Roberts, Christopher / C.J.
(2022)
Characterisation of alternative splice variants of PIP5K1A in the human metastatic prostate cancer cell line, LNCaP C4-2.
PhD thesis, University of Nottingham.
Abstract
PIP5KIα is lipid kinase of the type I phosphatidylinositol 4-phosphate 5-kinase subfamily, responsible for the generation of the majority of cellular phosphatidylinositol (4,5) bisphosphate, an important signalling molecule required for cellular membrane dynamics, vesicular transport, actin remodelling and motility. PIP5KIα functions in the growth and metastasis of prostate, breast and other tumours, and pharmacological targeting of PIP5KIα curtails these processes in mouse models. In prostate tumours there appears to be crosstalk between the PIP5KIα and androgen receptor (AR; a key driver of prostate cancer), although the molecular mechanisms and functions in disease are unclear.
Here we show that in LNCaP C4-2 prostate cancer cells CRISPR edited at the PIP5K1A locus, genes implicated in cell cycle regulation, K-Ras signalling, p53 signalling, metastasis, steroid hormone signalling and epithelial mesenchymal transition pathways are differentially expressed compared to wild type LNCaP C4-2 cells, highlighting the importance of PIP5KIα in these pathways in prostate cancer cells. Amongst the most significantly altered differentially expressed genes were androgen regulated genes such as KLK3, KLK4, UGT2B15 and UGT2B17, consistent with crosstalk between PI(4,5)P2 and androgen signalling. Exogenous expression of a fluorescently tagged form of PIP5KIα partially reversed the changes in KLK3 and UGT2B17 expression in KO cells, evidencing a causal link between PIP5KIα and expression of these genes.
Previous studies have shown that the several isoforms of PIP5KIγ, a paralog of PIPI5KIα, each have unique localisation and function facilitated by their variant C-termini. While five alternate splice variants of PIP5K1A have previously been identified, RNA sequencing data suggests the existence of many more. The existence in prostate and breast cancer cells of numerous alternate transcripts of PIP5K1A, resulting from complex alternate splicing, was confirmed by cloning and sequencing. Analysis of the conservation of such transcripts was conducted, finding conservation throughout the class Mammalia. Alternate function of a C-terminal variant isoform of PIP5KIα was investigated, finding alternate localisation compared to canonical isoforms, and a putative nuclear export signal was mapped to C-terminal sequence of this novel variant.
Finally, putative direct interaction of PIP5KIα and AR was assessed by yeast two-hybrid (2YH), finding no evidence toward the existence of such interaction. However, various positive controls for PIP5KIα function in the Y2H failed, suggesting that full length PIP5KIα is not suitable for use in this Y2H system.
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