Preliminary Study On The Significance Of Pokemon On Triple Negative Breast Cancer Stem Cells And Underlying Mechanisms

Objective: To clarify the significance of Pokemon on the stemness mentaince of triple negative breast cancer(TNBC)stem cells,and to preliminarily demonstrate the underlying mechanism,providing potential targets for the treatments of TNBC,especially refractory TNBC.Methods: Transwell assays were conducted on human TNBC cell lines MDA-MB-231 and HS-578 T to detect the changes of migration ability after Pokemon knockout.MammoCult assay were applied to determine the impact of on the quantity and volume of formed microspheres by Pokemon knockout MDA-MB-231 and HS-578 T cells.The ratio change of ALDH+ Stem/progenitor in MDA-MB-231 and HS-578 T cells after Pokemon knockout is demonstrated in ALDEFLUOR? assay.To reveal the preliminary mechanism on how Pokemon regulates TNBC stemness,the Global cDNA microarray was used.The result shows that ZEB1 is the factor which involved in TNBC stemness and change the most.Moreover,the correlation between ZEB1 and Pokemon was analyzed in TCGA database.The correlation is further verified by qRT-PCR and Western blot in Pokemon knockout TNBC cell lines.In addition,the mechanism by which Pokemon regulates ZEB1 was further explored by Cycloheximide assay and MG132 assay.It was preliminarily determined that Pokemon may regulate ZEB1 by proteasome pathway.Finally,targeted E3 ligase was screened out by comparing ZEB1 specific E3 ligase,Global cDNA microarray and chip-seq assay,then the result was verified by qRT-PCR and Western blot.Results: 1.The Transwell assay demonstrated that Pokemon knockout inhibited the invasion and migration of MDA-MB-231 and HS-578 T cells.2.The microsphere culture assay showed that the knockdown of Pokemon decreased the quantity and volume of microspheres of TNBC cells,and silencing Pokemon reduced the proportion of ALDH+ stem/progen-cell population in MDA-MB-231 and HS-578 T cells.3.Global cDNA microarray demostrated that knocking down Pokemon could down-regulate various stemness factors,among which ZEB1 change the most-downregulated by 2.16 times.4.It was found that the expression of Pokemon is positive correlated with the expression ZEB1 in TNBC.5.mRNA and protein levels of ZEB1 was decreased significantly after Pokemon knockout in MDA-MB-231 and HS-578 T cells.6.The half-life of ZEB1 degradation is much shortened in Pokemon-knockout MDA-MB-231 and HS-578 T cells by actinomycete ketone assay;The application of proteasome inhibitor MG132 could rescue the degradation of ZEB1 protein induced by Pokemon knockout in MDA-MB-231 and HS-578 T cells,suggesting that silencing Pokemon could reduce the stability of ZEB1 and is related to the degradation of proteasome pathway.7.Having analyzed and compared ZEB1 associated E3 ligase,global cDNA microarray and chip-seq assay,the E3 ligase NEDD4 L was screened out and was further verified by qRT-PCR and Western blot.Conclusion: Pokemon promotes stemness maintenance of TNBC cells,and the underlying mechanism may be related to the inhibition of ZEB1 ubiquitination proteasome pathway degradation induced by transcriptional repressing E3 ligase NEDD4 L.