De novo lipogenesis is a well-described androgen receptor (AR)–regulated metabolic pathway that supports prostate cancer tumor growth by providing fuel, membrane material, and steroid hormone precursor. In contrast, our current understanding of lipid supply from uptake of exogenous lipids and its regulation by AR is limited, and exogenous lipids may play a much more significant role in prostate cancer and disease progression than previously thought. By applying advanced automated quantitative fluorescence microscopy, we provide the most comprehensive functional analysis of lipid uptake in cancer cells to date and demonstrate that treatment of AR-positive prostate cancer cell lines with androgens results in significantly increased cellular uptake of fatty acids, cholesterol, and low-density lipoprotein particles. Consistent with a direct, regulatory role of AR in this process, androgen-enhanced lipid uptake can be blocked by the AR-antagonist enzalutamide, but is independent of proliferation and cell-cycle progression. This work for the first time comprehensively delineates the lipid transporter landscape in prostate cancer cell lines and patient samples by analysis of transcriptomics and proteomics data, including the plasma membrane proteome. We show that androgen exposure or deprivation regulates the expression of multiple lipid transporters in prostate cancer cell lines and tumor xenografts and that mRNA and protein expression of lipid transporters is enhanced in bone metastatic disease when compared with primary, localized prostate cancer. Our findings provide a strong rationale to investigate lipid uptake as a therapeutic cotarget in the fight against advanced prostate cancer in combination with inhibitors of lipogenesis to delay disease progression and metastasis.

Implications:

Prostate cancer exhibits metabolic plasticity in acquiring lipids from uptake and lipogenesis at different disease stages, indicating potential therapeutic benefit by cotargeting lipid supply.

The role of lipid metabolism in the incidence and progression of prostate cancer and several other cancer types has gained notable attention in an attempt to develop new therapeutic interventions. Lipids represent a diverse group of compounds derived from fatty acids and cholesterol that serve an essential role in many physiologic and biochemical processes. They function in energy generation and storage as well as intracellular signaling, protein modification, and precursor for steroid hormone synthesis. Additionally, fatty acids serve as the main building blocks for phospholipids that are incorporated together with free cholesterol into membranes and are critical for membrane function, cell signaling, and proliferation.

As a source of lipid supply, uptake of circulating exogenous lipids is sufficient for the requirements of most normal cells, and following development, lipogenic enzymes remain expressed at relatively low levels apart from a few specific biological processes (surfactant production in the lungs, production of fatty acids for milk lipids during lactation, and steroidogenic activity in tissues including prostate). However, lipogenic pathways, i.e., de novo lipogenesis (DNL) of fatty acids and cholesterol, are reactivated or upregulated in many solid cancer types, including prostate cancer. Enhanced lipogenesis is now acknowledged as a metabolic hallmark of cancer and is an early metabolic switch in the development of prostate cancer. It is maintained throughout the progression of prostate cancer and associated with poor prognosis and aggressiveness of disease (1–5). Yet, the contribution and identity of lipid uptake pathways as a supply route of exogenous lipids and their role in disease development and progression remain largely unknown.

Several lipogenic enzymes, including fatty acid synthase (FASN), are found to be overexpressed in prostate cancer (reviewed in ref. 1). Because increased FASN gene copy number, transcriptional activation, or protein expression are common characteristics of prostate cancer, fatty acid and cholesterol synthesis have become an attractive therapeutic target. However, the antineoplastic effects observed by inhibiting lipogenesis can be rescued by the addition of exogenous lipids (6, 7), highlighting lipid uptake as a mechanism of clinical resistance to lipogenesis inhibitors and that lipid uptake capacity is sufficient to substitute for the loss of lipogenesis. Indeed, it was recently reported that lung cancer cells expressing a strong lipogenic phenotype generated up to 70% of their cellular lipid carbon biomass from exogenous fatty acids and only 30% from de novo synthesis supplied by glucose and glutamine as carbon sources (8). Although altered cellular lipid metabolism is a hallmark of the malignant phenotype, prostate cancer is unique in that it is characterized by a relatively low uptake of glucose and glycolytic rate compared with many solid tumors subscribing to the “Warburg effect” phenotype (9, 10). Concordantly, prostate cancer cells showed a dominant uptake of fatty acids over glucose, with the uptake of palmitic acid measured at ∼20 times higher than uptake of glucose in both malignant and benign prostate cancer cells (11). Furthermore, exogenous fatty acids are readily oxidized by prostate cancer, reducing glucose uptake (12). Together, these studies demonstrate that exogenous uptake is a significant and previously underappreciated supply route of lipids in cancer cells with a lipogenic phenotype.

Both healthy and malignant prostate cells rely on androgens for a variety of physiologic processes, including several metabolic signaling pathways. Androgens, through binding to the androgen receptor (AR), transcriptionally regulate a multitude of pathways, including proliferation, differentiation, and cell survival of prostate cancer (13), with approximately equal numbers of genes activated and suppressed by androgen-activated AR. Targeting of the AR signaling axis is the mainstay treatment strategy for advanced prostate cancer. Although initially effective in suppressing tumor growth, patients inevitably develop castrate-resistant prostate cancer (CRPC), which remains incurable. Importantly, during progression to CRPC, survival and growth of prostate cancer cells remain dependent on AR activity, as demonstrated by treatment-resistance mechanisms involving AR mutation, amplification, and intratumoral steroidogenesis (reviewed in ref. 14). Thus, identifying critical pathways regulated by AR might provide novel therapeutic strategies to combat development of CRPC. Lipogenesis is a well-described AR-regulated metabolic pathway that supports prostate cancer cell growth by providing fuel, membrane material, and steroid hormone precursor (cholesterol). Androgens stimulate expression of FASN via activation of sterol regulatory element-binding proteins (SREBP; ref. 15), lipogenic enzymes ACACA and ACLY and cholesterol synthesis enzymes HMGCS1 and HMGCR (3, 16). In contrast, the role and expression of lipid transporters and their regulation by AR in prostate cancer remain largely uncharacterized (11, 17).

Our current understanding of lipid uptake is mostly derived from studies in nonmalignant cells and tissues. The hydrophobic properties of lipids allow for passive, nonspecific uptake via diffusion into the cell. Selective, protein-mediated lipid uptake involves receptor-mediated endocytosis of lipid transporters and their cognate lipoprotein cargo (18, 19), which contains various lipid components (phospholipids, cholesterol esters, triacylglycerol) that can be internalized via lipoprotein receptors (LDLR and VLDLR) or scavenger receptors (SCARB1 and SCARB2). Various scavenger receptors have also been shown to be associated with uptake of modified (acetylated or oxidized) LDL particles, including SCARF1, SCARF2, and CXCL16 (20, 21). Free fatty acids can be taken up by a family of six fatty acid transport proteins (SLC27A1-6) as well as fatty acid translocase (FAT/CD36) and GOT2/FABPpm (17).

Taken together, it is becoming evident that enhanced lipogenesis in prostate cancer development and progression is not the sole deregulated lipid supply pathway, and lipid uptake might play an important role in biochemical recurrence of prostate cancer. This warranted a comprehensive investigation and delineation of lipid uptake and the lipid transporter landscape in prostate cancer as well as its regulation by AR.

Cell culture

The following cell lines were acquired from ATCC in 2010: LNCaP (CVCL_0395), C4-2B (CVCL_4784), and VCaP (CVCL_2235). Fibroblast-free DuCaP cells were a generous gift from M. Ness (VTT Technical Research Centre of Finland). LNCaP and C4-2B cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific) supplemented with 5% fetal bovine serum (FBS) until passage 45. DuCaP and VCaP cells were cultured in RPMI supplemented with 10% FBS until passages 40 and 45, respectively. The medium was changed every 3 to 4 days. All cell lines were incubated at 37°C in 5% CO2. Cells were passaged at approximately 80% confluency by trypsinization. Cell lines were genotyped in March 2018 by Genomics Research Centre (Brisbane) and routinely tested for Mycoplasma infection.

For androgen and antiandrogen treatments, cells were seeded in regular growth medium for 72 hours before media were replaced with RPMI supplemented with 5% charcoal-dextran stripped serum (CSS; Sigma-Aldrich). After 48 hours, media were replaced with fresh 5% CSS RPMI, and cells were treated with either dihydroxytestosterone (DHT, 10 nmol/L) or synthetic androgen R1881 (1 nmol/L) for 48 hours to activate AR signaling. AR-antagonist enzalutamide or bicalutamide (Selleck Chemicals) was used at 10 μmol/L.

Measurement of lipid content by quantitative fluorescent microscopy

Prior to seeding of LNCaP and C4-2B cells in 5% CSS RPMI at a density of 4,000 and 3,000 cells/well, respectively, optical 96-well plates (IBIDI) were coated with 150 μL Poly-l-ornithine (Sigma-Aldrich) as described previously (22). DuCaP and VCaP cells were seeded in 10% CSS RPMI at a density of 15,000 cells/well. After treatment as indicated, media were removed, cells were washed with PBS once, fixed with 4% paraformaldehyde for 20 minutes at room temperature, and remaining aldehyde reacted with 30 mmol/L glycine in PBS for an additional 30 minutes. Nuclear DNA was then stained with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific) and lipids were stained with 0.1 μg/mL Nile Red (Sigma-Aldrich) overnight as described previously (23). Alternatively, free cholesterol was stained with 50 μg/mL filipin (Sigma-Aldrich) for 40 minutes. More than 500 cells/well were imaged using the InCell 2200 automated fluorescence microscope system (GE Healthcare Life Sciences). Quantitative analysis of 1,500 cells/treatment (3 wells) was performed in at least two independent experiments with Cell Profiler Software (Broad Institute; ref. 24).

