YTHDC1 Interacts With PKM2 To Regulate FOXO1-mediated Molecular Mechanism Of Autophagy And The Chemotherapy Resistance In Colorectal Cancer Cells

Colorectal cancer(CRC)is one of the common malignant tumors,and obesity,alcoholism,and lack of exercise are all predisposing factors for CRC.In recent years,the morbidity and mortality rate of CRC in China are increasing year by year,which seriously endangers people’s health.5-fluorouracil(5-FU)-based chemotherapy remains the cornerstone of treatment for advanced and metastatic CRC,however,chemotherapy resistance inevitably developed in the course of treatment.Chemotherapy resistance is a major obstacle to the successful treatment of CRC patients,Chemoresistance is a major obstacle to the therapeutic success of CRC patients,therefore,it is important to deeply explore the molecular mechanism of tumor chemoresistance for the development of novel strategies to overcome drug resistance in CRC patients.Autophagy is an intracellular lysosome-dependent degradation system that is usually activated in response to unfavorable microenvironmental stresses.Accumulating evidence suggests that cellular autophagy plays a critical role in regulating tumor chemoresistance.Pharmacologically inhibiting or genetically silencing autophagy impairs tumor initiation and progression and improves tumor chemosensitivity,this suggests that targeted intervention of autophagy holds promise as a potential therapeutic strategy to reverse chemotherapy resistance of the tumor.Epigenetic dysregulation has emerged as an important feature of virtually all human cancers,N6-methyladenosine(m~6A)modification is pervasive and widely dysregulated in tumors,the dynamically reversible nature of m~6A modification provides opportunities for therapeutic intervention in oncology.m~6A modification can regulate processes such as RNA splicing,translation,mRNA stability,and nuclear translocation,and it is a potential driver of tumor chemoresistance.Members of the YTH family of m~6A binding proteins are highly expressed in a variety of tumors,and its family member YTHDC1 can promote the development of cancers such as endometrial cancer,glioblastoma,acute myeloid leukemia.Our group previously showed that PKM2 promotes autophagy and induces chemoresistance by inducing expression of the transcription factor FOXO1 in CRC cells.GST pull down coupled with mass spectrometry revealed that PKM2 could interact with YTHDC1.However,the role and molecular mechanism of YTHDC1 remain unclear in PKM2 mediated autophagy and chemoresistance of CRC cells.In this topic,we investigated the role of YTHDC1 in PKM2 mediated FOXO1 expression and autophagy and chemoresistance of CRC cells,and the potential molecular mechanism is preliminarily clarified.The main research content of this topic is as follows:1.MTT,crystal violet staining and clonogenic assays revealed that overexpression of YTHDC1 could reverse the chemosensitization of CRC cells resulting from knockdown of PKM2,knockdown of YTHDC1 can reverse the chemoresistance of CRC mediated by overexpression of PKM2,this suggests that PKM2 induced CRC chemoresistance is dependent on YTHDC1 expression.Western blot showed that knockdown of PKM2 in CRC cells significantly inhibited the protein expression of autophagy related proteins ATG5,Beclin 1 and ULK1,and reduced expression of the transcription factor FOXO1,but YTHDC1 protein was unaffected,and knockdown of PKM2 mediated these changes could be addback by overexpression of YTHDC1.Flow cytometry experiments showed that simultaneous knockdown of YTHDC1 by overexpressing PKM2 could reverse the CRC cell killing effect of 5-FU inhibited by overexpressing PKM2,knockdown of PKM2 induced chemosensitization of CRC cells could be addback by overexpression of YTHDC1.These results suggest that YTHDC1plays a critical role in PKM2 mediated autophagy and chemoresistance in CRC cells.2.CoIP assay showed that PKM2 interacted with YTHDC1 in drug-resistant CRC cells,actinomycin D tracing experiments further confirmed that FOXO1 mRNA stability was significantly downregulated in CRC cells with PKM2 knockdown,overexpression of YTHDC1 could obviously reverse the decrease in FOXO1 mRNA stability mediated by knockdown of PKM2,this suggests that YTHDC1 interacts with PKM2 to regulate FOXO1 mRNA stability.QPCR showed that the decreased expression of FOXO1 and ATG5,Beclin 1,ULK1 mediated by knockdown of PKM2 could be reversed when YTHDC1 was overexpressed based on knockdown of PKM2,the increased expression levels of FOXO1 as well as autophagy related genes by overexpression of PKM2 could be reversed by knockdown of YTHDC1.These results suggest that YTHDC1 regulates FOXO1 mediated autophagy of CRC cells by interacting with PKM2.3.Rescue experiments were performed in CRC cells with PKM2 knocked down by constructing m~6A recognition functionally inactive mutants of YTHDC1,q PCR and Western blot showed that knockdown of PKM2 mediated downregulation of the mRNA and protein levels of FOXO1,which could be reversed by overexpression of wild-type YTHDC1 but not m~6A recognition functionally inactive mutant of YTHDC1.The MTT results showed that knockdown of PKM2 mediated killing effect of 5-FU on CRC cells could be adducted by overexpressing wild-type YTHDC1,but not the m~6A recognition function inactive mutant of YTHDC1.This suggests that PKM2 mediated autophagy and chemoresistance in CRC cells are dependent on the m~6A recognition function of YTHDC1.m~6A-RIP and YTHDC1-RIP results showed that there was m~6A modification in FOXO1 mRNA,YTHDC1 recognizes m~6A modification of FOXO1 mRNA.Knockdown of PKM2 in CRC cells did not affect the m~6A modification of FOXO1mRNA,but significantly reduced the enrichment of YTHDC1 at the m~6A modification region of FOXO1 mRNA.The above results suggest that PKM2 promotes autophagy and chemoresistance in CRC cells by recruiting YTHDC1 to the enrichment of m~6A modified region of FOXO1 mRNA through its interaction with FOXO1.In summary,this study clarified the role of YTHDC1 in inducing autophagy and chemoresistance in CRC cells.And this study initially reveals that PKM2 can regulate FOXO1 m~6A modification by YTHDC1 to promote chemoresistance in CRC.The results suggest that YTHDC1 may serve as an effective target for reversing CRC chemoresistance,targeted intervention of the protein interaction of YTHDC1 with PKM2holds promise as a potential novel strategy for CRC clinical treatment.