Genetic and epigenetic insights into colorectal tumorigenesis

Abstract

Colorectal cancer (CRC) is a major health burden with large numbers of new cases worldwide and high disease-specific mortality, despite great advances made towards improving patient clinical outcomes. It arises through the gradual acquisition of particular genetic and epigenetic alterations within normal cells, giving them selective advantage in driving malignant transformation. As such process takes over a decade, early cancer detection actions should strongly impact reducing morbidity. Identification of reliable CRC biomarkers is a permanent challenge for improving CRC management. Thanks to the emergence of new powerful technologies and the advances in the knowledge of the mechanistic bases of the disease, recent genetic and epigenetic markers are becoming promising candidates for early detection, risk stratification, prognosis, and prediction of treatment response. In this doctoral thesis, we have addressed mechanistic and clinical aspects of colorectal tumorigenesis: the deregulation and function of FOXD2 and FOXD2- AS1 in CRC tissues and cell lines (study I) and the role of precancerous mutations in normal colorectal mucosa (study II).

In study I, we characterized the transcriptomic and epigenetic profiles of FOXD2 and FOXD2-AS1 genes in normal and tumor colorectal samples. As bidirectional genes in head- to-head disposition, they showed a strong correlation at the transcriptomic level. However, in tumors they displayed an unbalanced bidirectional expression, whereas FOXD2 was strongly downregulated in association with higher methylation levels outside the promoter region. Interestingly, when we induced overexpression of such genes in CRC cell lines, FOXD2 behaved as a tumor suppressor by reducing migration and colony formation, while FOXD2-AS1 increased migration rates. Overall, our findings suggest the involvement of major mechanisms rewiring cancer, responsible for an altered bidirectional transcription of FOXD2 and FOXD2-AS1. In study II, we focused on the characterization of somatic mutation in normal colorectal mucosa of individuals with and without CRC, using an ultra-deep sequencing technology, CRISPR-Duplex Sequencing. We identified coding mutations in normal colon of most individuals on the 4 cancer genes included in the panel: BRAF, KRAS, PIK3CA, and TP53. However, TP53 and KRAS driver mutations were commonly found in normal colon of CRC patients, often displaying clonal expansions in early onset CRC. Additionally, we developed a primary and integrative mutational model based on the mutational analysis of normal biopsies with potential for CRC risk prediction. Overall, our results support a model where somatic evolution contributes to the expansion of mutated clones in the normal colon tissue, but this process is enhanced in young individuals with cancer.