Measurement of lipid uptake by quantitative fluorescent microscopy

Cells were seeded as described above. For quantifying C16:0 fatty acid uptake, growth medium was exchanged with 65 μL/well serum-free RPMI medium supplemented with 0.2% BSA(lipid-free; Sigma-Aldrich) and 5 μmol/L Bodipy-C16:0 (Thermo-Fisher), and cells were incubated at 37°C for 1 hour. Cellular uptake of cholesterol was measured as described recently (25). Briefly, medium was exchanged with serum-free 0.2% BSA RPMI media supplemented with 15 μmol/L NBD-cholesterol (22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino-23,24-bisnor-5-cholen-3β-Ol) (Thermo Fisher Scientific), and cells were incubated at 37°C for 2 hours. For quantifying lipoprotein complex uptake, serum-free 0.2% BSA RPMI medium was supplemented with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI)-labeled acetylated LDL (Thermo Fisher Scientific, 15 μg/mL) or DiI-labeled LDL (Thermo Fisher Scientific; 15 μg/mL) and incubated at 37°C for 2 hours. After incubation, cells were washed and fixed as described above. Cellular DNA and F-actin was counterstained with DAPI and Alexa Fluor 647 Phalloidin (Thermo Fisher Scientific). Image acquisition and quantitative analysis were performed as above.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Cells were seeded at a density of 9.0 × 104 (LNCaP and C4-2B) or 1.2 × 105 (DuCaP and VCaP) cells/well in 6-well plates (Thermo Scientific). Following completion of treatment, total RNA was isolated using the RNEasy mini kit (Qiagen) following the manufacturer's instructions. RNA concentration was measured using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific), and RNA frozen at −80°C until further use.

Up to 2 μg of total RNA was used to prepare cDNA with SensiFast cDNA synthesis kit (Bioline) according to the manufacturer's instructions and diluted 1:5. qRT-PCR was performed with SYBR-Green Master Mix (Thermo Fisher Scientific) using the ViiA-7 Real-Time PCR system (Applied Biosystems). Determination of relative mRNA levels was calculated using the comparative ΔΔCt method (26), where expression levels were normalized relative to that of the housekeeping gene receptor–like protein 32 (RPL32) for each treatment and calculated as fold change relative to the vehicle control treatment. All experiments were performed independently in triplicate. Primer sequences are listed in Supplementary Materials.

Protein extraction and Western blot analysis

Proteins for Western blotting were isolated by lysing cells in radio immunoprecipitation buffer [RIPA, 25 mmol/L Tris, HCl pH 7.6, 150 mmol/L NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, one cOmplete EDTA-free Protease Inhibitor Cocktail tablet (Roche) per 10 mL, phosphatase inhibitors NaF (30 μmol/L), sodium pyrophosphate (20 μmol/L), β-glycerophosphate (10 μmol/L), and Na vanadate (1 μmol/L)]. With cells on ice, media were carefully removed, and cells were washed with PBS. RIPA lysis buffer was added, and cells were incubated for 5 minutes on ice before collection of protein lysates. Protein concentration was measured using the Pierce BCA Protein Assay kit according to the manufacturer's instructions (Thermo Fisher Scientific).

Twenty micrograms of total protein/lane were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using NuPAGE 4-12% Bis-Tris SDS-PAGE Protein Gels (Thermo Fisher Scientific), and Western blot was completed using the Bolt Mini Blot Module (Thermo Fisher Scientific) according to the manufacturer's instructions. Membranes were reacted overnight at 4°C with primary antibodies raised against LDLR (Abcam, ab52818) and SCARB1 (Abcam, ab217318) at a dilution of 1:1,000 followed by probing with the appropriate Odyssey fluorophore-labeled secondary antibody and visualization on the LI-COR Odyssey imaging system (LI-COR Biotechnology). Protein expression levels were quantified using Image Studio Lite (LI-COR Biotechnology), normalized relative to the indicated housekeeping protein, and expressed as fold changes relative to the control treatment.

Cistrome analysis of AR ChIPseq peaks

AR ChIPseq analysis used BED files (hg38) downloaded from Cistrome (27) for the ± bicalutamide-treated vehicle controls for the ChIPseq data set, GSE49832 (28). The bedtools software tool (version 2.27.0) was used to identify AR ChIPseq peaks enriched in regions 5KB upstream and also in a 25KB window around Gencode transcripts (version 21).

RNA-seq analysis

For mRNA-seq, total cellular RNA was extracted using the Norgen RNA Purification PLUS kit #48400 (Norgen Biotek Corp.) according to the manufacturer's instructions, including DNase treatment. RNA quality and quantity were determined on an Agilent 2100 Bioanalyzer (Agilent Technologies) and Qubit. 2.0 Fluorometer (Thermo Fisher Scientific Inc.). Library preparation and sequencing was done at the Kinghorn Centre for Clinical Genomics (KCCG, Garvan Institute, Sydney) using the Illumina TruSeq Stranded mRNA Sample Prep Kit with an input of 1 μg total RNA (RIN > 8), followed by paired-end sequencing on an Illumina HiSeq2500 v4.0 (Illumina), multiplexing 6 samples per lane and yielding about 30M reads per sample. Raw data were processed through a custom-designed pipeline. Raw reads were trimmed using “TRIMGALORE” (29), followed by parallel alignments to the genome (hg38) and transcriptome (Ensembl v77/Gencode v21) using the “STAR” (30) aligner and read quantification with “RSEM” (31). Differential expression between two conditions was calculated after between sample TMM normalization (32) using “edgeR” (ref. 33; no replicates: Fisher exact test; replicates: general linear model) and is defined by an absolute fold change of >1.5 and a false discovery rate (FDR) corrected P < 0.05. Quality control of raw data included sequential mapping to the ERCC spike-in controls, rRNA, and a comprehensive set of pathogen genomes as well as detection and quantification of 3′ bias. Heat maps were generated with a hierarchical clustering algorithm using completed linkage and Euclidean distance measures.

Microarray gene-expression profiling and analysis

RNA from LNCaP tumor xenograft models of CRPC was collected as described previously (34). RNA was prepared for microarray profiling as described previously using a custom 180K Agilent oligo microarray (VPCv3, ID032034, GEO:GPL16604) (35). Probes significantly different between two groups were identified with an adjusted P ≤ 0.05, and an average absolute fold change of ≥1.5 (adjusted for an FDR of 5%).

Statistical analysis

Statistical analyses were performed with GraphPad Prism 7.0 (GraphPad Software) and R Studio (RStudio). Data reported and appropriate statistical tests are included in figure legends.

Androgens strongly increased cellular lipid content in AR-positive prostate cancer cells

Previous analysis by cellular Oil Red O staining and lipid chromatography of cellular extracts have demonstrated that androgens strongly enhance lipogenesis and cellular lipid content in prostate cancer cells, predominantly that of neutral lipids (triacylglycerols and cholesterol esters) stored in lipid droplets and phospholipids and free cholesterol present in membranes (16, 36). Consistent with these findings, our quantitative fluorescence microscopy (qFM) assay (23) of Nile Red-stained AR-positive prostate cancer cell lines LNCaP, C4-2B, VCaP, and DuCaP showed that synthetic androgen R1881 significantly increased cellular phospholipid and neutral lipid content as well as lipid droplet number (Fig. 1). This stimulatory effect of androgen was also observed with DHT (Supplementary Fig. S1) and mibolerone (data not shown) and blocked in the presence of enzalutamide (Supplementary Fig. S1). Furthermore, qFM of filipin-stained LNCaP cells confirmed that androgens also increased cellular levels of free, unesterified cholesterol (Fig. 1B), which was also blocked by enzalutamide. Although androgen-enhanced lipogenesis is a well-characterized fuel source for increased cellular lipid content, the role of lipid uptake in this process is still poorly understood.

Figure 1.

Androgens strongly increased lipid content of AR-positive prostate cancer cell lines. A, LNCaP, C4-2B, VCaP, and DuCaP cells were grown in CSS for 48 hours and treated with 1 nmol/L R1881 or vehicle (Ctrl) for 48 hours. Fixed cells were stained with fluorescent lipid stain Nile Red, and cellular mean fluorescent intensities (MFI) of phospholipid content (top left) and neutral lipid content (top right) as well as mean cellular number of lipid droplets (bottom left) and mean total cellular area of lipid droplets (bottom right) were measured by quantitative fluorescence microscopy (qFM). Representative 40× images of LNCaP cell are shown (blue, DNA, yellow, lipid droplets containing neutral lipids, scale bar, 20 μm). B, LNCaP cells were grown as described in A and treated with the indicated androgens in the presence or absence of enzalutamide (Enz, 10 μmol/L). Fixed cells were stained with filipin to label-free, unesterified cholesterol, and MFI of cellular-free cholesterol was measured by qFM (n ∼ 3,000 cells from 3 wells; significance calculated relative to vehicle (Ctrl): ns, not significant; ***, P < 0.001; or vehicle-treated LNCaP cells: #, P < 0.001, representative of 3 independent experiments). Representative 40× images of LNCaP cell are shown (blue, DNA, green, free cholesterol; scale bar, 10 μm).

Figure 1.

