Leave the fat out Prostate cancer is one of the most common tumors in men. Although characterized by slow growth rate, preventing prostate cancer progression to an aggressive stage is a major challenge. Watt et al. focused on cancer metabolism and showed increased fatty acid uptake in human malignant prostate cancer tissue. The increased uptake was mediated by up-regulation of the fatty acid translocase CD36. Silencing CD36 in human prostate cancer cells reduced fatty acid uptake and cell proliferation. In prostate cancer mouse and human preclinical models, Cd36 ablation or inhibition reduced prostate cancer severity. The data suggest that CD36 might be targeted for treating prostate cancer.

Abstract Metabolism alterations are hallmarks of cancer, but the involvement of lipid metabolism in disease progression is unclear. We investigated the role of lipid metabolism in prostate cancer using tissue from patients with prostate cancer and patient-derived xenograft mouse models. We showed that fatty acid uptake was increased in human prostate cancer and that these fatty acids were directed toward biomass production. These changes were mediated, at least partly, by the fatty acid transporter CD36, which was associated with aggressive disease. Deleting Cd36 in the prostate of cancer-susceptible Pten−/− mice reduced fatty acid uptake and the abundance of oncogenic signaling lipids and slowed cancer progression. Moreover, CD36 antibody therapy reduced cancer severity in patient-derived xenografts. We further demonstrated cross-talk between fatty acid uptake and de novo lipogenesis and found that dual targeting of these pathways more potently inhibited proliferation of human cancer-derived organoids compared to the single treatments. These findings identify a critical role for CD36-mediated fatty acid uptake in prostate cancer and suggest that targeting fatty acid uptake might be an effective strategy for treating prostate cancer.

INTRODUCTION Alterations of metabolic activities have been shown to support the malignant properties of cancer cells (1, 2). Recent studies highlighted the relevance of glucose, glutamate, and fatty acids derived from de novo lipogenesis in modulating the bioenergetic processes and macromolecule synthesis required to sustain growth and proliferation (1, 3). Fatty acids are also derived from adipose tissue lipolysis or the breakdown of triglycerides contained in circulating chylomicrons and lipoproteins. In most nontumorigenic cells, these exogenous fatty acids are a preferred source for adenosine 5´-triphosphate production, membrane biosynthesis, energy storage, and the generation of a wide array of signaling molecules (4). However, the role of exogenous fatty acids and their transporters in tumor biology has received relatively little attention. Prostate cancer is the second most commonly diagnosed cancer in men, representing 15% of male cancer diagnoses and 8% of all cancer cases (5). Although it is a common cancer, it is a relatively slow-growing malignancy, and many men have indolent or low-risk disease that takes decades to elicit clinical symptoms and is readily treated by active surveillance or curative intent therapy (6). The major clinical challenge is to prevent progression to aggressive disease in men with moderate- or high-risk prostate cancer. The slow disease progression and ineffective identification of prostate cancer by 18F-deoxyglucose positron emission tomography (7) indicate that prostate cancer may not be subjected to the typical metabolic reprogramming observed in rapidly proliferating tumors, where glucose is considered the dominant metabolic substrate. Heightened fatty acid production from de novo lipogenesis is reported in many cancers (3), including prostate cancer (8), and pharmacological blockade of this process limits tumor growth (9, 10). Mounting evidence also implicates adipose-derived fatty acids in tumor malignancy, as exemplified in ovarian and breast cancer, where adipocytes reside in close proximity to the tumor foci and their secreted products affect disease progression (11–13). In this regard, the prostate is covered by the prominent periprostatic adipose tissue, which has the capacity to supply substantial quantities of fatty acids to alter the prostate tumor microenvironment (14, 15). Moreover, fatty acids are the dominant metabolic substrate in immortalized prostate cancer cells (16, 17), and prostate cancer cell viability is reduced by blocking fatty acid oxidation via pharmacological and genetic inhibition of CTP1, the rate-limiting enzyme for mitochondrial fatty acid transfer (18). Preclinical and epidemiological studies also indicate that higher levels of dietary saturated fat increase the risk of all-cause mortality in men with localized prostate cancer (19–21). In linking these observations, the increased expression of fatty acid translocase (FAT)/CD36, a major transporter for exogenous fatty acids into cells (22), correlates with a poor prognosis in lung squamous cell, glioblastoma, bladder, and luminal A breast carcinomas, whereas inhibition of CD36 impairs epithelial-to-mesenchymal transition and metastases in human melanoma and breast cancer–derived tumors (23–25). In this study, we established CD36-mediated fatty acid uptake as a critical process for the production of lipid biomass and the generation of oncogenic signaling lipids in prostate cancer. Furthermore, we showed that CD36 monoclonal antibody (mAb) therapy reduced prostate cancer growth in patient-derived xenografts (PDXs) of high-risk localized disease and that the efficacy of CD36 blockade could be enhanced by combined inhibition of de novo lipogenesis. These data suggest that blocking fatty acid uptake might be a promising therapeutic approach for treating prostate cancer.

