ABSTRACT
The major processes in carcinogenesis include the inactivation of tumor-suppressor genes (TSGs). Although Knudson's two-hit model requires two independent inactivating mutations, perhaps more frequently, a TSG inactivation can occur through a loss of heterozygosity (LOH) of an inactivating mutation. Deletion and uniparental disomy (UPD) have been well documented as LOH mechanisms, but the role of gene conversion is poorly understood. Here, we developed a simple algorithm to detect somatic gene conversion from short-read sequencing data. We applied it to 6285 cancer patient samples, from which 4978 somatic mutations that underwent gene conversion to achieve LOH were found. This number accounted for 14.8% of the total LOH mutations. We further showed that LOH by gene conversion was enriched in TSGs compared with non-TSG genes, showing a significant contribution of gene conversion to carcinogenesis.
Subject(s)
Genes, Tumor Suppressor , Neoplasms , Carcinogenesis/genetics , Gene Conversion , Humans , Loss of Heterozygosity , Neoplasms/geneticsABSTRACT
BACKGROUND: Intratumor heterogeneity (ITH) in microsatellite instability-high (MSI-H) colorectal cancer (CRC) has been poorly studied. We aimed to clarify how the ITH of MSI-H CRCs is generated in cancer evolution and how immune selective pressure affects ITH. METHODS: We reanalyzed public whole-exome sequencing data on 246 MSI-H CRCs. In addition, we performed a multi-region analysis from 6 MSI-H CRCs. To verify the process of subclonal immune escape accumulation, a novel computational model of cancer evolution under immune pressure was developed. RESULTS: Our analysis presented the enrichment of functional genomic alterations in antigen-presentation machinery (APM). Associative analysis of neoantigens indicated the generation of immune escape mechanisms via HLA alterations. Multiregion analysis revealed the clonal acquisition of driver mutations and subclonal accumulation of APM defects in MSI-H CRCs. Examination of variant allele frequencies demonstrated that subclonal mutations tend to be subjected to selective sweep. Computational simulations of tumour progression with the interaction of immune cells successfully verified the subclonal accumulation of immune escape mutations and suggested the efficacy of early initiation of an immune checkpoint inhibitor (ICI) -based treatment. CONCLUSIONS: Our results demonstrate the heterogeneous acquisition of immune escape mechanisms in MSI-H CRCs by Darwinian selection, providing novel insights into ICI-based treatment strategies.
Subject(s)
Colonic Neoplasms , Colorectal Neoplasms , Humans , Microsatellite Instability , Colorectal Neoplasms/pathology , Colonic Neoplasms/genetics , Mutation , Antigen Presentation , Microsatellite Repeats/geneticsABSTRACT
During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as sister foci, and protects genome stability. To gain further insights into FANCD2 function and its regulatory mechanisms, we examined the genome-wide chromatin localization of FANCD2 in this setting by ChIP-seq analysis. We found that FANCD2 mostly accumulates in the central regions of a set of large transcribed genes that were extensively overlapped with known CFS. Consistent with previous studies, we found that this FANCD2 retention is R-loop-dependent. However, FANCD2 monoubiquitination and RPA foci formation were still induced in cells depleted of R-loops. Interestingly, we detected increased Proximal Ligation Assay dots between FANCD2 and R-loops following APH treatment, which was suppressed by transcriptional inhibition. Collectively, our data suggested that R-loops are required to retain FANCD2 in chromatin at the middle intronic region of large genes, while the replication stress-induced upstream events leading to the FA pathway activation are not triggered by R-loops.
Subject(s)
Chromatin/genetics , Chromosome Fragile Sites/genetics , DNA Replication/genetics , Fanconi Anemia Complementation Group D2 Protein/genetics , Genomic Instability/genetics , Aphidicolin/pharmacology , Cell Line, Tumor , Chromatin/metabolism , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Damage , DNA Repair , DNA Replication/drug effects , Enzyme Inhibitors/pharmacology , Fanconi Anemia Complementation Group D2 Protein/metabolism , Gene Expression Regulation/drug effects , Humans , Nucleic Acid Conformation , Signal Transduction/genetics , Ubiquitination/drug effectsABSTRACT
Duplication plays an important role in creating drastic changes in genome evolution. In addition to well-known tandem duplication, duplication can occur such that a duplicated DNA fragment is inserted at another location in the genome. Here, we report several genomic regions in the human genome that could be best explained by two types of insertion-based duplication mechanisms, where a duplicated DNA fragment was modified structurally and then inserted into the genome. In one process, the DNA fragment is turned into an extrachromosomal circular DNA, cut somewhere in the circle, and reintegrated into another location in the genome. And in the other, the DNA fragment forms a "lariat structure" with a "knot", the strand is swapped at the knot, and is then reintegrated into the genome. Our results suggest that insertion-based duplication may not be a simple process; it may involve a complicated procedures such as structural modification before reintegration. However, the molecular mechanism has yet to be fully understood.