extracellular fatty acids are the major contributor to ... · cancer cells exhibit increased de...

Metabolism

Extracellular Fatty Acids Are the MajorContributor to Lipid Synthesis in Prostate CancerSeher Balaban1, Zeyad D. Nassar2,3, Alison Y. Zhang4,5,6, Elham Hosseini-Beheshti1,Margaret M. Centenera2,3, Mark Schreuder1,7, Hui-Ming Lin4, Atqiya Aishah1,Bianca Varney1, Frank Liu-Fu1, Lisa S. Lee1, Shilpa R. Nagarajan1, Robert F. Shearer4,Rae-Anne Hardie5,8, Nikki L. Raftopulos1, Meghna S. Kakani1, Darren N. Saunders9,Jeff Holst5,8, Lisa G. Horvath4,5,6,10,11, Lisa M. Butler2,3, and Andrew J. Hoy1

Abstract

Prostate cancer cells exhibit altered cellularmetabolismbut,notably, not the hallmarks of Warburg metabolism. Prostatecancer cells exhibit increased de novo synthesis of fatty acids(FA); however, little is known about how extracellular FAs,such as those in the circulation, may support prostate cancerprogression. Here, we show that increasing FA availabilityincreased intracellular triacylglycerol content in culturedpatient-derived tumor explants, LNCaP and C4-2B spheroids,a range of prostate cancer cells (LNCaP, C4-2B, 22Rv1, PC-3),and prostate epithelial cells (PNT1). Extracellular FAs are themajor source (�83%) of carbons to the total lipid pool in allcell lines, compared with glucose (�13%) and glutamine(�4%), and FA oxidation rates are greater in prostate cancercells compared with PNT1 cells, which preferentially parti-tioned extracellular FAs into triacylglycerols. Because of the

higher rates of FA oxidation in C4-2B cells, cells remainedviable when challenged by the addition of palmitate to culturemedia and inhibition of mitochondrial FA oxidation sensi-tized C4-2B cells to palmitate-induced apoptosis. Whereas inPC-3 cells, palmitate induced apoptosis, which was preventedby pretreatment of PC-3 cells with FAs, and this protectiveeffect required DGAT-1–mediated triacylglycerol synthesis.These outcomes highlight for the first-time heterogeneity oflipid metabolism in prostate cancer cells and the potentialinfluence that obesity-associated dyslipidemia or host circu-lating has on prostate cancer progression.

Implications: Extracellular-derived FAs are primary buildingblocks for complex lipids and heterogeneity in FAmetabolismexists in prostate cancer that can influence tumor cell behavior.

IntroductionMetabolic changes during malignant transformation are rec-

ognized as a major hallmark of cancer (1). In general, cancer cellsexhibit increased glucose uptake and enhanced rates of glycolysis

resulting in lactate and energy production, termed the Warburgeffect (2). Other cancer-related alterations in intracellular meta-bolic pathways include elevated synthesis of nucleotides, pro-teins, and fatty acids (FA) to support increased rates of growth anddivision (3). Significant attention has centered on altered glucoseand glutaminemetabolism, including their roles as precursors forde novo FA synthesis/lipogenesis (4–6), yet the contribution of FAsto cancer cell biology remains elusive. FAs are the main structuralcomponents of biologicalmembranes and are building blocks forcomplex lipids such as triacylglycerols (TAG) and membranephospholipids, and signaling intermediates including diacylgly-cerol, phosphoinositols, sphingosine, and phosphatidic acid (7).The intracellular FA pool that acts as precursors for complex lipidsynthesis is supplied by extracellular sources such as lipoprotein-contained TAGs and adipose-derived free FAs, as well as throughde novo synthesis. Importantly, obese patients tend to have higherstage and a highmortality rate from a range of cancers (see ref. 8).These patients typically have increased adiposity and dyslipide-mia, resulting in a lipid-rich extratumoral environment.