Androgens strongly increased lipid content of AR-positive prostate cancer cell lines. A, LNCaP, C4-2B, VCaP, and DuCaP cells were grown in CSS for 48 hours and treated with 1 nmol/L R1881 or vehicle (Ctrl) for 48 hours. Fixed cells were stained with fluorescent lipid stain Nile Red, and cellular mean fluorescent intensities (MFI) of phospholipid content (top left) and neutral lipid content (top right) as well as mean cellular number of lipid droplets (bottom left) and mean total cellular area of lipid droplets (bottom right) were measured by quantitative fluorescence microscopy (qFM). Representative 40× images of LNCaP cell are shown (blue, DNA, yellow, lipid droplets containing neutral lipids, scale bar, 20 μm). B, LNCaP cells were grown as described in A and treated with the indicated androgens in the presence or absence of enzalutamide (Enz, 10 μmol/L). Fixed cells were stained with filipin to label-free, unesterified cholesterol, and MFI of cellular-free cholesterol was measured by qFM (n ∼ 3,000 cells from 3 wells; significance calculated relative to vehicle (Ctrl): ns, not significant; ***, P < 0.001; or vehicle-treated LNCaP cells: #, P < 0.001, representative of 3 independent experiments). Representative 40× images of LNCaP cell are shown (blue, DNA, green, free cholesterol; scale bar, 10 μm).

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Fatty acid, cholesterol, and lipoprotein uptake are increased by androgens

To directly measure the stimulatory effect of androgens on lipid uptake, a series of lipid uptake assays (Fig. 2) was used based on qFM of fluorophore-labeled lipid probes (Bodipy-C16:0, NBD-cholesterol, DiI-LDL, and DiI-acetylated LDL). As shown in Fig. 2A, androgen treatment (R1881) of four AR-positive prostate cancer cell lines (LNCaP, C4-2B, DuCaP, and VCaP) for 48 hours significantly increased uptake of Bodipy-C16:0. This effect was significantly blocked when cells were cotreated with enzalutamide. Notably, androgens also increased uptake of Bodipy-C12:0 but not Bodipy-C5:0 (data not shown and Supplementary Fig. S2), suggesting that cellular uptake of short-chain fatty acids is not androgen regulated. Similar to fatty acid uptake, androgens also significantly increased uptake of NBD cholesterol in AR-positive prostate cancer cells (Fig. 2B), and enzalutamide significantly suppressed this effect. Representative images of LNCaP cells show that Bodipy-C16:0 and NBD cholesterol were readily incorporated into lipid droplets (Fig. 2A and B).

Figure 2.

Androgens strongly increased lipid uptake. A, To measure fatty acid uptake, indicated cell lines were grown in CSS for 48 hours and treated with 1 nmol/L R1881 in the presence or absence of Enz (10 μmol/L) or vehicle (Ctrl) for 48 hours. Before fixation, cells were incubated with Bodipy-C16:0 for 1 hour, and lipid uptake was measured by qFM (n ∼ 3,000 cells from 3 wells, mean ± SD; one-way ANOVA with Dunnett multiple comparisons test relative to cell line–specific control (Ctrl), ns, not significant; ***, P < 0.001, representative of 2 independent experiments). Representative 40× images of DuCaP cell are shown (blue, DNA; red, F-actin; green, lipid droplets containing C16:0-Bodipy; scale bar, 20 μm). B, To measure cholesterol uptake, LNCaP cells were grown in CSS for 48 hours and treated with either 1 nmol/L R1881 or 10 nmol/L DHT in the presence or absence of Enz (10 μmol/L). Before fixation, cells were incubated with NBD cholesterol for 2 hours and cellular levels were measured by qFM [n ∼ 3,000 cells from 3 wells, mean ± SD; one-way ANOVA with Dunnett multiple comparisons test relative to cell line–specific vehicle (Ctrl), or unpaired t test between androgen treatment alone or in combination with enzalutamide; ns, not significant; ****, P < 0.0001, representative of 2 independent experiments]. Representative 40× images of LNCaP cells are shown (blue, DNA; red, F-actin; green, lipid droplets containing NBD-cholesterol; scale bar, 20 μm). C, To measure lipoprotein uptake, LNCaP cells were grown in CSS for 48 hours and treated with increasing concentrations of DHT or 1 nmol/L R1881. Before fixation, cells were incubated with DiI-LDL or DiI-acLDL for 2 hours, and lipoprotein uptake was measured by qFM [n ∼ 3,000 cells from 3 wells; mean ± SD; one-way ANOVA with Dunnett multiple comparisons test relative to control (Ctrl) in each respective cell line; ****, P < 0.0001, representative of 3 independent experiments].

Figure 2.

Androgens strongly increased lipid uptake. A, To measure fatty acid uptake, indicated cell lines were grown in CSS for 48 hours and treated with 1 nmol/L R1881 in the presence or absence of Enz (10 μmol/L) or vehicle (Ctrl) for 48 hours. Before fixation, cells were incubated with Bodipy-C16:0 for 1 hour, and lipid uptake was measured by qFM (n ∼ 3,000 cells from 3 wells, mean ± SD; one-way ANOVA with Dunnett multiple comparisons test relative to cell line–specific control (Ctrl), ns, not significant; ***, P < 0.001, representative of 2 independent experiments). Representative 40× images of DuCaP cell are shown (blue, DNA; red, F-actin; green, lipid droplets containing C16:0-Bodipy; scale bar, 20 μm). B, To measure cholesterol uptake, LNCaP cells were grown in CSS for 48 hours and treated with either 1 nmol/L R1881 or 10 nmol/L DHT in the presence or absence of Enz (10 μmol/L). Before fixation, cells were incubated with NBD cholesterol for 2 hours and cellular levels were measured by qFM [n ∼ 3,000 cells from 3 wells, mean ± SD; one-way ANOVA with Dunnett multiple comparisons test relative to cell line–specific vehicle (Ctrl), or unpaired t test between androgen treatment alone or in combination with enzalutamide; ns, not significant; ****, P < 0.0001, representative of 2 independent experiments]. Representative 40× images of LNCaP cells are shown (blue, DNA; red, F-actin; green, lipid droplets containing NBD-cholesterol; scale bar, 20 μm). C, To measure lipoprotein uptake, LNCaP cells were grown in CSS for 48 hours and treated with increasing concentrations of DHT or 1 nmol/L R1881. Before fixation, cells were incubated with DiI-LDL or DiI-acLDL for 2 hours, and lipoprotein uptake was measured by qFM [n ∼ 3,000 cells from 3 wells; mean ± SD; one-way ANOVA with Dunnett multiple comparisons test relative to control (Ctrl) in each respective cell line; ****, P < 0.0001, representative of 3 independent experiments].

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The majority of serum lipids are transported as lipoprotein particles (chylomicrons, VLDL, LDL, HDL), containing a complex mixture of apolipoproteins, phospholipids, cholesterol, and triacylglycerols which are taken up into cells by receptor-mediated endocytosis through cognate lipoprotein receptors such as the LDL receptor (LDLR) for LDL and scavenger receptor SCARB1 for acetylated LDL/HDL. Notably, in contrast to the covalent Bodipy and NBD fluorophore tags on C16:0 and cholesterol, the DiI label is a noncovalently bound dye infused into the lipoprotein particles that dissociates after cellular uptake and lysosomal processing. As shown in Fig. 2C, androgens significantly enhanced uptake of DiI-complexed LDL and acetylated LDL in LNCaP cells in a dose-dependent manner, indicating a potential role for their cognate receptors LDLR and SCARB1.

Androgen-enhanced lipid uptake is independent of cell-cycle progression and proliferation

Androgen-mediated activation of AR promotes G0/G1 to S phase progression of the cell cycle and proliferation in prostate cancer cells (reviewed in refs. 13, 37, 38). Because proliferation requires substantial membrane biogenesis for daughter cell generation, it was possible that androgen-enhanced lipid uptake was not mediated directly through AR signaling but indirectly as a result of androgen-stimulated proliferation. To address this possibility, LNCaP cells were synchronized in G0/G1 (>95% of cell population; Supplementary Fig. S3A) by incubation in CSS medium for 48 hours and treated for another 24 hours with three different cell-cycle inhibitors, which upon androgen (DHT) treatment-induced reentry into the cell cycle caused arrest in G0/G1 (tunicamycin), S phase (hydroxyurea), or G2/M (nocodazole; Supplementary Fig. S3A). As shown in Fig. 3, lipid uptake of Bodipy-C16:0 and NBD cholesterol was significantly and to a similar magnitude enhanced by androgen in the presence of all three cell-cycle inhibitors when compared with control. Flow cytometry of DNA content confirmed cell-cycle arrest (Supplementary Fig. S3B) by the inhibitors, and IncuCyte cell confluence analysis demonstrated growth inhibition (Supplementary Fig. S3C), respectively. Thus, androgen regulation of lipid uptake is directly mediated by AR throughout the cell cycle and is independent of cell-cycle progression and proliferation. Notably, a time course experiment of DHT-treated G0/G1 synchronized LNCaP cells in the presence of tunicamycin (Supplementary Fig. S3B; FACS) confirmed that the androgen-enhanced expression of classic AR-regulated genes KLK3/PSA (Supplementary Fig. S3D), TMPRSS2, and FKBP5 (data not shown) remained unaffected under the experimental conditions.

Figure 3.

Androgen-enhanced lipid uptake is independent of cell-cycle progression and proliferation. LNCaP cells were synchronized in G0–G1 by androgen deprivation (CSS for 48 hours) followed by treatment with tunicamycin (1 mg/mL), hydroxyurea (1 mol/L), or nocodazole (25 μg/mL) for another 24 hours, placing cell-cycle blocks in G0–G1, S phase, and mitosis, respectively. Cell-cycle reentry and progression to the respective cell-cycle block was stimulated by DHT (10 nmol/L). After 24 hours, cholesterol (NBD-cholesterol; left) and fatty acid uptake (Bodipy-C16:0; right) was measured by qFM [n ∼ 3,000 cells from 3 wells; mean ± SD; one-way ANOVA with Dunnett multiple comparisons test relative to cell line–specific vehicle (Ctrl), or unpaired t test between androgen treatment alone or in combination with cell-cycle inhibitor; ns, not significant; ****, P < 0.0001; **, P < 0.01, representative of 2 independent experiments].

Figure 3.