DISCUSSION Although the production of fatty acids via heightened lipogenesis is a well-known metabolic adaptation in prostate cancer (40), several lines of evidence indicated a prominent role for exogenous fatty acids to support prostate cancer pathogenesis (14, 17, 41). In this study, we have demonstrated increased fatty acid uptake and significant lipidomic remodeling in human prostate cancer that is, at least partly, mediated by CD36. Supporting this concept, CD36 is frequently gained or amplified in prostate cancer and is associated with poor patient prognosis (27). The present work further extends on other recent efforts examining CD36 in tumors (23). We show that CD36 deletion restricts fatty acid uptake from the tumor microenvironment, reduces cancer-mediated lipid biosynthesis from fatty acid precursors and the generation of oncogenic lipid signaling pathways, and attenuates cancer growth. These data provide the impetus to target CD36 as a therapy for prostate cancer. Tumors display altered metabolism relative to benign tissues (1). An important aspect of this work relates to the marked alterations in fatty acid metabolism in human prostate cancer. Recent studies have reported dependency on CD36 for oral cancer metastasis (23) and fatty acid oxidation for triple-negative breast cancer tumor growth (42); however, the bioenergetic requirement for fatty acid uptake and metabolism had not been formally tested in primary human cancer tissues. Here, we report substantial alterations in lipid metabolism in malignant prostate tissue from men with high-risk localized disease, highlighted by increased fatty acid uptake and preferential channeling of these exogenously derived fatty acids into lipid pools that support the requirement for cell division, bulk energy storage (triglycerides and sterol lipids), and membrane production (phospholipids and ceramides)—processes required for tumorigenicity. Some aggressive cancer cells co-opt lipolysis of intracellular triglycerides to enhance FFA availability, which induces high levels of malignancy (43). Although this hypothesis cannot be tested in human tissue, there are more triglyceride-rich lipid droplets in malignant compared with benign human prostatic tissue. Together, our results demonstrate that exogenous fatty acids are of equal importance as glucose for energy provision and contribute to the production of complex lipids in human prostate cancer. Extending on the human studies, we obtained several lines of evidence that blocking CD36 reduces fatty acid uptake and slows cancer progression. The present data show that reducing fatty acid uptake via loss of Cd36 modulates specific lipid metabolism nodes in tumors driven by loss of Pten, rather than a generalized remodeling of the entire prostate lipidome. In this regard, Pten deletion leads to the release of fatty acids and lysophospholipids from membrane phospholipids and ether lipids. Acyl and ether lysophospholipids, such as lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidic acid, have all been shown to fuel cancer cell proliferation, motility, and invasiveness by acting as signaling lipids or providing additional sources of fatty acids (43–48). Cd36 deletion reduces fatty acid uptake and prevents the accumulation of these lipids and their metabolites, strongly indicating blockade of these tumor-promoting pathways. Aside from fatty acid uptake, we also show that Cd36 deletion causes cPLA2α activation and production of arachidonic acid from phospholipids, which is linked to the severity of prostate cancer in men (49) and is associated with the Cd36 cancer phenotype shown here. Although these changes in lipid metabolism provide a likely molecular explanation for the antitumorigenic effects of CD36 deletion, CD36 also functions as a signaling receptor capable of activating SRC family kinases, mitogen-activated protein kinases, and reactive oxygen species pathways through its recognition of extracellular “danger” ligands, which include oxidized low-density lipoproteins, β-amyloid peptide, Staphylococcus aureus–derived microbial diacylglycerides and lipoteichoic acid, and Mycoplasma macrophage-activating lipopeptide-2 (50, 51). Each of these has oncogenic potential, so it is possible that changes aside from lipid metabolism may contribute to the antitumorigenic effects of CD36 ablation reported here. However, our findings showing reduced CD36 and cancer progression in culture cells (PC3 and LNCaP) and organoids, where danger ligands are absent or in very low abundance, provide strong support for fatty acid uptake and metabolism as an important component of CD36’s tumorigenic