Prostate cancer is the most common cancer in men andthe second leading cause of male cancer-related death. The main-stay of treatment for advanced prostate cancer is androgen dep-rivation therapy; however, this treatment is not curative, andpatients inevitably develop a lethal form of the disease termedcastration-resistant prostate cancer (9). Unlike most other carci-nomas, prostate cancer is characterized by a slow glycolytic rateand may be more reliant on FA oxidation to provide ATP forcell proliferation and growth, due to increased rates of citrate

1Discipline of Physiology, School of Medical Sciences & Bosch Institute, CharlesPerkins Centre, Faculty of Medicine and Health, The University of Sydney, NewSouth Wales, Australia. 2Adelaide Medical School and Freemasons FoundationCentre for Men's Health, University of Adelaide, Adelaide, South Australia,Australia. 3South Australian Health and Medical Research Institute, Adelaide,SouthAustralia, Australia. 4CancerDivision, TheKinghornCancerCentre/GarvanInstitute for Medical Research, Darlinghurst, New South Wales, Australia. 5Fac-ulty of Medicine and Health, University of Sydney, Sydney, New South Wales,Australia. 6Chris O'Brien Lifehouse, Camperdown, New South Wales, Australia.7Faculty of Medicine, University of Utrecht, Utrecht, the Netherlands. 8Origins ofCancer Program, Centenary Institute, University of Sydney, Camperdown, NewSouth Wales, Australia. 9School of Medical Sciences, University of New SouthWales, Sydney, New South Wales, Australia. 10School of Medicine, University ofNew South Wales Australia, Sydney, New South Wales, Australia. 11Royal PrinceAlfred Hospital, Camperdown, New South Wales, Australia.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

S. Balaban, Z.D. Nassar, and A.Y. Zhang contributed equally to this article.

Corresponding Author: Andrew J. Hoy, University of Sydney, Lab 3W, The Hub(D17), Charles Perkins Centre, Sydney, New South Wales 2006, Australia.Phone: 612-9351-2514; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-18-0347

�2019 American Association for Cancer Research.

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oxidation (10). Prostate cancer also exhibits a number of lipid-specific features, including increased lipid droplet numberand size in high-grade carcinomas compared with low-gradeprostate carcinomas and normal prostate tissue (11), andenhanced rates of de novo FA synthesis (12–14). While the func-tional significance of these phenotypes is yet to be elucidated, theylikely arise from aberrant activation of SREBPs and enhancedexpression of FASN (14–16). The resultant increase in de novoFA synthesis, which uses acetyl-CoA as a major substrate, is thebasis for evaluating 11C-acetate-PET for diagnosis and stag-ing (17); however, the influence of factors other than tumormetabolism on acetate uptake results in high false-positiveresults (17). One potential driving factor that influences 11C-acetate uptake and metabolism could be the extratumoral lipidenvironment. It is known that high extracellular lipid levelsinfluence glucose metabolism in type II diabetes (18) and inbreast cancer (19–21), and candirectly influence the rate of de novoFA synthesis from nonlipid sources (i.e. glucose- or glutamine-derived acetyl-CoA; ref. 22). It has also been reported that dietaryfat and metabolic disorders, including dyslipidemia and obesity,are adverse prognostic factors influencing disease behavior (seeref. 23). Recently, we identified a prognostic three-lipid signaturethat is associated with poor prognosis in men with lethal meta-static castration-resistant prostate cancer (24). Collectively, theseobservations suggest that the extracellular lipid environment mayinfluence prostate cancer FA metabolism and behavior, yet thisrelationship remains to be characterized.

The aim of this study was to assess FA metabolism in patient-derived explants and in a range of prostate cancer cell lines, andelucidate the role of FA metabolism in prostate cancer cell sur-vival. Insights into these situations may provide a greater under-standing into the potential role that elevated extracellular FAlevels may play in obesity-induced prostate cancer progression(see refs. 23, 25).

