The Molecular Mechanism Of SOX2-Beclin1/Autophagy Axis Regulating Colorectal Cancer Chemoresistance And Malignant Biological Phenotypes

Background and ObjectionColorectal cancer(CRC)is one of the most common malignant tumors in the world,with the second mortality rate.The main treatment is surgery,supplemented by comprehensive treatment of chemotherapy and radiotherapy.Chemoresistance and metastasis are the main reasons for the poor prognosis and low five-year survival rate of colorectal cancer patients.Stem cell transcription factor SOX2 can drive cancer stem cells characteristics and promote tumor occurrence and development.Autophagy,as an important mechanism of intracellular degradation of cytoplasmic proteins and organelles,can regulate a variety of proteins involved in tumorigenesis,proliferation and invasion,thereby promoting the malignant phenotypes of cancer.Our previous study has demonstrated that SOX2 promotes chemoresistance,stemness and epithelialmesenchymal transition(EMT)in CRC,but the underlying mechanism remains to be elucidated.In this study,we aim to explore whether SOX2 can promote CRC chemoresistance and malignant phenotypes via autophagy,providing new ideas for the diagnosis and treatment of CRC.Materials and methodsPlasmid transfection or lentiviral transfection was used to construct SOX2-overexpressing or silencing CRC cells.The expression of autophagy markers in transfected cells was detected by qRT-PCR and western blot.The number of LC3B fluorescent dots and autophagosomes was analyzed by confocal microscopy and transmission electron microscopy.Cell viability assay,sphere formation assay,scratch assay and transwell invasion assay were used to explore the effects of autophagy on the malignant phenotypes of CRC cells and whether autophagy was involved in SOX2-mediated chemoresistance and malignant phenotypes.Western blot was used to analyze autophagy genes regulated by SOX2.The role of Beclinl in SOX2-mediated autophagy activation,chemoresistance and malignant phenotypes in CRC was verified by cell function experiments.Chromatin immunoprecipitation,luciferase reporter assay,construction of promoter truncations and mutants were utilized to explore the mechanism of SOX2 regulating Beclinl.The clinical expression characteristics of SOX2 and Beclinl were analyzed through 90 pairs of CRC tissue microarrays.The role of SOX2-Beclinl/Autophagy axis on CRC growth and chemoresistance was explored in mouse xenograft.Results1.Overexpression of SOX2 significantly upregulated the expression of LC3II/I and promoted the formation of LC3B fluorescent dots and autophagosomes.2.Autophagy activation promoted malignant phenotypes of CRC cells;autophagy blockage inhibited SOX2-induced chemoresistance,stemness and EMT in CRC cells.3.Beclinl was an essential downstream molecule for SOX2 to activate autophagy;knockdown of Beclinl inhibited SOX2-mediated autophagy activation,chemoresistance and malignant phenotypes of CRC cells.4.Overexpression of SOX2 increased the luciferase activity of Beclinl full-length promoter;truncation of Beclinl promoter-1515～-1029 region or deletion mutation-1400～-1390 sequence(ATCATAACAGA)significantly decreased luciferase activity.5.Beclinl was upregulated in CRC tissues;and the overexpression of Beclinl was related to CRC AJCC staging,survival prognosis and SOX2 expression.6.Knockdown of SOX2 inhibited tumor growth;the combination of CQ and irinotecan significantly increased the chemotherapy effect;besides,the chemotherapy effect of the SOX2 knockdown group was greater than that of the control group.Conclusion1.SOX2 promotes chemoresistance,stemness and EMT of CRC cells by activating autophagy.2.SOX2 transcriptionally upregulates Beclinl expression by binding with Beclinl promoter(-1400～-1390,ATCATAACAGA),enhancing autophagy and chemoresistance,stemness and EMT of CRC cells.3.SOX2-Beclinl/Autophagy signaling axis promotes CRC growth and chemoresistance in vivo.