effects. We note the apparent discrepancy with respect to fatty acid oxidation in Pten−/− mice. The acute experiments in isolated prostate tissue using 14C-18:1 FFA show no effect of cancer on fatty acid oxidation, whereas the lipidomic analysis in the same mice demonstrates marked acetylcarnitine accumulation, which is indicative of increased fatty acid oxidation in vivo. In reconciling these data, we speculate that C18:1 FFA transported into prostate tumors is first distributed into phospholipid storage, where they can then be cleaved by cPLA2α to produce fatty acids for oxidation to support energetic requirements. Note that this reaction would produce lysophospholipids containing unsaturated fatty acids (18:1) and saturated fatty acid for oxidation. This biology would not be elucidated using the 14C-18:1 FFA tracer methodology. An in-depth understanding of metabolism has provided insights into the clinical utility of cancer therapeutics (52, 53). The translational relevance for using CD36 blockade as a therapeutic for patients with prostate cancer is highlighted by our studies using human PDXs of high risk, locally invasive disease, which shows that systemic CD36 mAb administration slows tumorigenicity in prostate cancer that is characterized by high rates of fatty acid uptake. Although CD36 was previously implicated in metastases in several other cancer types (23, 25, 28), the present studies define early localized disease with high-risk features as an appropriate therapeutic window for treatment. Cancer progression was delayed in Pten−/− Cd36 knockout mice despite a compensatory increase in fatty acid production by de novo lipogenesis. This raises the intriguing possibilities of cross-talk between these pathways to maintain fatty acid flux in transformed cells and/or the existence of an intracellular fatty acid–sensing mechanism/rheostat that could induce multiple pathways to ensure availability of fatty acids to maintain fitness of tumor cells. Such communication within a fatty acid regulatory network was previously described in immortalized prostate cancer cells (54), although it is not yet clear how this metabolic cross-talk is regulated. Our parallel finding that de novo lipogenesis was increased in response to reduced fatty acid uptake prompted our examination of dual targeting of fatty acid transport and de novo lipogenesis as a more effective therapeutic approach in prostate cancer. We confirmed that the combination therapy of CD36 and fatty acid synthase (FASN) inhibition reduces tumorigenesis in prostate cancer organoids more effectively than either treatment alone. There were several limitations to this study. Given that the focus of this study was on localized prostate cancer rather than metastatic or therapy-resistant disease, we did not assess the effect of Cd36 deletion or blockade on survival in mice. Typically, Pten−/− mice start to die between the ages of ~8 and 20 months depending on the laboratory and the background strain (55, 56). Although it was not tested in this study, given that we show reduced tumor burden and progression in Pten−/−.Cd36−/−, a longer latency of disease progression and prolonged survival in these mice is expected. In further support of CD36’s role in prostate cancer progression, it is worth noting that copy number gain and increased expression of the CD36 gene are evident in metastatic disease compared with localized disease. Another limitation was that we were unable to correlate fatty acid uptake rates in human prostate in vivo with clinical outcomes in patients; hence, it is currently unclear whether fatty acid uptake predicts a worse prognosis at diagnosis, a concept that will be tested in future studies. Assessing CD36 protein expression in primary tissue is unlikely to provide strong diagnostic value because CD36 protein is expressed in human prostate tissues in benign and tumor regions and there was no evidence for increased expression in tumor regions. Although this contrasts the transcript data from other datasets, showing elevated expression of CD36 in primary tumors from men who experienced more rapid disease recurrence, CD36 activity (CD36-mediated fatty acid uptake) is difficult to evaluate by immunohistochemistry because it is heavily dependent on translocation from the cytoplasm to the cell membrane and posttranslational modifications (57). In summary, we provided preclinical evidence that targeting the metabolic differences in fatty acid metabolism between tumor and normal cells might be an effective anticancer strategy and that CD36 might be a pharmacological target for early treatment in high-risk localized prostate cancer.