Androgen-enhanced lipid uptake is independent of cell-cycle progression and proliferation. LNCaP cells were synchronized in G0–G1 by androgen deprivation (CSS for 48 hours) followed by treatment with tunicamycin (1 mg/mL), hydroxyurea (1 mol/L), or nocodazole (25 μg/mL) for another 24 hours, placing cell-cycle blocks in G0–G1, S phase, and mitosis, respectively. Cell-cycle reentry and progression to the respective cell-cycle block was stimulated by DHT (10 nmol/L). After 24 hours, cholesterol (NBD-cholesterol; left) and fatty acid uptake (Bodipy-C16:0; right) was measured by qFM [n ∼ 3,000 cells from 3 wells; mean ± SD; one-way ANOVA with Dunnett multiple comparisons test relative to cell line–specific vehicle (Ctrl), or unpaired t test between androgen treatment alone or in combination with cell-cycle inhibitor; ns, not significant; ****, P < 0.0001; **, P < 0.01, representative of 2 independent experiments].

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Delineating the lipid transporter landscape in prostate cancer

Although the role of genes involved in DNL (e.g., ACLY, ACACA, FASN, HMGCR) is well described in prostate cancer, and their overexpression is associated with tumor development, disease progression, aggressiveness, and poor prognosis (reviewed in refs. 1, 2), very little is known about the expression and functional importance of lipid transporters in prostate cancer, their regulation by androgens, and their clinical relevance. To delineate the lipid transporter landscape in prostate cancer, a panel of 44 candidate lipid transporters was generated based on previous work describing their lipid transport function in various human tissues (19, 39–44).

Transcriptomic analysis by RNA-seq revealed that 41 candidate lipid transporters were expressed in five prostate cancer cell lines (LNCaP, DuCaP, VCaP, PC-3, and Du145) under normal culture conditions (a selection of 36 candidates is shown in Fig. 4A). Importantly, lipid transporters LDLR, SCARB1, SCARB2, and GOT2/FABPpm were robustly expressed at levels comparable with lipogenic genes HMGCR and FASN (Fig. 4A), whereas seven transporters, including CD36 and SLC27A6, displayed FPKM values <1 in the majority of cell lines. In addition, mRNA expression of these 41 lipid transporters was independently detected in LNCaP and Du145 cells and six additional prostate cancer cell lines (CWR22RV1, EF1, H660, LASCPC-01, NB120914, and NE1_3; ref. 45); John Lee, August 29, 2018), verifying mRNA expression of these transporters in a total of nine prostate cancer cell lines. Comparison of this list of lipid transporters with the recently delineated plasma membrane proteome of eight prostate cancer cell lines, including LNCaP, Du145, and CWR22Rv1 [ref. 45; John Lee, August 29, 2018) and previous work in LNCaP and CWR22Rv1 cells (17), confirmed the surface expression of LDLR, GOT2, LRPAP1, LRP8, and SCARB2. In addition, our proteomics analysis confirmed the exclusive expression of SCARB1, SCARB2, LRPAP1, SLCA27A1, and SLC27A2 in the membrane fraction of LNCaP cells, whereas GOT2 was also present in the soluble fraction (data not shown), which is consistent with its mitochondrial function. Western blot analysis demonstrated the expression of LDLR and SCARB1 in cell lysates of seven malignant and two nonmalignant prostate cell lines (Fig. 4B). Subsequently, expression of these lipid transporters in prostate cancer patient samples and clinical relevance was investigated by analyzing published tumor transcriptome data sets with Oncomine. Comparison of primary, localized prostate cancer versus normal prostate gland revealed that mRNA levels of only a few lipid transporters were significantly (P < 0.05) upregulated in primary prostate cancer, and no lipid transporter was significantly downregulated (Supplementary Fig. S4A–S4C). However, data mining of the reported proteome analysis of primary prostate cancer versus neighboring nonmalignant tissue (46) revealed that expression of 21 lipid transporters was lower in primary prostate cancer, whereas protein expression of both DNL enzymes FASN and HMGCR was increased by several magnitudes (Supplementary Fig. S4D). Although a measurable degree of discordance between mRNA and protein levels has been previously noted in integrated transcriptome and proteome studies of prostate cancer (46, 47), the proteomics data suggested that lipid uptake is reduced and DNL is increased in primary prostate cancer when compared with normal prostate gland. In contrast, mRNA levels of several lipid transporters were significantly upregulated in metastatic tumor samples compared with primary site (48), including SLC27A1, SLC27A3, SCARB1, and LDLR (Fig. 4C). Concordantly, analysis of the proteome comparison of localized prostate cancer versus bone metastasis (49) demonstrated that expression of 16 lipid transporters and FASN was higher in bone metastases (Fig. 4E), suggesting that tumor lipid supply from both uptake and DNL was increased. The lipoprotein transporters LDLR and SCARB1 were further investigated across other prostate cancer patient cohorts in Oncomine, including the Varambally (50) and La Tulippe (51) data sets. Both lipid transporter mRNAs were found to be significantly upregulated in samples from prostate cancer metastases when compared with primary tumors (Fig. 4E). Together, independently published data and our own analyses confirmed the mRNA, protein, and plasma membrane expression of several lipid transporters in prostate cancer cell lines and patient-derived tumor samples. Importantly, our analysis demonstrated that this route of lipid supply is clinically significant during disease progression and is associated with metastasis to the bone.

Figure 4.

Delineation of the lipid transporter landscape in prostate cancer (PCa; A) mRNA expression levels (mean FPKM, fragments per kilobase million; n = 2) of the indicated candidate lipid transporters and 2 lipogenic genes (FASN and HMGCR) were measured by RNA-seq in the five indicated prostate cancer cell lines grown in their respective maintenance media. B, Western blot confirmed the protein expression of LDLR and SCARB1 in the seven indicated prostate cancer cell lines and in two nonmalignant prostate cell lines (RWPE-1 and BHP-1) grown in their respective maintenance media. GAPDH was used as a loading control. A representative blot of two independent experiments is shown. C, Oncomine analysis of candidate lipid transporters in the Grasso data set (48) comparing gene expression of localized, primary prostate cancer versus metastatic prostate cancer. D, Protein expression analysis of indicated 18 lipid transporters and two lipogenesis enzymes (FASN and HMGCR) in paired patient samples of localized primary tumor and (blue) and bone metastasis (red) in the Iglesias-Gato proteome data set (49). E, Gene expression of LDLR (top) and SCARB1 (bottom) was compared in primary vs. metastatic prostate cancer in the Grasso, Varambally, and LaTulippe cohorts (mean ± SD; unpaired t test; ns, not significant; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05).

Figure 4.

Delineation of the lipid transporter landscape in prostate cancer (PCa; A) mRNA expression levels (mean FPKM, fragments per kilobase million; n = 2) of the indicated candidate lipid transporters and 2 lipogenic genes (FASN and HMGCR) were measured by RNA-seq in the five indicated prostate cancer cell lines grown in their respective maintenance media. B, Western blot confirmed the protein expression of LDLR and SCARB1 in the seven indicated prostate cancer cell lines and in two nonmalignant prostate cell lines (RWPE-1 and BHP-1) grown in their respective maintenance media. GAPDH was used as a loading control. A representative blot of two independent experiments is shown. C, Oncomine analysis of candidate lipid transporters in the Grasso data set (48) comparing gene expression of localized, primary prostate cancer versus metastatic prostate cancer. D, Protein expression analysis of indicated 18 lipid transporters and two lipogenesis enzymes (FASN and HMGCR) in paired patient samples of localized primary tumor and (blue) and bone metastasis (red) in the Iglesias-Gato proteome data set (49). E, Gene expression of LDLR (top) and SCARB1 (bottom) was compared in primary vs. metastatic prostate cancer in the Grasso, Varambally, and LaTulippe cohorts (mean ± SD; unpaired t test; ns, not significant; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05).

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Androgens regulate the expression of several lipid transporters

As shown above, androgens strongly enhance lipid uptake in AR-positive prostate cancer cell lines. However, our current understanding of the AR regulation of lipid transporters is very limited (17). We initiated a comprehensive analysis of androgen-regulated lipid transporters by searching for AR binding sites within a 25-kb window of the gene sequence and a 5-kb window upstream of the protein start codon of 45 candidate lipid transporters in the reported AR ChIPseq data set of LNCaP cells treated with AR-antagonist bicalutamide (28). As shown in Fig. 5A, 19 and 27 lipid transporters showed enrichment of AR ChIPseq peaks in the 5-kb and 25-kb windows, respectively, which was reduced in the presence of bicalutamide. Consistent with its reported androgen regulation (17), AR ChIPseq peaks were detected in the 25-kb window of the GOT2 gene, which were absent after bicalutamide treatment. For comparison, AR-regulated lipogenesis genes ACACA, FASN, and HMGCR (52) also showed reduced enrichment of AR ChIP peaks with bicalutamide. Notably, lipid transporter genes might contain additional AR ChIP peaks outside the cutoff of 25 kb. Alternatively, the absence of AR ChIP peaks might indicate that they are indirectly regulated by androgen-activated transcription factors, e.g., sterol element-binding proteins 1 and 2 (SREPB1/2). Indeed, the LDLR gene lacks AR ChIP peaks but contains flanking sterol regulatory elements and is positively regulated by SREBP1/2 (53, 54).

Figure 5.