Materials and MethodsCell culture

The human prostate epithelial cell line PNT1 and the humanprostate carcinoma cell lines LNCaP (androgen receptor pos-itive, androgen sensitive), C4-2B, 22Rv1 (androgen receptorpositive, androgen independent), and PC-3 (androgen receptornegative, androgen resistant) were obtained from the ATCC.Cell lines are validated annually by Garvan Molecular Geneticsusing a test based on the Powerplex 18D Kit (DC1808, Pro-mega) and tested for Mycoplasma every 3 months (MycoAlertMycoplasma Detection Kit, Lonza). All cell lines were culturedin RPMI1640 medium (Life Technologies Australia Pty Ltd.)supplemented with 10% FCS (HyClone, GE Healthcare LifeSciences) and 100 IU/mL penicillin and 100 IU/mL strepto-mycin (Life Technologies Australia Pty Ltd.). The passage num-bers of all cell lines were below 20 between thawing and use inthe experiments.

To create the spheroids, approximately 1 � 106 cells wereobtained by trypsinization from growing monolayer cultures.Cells (2�104/well)were seeded in96-well, ultra-lowattachment,round-bottom plates (Costar, Corning). These cells were thencentrifuged at 1,500� g for 10minutes and incubated at 37�C in ahumidified 5% CO2 incubator for 7 days. The cells were keptwithout agitation except when fresh growth medium was admin-istered every 72 hours.

To inhibit DGAT-1 activity, cells were treatedwith 60 nmol/L ofAZD3988 (Tocris Bioscience, Invitrogen; ref. 26) for 24 hours inRPMI, 10% FCS, and no antibiotics. After treatment, cells werewashed and sensitivity to palmitate-induced apoptosis or cellgrowth in fresh RPMI containing 10% FCS was assessed. Toinhibit FA oxidation, cells were treated with 100 mmol/L etomoxir(Sigma) in RPMI, 10% FCS, and no antibiotics.

Cell transfectionCells were seeded two days before the experiment and trans-

fected using RNAiMAX transfection reagent (#13778075; ThermoFisher Scientific) and 25 pmol Pooled CPT1A siRNA (ON-TAR-GETplus SMARTpool L-009749-00-0005; Thermo Fisher Scien-tific; ref. 27) or ON-TARGETplus Non-targeting Pool (D-001810-10-05) according to the manufacturer's instructions. After 48hours, cells were washed and sensitivity to palmitate-inducedapoptosis and FA oxidation assessed.

Patient-derived explantsHuman ethical approval for this project was obtained from the

University of Adelaide Human Research Ethics Committee, StAndrew's Hospital Research Ethics Committee (reference number80; Adelaide, South Australia) or St Vincent's Hospital's HumanResearch Ethics Committee (12/231; Sydney, New South Wales,Australia). Fresh prostate cancer specimens were obtained withwritten informed consent through the Australian Prostate CancerBioResource frommen undergoing robotic radical prostatectomyat either the St Andrew'sHospital (Adelaide, SouthAustralia) or StVincent's Clinic (Sydney, New South Wales, Australia). Patient-derived explants (PDE) were prepared and cultured as reportedpreviously (28, 29). Briefly, a 6-mm core of tissue was dissectedinto 1-mm3 pieces and cultured in triplicate on a presoakedgelatin sponge (Johnson & Johnson) in 24-well plates containingRPMI1640 with 10 % FBS, antibiotic/antimycotic solution(Sigma), 0.01 mg/mL hydrocortisone, and 0.01 mg/mL insulin(Sigma). PDEs were cultured at 37�C for up to 72 hours and thenrinsed twice in ice-cold PBS prior to being snap frozen.

Lipid loading of cells, spheroids, and PDEsCell lines, spheroids, and PDEs were incubated in RPMI medi-

um supplemented with differing concentrations of either oleateonly, or 1:2:1 palmitate:oleate:linoleate (FA Mix; Sigma) as indi-cated, plus 10% FCS (wt/vol) and no antibiotics for 24 hours.

