Prostate cancer cell proliferation is influenced by LDL-cholesterol availability and cholesteryl ester turnover

Raftopulos et al. Cancer & Metabolism (2022) 10:1 https://doi.org/10.1186/s40170-021-00278-1 RESEARCH Open Access Prostate cancer cell proliferation is influenced by LDL-cholesterol availability and cholesteryl ester turnover Nikki L. Raftopulos1†, Tinashe C. Washaya1†, Andreas Niederprüm2,3, Antonia Egert2, Mariam F. Hakeem-Sanni1, Bianca Varney1, Atqiya Aishah1, Mariya L. Georgieva2, Ellinor Olsson1, Diandra Z. dos Santos1,4, Zeyad D. Nassar5,6, Blake J. Cochran7, Shilpa R. Nagarajan1, Meghna S. Kakani1, Jordan F. Hastings8, David R. Croucher8,9, Kerry-Anne Rye7, Lisa M. Butler5,6, Thomas Grewal2 and Andrew J. Hoy1* Abstract Background: Prostate cancer growth is driven by androgen receptor signaling, and advanced disease is initially treatable by depleting circulating androgens. However, prostate cancer cells inevitably adapt, resulting in disease relapse with incurable castrate-resistant prostate cancer. Androgen deprivation therapy has many side effects, including hypercholesterolemia, and more aggressive and castrate-resistant prostate cancers typically feature cellular accumulation of cholesterol stored in the form of cholesteryl esters. As cholesterol is a key substrate for de novo steroidogenesis in prostate cells, this study hypothesized that castrate-resistant/advanced prostate cancer cell growth is influenced by the availability of extracellular, low-density lipoprotein (LDL)-derived, cholesterol, which is coupled to intracellular cholesteryl ester homeostasis. Methods: C4-2B and PC3 prostate cancer cells were cultured in media supplemented with fetal calf serum (FCS), charcoal-stripped FCS (CS-FCS), lipoprotein-deficient FCS (LPDS), or charcoal-stripped LPDS (CS-LPDS) and analyzed by a variety of biochemical techniques. Cell viability and proliferation were measured by MTT assay and Incucyte, respectively. * Correspondence: † Nikki L. Raftopulos and Tinashe C. Washaya These authors contributed equally. 1 School of Medical Sciences, Charles Perkins Centre, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia Full list of author information is available at the end of the article © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Raftopulos et al. Cancer & Metabolism (2022) 10:1 Page 2 of 15 Results: Reducing lipoprotein availability led to a reduction in cholesteryl ester levels and cell growth in C4-2B and PC3 cells, with concomitant reductions in PI3K/mTOR and p38MAPK signaling. This reduced growth in LPDScontaining media was fully recovered by supplementation of exogenous low-density lipoprotein (LDL), but LDL only partially rescued growth of cells cultured with CS-LPDS. This growth pattern was not associated with changes in androgen receptor signaling but rather increased p38MAPK and MEK1/ERK/MSK1 activation. The ability of LDL supplementation to rescue cell growth required cholesterol esterification as well as cholesteryl ester hydrolysis activity. Further, growth of cells cultured in low androgen levels (CS-FCS) was suppressed when cholesteryl ester hydrolysis was inhibited. Conclusions: Overall, these studies demonstrate that androgen-independent prostate cancer cell growth can be influenced by extracellular lipid levels and LDL-cholesterol availability and that uptake of extracellular cholesterol, through endocytosis of LDL-derived cholesterol and subsequent delivery and storage in the lipid droplet as cholesteryl esters, is required to support prostate cancer cell growth. This provides new insights into the relationship between extracellular cholesterol, intracellular cholesterol metabolism, and prostate cancer cell growth and the potential mechanisms linking hypercholesterolemia and more aggressive prostate cancer. Keywords: Prostate cancer, LDL, LDL-cholesterol, Cell proliferation, Cholesteryl ester, ACAT1, nCEH1, HSL Background The progression of prostate cancer, and other solid tumors, is supported by changes in cancer cell metabolism that are geared towards increasing biomass synthesis. One critical component is covering the increased demand for lipids in cellular membranes during proliferation [1], in particular cholesterol as it is an essential constituent of cellular membranes, comprising up to 30% of lipid content. Cholesterol metabolism in prostate cancer has received significant attention in recent years (see reviews [2, 3]). Beyond the role of cholesterol metabolism in oncogenesis and the differences in cholesterol biology observed between normal tissue and tumor, cholesterol metabolism has been suggested to play key roles in other aspects of prostate cancer pathophysiology including treatment resistance [2, 4]. Androgen deprivation therapy has remained the frontline strategy for clinical management of locally-recurrent and/or metastatic disease due to the dependence of prostate cancer cells on androgens for growth and survival. Although androgen deprivation therapy is initially successful in slowing prostate cancer progression, patients inevitably develop lethal castrate-resistant disease (CRPC), due to the emergence of adaptive survival pathways that reprogram androgen signaling and/or activate alternative tumor survival pathways [5]. Androgen deprivation therapy, by creating a low androgen environment, induces pronounced systemic metabolic changes including hypercholesterolemia [6], which may result in a plentiful supply of cholesterol for de novo steroidogenesis as an adaptive mechanism to promote the development of CRPC [7]. In fact, hypercholesterolemia is associated with a shorter time to the development of CRPC in patients who have undergone androgen deprivation therapy [8]. Several studies have also shown a relationship between elevated circulating cholesterol levels and a higher risk of prostate cancer development and progression [9–12]. Conversely, patients who use cholesterol-lowering agents such as statins have a lower risk of advanced prostate cancer and redu (...truncated)