Androgens regulated the expression of lipid transporters. A, AR ChIPseq peak enrichment analysis of 42 lipid transporter genes and six lipogenesis genes (ACLY, ACSS2, ACACA, FASN, HMGCS1, and HMGCR) in the Ramos-Montoya data set of LNCaP cells treated with bicalutamide (BIC) compared with vehicle control (VEH; 28). The number of peaks is highlighted by the bubble size and the enrichment score by the gray scale. B, LNCaP cells were grown in CSS for 48 hours and treated with 10 nmol/L DHT in the absence or presence of Enz (10 μmol/L) or vehicle (Ctrl) for 48 hours. mRNA expression of indicated lipid transporters was analyzed by RNA-seq, and heat maps were generated with a hierarchical clustering algorithm using completed linkage and Euclidean distance measures and scaled by z score (red, positive z score; blue, negative z score). C, mRNA expression of indicated lipid transporters was measured by qRT-PCR in LNCaP cells grown for 48 hours in CSS followed by treatment with 1 nmol/L R1881 in the presence or absence of Enz (10 μmol/L) for an additional 48 hours [n = 3; mean ± SD; one-way ANOVA with Dunnett multiple comparisons test relative to vehicle (Ctrl ns, not significant; ****, P < 0.0001)]. D, LNCaP cells were grown in CSS for 48 hours and treated with 10 nmol/L DHT in the presence or absence of Enz (10 μmol/L). Protein expression was measured by Western blot analysis and quantitated by densitometry analysis, and total protein levels were normalized to loading control [gamma tubulin; mean ± SD; one-way ANOVA with Dunnett multiple comparisons test relative to vehicle control (CSS); a representative blot of three independent experiments is shown]. E, Cells were treated as described above. After fixation, cells were incubated with LDLR primary antibody for 24 hours and counterstained with appropriate secondary antibody. Protein expression was measured by qFM (blue, DAPI; red, LDLR). F, mRNA expression analysis of indicated lipid transporters and lipogenic genes in paired LNCaP tumor xenografts before (intact) and 7 days after castration (nadir) of our previously reported longitudinal LNCaP tumor progression data set (34); *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 5.

Androgens regulated the expression of lipid transporters. A, AR ChIPseq peak enrichment analysis of 42 lipid transporter genes and six lipogenesis genes (ACLY, ACSS2, ACACA, FASN, HMGCS1, and HMGCR) in the Ramos-Montoya data set of LNCaP cells treated with bicalutamide (BIC) compared with vehicle control (VEH; 28). The number of peaks is highlighted by the bubble size and the enrichment score by the gray scale. B, LNCaP cells were grown in CSS for 48 hours and treated with 10 nmol/L DHT in the absence or presence of Enz (10 μmol/L) or vehicle (Ctrl) for 48 hours. mRNA expression of indicated lipid transporters was analyzed by RNA-seq, and heat maps were generated with a hierarchical clustering algorithm using completed linkage and Euclidean distance measures and scaled by z score (red, positive z score; blue, negative z score). C, mRNA expression of indicated lipid transporters was measured by qRT-PCR in LNCaP cells grown for 48 hours in CSS followed by treatment with 1 nmol/L R1881 in the presence or absence of Enz (10 μmol/L) for an additional 48 hours [n = 3; mean ± SD; one-way ANOVA with Dunnett multiple comparisons test relative to vehicle (Ctrl ns, not significant; ****, P < 0.0001)]. D, LNCaP cells were grown in CSS for 48 hours and treated with 10 nmol/L DHT in the presence or absence of Enz (10 μmol/L). Protein expression was measured by Western blot analysis and quantitated by densitometry analysis, and total protein levels were normalized to loading control [gamma tubulin; mean ± SD; one-way ANOVA with Dunnett multiple comparisons test relative to vehicle control (CSS); a representative blot of three independent experiments is shown]. E, Cells were treated as described above. After fixation, cells were incubated with LDLR primary antibody for 24 hours and counterstained with appropriate secondary antibody. Protein expression was measured by qFM (blue, DAPI; red, LDLR). F, mRNA expression analysis of indicated lipid transporters and lipogenic genes in paired LNCaP tumor xenografts before (intact) and 7 days after castration (nadir) of our previously reported longitudinal LNCaP tumor progression data set (34); *, P<0.05; **, P<0.01; ***, P<0.001.

Close modal

Next, mRNA transcript levels of our panel of 44 candidate lipid transporters were measured by RNA-seq in three AR-positive, androgen-sensitive prostate cancer cell lines (LNCaP, DuCaP, and VCaP) under conditions identical to the lipid content and uptake studies shown above (androgen deprivation in CSS for 48 hours and treatment with either vehicle or DHT (10 nmol/L) for 48 hours). As a control, AR function was blocked with enzalutamide in the presence and absence of DHT. As shown in Fig. 5B, RNA-seq analysis demonstrated that expression of 36 lipid transporter genes was altered by androgen treatment in LNCaP cells. Cholesterol efflux pump ABCA1 and scavenger receptor SCARF1 mRNA was significantly reduced by androgens, a response that was antagonized by enzalutamide. In contrast, DHT significantly increased the expression of fatty acid transporters (GOT2, SLC27A3, SLC27A4, SLC27A5, and CD36) and lipoprotein transporters (LDLR, LRP8, and SCARB1), which was also blocked by enzalutamide. Receptor-mediated endocytosis of lipoprotein particles through LDLR, VLDLR, SCARB1, SCARB2, and LDL receptor-related proteins (LRP1-12 and LRPAP1) converges in lysosomes for lipolysis and release of free cholesterol and fatty acids into the cytoplasm through their respective efflux pumps. Consistent with this, mRNA for lysosomal cholesterol efflux transporter NPC1 was also increased by DHT. Similar effects of DHT regulation of lipid transporter expression were observed in DuCaP and VCaP cells, with the exception of SLC25A5, LRP8, and SCARB1, which were repressed by DHT (Supplementary Fig. S5A). qRT-PCR showed significantly increased mRNA expression of the lipoprotein transport receptors LDLR (P < 0.0001) and VLDLR (P < 0.0001) in LNCaP cells treated with 1 nmol/L R1881 (Fig. 5C, top panel). Although R1881 also enhanced expression of SCARB1 and SLC27A4, there was no significant change in expression (P > 0.05), although both showed similar trends to LDLR and VLDLR (Fig. 5C, bottom). Cotreatment with enzalutamide (10 μmol/L) blocked the increase in lipid transporter expression (Fig. 5C). Analysis of DuCaP cells revealed similar results, demonstrating that the mRNA expression of the majority of tested lipid transporters was significantly enhanced by androgens (Supplementary Fig. S5B). Western blot analysis demonstrated that LNCaP cells exposed to 10 nmol/L DHT showed an almost 2-fold increase in LDLR protein expression, which was suppressed to levels similar to vehicle control when cotreated with enzalutamide (Fig. 5D, left). A similar trend of increased protein expression in cells treated with DHT was observed for SCARB1 (Fig. 5D, right). Cellular localization of LDLR protein in response to R1881 (1 nmol/L) using immunofluorescent microscopy showed that androgen treatment resulted in significantly increased expression of LDLR at the cellular periphery (plasma membrane; Fig. 5E), which was blocked by enzalutamide (10 μmol/L), confirming that AR signaling enhanced the abundance of LDLR protein at the cell surface. Finally, review of our previously reported longitudinal LNCaP xenograft study (34) revealed that mRNA levels of LDLR, VLDLR, SCARB1, SLC27A5, and SLC27A6 were reduced 7 days after castration (nadir) when compared with castration-naïve tumors (intact; Fig. 5F), which is consistent with their positive AR regulation of expression in LNCaP cells in vitro shown above.

Increased activation of de novo lipogenesis is a well-established metabolic phenotype in prostate cancer and other types of solid cancer; however, therapeutic inhibition of DNL alone has so far had only limited clinical success as therapy against neoplastic disease. Targeting DNL in preclinical cancer models, including our own work in prostate cancer, demonstrated that inhibition of DNL leading to lipid starvation can be efficiently rescued by exogenous lipids (55). Furthermore, obesity has been associated with more aggressive disease at diagnosis and higher rate of recurrence in prostate cancer patients (reviewed in refs. 56, 57). Thus, exogenous lipids may play a much more significant role in prostate cancer and other types of cancers than previously acknowledged. Indeed, recent estimates derived from studies in lung cancer cells with a similar lipogenic phenotype as prostate cancer cells suggested that 70% of lipid carbon biomass is derived from exogenous lipids and only 30% from DNL (8). Although androgens are known to activate DNL in prostate cancer (58), little is known about lipid uptake in this context.

In this study, we evaluated the effect of androgen treatment on lipid content (free cholesterol, neutral and phospholipids and lipid droplets) and lipid uptake of several lipid probes (C5:0, C12:0, and C16:0 fatty acids, cholesterol, LDL, and acetylated LDL) in a panel of prostate cancer cells. By applying cutting-edge automated quantitative fluorescence microscopy and image analysis, we provide the functionally most comprehensive analysis of lipid uptake in prostate cancer cells to date. Our work demonstrates that androgen significantly enhanced cellular uptake of LDL particles as well as free fatty acids and cholesterol and their subcellular storage in lipid droplets. Consistent with this, we showed a concordant increase in cellular phospholipids (membrane), neutral lipids (cholesterol-FA esters and TAGs stored in lipid droplets), and free cholesterol (Fig. 1A and B), which is a major component of cell membranes and essential for membrane structure and functional organization as well as a precursor for steroidogenesis reviewed in ref. 59. Although our work did not delineate the relative contributions of various anabolic and catabolic lipid metabolism processes to the net increase in cellular lipid content in response to androgen treatment, e.g., enhanced lipid uptake and lipogenesis (58) versus fatty acid oxidation, phospholipid degradation, steroidogenesis, and lipid efflux, it nevertheless shows that androgens caused a strong and expansive increase in lipid uptake across various lipid species. Our ongoing work suggests that lipid uptake has a higher supply capacity than DNL in prostate cancer cells (data not shown), which is consistent with the ability of exogenous lipids to efficiently recue DNL inhibition (55) and recent work estimating that 70% of carbon lipid biomass is derived from exogenous lipids in lung cancer cells expressing the lipogenic phenotype (8). Critically, we demonstrated that androgen-enhanced lipid uptake is directly mediated by AR signaling and independent of its stimulatory effect on cell-cycle progression and proliferation (13, 37), i.e., androgen-enhanced fatty acid and cholesterol uptake remained unaffected in prostate cancer cells arrested in G0/G1, S phase, or G2/M and in the absence of cell growth. This suggests that AR-regulated lipid uptake is maintained throughout the cell cycle, is not part of a cell-cycle–specific AR subnetwork (60) and is not indirectly caused by lipid biomass demand of daughter cell generation.

