The Role And Mechanism By Which DNA Hypomethylation Affects Colorectal Cancer Tumorigenesis

Global DNA hypomethylation is a most common epigenetic alteration in human neoplasia.Dnmt1 hypomorphic mouse models have been extensively used to study how DNA hypomethylation impacts tumorigenesis in various tissues.This has led to the concept that global DNA hypomethylation can promote or suppress tumorigenesis in a tissue-specific manner.However,the underlying mechanisms,especially how DNA hypomethylation suppresses tumorigenesis,remains largely unknown.In this thesis,we employed an Uhrf1 tandem Tudor domain knockin mutant mouse model(tyrosine and proline at 187/188 sites mutated to alanine,Uhrf1-TTD-KI,Uhrf1^ki/ki)which exhibits approximately 10%reduction of DNA methylation in intestine and colon to investigate how DNA hypomethylation affects intestinal tumorigenesis.Firstly,we found that UHRF1 is highly expressed in the intestinal crypt.Compared with wildtype mice,there is no significant difference in the growth curve and the expression levels of UHRF1,DNMT1,DNMT3A proteins in Uhrf1^ki/ki mice.In addition,there is no obvious difference on the structure and morphology of intestinal tissues,proliferation and differentiation of intestinal stem cells between the wild type and mutant mice.Secondly,we found that Uhrf1^ki/ki significantly inhibited the occurrence and development of intestinal tumors in Apc^Min/+and AOM/DSS intestinal tumor models.Consistent with observed suppression of tumorigenesis,Uhrf1^ki/ki/Apc^Min/+mice have a significantly prolonged survival time compared with the Uhrf1^+/+/Apc^Min/+mice.Thirdly,we found that the inhibition of intestinal cancer in Uhrf1^ki/ki/Apc^Min/+mice is not due to alteration in Wnt signaling or Hippo pathway,nor decreased cell proliferation.Instead,we found by immunochemistry and Western blot analyses that the level of active cleaved-caspase3 is markedly increased in intestinal tissues from Uhrf1^ki/ki/Apc^Min/+mice when compared with that from Uhrf1^+/+/Apc^Min/+mice.Further analysis demonstrated an increased level of caspase3m RNA and full-length caspase3 proteins in intestinal tissues from Uhrf1^ki/ki/Apc^Min/+mice.Western blot analysis also revealed an increased level of cleaved-caspase8.Moreover,the TUNEL assay demonstrated that apoptosis is substantially upregulated in intestinal tissues of Uhrf1^ki/ki/Apc^Min/+mice compared with that of Uhrf1^+/+/Apc^Min/+.Finally,we found that elevated caspase3 gene expression in Uhrf1^ki/ki/Apc^Min/+mice correlates with reduced DNA methylation levels in the caspase3 enhancer regions.In conclusion,we present a new mouse model for investigating the role of and molecular mechanisms by which DNA hypomethylation regulates tumorigenesis and provide evidence that increased caspase3 expression and apoptosis are at least in part the mechanism by which DNA hypomethylation suppresses intestinal tumorigenesis.