MATERIALS AND METHODS Study design The research objective of our study was to determine the effect of fatty acid metabolism in prostate tumorigenesis using analysis of human tissues, cell lines, genetically modified mice, and human PDXs and patient-derived organoids. Patients with prostate cancer undergoing radical prostatectomy were included in the human study (details provided in table S1). No randomization was performed for the human studies; however, investigators were blinded to the allocation of benign or malignant tissue during metabolic analyses. For mouse studies in genetically modified mice, 8- to 24-week-old male mice were used, and for PDX in vivo studies, 8- to 12-week-old male mice were used. One investigator was responsible for group allocation of mice, and subsequent investigators were blind to the genotypes or treatment at tissue collection, metabolic assays, and histological assessment. Experimental replicates were variable for each experiment and are detailed in the figure legend. Raw data for all the experiments are reported in table S2. Statistical analysis Statistical analysis was performed using unpaired one- or two-way Student’s t tests, two-way ANOVA, or repeated-measures ANOVA where appropriate. Multiple comparisons were performed using a Bonferroni post hoc analysis when required. Statistical significance was set a priori at P < 0.05. Data are reported as means ± SEM. In vitro experiments were performed in triplicate and expressed as a summary of the mean of replicates with SEM, unless specified otherwise. Histological quantitation of mouse prostate tissue was performed using an Aperio ScanScope digital scanner and viewed using ImageScope software (Aperio).

SUPPLEMENTARY MATERIALS www.sciencetranslationalmedicine.org/cgi/content/full/11/478/eaau5758/DC1 Materials and Methods Fig. S1. Survival curves for CD36 and other genes encoding proteins regulating fatty acid uptake. Fig. S2. Fatty acid uptake in prostate cancer cell lines. Fig. S3. shRNA knockdown of CD36 in PC3 and LNCaP cells. Fig. S4. Mouse phenotyping by flow cytometric analysis. Fig. S5. Phenotype of WT.Cd36−/− mice. Fig. S6. Assessment of metabolism and cancer pathology in Pten−/−.Cd36−/− mice fed a high-fat diet for 6 weeks. Fig. S7. Schematic depicting the changes in Pten−/−-induced lipid metabolism that link the production of oncogenic lipids to prostate cancer progression. Fig. S8. Linking cPLA2α inhibition to the antitumorigenic effects of Cd36 deletion. Fig. S9. Systemic effects of CD36 mAb treatment in mice. Table S1. Patient characteristics of specimens used for metabolism studies. Table S2. Raw data (Excel file). References (58–78)

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Acknowledgments: We thank R. Legaie, H. Nim, X. Chen, and M. Richards (Monash University) for technical and bioinformatics assistance, the Australian Prostate Cancer Bioresource, and the Melbourne Urological Research Alliance (MURAL). The results published here are, in part, based on data generated by TCGA, established by the National Cancer Institute and the National Human Genome Research Institute, and we are grateful to the specimen donors and relevant research groups associated with this project. Funding: This work was supported by the Prostate Cancer Foundation of Australia (ID: PCFA–NCG 3313, awarded to M.J.W. and R.A.T.) and the Diabetes Australia Research Trust (awarded to M.J.W.). M.J.W., M.K.M., L.A.S., and G.P.R. are supported by the National Health and Medical Research Council of Australia (APP1077703, APP1143224, APP1102752, and APP1121057), and R.A.T. is supported by the Victorian Cancer Agency (MCRF15023). L.F. is supported by the Department of Health and Human Services acting through the Victorian Cancer Agency (MCRF16007). Author contributions: M.J.W. and R.A.T. conceived and designed experiments. M.J.W., S.T.W., V.R.H., P.R.W., M.M., J.L., C.H., and B.N. performed metabolism experiments. K.E.A. and D.K.N. performed lipidomic experiments. L.A.S., C.H., and R.B.S. performed the omic analysis. A.K.C., S.T.W., P.R.W., M.M., N.L., L.H.P., B.N., R.R., L.F., and R.A.T. performed prostate cancer experiments including cell culture, PDX, and organoid experiments. M.K.M., G.P.R., M. Febbraio, M.P., S.N., and M. Frydenberg provided reagents, clinical materials, and mouse models. M.J.W. and R.A.T. wrote the paper. All authors edited the manuscript. Competing interests: D.K.N. is a cofounder, shareholder, and adviser of Frontier Medicines and Artris Therapeutics and the director of the Novartis-Berkeley Center for Proteomics and Chemistry Technologies. K.E.A. is a scientist from Frontier Medicines. Data and materials availability: All the data are included in the main text or in the Supplementary Materials.