Importantly, this work for the first time comprehensively elucidated the lipid transporter landscape in prostate cancer. Recent integrative omics studies of prostate cancer patient samples highlighted a measurable degree of discordance between genomics, epigenetics, transcriptomics, and proteomics, i.e., that gene copy number, DNA methylation, and mRNA levels did not reliably predict proteomic changes (46, 47). In addition, the plasma membrane localization of most candidate lipid transporters remains to be confirmed in prostate cancer (17, 61) despite recent progress in overcoming technical limitations challenging the comprehensive delineation of the surface proteome of prostate cancer cells (45). By comparing transcriptomic and proteomic analyses of cell lines, tumor xenografts, and patient samples, our work has conclusively demonstrated robust mRNA expression of 34 lipid transporters in multiple prostate cancer cell lines and expression of six lipid transporter proteins in the membrane fraction of LNCaP cells, of which plasma membrane expression was independently confirmed for LDLR, GOT2, LRPAP1, LRP8, and SCARB2 in eight prostate cancer cell lines (17, 45); John Lee, August 29, 2018]. Our data mining of previously reported prostate cancer tumor proteomes (normal gland vs. primary prostate cancer and primary prostate cancer vs. bone metastasis; refs. 46, 49) demonstrated that the expression of the lipid transporter landscape substantially changes during prostate cancer progression from localized disease (21 lipid transporters downregulated = low lipid uptake) to bone metastatic disease (16 lipid transporters upregulated = high lipid uptake). For comparison, the enhanced expression of lipogenic enzymes suggested that lipid synthesis was upregulated throughout prostate cancer progression from primary to metastatic disease. Our Oncomine analysis revealed a similar trend in the mRNA expression of lipid transporters in three prostate cancer patient sample cohorts reported previously (Grasso 2012, Varambally 2005, and La Tulippe 2002). If, and to what extent, the extremely lipid-rich environment of the bone marrow (50%–70% adiposity in adult men; ref. 62) is associated with enhanced lipid uptake in prostate cancer bone metastases remains to be investigated, including the possibility that the increased incidence of prostate cancer metastases to bone is linked to high levels of adiposity and specific lipid species within bone marrow, which provide increased stimulus for more aggressive growth and protumorgenic lipid signaling of metastatic prostate cancer. Of the 22 bone metastasis proteomes that were analyzed, 16 were from patients after long-term ADT and classified as CRPC, with one short-term ADT and five hormone-naïve cases, yet all shared the same general features, including enhanced lipid transport and fatty acid oxidation (49), suggesting that castration-resistant bone metastases rely on similar mechanisms for growth as hormone-naïve metastatic bone tumors. Contrary to above reports, an integrated transcriptomics and lipidomics study highlighted increased mRNA levels of SCARB1, GOT2, and SLC27As 2, 4, and 5 as well as polyunsaturated fatty acid (PUFA) accumulation in 20 paired localized primary tumors compared with matched adjacent nonmalignant prostate tissue (63). Although PUFA synthesis from essential fatty acids α-linolenic acid and linoleic acids remained transcriptionally unchanged, the authors proposed that increased phospholipid uptake through SCARB1 caused intratumoral PUFA enrichment in localized prostate cancer; however, this hypothesis still awaits experimental confirmation. The reason for discordance between both studies regarding lipid uptake in localized prostate cancer is unclear, but it is noteworthy that the activity of lipid transporters is also regulated through changes in their subcellular localization, highlighting the need for an integrated analysis of the cell-surface proteome and tumor lipidome in prostate cancer. We conclude that LDLR, GOT2, LRPAP1, LRP8, SCARB1, and SCARB2 are high-confidence lipid transporters that are associated with prostate cancer disease progression and bone metastasis, but further work is needed to fully delineate the lipid transporter proteome at the plasma membrane in prostate cancer.

We have provided the most comprehensive functional analysis of lipid uptake in prostate cancer cells to date and demonstrated that androgens strongly enhanced lipid uptake of fatty acids, cholesterol, and lipoprotein particles LDL and acetylated LDL in AR-positive prostate cancer cell lines (summarized in Fig. 6). Previous work indicated that expression of GOT2/FABPpm is enhanced by androgens and increases the cellular uptake of medium- and long-chain fatty acids in LNCaP and CWR22Rv1 prostate cancer cells (17). Our comprehensive analyses of AR binding sites (ChIPseq peaks), RNA-seq (of three DHT-treated AR-positive prostate cancer cell lines), qRT-PCR, Western blot, and DNA microarray of LNCaP tumor xenograft (34) revealed that an equal number of lipid transporters are activated and suppressed by androgens. AR-negative malignant and nonmalignant prostate cell lines (PC-3, Du145, and BHP-1) show avid lipid uptake (data not shown) and expression of transporters (Fig. 4A and B). Thus, it is likely that other signaling pathways regulate lipid supply in these cell lines. Furthermore, after using the independently confirmed plasma membrane expression (45) as a high-confidence filter, we conclude that LRPAP1 and SCARB2 are androgen-suppressed and LRP8, SCARB1, LDLR, and GOT2 are androgen-enhanced surface lipid transporters in prostate cancer cells. Interestingly, GOT2 (mitochondrial aspartate aminotransferase) is better known for its role in amino acid metabolism, the cytoplasm–mitochondria malate–aspartate shuttle, and the urea and tricarboxylic acid cycles. This suggests the moonlighting of metabolic enzymes in other subcellular compartments (64), e.g., the plasma membrane (17, 61) and, strikingly, with additional substrate specificities and catalytic activity (65). Thus, there is a possibility that future studies will discover additional proteins involved in lipid uptake due to their plasma membrane expression.

Figure 6.

AR regulates lipid uptake and lipogenesis. Schematic representation of cellular supply pathways of cholesterol and fatty acids in prostate cancer cells (transporter-mediated uptake, lipogenesis, passive diffusion, tunneling nanotubes). Lipid transporters and lipogenic enzymes whose expression is increased or decreased by androgens are highlighted in red and blue, respectively. Lipid transporters without confirmed surface expression in prostate cancer are marked by lighter shades of red and blue. Only lipid transporters with confirmed mRNA and protein expression in cell lines and patient samples are shown.

Figure 6.

AR regulates lipid uptake and lipogenesis. Schematic representation of cellular supply pathways of cholesterol and fatty acids in prostate cancer cells (transporter-mediated uptake, lipogenesis, passive diffusion, tunneling nanotubes). Lipid transporters and lipogenic enzymes whose expression is increased or decreased by androgens are highlighted in red and blue, respectively. Lipid transporters without confirmed surface expression in prostate cancer are marked by lighter shades of red and blue. Only lipid transporters with confirmed mRNA and protein expression in cell lines and patient samples are shown.

Close modal

Targeting cholesterol homeostasis in prostate cancer as a therapeutic strategy to delay development of CRPC has recently received increasing attention (66–68). Cholesterol is a precursor of steroid hormone synthesis, and we previously showed that progression to CRPC is associated with increased intratumoral steroidogenesis of androgens (34). Hypercholesterolemia has been reported to enhance LNCaP tumor xenograft growth and intratumoral androgen synthesis (69), and monotherapy against dietary cholesterol adsorption in the intestine with ezetimibe (67) or de novo cholesterol synthesis with simvastatin (66) reduced LNCaP tumor xenograft growth, androgen steroidogenesis, and delayed development of CRPC. Furthermore, targeting cholesterol uptake via SCARB1 antagonism with ITX5061 reduced HDL uptake (but not LDL) in LNCaP, VCaP, and CRW22Rv1 cells and sensitized CWR22Rv1 tumor orthografts to ADT (68). Comparatively, the same study showed that ITX5061 conferred stronger growth inhibition than simvastatin in LNCaP and CWR22Rv1 cells (68) under hormone-deprived conditions, suggesting that cholesterol uptake via SCARB1 is a significant supply route in this prostate cancer model. Due to the coexpression of multiple lipoprotein transporters (LDLR, VLDLR, SCARB1, and LRP1-12) in conjunction with increased cholesterol synthesis in prostate cancer, novel cotargeting strategies antagonizing this cholesterol supply redundancy might have profound synergies in extending the efficacy of ADT and delaying the development of CRPC. Such cotreatment strategies could include simvastatin in combination with specific inhibitors of lipid processing in the lysosome, which is a critical hub for lipid uptake through endocytosis, including phagocytosis, pinocytosis, and receptor-mediated endocytosis. The latter pathway is used by all major lipoprotein receptors, including LDLR, VLDLR, SCARB1, and the LRPs 1–12, and was the focus of a recently started phase I/II clinical trial (NCT03513211). Strategies of cotargeting lipid uptake and synthesis with repurposed drugs are currently under investigation by our group and show very promising and potent antineoplastic synergies in preclinical models of advanced prostate cancer.

No potential conflicts of interest were disclosed.