Substrate metabolismPDE fatty acid uptake. Explants were cultured in assay mediacontaining 2% FA-free BSA, 0.2 mmol/L cold oleate (Sigma), and0.2 mCi/mL [1-14C]oleate (PerkinElmer) in the presence orabsence of 100 nmol/L insulin. Snap-frozen tissues were homog-enized in 100 mL PBS using a Precellys24 tissue homogenizer(Bertin Technologies) and lysate transferred to 900 mL UltimaGold scintillation fluid for counting on a Tri-Carb 2810TR liquidscintillation analyzer (PerkinElmer).

Extracellular-derived FA metabolism. Cells were maintained inFCS-containing media prior to experimentation. Cells werewashed inwarmed PBS, then incubated in assaymedium contain-ing 0.5 mmol/L cold oleate or palmitate, [1-14C]oleate or[1-14C]palmitate (0.5 mCi/mL; PerkinElmer) conjugated to 2%(wt/vol) FA-free BSA and 1 mmol/L L-carnitine in low-glucose

Balaban et al.

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DMEM for 4 hours. Mitochondrial oxidation was determinedfrom 14CO2 production as described previously (30). Cells wereharvested on ice-cold PBS to determine 14C-oleate incorporationinto intracellular lipid pools and protein content. FA uptake wascalculated as the sum of 14CO2,

14C activity in the aqueous phase,and 14C incorporation into lipid-containing organic phase of celllysates.

Intracellular (TAG)-derived FAmetabolism.Cells weremaintainedin FCS-containing media prior to experimentation. Cells werewashed in warmed PBS, then pulsed overnight for 18 hours inassay media containing 2% FA-free BSA, with [1-14C]oleate (1mCi/mL; PerkinElmer) and cold oleate (C4-2B: 20 or 150 mmol/L;PC-3: 80 or 300 mmol/L) to prelabel the endogenous TAG pool.Following the pulse, the specific activity of the TAG pool wasdetermined in a cohort of cells bymeasuring the 14C activity in theTAG following lipid extraction and TLC aswell as the biochemicalassessment of the TAG pool (for details, see Biochemical mea-sures). TAG-derived FAoxidation (endogenous FAoxidation)wasdetermined by measuring 14CO2 production in another cohortrun in parallel where cells were chased for 4 hours in RPMImediacontaining 0.5% FA-free BSA and 1 mmol/L L-carnitine.

Glucose and glutamine metabolism. Cells were maintained inFCS-containing media prior to experimentation. Cells werewashed in warmed PBS, then incubated in the same media foroleate metabolism but with the either U-[14C]-D-Glucose or1-[14C]-L-Glutamine (0.5 mCi/mL, PerkinElmer) in place of[1-14C]oleate and placed on cells for 4 hours. Substrate uptakewas calculated as the sum of 14CO2,

14C activity in the aqueousphase and 14C incorporation into lipid-containing organicphase of cell lysates.

Cellular lipids were extracted using the Folch method (31).Lipidswere separatedbyTLCusingheptane-isopropyl ether-aceticacid (60:40:3, v/v/v) as developing solvent for TAG and phos-pholipids or by a two-step solvent system for ceramides whereTLC plates were developed to one-third of the total length of theplate in chloroform:methanol: 25%NH3 (20:4:0.2, v/v/v), dried,then rechromotographed in heptane: isopropyl ether: acetic acid(60:40:3, v/v/v). 14C activity in the TAG, phospholipid andceramide bands was determined by scintillation counting.

The contribution of oleate, glucose and glutamine to lipidsynthesis was calculated by summing the 14C activity from theorganic phase following lipid extraction for each substrate,expressed as pmol/min/mg, then calculating the percent contri-bution for each substrate.

Biochemical measuresMonolayer cultured cell and spheroid TAGs were extracted

using the method of Folch and colleagues (31) and quantifiedusing an enzymatic colorimetric method (GPO-PAP reagent,Roche Diagnostics). Cell protein content was determined usingPierce Micro BCA protein assay (Life Technologies Australia PtyLtd.).

Visualization of lipid dropletsPDEs were washed twice in ice-cold PBS and snap frozen.

