Morris, Ryan
(2023)
Transcriptome Alterations Following Loss of PIP5K1α Function in Prostate Cancer Cells.
PhD thesis, University of Nottingham.
Abstract
The human genome encodes three membrane-targeted phosphatidylinositol-4-phosphate 5-kinases (PIP5K1α, PIP5K1β and PIP5K1γ) that generate the signalling molecule phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. These lipid kinases target the 5th position of the inositol ring structure located on phosphatidylinositol 4-phosphate [PI(4)P]. PI(4,5)P2 acts as a key second messenger in cells, involved in regulation of many cellular processes, a major aspect of which is through the activation of PI3K/AKT and PKC signalling pathways. Dysregulation of these pathways through gene alterations is commonly associated with oncogenesis, resulting in sustained cell proliferation, aberrant cell migration, evasion of apoptosis and increased angiogenic potential. In the context of prostate cancer, inactivation of the tumour suppressor gene PTEN occurs in ~50% of advanced malignancies, with this a main driver in oncogenesis. More recently, it has been identified that PIP5K1A gene amplifications are found in ~10% of advanced prostate cancers, however the role that this kinase exerts in prostate tumorigenesis has been less studied.
This research project has set out to explore whether altered expression of PIP5K1α may be associated with transcriptome changes in cancer cells, using human LNCaP C4-2 cells as a model for castration- resistant prostate cancer. Two previously generated CRISPR edited cell lines were used in this work (ΔN and KO) which have abrogated or severely reduced PIP5K1α protein expression. Targeting of Exon 1 in ΔN LNCaP C4-2 cells impaired PIP5K1α protein expression, with translation initiation pursuing from downstream AUG codons. In KO C4-2 cells, both Exons 1 and 6 in PIP5K1A were targeted through CRISPR editing, which heavily impacted protein expression. Exon 1 and Exon 6 encode the N terminus and part of the PIP5K1A kinase domain respectively.
RNA-Sequencing the CRISPR clones relative to WT LNCaP C4-2 revealed significant transcriptome alterations. Approximately 2 fold more differentially expressed genes were observed in ΔN-WT in comparison to KO-WT. However enrichment analysis revealed that both CRISPR clones were associated with genes upregulated with androgen signalling and cell cycle progression. In regards to the former, upon rescuing PIP5K1A depleted cells with a PIP5K1α-mVenus-N1 construct, this negatively regulated the expression of KLK3, a classical androgen regulated gene. This suggests that the expression of PIP5K1α is needed to coordinate the expression of KLK3.
With enrichment analysis suggesting that both cell lines undergo increased expression of genes implicated with cell cycle progression, it was interesting to observe that both ΔN and KO C4-2 cells showed defects in cell proliferation. Through BrdU incorporation assays it was observed that both clones exhibited G1 arrest, with this coupled with increased formation of γ-H2AX foci, indicative of double stranded DNA breaks. Furthermore, both ΔN and KO LNCaP C4-2 cells showed increased p21 levels, a protein which triggers cell cycle arrest. Collectively, this suggests that PIP5K1α is necessary in orchestrating progression through the cell cycle.
Another unexpected observation was that activation of AKT phosphorylation was increased in the ΔN clone compared to WT or KO, despite reduced expression of PIP5K1α. The levels of AKT activating phosphoinositides were not elevated in this clone, with this assayed using fluorescent biosensors. Upon further scrutinising the DEGs from ΔN LNCaP C4-2 cells, a loss of PIK3R1 expression was observed. Subsequently the role of PIK3R1 in regulating AKT phosphorylation in ΔN C4-2 cells underwent preliminary exploration in this work. In this, ΔN cells were rescued with a fluorescent PIK3R1 construct with phospho-AKT levels assayed by flow cytometry.
These assays suggested that rescuing PIK3R1 expression in clone ΔN may reduce levels of phosphorylated AKT, albeit with these results being statistically insignificant. Overall, the elevations in AKT phosphorylation in ΔN cells may be linked to loss of PIP5K1α expression, implying that this kinase may be influencing PI3K/AKT signalling at a transcriptional level.
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