The Inhibitory Effect And Regulatory Activity Of Metformin On The Epithelial-mesenchymal Transition For Colorectal Cancer

Colorectal cancer(CRC)is the third most common cancer and the fourth leading cause of cancer-related death worldwide.Several studies have indicated that CRC cancer patients with diabetes melhtus ?(DM ?)tend to have a worse prognosis than patients without DM ?.As one of the classical oral hypoglycemic agents,metformin has been discovered to decrease cancer risks and has been proven effective in survival improvement for several cancers including CRC in recent years.However,there are still vacancies in such field.Epithelial-mesenchymal transition(EMT)is a process by which epithelial cells lose epithelial characteristics,such as polarity and adhesion,gain migratory properties and transform into mesenchymal cells.EMT is widely occuring in embryonic development,tissue regeneration,fibrosis,and cancer.For cancer,EMT has been found to be associated with several malignant characters such as invasion,metastasis,recurrence and drug resistance.In a few studies,metformin appeared to inhibit the EMT of prostate,thyroid,lung and breast cancer cells.However,the anti-EMT mechanism is not clear and there 1s still little analogue laboratory and clinical evidence for CRC.Previous studies found that EMT can be triggered by activating signaling pathways such as Wnt,Notch,TGF,JAK-STAT,etc.,and signals from the upstream pathways eventually pass through the regulation of two double negative feedback chimaeric regulatory circuits:the upstream circuit Snail/miR-34 and the downstream circuit Zeb/miR-200.In each circuit,the transcription factor family(Zeb/Snail)component and the miRNA family(miR-200/miR34)component act as mutual inhibitors to each other.Based on the interactive mechanism,the circuit Snail/miR-34 and Zeb/miR-200 receives the input signals from upstream pathways and outputs signals and with the acts of other factors such as Twist?GRHL2 and OVOL2,affects cells' EMT phenotypes eventually,thus acts as the key regulations of the EMT process.To further elucidate the anti-neoplastic activities of metformin,this study analyzed the metformin,EMT phenotype and survival for CRC patients with DM ?,explored the metformin's regulatory effects on the feedback circuits Snail/miR-34 and Zeb/miR-200,and the related factors Twist?GRHL2 and OVOL2,thus further elucidated the underlying pharmacological mechanisms of metfonnin in the EMT inhibition and anti-cancer actions.Part ? Metformin,Epithelial-mesenchymal transition(EMT)phenotype and survival for colorectal cancer(CRC)patients with diabetes mellitus ?(DM?)ObjectivesWe aimed to explore the association between metformin treatment and the epithelial-mesenchymal transition(EMT)phenotype and further appraise the prognostic values of metformin and EMT markers E-Cadherin and Vimentin for colorectal cancer(CRC)in clinical practice.Methods1)We collected specimens and evaluated chnicopathological parameters of 102 stage ? to ? CRC patients with pre-diagnosed type 2 diabetes mellitus(DM ?).2)Expression of E-Cadherin and Vimentin in tumors were detected by immunohistochemistry(IHC),and was performed statistical analysis was performed using SPSS 19.0.3)Student's t-test was used for continuous variables analysis and Pearson's chi-squared(?2)test was used for comparison of categorical variables.Survival data were analyzed using the Kaplan-Meier method and compared by log-rank statistics.Cox regression was used for multivariate survival analysis.ResultsAmong 102 patients,28 were metformin users.The expression of E-Cadherin and Vimentin was detected as positive in 48.04%(49/102)and 53.92%(55/102)of patients respectively.1)In correlation tests,we found a lower tumor cell EMT degree(more E-Cadherin(P=0.014)and less Vimentin(P=0.01l)expression)in patients who used metfonmin,and the expression of E-Cadherin and Vimentin was associated with serum CA19-9(P=0.048,P=0.009),tumor invasive depth(T)P<0.001,P=0.045)and lymph invasion(N)(p=0.013,p=0.001).2)Univariate survival analysis using the Kaplan-Meier method showed that normal pre surgery serum CA19-9 values(P=0.006,P=0.005),metformin use(P=0.003,P=0.004),shallow tumor invasive depth(T1-3)(P=0.004,P=O.010),less lymph mvasion(NO)(P<0.001,P<0.001),positive E-Cadherin expression(P=0.001,P?0.003)and negative Vimentin expression(P=0.019,P=0.012)contributed to both longer DFS and OS.3)In Cox multivariate regression analysis,E-Cadherin was identified as a prognostic factor for disease-free survival(DFS)(P=0.038)and metformin use(P=0.015,P=0.044)and lymph invasion(P=0.016,P=0.023)were considered as the prognostic factors for both DFS and overall survival(OS).Conclusion1)Metformin may improve the prognosis for the colorectal cancer patients with type 2 diabetes mellitus;2)High expression of E-cadherin in primary tumor and tumor-free local lymph nodes indicate better prognosis for the colorectal cancer patients.with type 2 diabetes mellitus;3)Metformin may inhibit the invasion and dissemination and impede the epithelial-mesenchymal transition of colorectal cancer.Part ? The regulatory effects of metformin on the epithelial-mesenchymal transition(EMT)for colorectal cancer(CRC)ObjectivesThe epithelial-mesenchymal transition(EMT)plays a critical role in cancer progression,metastasis and drug resistance.The negative feedback circuits Snail/miR-34 and Zeb/miR-200 express key regulatory functions for the EMT process.In this system,the TF components Snail and Zeb promotes the EMT process and are classified as the positive regulatory elements,while the miRNA components miR-34 