Frozen sections of 5-mm thickness were dried then washed withpropylene glycol prior to stainingwithwarm (60�C)Oil RedO for10 minutes. Tissues were differentiated in 85% propylene glycol,rinsed twice in distilled water, and counterstained with hema-

toxylin for 30 seconds. Tissues were rinsed in distilled water andmounted with aqueous mounting medium. Stained tissues werevisualized using a Panoramic 250 Digital Slide Scanner (3DHistech) and staining quantitated using ImageJ software (v1.49t).

Spheroids were washed in PBS and fixed with 4% PFA. Spher-oids were then washed with 60% isopropanol and stained withOil Red O for 15 minutes at room temperature. Spheroids werewashed with isopropanol, distilled water, and then embedded inoptimal cutting temperature, before cryosectioning, and counter-stained with hematoxylin. The stained spheroid droplets wereobserved using a Leica DM4000 microscope.

PDE IHCATGL staining was performed on PDEs from 10 patients on the

Leica BOND RX platform. Sections of 4-mm–thick paraffin-embedded blocks were dewaxed and rehydrated with Bond washsolution (ref: AR9222). The slides underwent antigen retrievalusing a standardized heat-induced epitope retrieval protocol(HIER 20minutes with ER2). The primary antibody against ATGL(#2138S, Cell Signaling Technology) was applied and incubatedfor 15 minutes, followed by application of a post-primary mouseantibody for 8 minutes, followed by a secondary rabbit antibodyfor 8 minutes (both part of Leica Bond Polymer Refine DetectionSystem). Bound antibody was stained with 3, 3-diaminobenzi-dine tetrahydrochloride (Mixed DAB Refine) and then counter-stained with hematoxylin. Optimal primary antibody concentra-tion was determined by serial dilutions, optimizing for maximalsignal without background interference. The final ATGL antibodydilution used was 1:500. ATGL staining was quantified onlyin areas of tumor as determined by a clinical pathologist. Only5 of the 10 PDEs that were sectioned and stained for ATGLcontained tumor.

Western blot analysisProtein extraction from monocultures was performed as

described previously (32). Cell lysates were subjected to SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) mem-branes (Merck Millipore), and then immunoblotted with anti-bodies for anti-ATGL (2138S), anti-PARP (9532S), anti-ATF4 (11815S), and anti-GAPDH (2118S) obtained from CellSignaling Technology, anti-CPT1A (ab128568), anti-DGAT-1(ab54037), and anti-DGAT-2 (ab59493) from Abcam. Chemilu-minescence performed using Luminata Crescendo Western HRPSubstrate (Merck Millipore) and imaged using the Bio-Rad Che-miDoc MP Imaging System (Bio-Rad Laboratories) using ImageLab software 4.1 (Bio-Rad Laboratories).

Palmitate treatment and cell viabilityPC-3 and C4-2B cells were plated in triplicate in 96-well plates

(3� 103 cells/well) and a group of cells were then lipid loaded for24 hours, with ethanol used as a vehicle control. The followingday, the media were removed, cells were washed, and fresh RPMImedia containing 10 % FCS supplemented with 250 mmol/Lpalmitate (Sigma-Aldrich) or ethanol as a vehicle control added.In separate experiments, a palmitate dose responsewas performedusing fresh RPMImedia containing 10% FCS supplemented with62.5, 125, 250, or 500 mmol/L palmitate or ethanol as a vehiclecontrol. At defined timepoints stated infigure legends,MTT assayswere performed as described previously (33), cells counted, andviability assessed by Trypan blue dye exclusion at indicated timepoints. In another cohort, cells were lysed for protein content

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determination after 4 days of treatment or immunoblot analysisafter 24 hours of palmitate treatment.

Statistical analysisStatistical analyses were performed with GraphPad Prism 7.03

(Graphpad Software). Differences among groups were assessedwith appropriate statistical tests noted in figure legends. P� 0.05was considered significant. Data are reported asmean� SEMof atleast three independent determinations.