Conception and design: K.D. Tousignant, L.K. Philp, C.C. Nelson, M.C. Sadowski

Development of methodology: M.C. Sadowski

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K.D. Tousignant, A. Rockstroh, S.J. McPherson, M.E. Dinger, C.C. Nelson, M.C. Sadowski

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K.D. Tousignant, A. Rockstroh, A. Taherian Fard, M.L. Lehman, C. Wang, L.K. Philp, M.C. Sadowski

Writing, review, and/or revision of the manuscript: K.D. Tousignant, M.L. Lehman, S.J. McPherson, L.K. Philp, C.C. Nelson, M.C. Sadowski

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L.K. Philp, N. Bartonicek, M.C. Sadowski

Study supervision: L.K. Philp, M.E. Dinger, C.C. Nelson, M.C. Sadowski

This study was supported by the Movember Foundation and the Prostate Cancer Foundation of Australia through a Movember Revolutionary Team Award (K.D. Tousignant, A. Rockstroh, A. Taherian Fard, M.L. Lehman, C. Wang, S.J. McPherson, L. Philp, C.C. Nelson, and M.C. Sadowski).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Swinnen
JV
,
Brusselmans
K
,
Verhoeven
G
. 
Increased lipogenesis in cancer cells: new players, novel targets
.
Curr Opin Clin Nutr Metab Care
2006
;
9
:
358
65
.
2.
Menendez
JA
,
Lupu
R
. 
Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis
.
Nat Rev Cancer
2007
;
7
:
763
77
.
3.
Fritz
V
,
Benfodda
Z
,
Rodier
G
,
Henriquet
C
,
Iborra
F
,
Avancès
C
, et al
Abrogation of de novo lipogenesis by stearoyl-CoA desaturase 1 inhibition interferes with oncogenic signaling and blocks prostate cancer progression in Mice
.
Mol Cancer Ther
2010
;
9
:
1740
54
.
4.
Deep
G
,
Schlaepfer
I
. 
Aberrant lipid metabolism promotes prostate cancer: role in cell survival under hypoxia and extracellular vesicles biogenesis
.
Int J Mol Sci
2016
;
17
:
1061
.
5.
Flavin
R
,
Zadra
G
,
Loda
M
. 
Metabolic alterations and targeted therapies in prostate cancer
.
J Pathol
2011
;
223
:
283
94
.
6.
Kuemmerle
NB
,
Rysman
E
,
Lombardo
PS
,
Flanagan
AJ
,
Lipe
BC
,
Wells
WA
, et al
Lipoprotein lipase links dietary fat to solid tumor cell proliferation
.
Mol Cancer Ther
2011
;
10
:
427
36
.
7.
Griffiths
B
,
Lewis
CA
,
Bensaad
K
,
Ros
S
,
Zhang
Q
,
Ferber
EC
, et al
Sterol regulatory element binding protein-dependent regulation of lipid synthesis supports cell survival and tumor growth
.
Cancer Metab
2013
;
1
:
3
.
8.
Hosios
AM
,
Hecht
VC
,
Danai
LV
,
Johnson
MO
,
Rathmell
JC
,
Steinhauser
ML
, et al
Amino acids rather than glucose account for the majority of cell mass in proliferating mammalian cells
.
Developmental Cell
2016
;
36
:
540
9
.
9.
Effert
PJ
,
Bares
R
,
Handt
S
,
Wolff
JM
,
Büll
U
,
Jakse
G
. 
Metabolic imaging of untreated prostate cancer by positron emission tomography with sup 18 fluorine-labeled deoxyglucose
.
The J Urol
1996
;
155
:
994
8
.
10.
Zadra
G
,
Photopoulos
C
,
Loda
M
. 
The fat side of prostate cancer
.
Biochim Biophys Acta
2013
;
1831
:
1518
32
.
11.
Liu
Y
,
Zuckier
LS
,
Ghesani
NV
. 
Dominant uptake of fatty acid over glucose by prostate cells: a potential new diagnostic and therapeutic approach
.
Anticancer Res
2010
;
30
:
369
74
.
12.
Schlaepfer
IR
,
Glodé
LM
,
Hitz
CA
,
Pac
CT
,
Boyle
KE
,
Maroni
P
, et al
Inhibition of lipid oxidation increases glucose metabolism and enhances 2-Deoxy-2-[(18)F]Fluoro-D-glucose uptake in prostate cancer mouse xenografts
.
Mol Imaging Biol
2015
;
17
:
529
38
.
13.
Lonergan
PE
,
Tindall
DJ
. 
Androgen receptor signaling in prostate cancer development and progression
.
J Carcinogen
2011
;
10
:
20
.
14.
Dutt
SS
,
Gao
AC
. 
Molecular mechanisms of castration-resistant prostate cancer progression
.
Fut Oncol
2009
;
5
:
1403
13
.
15.
Heemers
H
,
Verrijdt
G
,
Organe
S
,
Claessens
F
,
Heyns
W
,
Verhoeven
G
, et al
Identification of an androgen response element in intron 8 of the sterol regulatory element-binding protein cleavage-activating protein gene allowing direct regulation by the androgen receptor
.
J Biol Chem
2004
;
279
:
30880
7
.
16.
Swinnen
JV
,
Van Veldhoven
PP
,
Esquenet
M
,
Heyns
W
,
Verhoeven
G
. 
Androgens markedly stimulate the accumulation of neutral lipids in the human prostatic adenocarcinoma cell line LNCaP
.
Endocrinology
1996
;
137
:
4468
74
.
17.
Pinthus
JH
,
Lu
JP
,
Bidaisee
LA
,
Lin
H
,
Bryskine
I
,
Gupta
RS
, et al
Androgen-dependent regulation of medium and long chain fatty acids uptake in prostate cancer
.
Prostate
2007
;
67
:
1330
8
.
18.
Sahoo
S
,
Aurich
MK
,
Jonsson
JJ
,
Thiele
I
. 
Membrane transporters in a human genome-scale metabolic knowledgebase and their implications for disease
.
Front Physiol
2014
;
5
:
91
.
19.
Doege
H
,
Stahl
A
. 
Protein-mediated fatty acid uptake: novel insights from in vivo models
.
Physiology
2006
;
21
:
259
68
.
20.
Tamura
Y
,
Osuga
J
,
Adachi
H
,
Tozawa
R
,
Takanezawa
Y
,
Ohashi
K
, et al
Scavenger receptor expressed by endothelial cells I (SREC-I) mediates the uptake of acetylated low density lipoproteins by macrophages stimulated with lipopolysaccharide
.
J Biol Chem
2004
;
279
:
30938
44
.
21.
Miller
YI
,
Choi
SH
,
Fang
L
,
Tsimikas
S
. 
Lipoprotein modification and macrophage uptake: role of pathologic cholesterol transport in atherogenesis
.
Subcell Biochem
2010
;
51
:
229
51
.
22.
Liberio
MS
,
Sadowski
MC
,
Soekmadji
C
,
Davis
RA
,
Nelson
CC
. 
Differential effects of tissue culture coating substrates on prostate cancer cell adherence, morphology and behavior
.
PLoS One
2014
;
9
:
e112122
.
23.
Levrier
C
,
Sadowski
MC
,
Nelson
CC
,
Healy
PC
,
Davis
RA
. 
Denhaminols A-H, dihydro-beta-agarofurans from the endemic Australian rainforest plant Denhamia celastroides
.
J Nat Prod
2015
;
78
:
111
9
.
24.
Kamentsky
L
,
Jones
TR
,
Fraser
A
,
Bray
MA
,
Logan
DJ
,
Madden
KL
, et al
Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software
.
Bioinformatics
2011
;
27
:
1179
80
.
25.
Egbewande
FA
,
Sadowski
MC
,
Levrier
C
,
Tousignant
KD
,
White
JM
,
Coster
MJ
, et al
Identification of gibberellic acid derivatives that deregulate cholesterol metabolism in prostate cancer cells
.
J Nat Prod
2018
;
81
:
838
45
.
26.
Schmittgen
TD
,
Livak
KJ
. 
Analyzing real-time PCR data by the comparative C(T) method
.
Nat Protoc
2008
;
3
:
1101
8
.
27.
Mei
S
,
Qin
Q
,
Wu
Q
,
Sun
H
,
Zheng
R
,
Zang
C
, et al
Cistrome Data Browser: a data portal for ChIP-Seq and chromatin accessibility data in human and mouse
.
Nucleic Acids Res
2017
;
45
:
D658
62
.
28.
Ramos‐Montoya
A
,
Lamb
AD
,
Russell
R
,
Carroll
T
,
Jurmeister
S
,
Galeano-Dalmau
N
, et al
HES6 drives a critical AR transcriptional programme to induce castration‐resistant prostate cancer through activation of an E2F1‐mediated cell cycle network
.
EMBO Mol Med
2014
;
6
:
651
61
.
29.
Krueger
F
.
Trim Galore
. 
2012
;
Available from
: http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/.
30.
Dobin
A
,
Davis
CA
,
Schlesinger
F
,
Drenkow
J
,
Zaleski
C
,
Jha
S
, et al
STAR: ultrafast universal RNA-seq aligner
.
Bioinformatics
2013
;
29
:
15
21
.
31.
Li
B
,
Dewey
CN
. 
RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome
.
BMC Bioinformatics
2011
;
12
:
323
.
32.
Robinson
MD
,
Oshlack
A
. 
A scaling normalization method for differential expression analysis of RNA-seq data
.
Genome Biol
2010
;
11
:
R25
.
33.
Robinson
MD
,
McCarthy
DJ
,
Smyth
GK
. 
edgeR: a Bioconductor package for differential expression analysis of digital gene expression data
