Analysis of 14-3-3σ methylation and associated changes in gene expression and function in colorectal carcinoma
Roberts, Kirsty Anne
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The aims of the work presented in this thesis were: to investigate the role of methylation of 14-3-3σ (a key regulator of p53-mediated G2/M arrest and of translational control during mitosis) in colorectal cancer using colorectal cancer cell lines and fresh colorectal tumours; to investigate any relationship between 14-3-3σ methylation status and gene expression; to determine whether aberrant methylation is associated with cell cycle defects and other factors known to contribute to colorectal carcinogenesis. PCR bisulphite sequencing showed that 78% (7/9) of colorectal cancer cell lines were unmethylated in the 14-3-3σ upstream promoter region (UPR). The unmethylated cell lines expressed high levels of 14-3-3σ, while methylated cell lines expressed negligible levels of 14-3-3σ protein or mRNA. Methylated colorectal cancer cell lines were treated with 5-aza-2’-deoxycytidine and demethylation was confirmed by MSP analysis. However, demethylation did not induce 14-3-3σ re-expression in the methylated cell lines, suggesting that CpG methylation may not be the only mechanism of transcriptional control. In contrast to colorectal cancer cell lines, 90% (89/99) of fresh colorectal tumours were methylated at CpG dinucleotides within the 14-3-3σ UPR. Bisulphite sequencing analysis of individual clones from 14-3-3σ methylated tumours (n =3) demonstrated that the clones displayed methylated CpG levels of approximately 41%. In agreement with previous PCR bisulphite sequencing analysis, there were a low percentage of methylated CpG dinucleotides (~ 15%) in clones from the 14-3-3σ unmethylated tumours (n =3). Unmethylated tumours expressed significantly higher levels of 14-3-3σ in comparison to methylated tumours (p =0.03), indicating that 14-3-3σ methylation may be associated with expression. PCR bisulphite sequencing analysis of matched normal mucosa tissues indicated that the 14-3-3σ UPR was methylated in all samples. Preliminary studies therefore suggest that there is tumour-specific loss of 14-3-3σ methylation in colorectal tumours within the 14-3-3σ UPR and CpG island. There were no apparent clinico-pathological correlations with 14-3-3σ methylation status. Whilst 14-3-3σ methylation was associated with expression in fresh colorectal tumours, there was no significant difference in expression levels between unmethylated colorectal tumours and matched methylated normal tissue. Bisulphite sequencing analysis of individual clones from normal tissues (from patients free of cancer) revealed that the 14-3-3σ UPR and CpG island was methylated at the majority of CpG sites analysed in colonic tissue (422/495, ~85.2%) and approximately half (795/1557, 51.1%) of CpG sites in skin samples (n =3). Furthermore, higher levels of 14-3-3σ protein were observed in skin tissue samples compared to normal colonic tissue, suggesting that 14-3-3σ CpG island methylation may be associated with tissue-specific expression. Experiments to assess the relationship between 14-3-3σ methylation and general methylation defects, suggest that methylation differences in 14-3-3σ were not simply a consequence of more general methylation phenomena well described in colorectal cancer. Nearest Neighbor analysis showed no evidence of generalised hypomethylation. Furthermore, MethyLight analysis of the CpG Island Methylator Phenotype (CIMP) showed no relationship between 14-3-3σ methylation status and CIMP; since, 1/5 (20%) tumours methylated at 14-3-3σ UPR and 1/5 (20%) tumours unmethylated at 14-3-3σ UPR were CIMP positive. In vitro functional assays showed that overexpression of 14-3-3σ in SW480 cells (14-3-3σ methylated) delayed the apoptotic response to UV-C, compared to control SW480 cells. This suggests that 14-3-3σ may protect colorectal cancer cells from apoptosis. MTT assays showed that overexpression of 14-3-3σ in SW480 cells resulted in a trend of increasing proliferation with a significant increase on day 4, compared to controls SW480 cells (p <0.01). Furthermore, FACS-sorted SW480 cells overexpressing 14-3-3σ, showed a significant shift to S-phase from G1 compared to control SW480 cells (p <0.01). Western blot analysis and immunohistochemistry revealed no relationship between p53 status and methylated 14-3-3σ in fresh tumours, while there was no relationship between published p53 status for colorectal cancer cell lines and 14-3-3σ methylation status defined experimentally. I have presented data which shows that methylation status of 14-3-3σ varies between colorectal cancer tissue, colorectal cancer cell lines and normal colonic tissue. Overexpression of 14-3-3σ appears to contribute to colorectal cancer carcinogenesis, raising the hypothesis that 14-3-3σ expression and function may at least in part be dependent on CpG methylation.