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Colorectal cancer is one of the most frequent neoplasms and an important cause of mortality in the developed world. Mendelian syndromes account for about 5% of the total burden of CRC, being Lynch syndrome and familial adenomatous polyposis the most common forms. Lynch syndrome tumors develop mainly as a consequence of defective DNA mismatch repair associated with germline mutations in MLH1, MSH2, MSH6 and PMS2. A significant proportion of variants identified by screening these genes correspond to missense or noncoding changes without a clear pathogenic consequence, and they are designated as "variants of uncertain significance'', being the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene a clear example. The implication of this variant as a low-penetrance risk variant for CRC was assessed in the present study by performing a case-control study within a large cohort from the COGENT consortium-COST Action BM1206 including 18,723 individuals (8,055 colorectal cancer cases and 10,668 controls) and a case-only genotype-phenotype correlation with several clinical and pathological characteristics restricted to the Epicolon cohort. Our results showed no involvement of this variant as a low-penetrance variant for colorectal cancer genetic susceptibility and no association with any clinical and pathological characteristics including family history for this neoplasm or Lynch syndrome.

The common 2652 6N del variant in the CASP8 promoter (rs3834129) has been described as a putative low-penetrance risk factor for different cancer types. In particular, some studies suggested that the deleted allele (del) was inversely associated with CRC risk while other analyses failed to confirm this. Hence, to better understand the role of this variant in the risk of developing CRC, we performed a multi-centric case-control study. In the study, the variant 2652 6N del was genotyped in a total of 6,733 CRC cases and 7,576 controls recruited by six different centers located in Spain, Italy, USA, England, Czech Republic and the Netherlands collaborating to the international consortium COGENT (COlorectal cancer GENeTics). Our analysis indicated that rs3834129 was not associated with CRC risk in the full data set. However, the del allele was under-represented in one set of cases with a family history of CRC (per allele model OR = 0.79, 95% CI = 0.69-0.90) suggesting this allele might be a protective factor versus familial CRC. Since this multi-centric case-control study was performed on a very large sample size, it provided robust clarification of the effect of rs3834129 on the risk of developing CRC in Caucasians.

The aim of this project was to investigate the anticancer activity of the anti-diabetic drug metformin, individually or combined in various settings with chemotherapy, on in vitro models of colorectal cancer (CRC). I found that metformin reduced cell proliferation by inducing cell cycle arrest in the G0/G1 phase, but did not lead to cell death. The anti-proliferative action of metformin resulted mediated by the inactivation of the mTOR pathway and of IGF1R protein. However, the drug only transiently arrested cell growth, since its effects were reversed after drug removal. When I combined the biguanide with the chemotherapy drugs commonly used to treat CRC I observed different responses in the cell lines analysed that reflected their genetic background and their different sensitivities to both the biguanide and chemotherapy. I found that metformin added before chemotherapy drugs antagonised their effects in the majority of the treatments. On the contrary, its administration after long chemotherapeutic treatments significantly reduced the cell viability. I noted that metformin better inhibits cell proliferation in cell lines with rapid growth. I have also assessed the dual treatment combination of metformin with different drugs that target specific genes or proteins (targeted therapies), focusing on BRAF-mutant cell lines. For most of the tested treatments the simultaneous administration of metformin and target drugs gave no advantages over the single drugs, and often resulted in antagonism. Overall, our results show that although further investigations are still needed to elucidate the results of the treaments including metformin, these data suggest that caution should be used in administering chemotherapy to indviduals taking metformin.

The aim of this project was to investigate the anticancer activity of the anti-diabetic drug metformin, individually or combined in various settings with chemotherapy, on in vitro models of colorectal cancer (CRC). I found that metformin reduced cell proliferation by inducing cell cycle arrest in the G0/G1 phase, but did not lead to cell death. The anti-proliferative action of metformin resulted mediated by the inactivation of the mTOR pathway and of IGF1R protein. However, the drug only transiently arrested cell growth, since its effects were reversed after drug removal. When I combined the biguanide with the chemotherapy drugs commonly used to treat CRC I observed different responses in the cell lines analysed that reflected their genetic background and their different sensitivities to both the biguanide and chemotherapy. I found that metformin added before chemotherapy drugs antagonised their effects in the majority of the treatments. On the contrary, its administration after long chemotherapeutic treatments significantly reduced the cell viability. I noted that metformin better inhibits cell proliferation in cell lines with rapid growth. I have also assessed the dual treatment combination of metformin with different drugs that target specific genes or proteins (targeted therapies), focusing on BRAF-mutant cell lines. For most of the tested treatments the simultaneous administration of metformin and target drugs gave no advantages over the single drugs, and often resulted in antagonism. Overall, our results show that although further investigations are still needed to elucidate the results of the treaments including metformin, these data suggest that caution should be used in administering chemotherapy to indviduals taking metformin.