ROLE OF HIGH DENSITY LIPOPROTEINS AGAINST PROSTATE CANCER

Background. Prostate cancer (PCa) is the second leading cause of cancer-related death in men. Although androgen deprivation therapy (ADT) is initially effective, many patients progress to castration-resistant disease with poor prognosis and limited treatment options, highlighting the need for new therapeutic strategies. The tumor microenvironment (TME) contains pro-oxidant, pro-inflammatory mediators and lipids such as cholesterol and fatty acids, which sustain proliferation, survival, and angiogenesis. Cholesterol can also be converted into oxysterols, endogenous LXR ligands that generally exert antiproliferative effects. However, 27-hydroxycholesterol (27-HC) has been shown to promote tumor growth in breast cancer, although its role in PCa remains controversial. Several studies report an inverse correlation between HDL-cholesterol levels and cancer prognosis, including in PCa. Beyond their atheroprotective role, HDL promote cholesterol efflux and exert anti-inflammatory, antioxidant, and immunomodulatory effects. Synthetic HDL (sHDL), currently under clinical evaluation as anti-atherosclerotic agents, retains these activities and can serve as drug delivery vehicles by targeting tumors through scavenger receptor BI (SR-BI), that is overexpressed in different tumor types, including PCa. Aim. Previous in vitro and in vivo studies have shown that HDL can inhibit PCa tumour growth by reducing intracellular cholesterol content and oxidative stress, inflammation and angiogenesis. However, the overall effects of HDL on cholesterol metabolism and their anti-inflammatory activities, especially in the context of the TME, should be deeply investigated. Thus, this study aims to characterize the effect of plasma-derived and sHDL on PCa proliferation and metabolism, focusing on oxysterols, and to better characterize the anti-inflammatory activities of HDL in the context of TME. Methods. Androgen-sensitive and -resistant PCa cell lines (LNCaP and PC-3, respectively) were tested and compared with non-tumour prostate epithelial cells (PNT2). Lipoproteins were isolated by ultracentrifugation from the plasma of healthy volunteers. Synthetic HDL particles (sHDL) were formulated using apoA-I and POPC, with or without cholesterol. Cell proliferation of PCa cell lines and human-derived organoids was assessed by cell count, colony formation assay, Incucyte and MTS assay. GC-MS was used to determine the intracellular levels of oxysterols. Gene and protein expression were quantified through RT-PCR and WB, respectively. Single-cell RNA sequencing (scRNA-seq) dataset of prostate from WT and Ptenpc-/-, either castrated or not, was analysed to assess immune infiltration and metabolic pathway enrichment both in prostate epithelial and immune cells. Results. Both plasma-derived and sHDL, composed of POPC:apoA-I:cholesterol at a 2.17:1:0.22 weight ratio, significantly reduced colony formation in LNCaP and PC3 cells and suppressed the growth of patient-derived organoids, derived from mCRPC and NE-CRPC biopsies. Gene and protein expression analyses revealed that PCa cells overexpress LDL-R and LRP1, suggesting a reliance on extracellular cholesterol uptake rather than de novo synthesis. This is supported by functional data showing that HMG-CoA reductase inhibition did not alter cellular cholesterol levels in PC3 cells, and by scRNA-seq data of epithelial cells isolated from the prostate of WT mice and prostate tumours of Ptenpc-/- mice, either castrated or not. Furthermore, HDL treatment significantly reduced intracellular 27-HC levels, that were particularly elevated in PC3 cells, and abrogated 27-HC–induced proliferation in both LNCaP and PC3. Regarding the anti-inflammatory capacity of HDL, both plasma-derived and sHDL counteracted IL-6–driven proliferation, blunting the activation of p38 and Akt signaling pathways. Finally, scRNA-seq analysis of prostates from Ptenpc-/-mice revealed that NK cells exhibited significant enrichment in gene sets involved in lipoprotein and lipid uptake, suggesting a shift toward extracellular lipid and cholesterol dependency, as observed in PCa cell lines. Preliminary in vitro experiments showed that plasma-derived HDL had no major effect on human NK cell activation or cytotoxicity, apart from a slight reduction in granzyme B upon stimulation. In line with this, the expression of surface activation markers was unchanged, indicating that under these conditions HDL do not significantly affect NK cell functionality. Conclusions. This study highlights lipid metabolism, and cholesterol in particular, as a key driver of prostate cancer, involving both tumor cells and immune components of the microenvironment. HDL emerge as modulators of metabolic and inflammatory pathways, exerting antiproliferative effects by lowering intracellular cholesterol and counteracting 27-HC– and IL-6–induced growth. Synthetic HDL (sHDL) preserved these activities, supporting their potential as innovative therapeutic tools in prostate cancer. Further validation in preclinical models will be required to confirm these findings and to assess the translational relevance of HDL- and sHDL-based strategies in PCa.