The Study Of The Mechanism Of Abhd5/HOXA13 In Promoting The Malignant Phenotype Of Colon Cancer

Background:Colorectal cancer(CRC)is one of the most common malignant tumors in the world.Tumor growth and relapse are driven by tumor?stemness?,and targeting self-renewal,the key property unique to?stemness?,may represent a new paradigm in cancer therapy.However,mechanisms regulating the self-renewal capacity of cancer cells are poorly understood.Epigenetics modifications(methylation,acetylation,phosphorylation,ubiquitination,etc.)play an important role in protein activity.The methylation modification of multiple tumor suppressor genes is closely related to their tumor suppressor activity.Similarly,the methylation modification of many oncogenes is closely related to their carcinogenic activity.However,its mechanism is not clear.Therefore,revealing the mechanism of oncogene or tumor suppressor gene epigenetic modification can further clarify the molecular mechanism of tumorigenesis and development,which is of great significance for the development of targeted medicine.At present,the abnormal activation or inactivation of metabolic genes are becomingthe sign of malignant tumors,and a large number of studies have found that metabolic genes also play a key role in the occurrence of colorectal cancer.We previously identified lipid metabolism-related genes,which contain hydrolase domain protein 5,also known as Abhd5(Abhydrolase domain containing 5,Abhd5)as an important tumor suppressor factor for colon cancer.The previous study of our research group found that the deficiency of Abhd5significantly promoted the occurrence and metastasis of colon cancer,but the underlying molecular mechanism has not been fully elucidated.Abhd5 is an important cofactor in the process of triglyceride decomposition in the body.Although it has no lipase activity,it can participate in regulating the fat decomposition process in non-fat tissue and adipose tissue by activating other triglyceride lipase.This study found that the absence of Abhd5 promotes the activation of the Wnt signaling pathway,increasing and maintaining the"stemness"of colon cancer cells in a?-Catenin independent manner,speculating that Abhd5-mediated change in self-renewal capacity of colon cancer cell is an important reason for promoting the development and progression of colon cancer,further exploring the molecular mechanism of the interaction between Abhd5 and DPY30,which affects the activity of histone methyltransferase SET1A and maintains the"stemness"of colon cancer,and further clarifying the molecular pathways by which this gene plays the role of tumor suppressor.It is known that 90%of colon cancers and all familial adenomatous polyposis are related to the excessive activation of the Wnt signaling pathway.However,the strategy of simply blocking the Wnt signaling pathway in colon cancer has no significant effect.Therefore,revealing new target genes,especially regulating the Wnt/?-Catenin signaling pathway,will have important significance and value in the diagnosis and treatment of clinical tumors.In recent years,a large number of studies have confirmed that development-related genes participate in the regulation of Wnt signaling pathway and promote the occurrence and development of colon cancer.Our research found that development-related genes,homeobox gene A13(Homeobox A13,HOXA13),are significantly up-regulated in colon cancer,and their high gene expression significantly promotes the proliferation of colon cancer cells.In order to further clarify the cancer-promoting effect of HOXA13 in colon cancer,we established HOXA13 knockdown and overexpression colon cancer cell models through lentiviral vectors to explore the specific molecular mechanism of HOXA13 in regulating the Wnt signaling pathway and promoting the occurrence and development of colon cancer.Objective1.To clarify that Abhd5 affects SET1A activity by interacting with DPY30,thereby regulating the molecular pathways of important oncogene YAP methylation activation,revealing the new function of Abhd5 in epigenetic regulation.2.To further clarify the molecular pathway of Abhd5 exerting the effect of tumor suppressor,and provide a new target for the clinical treatment of colon cancer.3.To explore the role and molecular mechanism of HOXA13 in the malignant proliferation of colon cancer.Material and methoes:1.Generate Abhd5 knockout human colonic cancer cell lines via lentiviral technique and Abhd5 knockout mice under APC^min/+genetic background,combine with database analysis,stem cell spheroidization experiment,soft agar clone formation Experiments,flow cytometry techniques and other methods to verify the influence of Abhd5 deletion which activates YAP signal on the"stemness"of colon cancer.2.Use human colon cancer cell HCT-116 as a model,combined with experimental methods such as mass spectrometry,immunohistochemistry,immunofluorescence staining,Western Blot,Co-IP,Chip and other methods to clarify the to clarify the influence of Abhd5's regulation of YAP post-translational modification on YAP cell location and activity.3.Use luciferase reporter gene experiment,immunofluorescence,Co-IP and other experimental techniques to explore the possible mechanism of Abhd5 regulating SET1A.4.Nucleoplasmic separation experiments,mass spectrometry,IP and other experiments were used to clarify the molecular mechanism of Abhd5 regulating DPY30 nuclear translocation and affecting SET1A activity.5.Colon cancer tissue chip immunohistochemical staining was used to detect the expression and clinical relevance of HOXA13 in colon cancer and adjacent tissues.6.Lentiviral vector technology to construct HOXA13 gene knockout and overexpression human colon cancer cell line for in vitro proliferation detection and mechanism study.Results:1.Loss of Abhd5 activates MET to sustain the stemness capacity of CRC cells independent of?-Catenin and loss of Abhd5 activates MET transcription by promoting YAP nuclear localization and activation.2.Abhd5 deficiency promotes nuclear localization and activation of YAP via inducing SET1A-mediated YAP methylation at K342.3.Abhd5 impedes SET1A-induced YAP methylation by a non-canonical mechanism involving sequestering DPY30 in the cytoplasm for ubiquitin-mediated degradation.4.Pharmacological inhibition of Met reduces the stemness of primary CRC cells derived from Abhd5low DPY30high Methigh tumors.5.The expression of HOXA13 in human colon cancer tissues is higher than that in normal tissues,and the high expression of HOXA13 in cancer tissues is closely related to poor clinical prognosis.6.HOXA13 has the ability to regulate the malignant proliferation of tumor cells.7.HOXA13 promotes the malignant proliferation of colon cancer through the?-Catenin-dependent Wnt signaling pathway.Further research found that the Wnt signaling pathway was significantly enriched in colon cancers with high expression of HOXA13,and the activity of?-Catenin was significantly increased in colon cancer cells with overexpression of HOXA13.There is a strong interaction between HOXA13 and?-Catenin.Conclusion:1.The deficiency of Abhd5 activates the target gene Met of Wnt signaling pathway in a?-Catenin independent manner,promoting regeneration of colorectal cancer.2.The deficiency of Abhd5 suppresses the ubiquitylation and degradation of DPY30 to promote translocation of DPY30 into the nucleus,activating methylase SET1A and inducing methylation and nuclear localization of YAP K342,which continuingly activates the expression of downstream gene Met.3.HOXA13 is high expression in human colorectal cancer tissues,and its high expression is correlated with poor clinical prognosis.4.HOXA13 combines with?-Catenin and promotes the nuclear accumulation of it,resulting in upregulation of genes in Wnt signaling pathway such as Cyclin D1 and Met,thereby enhancing the activity of Wnt/?-Catenin signaling pathway,ultimately promotes the malignant proliferation of colorectal cancer cells.