Cancer-preceding Gene Expression Changes in Mouse Colon Mucosa

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in the Western world and interactions between genetic and environmental factors, including diet, are suggested to play a critical role in its etiology. Yet, the mechanisms by which diet impacts colorectal tumorigenesis remain largely unknown. Colorectal cancer evolves as a multistep process, which requires a series of genetic and epigenetic alterations in growth regulatory genes. The process is accelerated in individuals with inherited cancer predisposition such as Lynch syndrome (LS) which is one of the most common inherited cancer susceptibility syndromes and caused by inherited mutation in one of the DNA mismatch repair (MMR) genes. CRC is thought to develop via the so called adenoma-carcinoma sequence. However, the early events that occur in colon mucosa prior to polyp formation remain unknown. The research presented here investigates the gene expression changes arising in histologically normal colonic mucosa as putative cancer-preceding events available for early detection. This was achieved by pursuing a long-term feeding experiment in the mouse model for human Lynch syndrome (Mlh1+/-), and the wild type (Mlh1+/+) littermates, fed with either Western-style (WD) diet or healthy AIN-93G control diet. Carcinomas developed mainly in WD fed mice. WD also accelerated the progression of carcinogenesis. In the first study, the expression of 94 growth-regulatory genes previously linked to human CRC was studied for 5 weeks and 12 months old mice. Promoter CpG island methylation status was also studied for the genes which showed reduced expression. In mice fed for 12 months with WD, proximal colon mucosa, the predominant site of cancer formation in LS, exhibited a significant expression decrease in several tumor suppressor genes. Furthermore, a reduced mRNA expression was accompanied by an increased CpG dinucleotide promoter methylation of the respective genes suggesting a cause for the mRNA down regulation. The strongest expression decrease together with a significant increase in its promoter methylation was seen in Dkk1, an antagonist of the canonical Wnt signaling pathway. Furthermore, the inactivation of Dkk1 seemed to predispose to neoplasias in the proximal colon. The findings suggest that the inactivation of Dkk1 is a prominent early marker for colon oncogenesis. In study 2 the aim was to comprehensively clarify the role of Mlh1 expression during colon tumorigenesis, which is usually associated with Lynch syndrome and MSI. Here, the same mouse model and diets were used to study cancer-preceding expression changes in the colon mucosa of 12 and 18-month-old mice. The Mlh1 protein expression and MSI status were studied in the colon carcinomas, and the effect of inherited predisposition (Mlh1+/) and Western-style diet on those. CRC development always includes a lack of genomic integrity and the different types of genomic instabilities, such as chromosomal instability and MSI are thought to reflect distinct cancer initiating mechanisms. In the present study neither wildtype Mlh1+/+ nor heterozygote Mlh1+/- mice lacked the Mlh1 protein or showed MSI in their CRCs, while Mlh1 RNA expression was already significantly decreased in their normal mucosa. Instead, CRC mice showed a distinct expression profile with shortage of Mlh1 and several other chromosomal segregation gene-specific transcripts in mucosa and aberrant mitosis in tumors. The genome wide expression profiling experiment demonstrated that cancer-preceding changes are already seen in histologically normal colon mucosa and a that decreased expression of Mlh1 together with other chromosomal segregation genes may form a field-defect in mucosa and trigger MMR-proficient, chromosomally unstable CRC.