Unit:
Faculty of MedicineLibrary of the School of Health Sciences
Author:
Dionellis Vasileios-Stamatios
Dissertation committee:
Βασίλειος Γοργούλης, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Αθανάσιος Κοτσίνας, Επίκουρος Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Αθανάσιος Χαλαζωνίτης, Καθηγητής, Σχολή Επιστημών, Τμήμα Μοριακής Βιολογίας, Πανεπιστήμιο Γενεύης
Jiri Bartek, Καθηγητής, Τμήμα Ιατρικής Βιοχημείας και Βιοφυσικής, Ινστιτούτο Karolinska
Μαρία Γαζούλη, Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Κωνσταντίνος Ευαγγέλου, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Σοφία Χαβάκη, Επίκουρη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Original Title:
Βιοπληροφορική ανάλυση της γονιδιωματικής αστάθειας κατά την καρκινογένεση
Translated title:
Bioinformatics analysis of genomic instability during carcinogenesis
Summary:
Colorectal cancer (CRC) is one of the leading causes of cancer mortality; it typically originates as adenomas that progress over time to carcinomas. Only in the last decade, with the advent and development of High-throughput Next Generation Sequencing technology, the genomes of thousands of human CRCs have been sequenced, revealing a large number of genetic alterations. Additionally, studies in mouse cancer models have been used and are being used to understand the mechanism of colorectal cancer development. However, the molecular mechanisms governing the accumulation of these mutations are still under investigation. In most cancer sequencing studies, the DNA being sequenced is derived from many cancer cells, typically in the order of millions of cells. This means that, effectively, only mutations that are present in the majority of the cells of a tumor can be identified. However, mutations arise in single cells, and they will become detectable only if the cell in which they arose has a selective advantage and overtakes the tumor. Thus, the sequencing of the genome of single cells is essential for understanding cancer development and evolution.
Currently, single-cell genome sequencing is technically very challenging. To overcome this challenge, in this doctoral dissertation, which was conducted in two separate stages, single crypts were initially isolated from both morphologically normal intestine and intestinal adenomas of cancer-prone Apcmin/+ mice, and then from tumors developed in the colon of mice with differentially combined Apclox/lox, KrasLSL-G12D and Tp53lox/lox targetable alleles. Normal, adenomatous and single crypt cancer cells were isolated and expanded through a 3D-organoid culture system, and their exomes were sequenced.
In the first phase, analysis of the sequencing data revealed that the adenoma-derived cells had, on average, almost 11 times more SNSs in the protein coding genic regions than the normal cells. Most of the SNSs were unique to each organoid, even for organoids that were derived from the same tumor. These results indicate that point mutations in mouse intestinal adenomas—and, hence, in cancers derived from these adenomas—arise after transformation of a normal cell into a precancerous cell and are, thus, in their majority, not due to normal aging.
The analysis of the sequencing data of the second phase showed that the number of single nucleotide substitutions (SNSs) correlated with the age of the tumour but was unaffected by the number of targeted cancer-driver genes. Thus, tumours that expressed mutant Apc, Kras, and Tp53 alleles had as many SNSs as tumours that expressed only mutant Apc. In contrast, the presence of large-scale copy number alterations (CNAs >10 Mb) correlated strongly with Tp53 inactivation. Comparison of the SNSs and CNAs present in organoids derived from the same tumour revealed intratumoural heterogeneity consistent with genomic lesions accumulating at significantly higher rates in tumour cells compared to normal cells. The rate of acquisition of SNSs increased from the early stages of cancer development, whereas large-scale CNAs accumulated later, after Tp53 inactivation. Thus, a significant fraction of the genomic instability present in cancer cells cannot be explained by aging processes occurring in normal cells before oncogenic transformation.
Main subject category:
Health Sciences
Keywords:
Colorectal cancer, Genomic instability, Exome sequencing, Single nucleotide variants, Copy number alterations, Mutational signatures, Organoids
Number of references:
285
File:
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Dionellis_PhD_thesis.pdf
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File access is restricted only to the intranet of UoA.
