Investigating the effect of age-associated mitochondrial alterations in intestinal tumour development

Abstract

During ageing there is clonal expansion of mitochondrial DNA (mtDNA) point mutations in colonic epithelial crypts, leading to defects in mitochondrial oxidative dysfunction (OXPHOS). In addition, OXPHOS dysfunction has been shown to be caused by mtDNA mutations in human colorectal adenocarcinomas. However, the effect of age-related mtDNA mutations has on intestinal tumour development remains unknown. To address this question, a mouse model (PolgAmut/mut), which has accelerated accumulation of mtDNA mutations leading to OXPHOS deficiency in the intestine with age, was crossed with an inducible mouse model of intestinal adenoma development (PolgAmut/mut/Lgr5-creER/Apcfl/fl). These mice have a shorter life expectancy due to accelerated tumour growth compared with control mice (PolgA+/+/Lgr5-creER/Apcfl/fl) (unpublished data). The aim of this thesis was to investigate which molecular pathways are altered by age-associated OXPHOS dysfunction in normal intestine, and whether these provide an advantage during intestinal tumour development. Whole transcriptome, unbiased, RNA sequencing was performed on small intestinal (SI) crypts from PolgAmut/mut and PolgAmut/mut mice to identify differentially expressed genes. Results showed upregulation of expression of genes in the de novo serine synthesis pathway (SSP) and serine-uptake and metabolism. Upregulation of these genes was also seen in SI adenomas from PolgAmut/mut/Lgr5-creER/Apcfl/fl mice and was confirmed at the protein level by immunohistochemistry. SI adenoma organoid ex vivo culture from PolgAmut/mut/Lgr5- creER/Apcfl/fl and PolgA+/+/Lgr5-creER/Apcfl/fl mice allowed functional analyses to be performed. Extracellular flux analysis and metabolomics confirmed a reduced oxidative respiratory capacity and an upregulation of de novo serine synthesis in the PolgAmut/mut/Lgr5- creER/Apcfl/fl organoids; which displayed a resilience to serine and glycine starvation compared to controls. These data show an upregulation of the SSP and generation of onecarbon units in normal intestinal crypts in response to OXPHOS dysfunction. These pathways provide substrates for anabolic and antioxidant pathways, providing a selective advantage for tumour growth and survival.