The work performed involved model development, metabolic assessment, and in vitro validation of observations of PI5P4K in prostate tissue and cancer models. I was successful in generating and characterizing multiple new mouse models during PCAPIP. Specifically, the first prostate-specific knock out of Pip4k2a in prostate luminal cells. Based on the novel observation that PI5P4K was highly expressed in the basal cell layer of the prostate, I also generated the first basal cell-specific knock out mouse for inducibility targeting Pip4k2a. I also generated mice with gene combinations that will lead to prostate tumor development, specifically through deletions of the cancer gene Pten.
A major objective of PCAPIP was to uncover what metabolism processes are influenced by PI5P4K in normal cells and prostate cancer. I used experimental approaches to characterize multiple metabolic pathways when PI5P4K is depleted in PCa cell models. I used a large-scale metabolomics approach that detected relative levels of 153 metabolites from 37 pathways. I also ran a state-of-the-art lipidomic analysis that characterized 282 lipid species in samples with and without PI5P4K depletion. These experiments were paired with RNA sequencing analysis, which enabled the identification of changes to metabolic signaling pathways.
Finally, I performed validation experiments using in vitro models to confirm metabolic phenotypes from PI5P4K depletion. This involved previously characterized human prostate cancer cell lines and newly generated mouse organoid cells. I verified that the mouse models were effective at genetically eliminating Pip4k2a expression in animals in the cells of interest (luminal and basal). This confirms the molecular changes are indeed being activated in the in vivo setting. I also found that normal mouse luminal prostate cells have much lower relative expression of PI5P4K compared to tissue from Pten mutant tumors. Using human prostate cancer cells, I validated the inverse expression changes of AR transcript signature genes with depletion of PI5P4K. As well, I found that depletion of PI5P4K in various stress conditions could induce lipid droplet accumulation and increases in the level of reactive oxygen species. I discovered that genetically altered LNCaP cells that lack PI5P4K are significantly more vulnerable to stress conditions such as drug treatments (enzalutamide) and lipid overdose compared to controls.
These results are being composed into a high-impact publication from our group at the University of Bern. As well, were incorporated into a co-authored article submitted to the journal Cell Metabolism with collaborators. PCAPIP results were additionally disseminated in local seminars across Switzerland and at international conferences in France, USA, and Canada in the format of talks and posters.
