Author: Reham Mounzer
Mounzer, Reham, 2023 Investigating the metabolic regulation of extracellular vesicles in colorectal cancer, Flinders University, College of Medicine and Public Health
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Colorectal cancer (CRC) is the second most diagnosed cancer in Australia. Metabolic dysregulation is a hallmark of CRC, therefore, further understanding of its effect on disease pathogenesis is vital in increasing overall survival. Recently, extracellular vesicles (EVs), have been implicated in the progression of cancer, including CRC. These tumour-released EVs transport nutrients and miRNAs to the tumour and aid its survival through metastasis and immune evasion. Altered metabolic conditions have been found to increase EV release in breast cancer cells, and although EV biogenesis, release, and cargo sorting mechanisms are generally well-understood, the exact mechanism by which metabolism alters EVs is not. Understanding the metabolic regulation of the aforementioned EV processes could allow for better therapeutic outcomes in CRC treatment.
Intracellular metabolism is mainly governed by the major nutrient sensing protein, the mechanistic target of rapamycin (mTOR). mTOR exists in two subtypes, mTORC1 and mTORC2, each regulating various metabolic pathways in cells, such as protein synthesis, glycolysis, and lipogenesis. A few studies have supported a role for mTORC1 in EV secretion, however, the data is conflicting and the role of mTORC2 is not known. In this study, the role of mTOR in EV release in CRC was investigated. mTOR was inhibited in the human colorectal cancer cell line, HCT116, with torin1 and rapalink-1 to investigate the effect on EV release, sorting, and cargo. EVs were quantified and RNA sequencing performed to determine the effect of mTOR inhibition on cellular gene expression, and miRNA composition in EVs. mTORC1 and mTORC2 inhibition reproducibly increased EV release in CRC cells, with substantial cellular gene expression changes observed. Investigation into the gene expression changes highlighted a subset of genes that could be involved in mTOR-regulated EV release, and a possible role for mTOR in selective miRNA sorting into EVs was identified. Furthermore, differential expression of non-coding RNAs identified novel roles for mTOR in the regulation of various cellular processes contributing to CRC biology.
Evidence has shown the type II diabetes drug, metformin, to reduce CRC progression and cancer development risk, but the exact mechanisms by which it does so are unknown. HCT116 cells were also treated with metformin, to determine its effect on EV release, miRNA sorting, and cargo, to ultimately investigate whether metformin reduces CRC progression by acting on EV pathways. Although metformin did not alter EV release, significant changes to miRNA cargo were observed, resulting in reduced proliferation of cells receiving those EVs. RNA sequencing of metformin-treated HCT116 cells and their EVs showed a possible role for metformin in selective sorting of miRNAs into EVs.
In this study, a novel role for mTORC1 and mTORC2 in EV release in CRC and selective miRNA sorting into EVs was identified. Furthermore, metformin altered selective sorting of miRNAs into EVs causing a reduction of recipient HCT116 cell proliferation. These findings provide the opportunity to improve future therapeutic outcomes in the treatment of CRC, via the targeting of mTOR and the use of metformin in reducing EV-mediated cancer progression by altering number and cargo, respectively.
Keywords: Extracellular vesicles, metformin, mTOR, colorectal cancer.
Subject: Medical Science thesis
Thesis type: Doctor of Philosophy
Completed: 2023
School: College of Medicine and Public Health
Supervisor: Prof Janni Petersen