ResultsIncreasing FA availability increases triacylglycerol content inpatient-derived explants and a range of human prostate cancercell lines

First, we assessed the influence of the extracellular lipidenvironment on neutral lipid levels in clinical prostate tumorscultured as patient-derived explants (PDE). PDEs incubatedin FCS-containing media supplemented with 500 mmol/L ofthe monounsaturated FA oleate for 72 hours displayedincreased intracellular neutral lipid levels (as determined byOil Red-O staining; Fig. 1A), and a trend for increased proteinlevels (P ¼ 0.09) of the lipid droplet lipase adipose triglyceridelipase (ATGL; Fig. 1B and C). PDEs also accumulated radi-olabeled oleate in a time-dependent manner (Fig. 1D). FAuptake is stimulated by insulin in many tissues (34) and herethis was also evident in PDEs (Fig. 1D). Collectively, these datademonstrated that castrate-sensitive prostate cancer was sen-sitive to the extracellular lipid environment and accumulatesFAs as neutral lipids.

We further explored these observations using 3D spheroidmodels to assess the time- and dose-dependent effects ofextracellular FAs on neutral lipid levels. Incubating andro-gen-sensitive LNCaP spheroids in 500 mmol/L oleate increasedOil Red-O staining, with stronger staining observed following72 hours of culture (Fig. 1E). Increased Oil Red-O staining inLNCaP spheroids was associated with an increase in the neutrallipid TAG in a dose- and time-dependent manner (Fig. 1F).Similar patterns were observed in androgen-insensitive C4-2Bspheroids (Fig. 1G and H).

We next assessed the effects of the extracellular lipid environ-ment on intracellular TAG levels in a range of prostate cancer celllines and normal prostate epithelial cells. Nonmalignant prostateepithelial PNT1 cells (Fig. 1I), AR-positive and androgen-sensitiveLNCaP cells, AR-positive and androgen-independent C4-2B and22Rv1 cells (Fig. 1J–L), and AR-negative PC-3 cells (Fig. 1M)accumulated intracellular TAG in adose-dependentmannerwhencultured in FCS-containing media supplemented with increasingextracellular levels of oleate. Importantly, this pattern was alsoobserved in media containing a 1:2:1 palmitate: oleate:linoleatemixture of FAs, representing a physiologicmixture that reflects themost prominent circulating free FAs (35). Collectively, theseexperiments demonstrated that increasing extracellular FA levelsenhanced intracellular TAG levels in a range of prostate cancermodel systems, and this was not restricted to a specific FA species.

Human prostate cancer cells have greater oxidation ofextracellular FAs

The esterification of extracellular FAs into TAG for storage inlipid droplets is only one intracellular fate for these FAs. Usingestablished radiometric approaches (36), we defined in detail the

intracellular handling of extracellular FAs across a range of pros-tate cancer cells. We observed that FA uptake occurred in all celllines analyzed, butwas faster in LNCaP, C4-2B, andPC-3 cells (P<0.05), and tended tobe slower in 22Rv1 cells (P¼0.06) comparedwith PNT1 cells (Fig. 2A). While it has been long assumed thatprostate cancer cells activate FA oxidation (10), little direct evi-dence of catabolism of FAs in prostate cancer cells has beenreported. The generation of CO2 from extracellular oleate wasapproximately 11-foldhigher in all prostate cancer cells comparedwith PNT1 cells (Fig. 2B). Finally, the incorporation of extracel-lular FAs into TAG was increased in LNCaP and C4-2B, but lowerin 22Rv1 cells compared with PNT1 cells (Fig. 2C).

