title: Integrative multi-dimensional omics profiling for the in-depth understanding of the mechanisms of ω-3 polyunsaturated fatty acids metabolism in prostate cancer
Creators: name: He, Chao
Publisher: name: University of Liverpool Repository
Year: 2029

description: Prostate cancer (PCa) affects a substantial number of men, and current therapies are in urgent need of improvement. The dietary intervention of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) has shown promise in the prevention of cancer development. This project aims to unveil the underlying mechanisms and provide a potentially novel approach to treat prostate cancer. In addition to conventional biochemical and biophysical studies in this project, we also applied large-scale proteomic, lipidomic, and transcriptomic profiling techniques to study the changes in acetylome, lipidome and transcriptome, respectively, after the ω-3 PUFAs treatment in PCa cells.
 Results from colony formation assays and Calcein AM viability assays indicate that ω-3 PUFAs have the ability to inhibit the proliferation of prostate cancer in vitro. Consistent with our previous findings, ω-3 PUFAs treatment decreases the phosphorylation of pyruvate dehydrogenase A1 (PDHA1), a gatekeeper enzyme link between glycolysis and the mitochondrial oxidative phosphorylation, and therefore enhances its activity. Likewise, the treatment of JX06, a potent pyruvate dehydrogenase kinase 1 (PDHK1) inhibitor, also leads to a decrease in the phosphorylation of PDHA1 and inhibits the proliferation of prostate cancer cells. However, the mechanism of action may be quite different. ω-3 PUFAs activate AMPK to impede the interaction between PDHA1 and PDHK1, leading to decreased PDHA1 phosphorylation, while JX06 directly inhibits the PDHK1 activity through the formation of a disulfide bond to induce conformational changes via Van der Waals forces, therefore hindering access of ATP to its binding pocket and impairing PDHK1’s enzymatic activity. Furthermore, our findings also suggest that ω-3 PUFAs trigger ferroptosis rather than apoptosis in PCa.
 For a further in-depth understanding of ω-3 PUFAs metabolism in PCa, we conducted more investigations from the perspective of fatty acid metabolism.
 Firstly, findings suggest that oleic acid (OA, ω-9 MUFA) elevates while eicosapentaenoic acid (EPA, ω-3 PUFA) reduces the cellular acetyl-CoA level, resulting in alterations of the global acetylome. The acetylome profiling identified 538 acetylated peptides, representing 387 acetylated proteins. Gene Ontology (GO) analysis suggested that some candidates are involved in cadherin binding, which may affect cell motility. Thus, PFN1 and FLNA were selected for further investigation due to their critical role in cancer motility modulation. Downregulation of PFN1 and FLNA indicated that they are essential in cell migration and invasion of prostate cancer cells. Furthermore, OA promotes while EPA inhibits prostate cancer migration and invasion. Immunofluorescence assay indicated that EPA impedes the assembly of actin skeleton and the formation of lamellipodia or filopodia through reduced localization of PFN1 and FLNA to the leading edge of cells. Therefore, perturbed acetylome may be one critical step in fatty acid-affected cancer cell motility.
 Secondly, we studied the anabolism of different fatty acids in PCa. The alterations in the cellular lipid pool were examined through lipidomic profiling. Results suggested that ω-3 PUFAs elevated the generation of PUFA-derived Phosphatidylethanolamine (PE) and Phosphatidylcholine (PC), which may play a critical role in ferroptosis execution. Moreover, a cell-derived xenograft study was carried out and indicated that EPA initially exhibited a tumour-inhibition effect. However, it was followed by a sharp acceleration in growth rate. Further investigations are designed to study the mechanism.
 Thirdly, to unveil the response of PCa to EPA treatment, the transcriptome study was conducted. Results indicated that EPA treatment induced the expression of genes involved in ferroptosis, stress response and mitophagy. Further evidence suggested that SQSTM1 (p62) and SESN2 regulate mitophagy, a process that scavenges damaged mitochondria to protect cancer cells from ferroptosis. 
 In conclusion, the investigations of this study have shown that prostate cancer incorporates ω-3 PUFAs into phospholipids, resulting in ferroptosis and mitochondria damage. As a result, cellular acetyl-CoA level was decreased, leading to impeded prostate cancer cell migration and invasion. Moreover, prostate cancer cells employed mitophagy to mitigate ω-3 PUFAs induced mitochondrial damage and ferroptosis.
This project provides us with more insights into the response of PCa when treated by ω-3 PUFAs from the viewpoint of multi-dimensional omics profiling. This would facilitate the development of more effective and potent therapeutic targets and strategies for prostate cancer therapy.