Hadia, Shuaib2023-02-202023-02-202022https://hdl.handle.net/10539/34611A dissertation submitted in fulfilment of the requirements for the degree of Master of Science to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2022Pancreatic cancer (PC) is the 11th most common cancer globally and the 7th leading cause of cancer related deaths worldwide. The incidence of PC is expected to rise, especially in the developing world. The most common type of PC is pancreatic ductal adenocarcinoma (PDAC), accounting for more than 85% of all PC cases. PDAC is an aggressive malignancy, presenting with a 5-year survival rate of only 9%. Transcriptional profiling has sorted PDAC into distinct subtypes with the majority being classified as either classical epithelial (E) or quasi-mesenchymal (QM). Research has shown the two subtypes to exist on a continuum with features of both subtypes present in PDAC cells, suggesting an interconversion between the cell states. This interconversion has been linked to PDAC plasticity and a heterogeneous tumour, which together contribute to the poor clinical outcome of PDAC. Vitamin D and its receptor, the vitamin D receptor (VDR), function as a transcription factor and regulator of gene expression. Vitamin D deficiency has been associated with an increased risk of developing PC. Vitamin D supplementation has been shown to modulate PDAC E and QM subtypes, and hence PDAC plasticity. Here we look at the transcriptional profile of PDAC cells taken from PDAC patients, supplemented with 10 nM biologically active vitamin D - 1,25(OH)2D3. We quantified transcript and gene-level abundance estimates from publicly available RNA-sequence data, and then performed differential gene expression (DGE) and pathway analysis on the RNA expression profiles of the PDAC samples (E: n = 3; QM n = 3). We identified genes differentially expressed in each PDAC subtype, as well as biological processes enriched from these differentially expressed genes in response to 10 nM 1,25(OH)2D3 supplementation. In total, DGE analysis yielded 1991 and 1744 DEGs (Wald Test p-value < 0.05) in the E and QM subtypes respectively in response to 10 nM 1,25(OH)2D3 supplementation. Comparing the DEGs of the PDAC subtypes revealed 335 commonly DEGs, of which 270 were contra-regulated, indicating a differential response to 1,25(OH)2D3 between the subtypes. Notably, the gene encoding the vitamin D metabolising enzyme, CYP24A1, was up-regulated in the E subtype in response to 1,25(OH)2D3 supplementation only. CYP24A1 contains a vitamin D response element (VDRE) and is known to be a direct transcriptional target of liganded VDR. Various cancer-related genes, including RAS family genes, the MYC oncogene, TGFB1 and TGFB2, HIF1A and CCND2 were differentially expressed in both the E and QM PDAC subtypes, in response to 1,25(OH)2D3 supplementation. Biological pathways relating to activating immune 14 responses were up-regulated from the DEGs of both subtypes, and pathways relating to down regulation of cell cycle were enriched from the DEGs of the E subtype. Both subtypes presented with mixed expression of epithelial and mesenchymal markers in response to 1,25(OH)2D3 supplementation, showing 1,25(OH)2D3 to not promote the intrinsic cell type markers of that subtype only. We noted the down-regulation of TGFB1 and TGFB2 in the E subtype, but up regulation of TGFB2 in the QM subtype. This suggests that differential TGFB gene expression may possibly modulate the differential response to 1,25(OH)2D3 observed between the E and QM PDAC subtypes, through differential regulation of the VDR and EMT-related transcription factors.enDissertation