Caveolin-1-mediated sphingolipid oncometabolism underlies a metabolic vulnerability of prostate cancer

Plasma and tumor caveolin-1 (Cav-1) are linked with disease progression in prostate cancer. Here we report that metabolomic profiling of longitudinal plasmas from a prospective cohort of 491 active surveillance (AS) participants indicates prominent elevations in plasma sphingolipids in AS progressors that, together with plasma Cav-1, yield a prognostic signature for disease progression. Mechanistic studies of the underlying tumor supportive onco-metabolism reveal coordinated activities through which Cav-1 enables rewiring of cancer cell lipid metabolism towards a program of 1) exogenous sphingolipid scavenging independent of cholesterol, 2) increased cancer cell catabolism of sphingomyelins to ceramide derivatives and 3) altered ceramide metabolism that results in increased glycosphingolipid synthesis and efflux of Cav-1-sphingolipid particles containing mitochondrial proteins and lipids. We also demonstrate, using a prostate cancer syngeneic RM-9 mouse model and established cell lines, that this Cav-1-sphingolipid program evidences a metabolic vulnerability that is targetable to induce lethal mitophagy as an anti-tumor therapy.


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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Jeri Kim Samir Hanash Timothy C. Thompson Jun 5, 2020 Gene expression data for prostate cancer cell lines were obtained from Cancer Cell Line Encyclopedia (CCLE) (www.broadinstitute.org/ ccle). Gene expression data and clinical data were additionally downloaded from The Cancer Genome Atlas (TCGA) network project webpage (https://tcga-data.nci.nih.gov/tcga/) and the cBioPortal public Data Portal (cbioportal.org). Networks were visualized using cytoscape (https://cytoscape.org/). TCGA prostate adenocarcinoma (PRAD) cases were previously classified by TCGA network using the "iCluster" multi-platform based method as previously described by the Cancer Genome Atlas Research Network.
Statistical analyses were carried out using R statistical software program version 3.6.3 (https://www.r-project.org/) or GraphPad Prism V7 (San Diego, CA) The MS metabolomics data generated and analyzed during this study have been deposited to the NIH Common Fund's National Metabolomics Data Repository Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
cytoscape.org/). TCGA prostate adenocarcinoma (PRAD) cases were previously classified by TCGA network using the "iCluster" multi-platform based method as previously described by the Cancer Genome Atlas Research Network. Other relevant data supporting the findings of this study are available within the Article and Supplementary Information, or are available from the authors upon reasonable request.
Human biospecimen (plasma) was obtained from patients participating in a prospective clinical cohort that included men diagnosed with localized prostate cancer and that were enrolled on an AS trial protocol between February 2006 and February 2014 (n=825). Of these, 616 patients had at least 1 year follow-up and 491 patients had baseline plasma samples, enabling inclusion in the study. All samples that met inclusion criteria were included in this study.
In vitro experiments were performed in biological triplicate unless otherwise stated. For in vivo experiments, subconfluent mouse RM-9 prostate cancer cells, previously transduced with luciferase lentivirus, were injected into 8 week-old C57BL/6N mice (Jackson Labs, Main). A total of 32 mice were used, 9 mice in the treatment arm (60mg/kg of eliglustat) and 23 mice in the control arm (saline). ). A sample size of 9 eliglustat (case) and 23 saline control yields >99% power at a significance level (!) of 0.05 to detect a difference in tumor volume of "_1-"_2=300 using a one-sided two-sample t-test assuming that tumor volumes are normally distributed with standard deviation of 100 and 150 in eliglustat and saline groups, respectively.
Of the 825 samples on the AS trial protocol, 334 patients had less than 1 year follow-up or did not have baseline samples available and were therefore excluded from the analyses described in the current study. Individuals were additionally excluded if they presented with active noncutaneous malignancy at any site, had prior radiation therapy for treatment of the primary tumor, or Planned concomitant immunotherapy, hormonal therapy, chemotherapy, or radiation therapy while on protocol.
Mass spectrometry based analyses were conducted using standardize operating procedures; quality control samples (reference quality control samples as well as batch-specific pooled quality control samples) were included in all analytical runs. Individual human and mouse plasmas were assayed as singlets. Gene expression data was derived from open-source databases (TCGA and CCLE).
All data generated from in vitro and in vivo replicate experiments are included in the current study. Readouts are reported as mean +/-error (standard deviation or standard error of the mean) unless otherwise specified. In vitro experiments were assessed in biological triplicates unless otherwise stated as to ensure reproducibility. Confocal microscopy images were based on single experiments; however, we note the use of multiple orthogonal techniques and multiple fields to showcase concordance in claims reported herein.
Samples analyzed by mass spectrometry were assayed in a blinded randomized fashion. For in vivo experiments, subconfluent mouse RM-9 prostate cells, previously transduced with luciferase lentivirus, were injected subcutaneously into 8 week-old C57BL/6N mice (Jackson Labs, Main). After 3 days, tumor growths were confirmed by bioluminescence and mice were randomly distributed into 2 treatment arms, saline or eliglustat (daily intra-peritoneal injection at 60mg/kg). A separate cohort of non-tumor bearing mice were randomly divided and treated with either saline or eliglustat (daily intra-peritoneal injection at 60mg/kg). Randomization does not apply to in vitro experiments.
Samples analyzed by mass spectrometry were assayed in a blinded randomized fashion. Note that full information on the approval of the study protocol must also be provided in the manuscript.

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