and miR-200 inhibits the EMT process and are classified as the negative regulatory elements.From upstream to downstream,the TFs interact with the corresponding miRNAs and compose two feed-back regulatory circuits:the Snail/miR-34 circuit and the Zeb/miR-200 circuit.By the intracircuit mutual inhibitions and the intercircuit hierarchical managements,the EMT process is regulated.The EMT-related signals from upstream pathways are regulated by the Snail/miR-34 and Zeb/miR-200 circuit successively,and finally decide the cells' fate in the EMT under the impacts of other factors such as Twists GRHL2 and OVOL2.The anti-EMT effect of metformin had been studied in series of tumors other than colorectal cancer,and the inner mechanism of such inhibition was still elusive.Here,we proposed to assess the anti-EMT abilities and explore the inherent pharmacological mechanisms of the classic hypoglycaemic agent metfommin for colorectal cancer(CRC).MethodsFor the EMT model,the TGF-p-induced CRC cell lines SW480 and HCTll6 were treated with metformin.The viability,migration and invasion abilities of the cells were evaluated with the Cell Counting Kit-8,wound-healing and trans-well assay.The alterations of the the Snail/miR-34 and Zeb/miR-200 circuit and the EMT markers E-cadherin and vimentin were detected by western blot,qPCR and immunofluorescent staining.Genes encoding ?-catenin,GRHL2,OVOL2 were knocked down with siRNAs in mechanical study.SPSS 19.0 was used for statistic analysis and the Toggle switch model theory of the miRNA-TF chimera was adopted to further discussion.Results1)Metformin exhibited inhibitory effects on the proliferation,migration and invasion of the CRC SW480 cells:The OD values in CCK-8 assay showed that SW480 cells' growth and proliferation were chrono-(0,1,2,5,10 mM)and dose-(0,24,48,72 h)dependently inhibited by metformin;In wound-healing assay,themigratory abilities of metformin(5 mM)and TGF-p(10n g/ml,48 h)-treated cells were significantly decreased compared with cells treated only with TGF-?(10 ng/ml,48 h,positive control);In trans-well assay,the invaded cells of metformin(5 mM)and TGF-?(10 ng/ml,48 h)-induced cells were notably less than TGF-?(10 ng/ml,48 h)-induced cells.2)Metformin revealed the anti-EMT abilities for both SW480 and HCT116 cells:Western blot and qPCR assays revealed that the protein and mRNA levels of the key EMT markers E-cadherin and vimentin in metformin(5 mM)and TGF-?(10 ng/ml,48 h)-treated samples were significantly different(higher E-cadherin and lower vimentin)from only TGF-?(10 ng/ml,48 h)-treated samples.3)Meformin presented different regulations for the Snail/miR-34 and Zeb/miR-200 circuit:for the TGF-? induced SW480 and HCTll6 cells,metformin increased miR-200a,miR-200c and miR-429 levels and decreased miR-34a level of the EMT-inhibiting miRNAs compoent,and decreased Snail1 and Zebl levels of the EMT-promoting transcription factors(TF)compoent in Snail/miR-34 and Zeb/miR-200 circuit.4)Metformin induced the increase of the epithelial marker E-cadherin signals and the decrease of the mesenchymal marker Zebl signals in general,and enlarge the proportion of epithelial-mesenchymal hybrid cells at the cell level in EMT process:from immunofluorescence,we observed increased proportion of E-cadherin and Zebl co-expression cells in metfonmin(5 mM)and TGF-B(10 ng/ml,48 h)-treated slides than in only TGF-p(10 ng/ml,48 h)-treated slides.5)Based on the Toggle switch model theory and combined with the results of qPCR,western blot and immunofluorescent staining,the increased proportion of E-cadherin and Zebl co-expression cells may be attributed to the inhibition from metfromin in the positive EMT direction.And metformin may not able to facilitate the reverse EMT(the MET process)of the colorectal cells.6)After knocking-down the gene encoding p-catenin for the TGF-? induced SW480 and HCT116 cells,the concentration Twistl and nuclear ?-catenin decreased and GRHL2 and OVOL2 increased.7)After knocking-down the GRHL2 gene,the OVOL2 was down-regulated and Zebl was up-regulated in both SW480 and HCT116 cells;The GRHL2 and Zebl increased in both OVOL2-knocked-down SW480 and HCT116 cells.8)In western blot and immunofluorescent staining,p-catenin induced and metfonmin exposed SW480 and HCTll6 cells exhibited the increase of Twistl and nuclear[3-catenin and the decrease of GRHL2 and OVOL2.Conclusion1)Metformin inhibits the proliferation,migration and invasion of the colorectal cancer cells;2)Metformin may play the bidirectional regulation on the Snail/miR-34 and Zeb/miR-200 circuit,in which the inhibition is stronger than the promotion;3)Metformin may induce the colorectal cancer cells to lag in the E/M hybrid phase of EMT process;4)The inhibition of the EMT and the regulation on the Snail/miR-34 and Zeb/miR-200 circuit system for the colorectal cancer by metfonmin express as the upregulation of the epithelial marker E-cadherm and the downregulation of the mesenchymal marker Vimentin in the individual level,and exhibit as the numerical increase of the E/M hybrid cells in the group level.5)The ?-catenin is involved in the transduction of the TGF-? activated EMT signal,and metformin may negatively regulate the Snail 1 in the Snail/miR-34 circuit by inhibiting the p-catenin/Twist 1/Snail1?pathway during the EMT inhibition.6)The GRHL2 may directly repress or indirectly inhibit the Zebl in the Zeb/miR-200 circuit by activating the OVOL2.The OVOL2 may feed negative signals back to the GRHL2.Metformin may negatively regulate the Zebl element by inhibiting the ?-catenin factor and activating GRHL2/OVOL2/Zebl pathway.