Extracellular FAs are the major source of carbons for lipidsynthesis inhumanprostate cancer cells comparedwith glucoseand glutamine

The assessment of cancer cell FAmetabolism is often limited tothe generation of new FAs from nonlipid sources such as glucoseand glutamine (i.e., de novo lipogenesis), which has been pro-posed to meet the increased requirement for phospholipid (37).As such, we next assessed the contribution of glucose and gluta-mine carbons to total lipid synthesis in a range of prostate cancercells and nonmalignant prostate epithelial cells and comparedthese to the contribution of extracellular FAs. First, we observedthat all cells tested took up glucose and glutamine at a greater ratecompared with oleate (Fig. 2D–H). Only a very small proportionof the glucose (�5%) and glutamine (�2%) was partitioned tocellular lipids via de novo lipogenesis, whereas the vast majority ofoleate was incorporated into lipids (�95%; Fig. 2D–H). Wefurther examined the relative contributions of glucose, glutamine,and oleate as substrates for total lipid synthesis by summing theabsolute rates of lipid synthesis for each. Oleate contributed anaverage of 83% of carbons to the total lipid pool in all cell lineswith glucose providing approximately 13% and glutamine con-tributing approximately 4% (Fig. 2I). However, LNCaP cellstended to have a greater contribution to lipid carbons fromglucose compared with PNT1 cells (PNT1: 11%; LNCaP: 17%,P ¼ 0.1), evidence of increased de novo lipogenesis as reportedpreviously (12–14). Collectively, these data clearly demonstratedthat lipid synthesis from glucose and glutamine carbons contrib-uted only a minor fraction (�17%) of the total lipid synthesis inthe basal state.

Increasing FA availability enhances fatty acid flux into and outof lipid droplets in human prostate cancer cells

A major destination for extracellular FAs is TAG stored incytosolic lipid droplets (Fig. 1). This TAG is not a terminaldestination for FAs and can bemobilized via the actions of neutrallipases (38, 39). As such, we next assessed the catabolism ofintracellular-derived FAs. Overnight exposure to media contain-ing higher amounts of oleate and 0.2 mCi/ml 14C-oleate increasedTAG levels in both C4-2B and PC-3 cells compared with cellstreated with lower amounts of oleate and 0.2 mCi/mL 14C-oleate(data not shown, similar to Fig. 1). The oxidation of intracellularTAG-derived FAs was increased in cells that were incubatedovernight in media containing more oleate (Fig. 2J and K).Collectively, these experiments demonstrated that a larger intra-cellular FA store not only promoted FA oxidation to generate ATP,NADH, and othermetabolites, but also increasedmobilization ofFAs, potentially for provisioning into phospholipid synthesis.

Balaban et al.





Increasing intracellular TAG levels protects PC-3 cells fromapoptosis induced by palmitate or serum starvation

We have demonstrated that a range of prostate cancer modelsystems accumulate lipid droplets, which is supported by recentin vivo data (16). Lipid accumulation is associated withincreased tumor burden (11, 16) and therefore it is possiblethat these higher intracellular lipid levels provide a survivaladvantage. Next we assessed the responsiveness of lipid-loadedcastration-resistant C4-2B and PC3 cells to apoptotic stimuli by

challenging these cells to either high levels of palmitate in FCS-containing media (21, 40, 41) or with serum-free media. BothC4-2B and PC3 cells are androgen-independent metastaticprostate cancer cell lines that model CRPC, which is currentlyincurable. The addition of 250 mmol/L palmitate to FCS-con-taining media reduced MTT absorbance within 2 days and thiseffect was further enhanced by 4 days compared with cellscultured in FCS-containing media (Fig. 3A). This reducedmetabolic activity was consistent with a striking reduction in

Figure 1.