.
Bioinformatics
2010
;
26
:
139
40
.
34.
Locke
JA
,
Guns
ES
,
Lubik
AA
,
Adomat
HH
,
Hendy
SC
,
Wood
CA
, et al
Androgen levels increase by intratumoral de novo steroidogenesis during progression of castration-resistant prostate cancer
.
Cancer Res
2008
;
68
:
6407
15
.
35.
Sieh
S
,
Taubenberger
AV
,
Rizzi
SC
,
Sadowski
M
,
Lehman
ML
,
Rockstroh
A
, et al
Phenotypic characterization of prostate cancer LNCaP cells cultured within a bioengineered microenvironment
.
PloS One
2012
;
7
:
e40217
.
36.
Swinnen
JV
,
Esquenet
M
,
Goossens
K
,
Heyns
W
,
Verhoeven
G
. 
Androgens stimulate fatty acid synthase in the human prostate cancer cell line LNCaP
.
Cancer Res
1997
;
57
:
1086
90
.
37.
Heinlein
CA
,
Chang
C
. 
Androgen receptor in prostate cancer
.
Endocrine Rev
2004
;
25
:
276
308
.
38.
Balk
SP
,
Knudsen
KE
. 
AR, the cell cycle, and prostate cancer
.
Nucl Receptor Signal
2008
;
6
:
e001
.
39.
Anderson
CM
,
Stahl
A
. 
SLC27 fatty acid transport proteins
.
Mol Aspects Med
2013
;
34
:
516
28
.
40.
Goldstein
JL
,
Anderson
RG
,
Brown
MS
. 
Receptor-mediated endocytosis and the cellular uptake of low density lipoprotein
.
Ciba Found Symp
1982
:
77
95
.
41.
Go
GW
,
Mani
A
. 
Low-density lipoprotein receptor (LDLR) family orchestrates cholesterol homeostasis
.
Yale J Biol Med
2012
;
85
:
19
28
.
42.
Wang
MD
,
Kiss
RS
,
Franklin
V
,
McBride
HM
,
Whitman
SC
,
Marcel
YL
. 
Different cellular traffic of LDL-cholesterol and acetylated LDL-cholesterol leads to distinct reverse cholesterol transport pathways
.
J Lipid Res
2007
;
48
:
633
45
.
43.
Kennedy
BE
,
Charman
M
,
Karten
B
. 
Niemann-pick type C2 protein contributes to the transport of endosomal cholesterol to mitochondria without interacting with NPC1
.
J Lipid Res
2012
;
53
:
2632
42
.
44.
Nath
A
,
Chan
C
. 
Genetic alterations in fatty acid transport and metabolism genes are associated with metastatic progression and poor prognosis of human cancers
.
Sci Rep
2016
;
6
:
18669
.
45.
Lee
JK
,
Bangayan
NJ
,
Chai
T
,
Smith
BA
,
Pariva
TE
,
Yun
S
, et al
Systemic surfaceome profiling identifies target antigens for immune-based therapy in subtypes of advanced prostate cancer
.
Proc Nat Acad Sci U S A
2018
;
115
:
E4473
82
.
46.
Iglesias-Gato
D
,
Wikström
P
,
Tyanova
S
,
Lavallee
C
,
Thysell
E
,
Carlsson
J
, et al
The proteome primary prostate cancer
.
Eur Urol
2016
;
69
:
942
52
.
47.
Latonen
L
,
Afyounian
E
,
Jylhä
A
,
Nättinen
J
,
Aapola
U
,
Annala
M
, et al
Integrative proteomics in prostate cancer uncovers robustness against genomic and transcriptomic aberrations during disease progression
.
Nat Commun
2018
;
9
:
1176
.
48.
Grasso
CS
,
Wu
YM
,
Robinson
DR
,
Cao
X
,
Dhanasekaran
SM
,
Khan
AP
, et al
The mutational landscape of lethal castration-resistant prostate cancer
.
Nature
2012
;
487
.
49.
Iglesias-Gato
D
,
Thysell
E
,
Tyanova
S
,
Crnalic
S
,
Santos
A
,
Lima
TS
, et al
The proteome of prostate cancer bone metastasis reveals heterogeneity with prognostic implications
.
Clin Cancer Res
2018
;
24
:
5433
44
.
50.
Varambally
S
,
Yu
J
,
Laxman
B
,
Rhodes
DR
,
Mehra
R
,
Tomlins
SA
, et al
Integrative genomic and proteomic analysis of prostate cancer reveals signatures of metastatic progression
.
Cancer Cell
2005
;
8
:
393
406
.
51.
LaTulippe
E
,
Satagopan
J
,
Smith
A
,
Scher
H
,
Scardino
P
,
Reuter
V
, et al
Comprehensive gene expression analysis of prostate cancer reveals distinct transcriptional programs associated with metastatic disease
.
Cancer Res
2002
;
62
:
4499
506
.
52.
Swinnen
JV
,
Ulrix
W
,
Heyns
W
,
Verhoeven
G
. 
Coordinate regulation of lipogenic gene expression by androgens: evidence for a cascade mechanism involving sterol regulatory element binding proteins
.
Proc Natl Acad Sci U S A
1997
;
94
:
12975
80
.
53.
Yokoyama
C
,
Wang
X
,
Briggs
MR
,
Admon
A
,
Wu
J
,
Hua
X
, et al
SREBP-1, a basic-helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene
.
Cell
1993
;
75
:
187
97
.
54.
Streicher
R
,
Kotzka
J
,
Müller-Wieland
D
,
Siemeister
G
,
Munck
M
,
Avci
H
, et al
SREBP-1 mediates activation of the low density lipoprotein receptor promoter by insulin and insulin-like growth factor-I
.
J Biol Chem
1996
;
271
:
7128
33
.
55.
Sadowski
MC
,
Pouwer
RH
,
Gunter
JH
,
Lubik
AA
,
Quinn
RJ
,
Nelson
CC
. 
The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer
.
Oncotarget
2014
;
5
:
9362
81
.
56.
Balaban
S
,
Lee
LS
,
Schreuder
M
,
Hoy
AJ
. 
Obesity and cancer progression: is there a role of fatty acid metabolism?
BioMed Res Int
2015
;
2015
:
274585
.
57.
Taylor
RA
,
Lo
J
,
Ascui
N
,
Watt
MJ
. 
Linking obesogenic dysregulation to prostate cancer progression
.
Endocrine Connections
2015
;
4
:
R68
80
.
58.
Swinnen
JV
,
Van Veldhoven
PP
,
Esquenet
M
,
Heyns
W
,
Verhoeven
G
. 
Androgens markedly stimulate the accumulation of neutral lipids in the human prostatic adenocarcinoma cell line LNCaP
.
Endocrinology
1996
;
137
:
4468
74
.
59.
Subczynski
WK
,
Pasenkiewicz-Gierula
M
,
Widomska
J
,
Mainali
L
,
Raguz
M
. 
High cholesterol/low cholesterol: effects in biological membranes: a review
.
Cell Biochem Biophys
2017
;
75
:
369
85
.
60.
McNair
C
,
Urbanucci
A
,
Comstock
CE
,
Augello
MA
,
Goodwin
JF
,
Launchbury
R
, et al
Cell-cycle coupled expansion of AR activity promotes cancer progression
.
Oncogene
2017
;
36
:
1655
68
.
61.
Stump
DD
,
Zhou
SL
,
Berk
PD
. 
Comparison of plasma membrane FABP and mitochondrial isoform of aspartate aminotransferase from rat liver
.
Am J Physiol Gastrointest Liver Physiol
1993
;
265
:
G894
902
.
62.
Blebea
JS
,
Houseni
M
,
Torigian
DA
,
Fan
C
,
Mavi
A
,
Zhuge
Y
, et al
Structural and functional imaging of normal bone marrow and evaluation of its age-related changes
.
Sem Nuclear Med
2007
;
37
:
185
94
.
63.
Li
J
,
Ren
S
,
Piao
HL
,
Wang
F
,
Yin
P
,
Xu
C
, et al
Integration of lipidomics and transcriptomics unravels aberrant lipid metabolism and defines cholesteryl oleate as potential biomarker of prostate cancer
.
Scientific Rep
2016
;
6
:
20984
.
64.
Boukouris
AE
,
Zervopoulos
SD
,
Michelakis
ED
. 
Metabolic enzymes moonlighting in the nucleus: metabolic regulation of gene transcription
.
Trends Biochem Sci
2016
;
41
:
712
30
.
65.
Bradbury
MW
,
Stump
D
,
Guarnieri
F
,
Berk
PD
. 
Molecular modeling and functional confirmation of a predicted fatty acid binding site of mitochondrial aspartate aminotransferase
.
J Mol Biol
2011
;
412
:
412
22
.
66.
Gordon
JA
,
Midha
A
,
Szeitz
A
,
Ghaffari
M
,
Adomat
HH
,
Guo
Y
, et al
Oral simvastatin administration delays castration-resistant progression and reduces intratumoral steroidogenesis of LNCaP prostate cancer xenografts
.
Prostate Cancer Prostatic Dis
2016
;
19
:
21
7
.
67.
Allott
EH
,
Masko
EM
,
Freedland
AR
,
Macias
E
,
Pelton
K
,
Solomon
KR
, et al
Serum cholesterol levels and tumor growth in a PTEN-null transgenic mouse model of prostate cancer
.
Prostate Cancer Prostatic Dis
2018
;
21
:
196
203
.
68.
Patel
R
,
Fleming
J
,
Mui
E
,
Loveridge
C
,
Repiscak
P
,
Blomme
A
, et al
Sprouty2 loss-induced IL6 drives castration-resistant prostate cancer through scavenger receptor B1
.
EMBO Mol Med
2018
;
10pii
:
e8347
.
69.
Mostaghel
EA
,
Solomon
KR
,
Pelton
K
,
Freeman
MR
,
Montgomery
RB
. 
Impact of circulating cholesterol levels on growth and intratumoral androgen concentration of prostate tumors
.
PLoS One
2012
;
7
:
e30062
.