Periodic Reporting for period 1 - PCAPIP (Towards understanding non-canonical phosphatidylinositol kinases in the maintenance of prostate metabolism.)
Période du rapport: 2018-06-01 au 2020-05-31
My hypothesis is that PI5P4K is a critical regulator of AR signaling that supports PCa cell survival by influencing metabolic stress cooping mechanisms. To test this I have the following objectives: (1) Establish the biological phenotype associated with PI5P4K expression in prostate cells, (2) Examine how targeting PI5P4K influences the molecular and tumorigenic characteristics of PCa, and (3) Uncover how PI5P4K supports changes to androgen dependence through control of prostate cell metabolism. In attempt to answer these questions I generated the first prostate-specific knock-out mouse model PI5P4K in both normal prostate and in combination with an established model of aggressive cancer. As well, advanced metabolomics technology was used to measure how PI5P4K influences metabolic networks that drive the survival of cancer cells. This first-in-class study aims to establish the role of a novel pathway that could be a key control in the progression to untreatable PCa.
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.
I expect to find that depletion of PI5P4K impacts the ability of PCa to coup with metabolic stress. This will be apparent in changes to cellular metabolite abundance, cell cycle, cancer survival, and reactive oxygen species. By developing novel mouse models, I will characterize PI5P4K expression patterns in normal and cancer tissue to provide insights into tissue-specific phenotypes of type II PI kinases. Most importantly, I aim to characterize an inverse relationship of PI5P4K and the AR pathway. I expect to find that PI5P4K is upregulated in AR-indifferent cancer models that require additional metabolic stress pathways to survive the stress of cancer progression.
The results of PCAPIP have potential impacts on the field of medical biology and pharmaceutical development. By uncovering non-canonical pathways that may have interplay with AR and PI3K signaling, drug development for PI5P4K may prove to be attractive in a clinical setting. This extends beyond cancer biology, but also to other diseases such as diabetes or rare metabolic disorders. Through this understanding, we hope to determine better how to improve the efficacy of AR targeted drugs and establish a new treatment option that has previously been unexplored.