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Unravelling molecular mechanisms underlying genetic and epigenetic instabilities in colorectal cancer

Weis, Stefan. Unravelling molecular mechanisms underlying genetic and epigenetic instabilities in colorectal cancer. 2015, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_11139

Abstract

Colorectal cancers (CRCs) show extensive genetic and epigenetic aberrations, including DNA sequence changes, chromosomal alterations and abnormal epigenetic chromatin modifications. These alterations often reflect defects in molecular mechanisms establishing and maintaining (epi-) genome integrity. Epigenetic alterations in CRC are well documented, both, at the levels of histone modifications and DNA methylation. Epigenetic mechanisms play critical roles in the regulation of DNA-templated processes such as transcription, DNA repair but also replication. Shaping higher order chromatin structure, they regulate DNA accessibility and, thereby, assure proper genome function. Although many epigenetic alterations in CRC and other cancers are described and, in specific cases, their role in carcinogenesis established, the origin of and the molecular mechanisms underlying epigenetic instability in CRC remained elusive.
The aims of my thesis were to elucidate the molecular basis of DNA methylation aberrations in CRC in a first part, and to investigate mechanisms by which CRC-associated DNA methylation alterations contribute to colorectal carcinogenesis in a second part.
A subset of human CRCs is characterized by genome-wide hypermethylation of promoter CpG islands (CGIs) and therefore referred to as displaying a CGI-methylator phenotype (CIMP). Mutational inactivation and metabolic inhibition of the Ten Eleven Translocation (TET) DNA dioxgenases, key factors of active DNA demethylation, have been reported to be causal for DNA hypermethylation phenotypes in leukemias and gliomas. We investigated whether deregulated, TET-mediated DNA demethylation can account for CIMP in CRC. We discovered that TET1 is significantly downregulated, both at the mRNA and protein level, in CIMP-displaying CRCs. Knock-down of TET1 in the non-CIMP CRC cell line SW620 confirmed that the loss of TET1 results in genome-wide CGI hyper- and hypomethylation in cell culture, which, upon xenograft tumor formation, converted into a hypermethylation phenotype. Notably, the loss of TET1 in the CRC cell line resulted in increased promoter methylation and downregulation of MLH1 and CDH1, a characteristic feature of CRC-CIMP. These results thus showed that depletion of TET1 and selection during tumor growth can recapitulate features of CIMP in non-CIMP cancer cells. Thus, TET1 activity in colon epithelial cells contributes to maintenance of cell type specific CGI-methylation patterns and prevents the development of CRC through the CIMP-pathway.
Beside cancer-specific defects in the maintenance of CpG methylation, aging constitutes a major source of methylation alterations associated with CRC. Lifestyle-related factors including overweight, aspirin use, hormone replacement therapy (HRT) and smoking have been reported to influence the risk of CRC over long periods of time. We investigated whether these factors exert their effects on cancer risk via modulation of age-related DNA methylation changes. Genome-wide and detailed target-wise methylation analysis in a cohort of 546 healthy women revealed that promoter-associated DNA methylation increases over age. We observed that smoking and obesity promote, whereas aspirin and HRT reduce the genome-wide methylation drift. A significant fraction of promoter-associated CpG sites, whose age-related hypermethylation was modified by lifestyle factors, also occurred hypermethylated in CRC. Lifestyle factors affected the methylation drift of CRC-related CpGs concordant with the effects of these factors on CRC risk. Our results suggest that the effect of the investigated lifestyle factors on CRC-risk can be accounted for by the modulation of age-related DNA methylation changes.
The promoter methylation of cancer-relevant genes detectable in the healthy colorectal mucosa allows the definition and monitoring of early epigenetic events in colorectal carcinogenesis. To explore this possibility, we selected five genes (FOXF1, CA4, NPY1R, GREM1 and IFITM1) downregulated in colorectal adenomas and quantified their promoter methylation levels in CRC tissue as well as in the matched healthy appearing colonic mucosa. We included MLH1 and MGMT, two genes with established roles in colorectal carcinogenesis, in this analysis. With different incidence all seven markers were found hypermethylated in 106 CRC samples tested. Interestingly, methylation of the two novel markers CA4 and GREM1 associated with CRC-CIMP. We found that these two markers are methylated at low levels also in cancer-associated mucosa as well as in colonic mucosa from healthy individuals, as it was reported previously for the CIMP marker MLH1. These results thus suggest that features of CIMP-specific DNA methylation are present and, hence, detectable already in the mucosa of healthy individuals.
DNA hypermethylation in gene promoters often interferes with transcription of the associated gene. We identified SHOCA-2 as a regulator of Epidermal Growth Factor Receptor (EGFR) signaling and observed its loss of expression in advanced CRCs. The loss of SHOC-2 expression was associated with different genetic and epigenetic alterations. We detected chromosomal deletions affecting the SH2D4A gene (encoding for SHOCA-2) as well as mutations in the coding region of the gene. Finally, we found the cancer-specific hypermethylation of two CpG sites in the 5´-untranslated region of SH2D4A, one of which overlapped with a binding site for the transcription factor Sp1. We found that methylation of this CpG site in the SH2D4A sequence context interferes with Sp1 binding in vitro and therefore might be responsible for SHOCA-2 expression loss in some CRCs. This describes a molecular mechanism by which site-specific DNA methylation may contribute to cancerous transformation.
Advisors:Schär, Primo-Leo
Committee Members:Spagnoli, Giulio C.
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Division of Biochemistry and Genetics > Molecular Genetics (Schär)
Item Type:Thesis
Thesis no:11139
Bibsysno:Link to catalogue
Number of Pages:65 p.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:57
Deposited On:27 Feb 2015 12:28

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