1.A CRISPR Knockout Negative Screen Reveals Synergy Between CDKs Inhibitor And Metformin In The Treatment Of Human Cancer 2.Methylation Silencing Of TGF-? Receptor Type ? Is Involved In Malignant Transformation Of Esophageal Squamous Cell Carcinoma

Background:In recent years,numerous laboratory research and pharmacoepidemiology studies provide support for metformin,an oral biguanidine hypoglycemic agent,as a potential antitumor agent.However,the lack of a clear understanding of the indications and the mechanisms underlying metformin activity limit its efficacy in the treatment of human cancer.Method:In this study,we performed a genome-wide CRISPR knockout negative screen with the Brunello pooled sgRNA library to identify potential targets that might synergize with the antitumor properties of metformin.Next generation sequencing of pooled genomic DNAs isolated from surviving cells after 18 days of metformin treatment(T18)compared to untreated cells at day 0(T0)yielded candidate genes(sgRNAs)depleted in the T18 relative to the T0 cell population.Moreover,using biochemistry,molecular biology,metabolomics and in vivo experiments to illuminate the molecular mechanisms underlying the synergy.Results:SiRNA knockdown of a group of cyclin-dependent kinases(CDKs),including CDK1,CDK4 and CDK6,confirmed the results of the screen.Combination treatment of the CDKs inhibitor abemaciclib,which is already under clinical investigation,with metformin profoundly inhibited the tumor viability in vitro and in vivo.Although cell cycle parameters were not further altered under combination treatment compare to metformin or abemaciclib treatment,investigation of the metabolome revealed significant changes in cell metabolism,especially with regard to fatty acid oxidation,the tricarboxylic acid cycle and aspartate metabolism.Such changes appeared to be mediated through inhibition of the mTOR pathway.Moreover,mTOR inhibition was involved in the synergetic effect of metformin and CDKs inhibitor.Conclusion:Collectively,our study suggests that metformin has a wider application in clinical oncology,and that the combination of CDK4/6 inhibitors with metformin could be recognized as an attractive treatment option for cancer and potential therapy in future clinical applications.Background:Although massive studies have been conducted to investigate the mechanisms of esophageal squamous cell carcinoma(ESCC)carcinogenesis,the understanding of molecular alterations during the malignant transformation of epithelial dysplasia is still lacking,especially regarding epigenetic changes.Methods:A whole-genome bisulfite sequencing(WGBS)analysis was performed on a series of tumor,dysplastic and non-neoplastic epithelial tissue samples from ESCC patients to identify differentially methylated regions between successive stages.We also used The Cancer Genome Atlas multiplatform molecular and clinical data as well as immunohistochemistry to evaluate the methylation and expression of TGFBR2 in tumor samples.The mechanism of TGFBR2 methylation was elucidated by RT-qPCR,western blot,flow cytometry and animal models.Results:Using WGBS,we identified 969 differentially methylated regions(DMRs)between non-neoplastic and tumor samples,1293 DMRs between non-neoplastic and dysplastic samples and 1838 DMRs between dysplastic and tumor samples.Promoter hypermethylation in one candidate gene,TGF-? Receptor type ?(TGFBR2),an important mediator of transforming growth factor-B(TGF-?)signaling,was identified.Immunohistochemical staining of primary tissue samples confirmed down-regulation of TGFBR2 protein in ESCC compared to dysplastic and normal tissue.TGFBR2 mRNA was significantly downregulated in several ESCC cell lines compared to the immortalized esophageal epithelial cell line Het-1A.Treatment of ESCC cell lines with a DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine reactivated the expression of TGFBR2.Both 5-Aza-2'-deoxycytidine and the lentiviral mediating the overexpression of TGFBR2 inhibited the proliferation of ESCC cell line KYSE-150 by inducing cell cycle G2/M arrest.Furthermore,xenografts derived from KYSE-150-TGFBR2-overexpressed cells showed reduced growth in nude mice relative to controls.Conclusions:The identification of methylation silencing of TGFBR2 in ESCC will enable us to further explore whether this epigenetic change could be considered as a predictor of malignant transformation in esophageal epithelial dysplasia and whether use of a TGFBR2 agonist may lead to a new therapeutic strategy in patients with ESCC.