Targeting the intestinal stem cell niche through energy and metabolism signaling, for colorectal cancer chemoprevention using aspirin and metformin

Abstract

Colorectal cancer (CRC) although largely preventable, remains the second most common cause of cancer-related deaths in the United Kingdom. Obesity, type 2 diabetes and physical inactivity are CRC risk factors that alter metabolism. Growing evidence indicates that overnutrition increases ‘stemness’: the ability of intestinal stem cells to self-renew or differentiate. The mechanistic target of rapamycin (mTOR) pathway, a central metabolic regulator, is elevated in CRC and modulates stem cell response to nutrients. Therefore, chemopreventive agents which regulate nutrient signalling may be used to combat the increase in obesity-related CRC. Aspirin and metformin have been independently shown to decrease CRC incidence. Aspirin has been shown to have a differential protective role in obese individuals. Both drugs reduce stemness in CRC cell lines independently, however, the potential of the combination of aspirin and metformin for the chemoprevention of obesity- driven CRC remains to be explored. Hence, this thesis aims to understand whether aspirin and metformin decrease overnutrition-driven stemness in CRC, while exploring their synergistic potential and mechanism.

In CRC cell lines, aspirin and metformin treatment reduced transcript levels of stem cell markers LGR5 and CMYC, and inhibited mTORC1 downstream target phosphorylation levels. Utilizing 3-dimentional (3D) sphere formation assays revealed that aspirin and metformin also reduced stem cell function in the HCT116 cell line. Drug combination showed a stronger effect compared to either drug alone, and synergy assays confirmed that drug combination has a synergistic effect. These effects on the stem cell niche were further validated in mouse Apcfl/fl intestinal organoids, whereby aspirin and metformin reduced clonogenicity capacity. Patient organoids from different genetic backgrounds were established: sporadic, familial adenomatous polyposis (FAP), and Peutz- Jeghers syndrome (PJS). Upon treatment with aspirin and metformin, LGR5 and CMYC transcript levels were reduced in all normal mucosa patient-derived organoids. Aspirin and metformin attenuated the effects of lipid mixture overnutrition in mouse Apcfl/fl organoids and normal mucosa patient-derived organoids. Transcriptome analysis of aspirin, metformin or combination treated HCT116 cells further demonstrated the synergistic effect and revealed potential pathways modulated by these drugs: ER stress and the spliceosome. My findings provide a novel insight for the potential role of the combination of aspirin and metformin for CRC chemoprevention, particularly in ‘at risk’ obese individuals.