Dissecting the roles of cholesterol and caveolin in castration-resistant prostate cancer

Abstract

Prostate cancer is one of the most common causes of cancer related death in men, mainly due to aggressive tumour metastases. In epidemiological studies, hypercholesterolemia has been associated with advanced prostate cancer risk, while use of cholesterol-lowering drugs, statins, inversely correlated with advanced prostate cancer risk. However, a causal relationship between hypercholesterolemia and progression of advanced prostate cancer including castration-resistant prostate cancer needs to be clearly demonstrated. One clue on the potential mechanism of hypercholesterolemia in prostate cancer comes from the demonstrated role of caveolin-1 as a prostate cancer promoter. Caveolin-1 is a cholesterol-binding membrane protein that is essential for formation of caveolae, cell surface pits enriched in cholesterol. Caveolae have been suggested to mediate a myriad of cellular functions, and caveolin-1 action has been directly equated to caveolae function in previous studies. However, it has been recently demonstrated that PTRF/cavin-1 is also required for caveola formation, in addition to caveolin-1. Prior to this study, the role of PTRF/cavin-1 in prostate cancer has not been examined. The aims of this study were 1) to clarify differential roles of caveola forming proteins, caveolin-1 and PTRF/cavin-1, 2) to determine the effect of diet-induced hypercholesterolemia on advanced prostate tumour progression mediated through androgen-independent mechanisms using an orthotopic human prostate cancer PC-3 xenograft mouse model, and 3) to unveil potential mechanisms between hypercholesterolemia and advanced prostate tumour progression. For these studies, bioluminescence based in vivo and ex vivo optical imaging have been optimised using an orthotopic PC-3 xenograft mouse model. To address aim 1, the expression of caveolin-1 and PTRF/cavin-1 in prostatic epithelia and stroma has been determined using tissue microarrays containing normal, non- and malignant human prostatic tissues. Caveolin-1 overexpression was found in around 70% of highly aggressive prostatic carcinoma (≥ Gleason score 8) without the presence of PTRF/cavin-1 expression, but not in normal tissues. Utilising PC-3 as a suitable model of caveolin-1 positive, but PTRF/cavin-1 negative prostate cancer cells, this study found that PTRF/cavin-1 neutralised the tumour promoting effects of caveolin-1 microdomains on PC-3 xenograft tumour progression. PTRF/cavin-1 positive PC-3 tumours had lower expression of IL-6 with decreased α-smooth muscle actin expression in stroma tissues compared with control PC-3 xenograft tumours. To support the differential effects of PTRF/cavin-1 and caveolin-1 on prostate cancer progression, prostate cancer LNCaP and 22Rv1 cells which express neither caveolin-1 nor PTRF/cavin-1 were used. In these cells, caveolin-1 but not PTRF/cavin-1 significantly increased anchorage-independent growth. Further studies demonstrated reversed effects of caveolin-1 in caveolin-1 positive LNCaP cells by co-expression of PTRF/cavin-1. While specific caveolin-1 membrane microdomains have tumour promoting effects, cellular cholesterol contents may directly modulate tumour promoting effects. To investigate this (aim 2), mice were randomly assigned to normal or hypercholesterolemic diet groups, and xenografted tumour progression was determined using in vivo and ex vivo imaging. This study showed that diet-induced hypercholesterolemia predominantly accelerated tumour metastases to lymph nodes, lung and proximal (femur, tibia/fibula) and distant (humerus, ulna/radius) bones, and joint metastases. These intriguing findings revealed additional effects of cholesterol on advanced prostate tumour metastases through androgen-independent mechanisms. In other words, this study strongly suggests the importance of cholesterol management to suppress the tumour promoting effects of cholesterol through androgen-sensitive (de novo androgen synthesis) and androgen-independent (current study) mechanisms in advanced prostate cancer, especially for patients with castration-resistant prostate cancer. To probe the molecular mechanisms involved in these findings (aim 3), in vitro quantitative proteomics was performed to identify cholesterol-induced proteome alterations. The cholesterol-induced protein expression of IQGAP1, a membrane scaffolding protein, was identified and verified. Both caveolin-1 and IQGAP1 were highly expressed in aggressive prostate cancer cell lines, PC-3 and DU145, and the expression of these proteins was up-regulated by increased cellular cholesterol levels. Consistently, knockdown of IQGAP1 and/or caveolin-1 reduced the transmigration and invasion of PC-3 cells in vitro and metastases in vivo. In summary, this thesis presents the first direct demonstration of a causal relationship between hypercholesterolemia and aggressive prostate tumour metastases mediated through androgen-independent mechanisms. In addition to highlighting the need to monitor serum cholesterol in prostate cancer patients, results from this thesis also reveal potential mechanisms and a novel molecular target, IQGAP1.