The gene set enrichment analysis(GSEA)of the expression profile was performed to validate the correlation between p62 and important gene.(5)(1)p62 was transiently knocked down(two different RNAi sequences)or overexpressed in breast cancer cells.RT-q PCR,RT-PCR and Western blot assays were performed to verify the relationship between p62 and the significant gene in vitro.(2)Epirubicin-resistant MCF-7(Epi R-MCF-7)cells worked as a cancer stem cell model.Mammosphere formation assay was conducted to explore the potential role of important gene in p62-mediated breast cancer stem-like properties.(6)(1)Luciferase reporter of the significant gene was constructed.Dual luciferase reporter assay was performed to test whether p62 would influence the promoter activity of the significant gene.(2)Actinomycin D was used to inhibit genome transcription(0,30,60,90 minutes).RT-q PCR was performed to test whether p62 would influence the mRNA half-life of the significant gene.(3)RNA-IP assay was performed to see whether p62 could act as a mRNA-binding protein to bind with the mRNA of the significant genedirectly.(4)If p62 could not bind with the mRNA of the important gene,other signaling molecules need to be found.RT-q PCR and Western blot assays were employed to verify this.3.Results(1)p62 was overexpressed in both ALDH1+ populations and sphereroids compared with ALDH1-populations and monolayer cells,respectively.(2)(1)The ALDH1+ population was markedly decreased following depletion of p62 and increased by p62 overexpression in MDA-MB-231 cells.(2)Knockdown of p62 decreased the mammosphere-forming abilities(both decreased diameters and numbers)in MDA-MB-231 cells,while overexpression of p62 increased the mammosphere-forming abilities in MCF-7 cells.(3)(1)The ALDH1+ cells in shp62 group exhibited lower p62 mRNA expression level than sh NC group,along with lower expression of NANOG,SOX2 and POU5F1.(2)Compared with sh NC group(6/6),mice inoculated with the shp62 cells exhibited a lower tumor incidence(5/6),along with reduced tumor volumes.(4)(1)(2)According to the results that the expression microarray data compared with one stem cell gene set,28 genes(NLE1,SRSF3,POLD2,TDP2,HSPA9,CHEK1,NUDT1,RFC5,ITGB3 BP,MCM6,ARL4 A,LUC7L3,CUL4 A,MCM3,SET,NOG,SP1,HMGB1,MRE11 A,POLR1C,RFC3,SNCRIP,MCM4,GMNN,MYC,PBX1)were found.(3)Subsequent verification by RT-q PCR and Western blot,knockdown of p62 significantly decreased MYC mRNA level.(4)We performed the gene set enrichment analysis(GSEA)of the expression profile and found that suppression of p62 significantly diminished the enrichment of genes that are regulated by c-Myc.(5)(1)Transient knock-down of p62(both RNAi sequences)decreased MYC mRNA level as well as c-Myc protein level,while overexpression of p62 increased both MYC mRNA and c-Myc protein levels.(2)Re-constitution of MYC expression in Epi R-MCF-7-shp62-2 cells abolished the reduced mammosphere-forming ability(6)(1)Dual luciferase reporter assay showed that p62 overexpression did not increase luciferase activities of p GL3-MYC.(2)Actinomycin D inhibitory results showed that MYC mRNA half-life for sh NC and shp62 MDA-MB-231 cells were 38.68 minutes and 70.88 minutes,respectively;MYC mRNA half-life for sh NC and shp62 MCF-7 cells were 47.33 minutes and 86.29 minutes,respectively.(3)RT-PCR followed RNA-IP showed that both p62 and Ig G did not bind with MYC mRNA directly.(4)RT-q PCR analysis of the let-7 cluster in the transfected cells showed that let-7a/b were significantly repressed by p62 overexpression.Re-constitution of let-7a/b activity by the mimics reversed the p62-mediated elevation of MYC at the mRNA and protein levels.4.Conclusions(1)p62 expression is elevated in BCSC-enriched cell populations.(2)p62 is essential for promoting breast cancer stem-like properties both in vivo and in vitro.(3)MYC is necessary for the maintenance of p62-mediated stem-like properties in breast cancer.(4)p62 specially promotes MYC mRNA stability,mostly by repressing the expression of let-7a/b. |
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