Investigation of tumour suppressor microRNA-424(322)/503 in the regulation of mammary epithelial cell stemness and metabolism

Ph. D. Thesis.Mammary glands are composed of heterogeneous cells that coordinate remodelling throughout a woman’s lifetime. Molecular networks underlying changes in the normal mammary gland become increasingly complicated upon malignant transformation. Part of the complexity is due to breast cancer cells that adopt features similar to embryonic stem cells. In relation to more differentiated cells within a tumour bulk, stem-like cells are self-renewing and highly adaptable. Cellular metabolism largely influences the promotion and maintenance of stemness in breast cancer. The specific events, and the mechanisms by which they are controlled, require further exploration. MicroRNAs (miRNAs) are noncoding RNA molecules that regulate gene expression through translational repression of target messenger RNAs. The genomic region of tumour suppressive miR-424(322)/503 is deleted in a subset of primary breast tumours, and knockout mice form invasive carcinomas. The mechanisms by which loss of this miRNA cluster contributes to tumorigenesis are undefined. The current PhD thesis investigated the acquisition of stemness and metabolic reprogramming upon loss of miR-424(322)/503. Knockout and knockdown of miR-424/503 in MCF-10A cells was achieved using CRISPR and miRNA inhibitors, respectively. Stemness was determined by relative expression of pluripotency transcription factors, and mammosphere formation. Metabolism was investigated using a Seahorse XF Analyser and quantifying relative activity of metabolic enzymes. To identify targets, computational algorithms and gene expression tools were applied. Reduction of miR-424(322)/503 in mammary epithelial cells increased the expression of stemness markers and mammosphere formation. Mitochondrial respiration and glycolysis were enhanced in cells with reduced or deleted miR-424(322)/503. Depletion of miR-424(322)/503 impaired pyruvate oxidation, rendering cells dependent on glutamine to maintain high rates of oxygen consumption. Mechanistically, miR-424(322)/503 regulated pluripotency and metabolism via direct targeting of zinc finger protein 217 (ZNF217). Repression of the transcription factor was sufficient to reverse the stem-like and metabolic phenotypes. The current thesis revealed previously unknown roles for miR-424(322)/503 and ZNF217 in regulating the metabolism of mammary epithelial cells. Correlations and mechanisms discovered here offer insight into the major phenotype of knockout mice - breast tumorigenesis. If repression of ZNF217 and/or glutamine deprivation impairs the ability of cells to transform and progress into mammary tumours in vivo, these vulnerabilities may establish an efficient strategy for targeting stemness in miR-424(322)/503-negative/low breast tumours.The JGW Patterson Foundation, The Academy of Medical Sciences at Newcastle University