Prostate cancer cells, spheroids, and patient-derived tumor explants take up and store FA as TAG. Patient-derived tumor explant neutral lipid levels by Oil Red-Ostaining (A), representative image (B), and intensity of ATGL immunostaining after 72-hour incubation in media (C) containing 500 mmol/L oleate, where 0, nostaining; 1, 1þ (moderate) immunostaining; and 2, 2þ (high) immunostaining, and 3H-oleate uptake (D). Scale bars, 100 mm (A) and 200 mm (B). Oil Red-Ostaining is representative of n¼ 5 patient-derived explants and ATGL IHC is representative of n¼ 5 patient-derived explants that contained cancer. ATGL IHCquantification is paired for multiple explants, P determined by paired Student t test. 3H-oleate uptake data are paired for multiple explants from 3 individuals[patient 1 (empty circles), patient 2 (gray circles), and patient 3 (filled circles)]. � , P� 0.05 main effect for time; #, P� 0.05 main effect for insulin by two-wayANOVA. LNCaP spheroid neutral lipid levels by Oil Red-O staining (E) and TAG content after 72-hour incubation in oleate (F). C4-2B spheroid neutral lipid levelsby Oil Red-O staining (G) and TAG content (H) after 72-hour incubation in 0.5 mmol/L oleate. Scale bars, 200 mm. Oil Red-O staining is representative of n¼ 18spheroids. TAG data are presented as single measure of n¼ 18 spheroids combined. TAG content of PNT1 (I), LNCaP (J), C4-2B (K), 22Rv1 (L), and PC-3 cells (M)following overnight incubation in either oleate alone or 1:2:1 mixture of palmitate:oleate:linoleate [FA Mix; three (PNT1, 22Rv1, LNCaP) or five (C4-2B, PC-3)independent experiments performed in triplicate]. Data are presented as mean� SEM [� , P� 0.05 for main effect for (FA); #, P� 0.05 vs. oleate at same (FA)]by two-way ANOVA followed by Tukeymultiple comparisons test.






cell number after 4 days (Fig. 3B) and preceded by activation ofPARP and ATF4 signaling after 1 day of palmitate treatment(Fig. 3C). Interestingly, PC-3 cells lipid-loaded with eitheroleate alone (Fig. 3A), or FA mixture (Supplementary Fig.

S1A), were partly protected from palmitate-induced apoptosis(Fig. 3B) and displayed blunted activation of PARP and ATF4signaling (Fig. 3C). Lipid-loaded PC-3 cells were similarly, butless strikingly, protected from serum starvation–induced

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Figure 2.

Comparison of substrate metabolism in a range of prostate cancer cells. 14C-oleate uptake (A), oxidation (B), and incorporation into TAG in PNT1, LNCaP, C4-2B,22Rv1, and PC-3 cells (three independent experiments performed in duplicate; C). D–H,Absolute rates of 14C-labeled substrate incorporation into intracellularlipids (lipid synthesis) and total uptake (sum of media 14CO2,

14C activity in both the aqueous and organic phases of a Folch extraction) of various substrates inPNT-1 (D), LNCaP (E), C4-2B (F), 22Rv1 (G), and PC-3 cells (H), and percent contribution of substrates to lipid synthesis in all cell lines in the basal state (threeindependent experiments performed in duplicate; I). Total lipid synthesis was defined as the sum of the gray bars in panelsD–H. Oxidation of TAG-derived14C-oleate in C4-2B (J) and PC-3 cells (K) following overnight incubation in either low (20 or 80 mmol/L oleate, respectively) or high (150 or 300 mmol/L oleate,respectively; three independent experiments performed in triplicate). Data are presented as mean� SEM. � , P� 0.05 versus PNT1 by one-way ANOVA followedby Dunnett multiple comparisons test (A–C), � , P� 0.05 versus oleate by two-way ANOVA followed by Tukey multiple comparisons test (D–H). J and K,� , P� 0.05 versus 20 or 80 mmol/L, respectively, by Student t test.

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reduction in viable cells (Supplementary Fig. S1B) and cellnumber (Supplementary Fig. S1C).

C4-2B cells have low sensitivity to palmitate-induced apoptosisPC-3 and C4-2B cells are castrate-resistant prostate cancer cells

that accumulate similar amounts of TAG following incubation inFA-richmedia (Fig. 1), but onlyC4-2B cells express AR.C4-2B cellsincubated in FCS-containingmedia supplementedwith palmitatehad only a mild attenuation of MTT absorbance compared withcontrol cells grown in FCS-only media (Fig. 3D). This response topalmitate supplementation by C4-2B cells was different from PC-3 cells (Fig. 3A), and this difference was also observed in responseto dose-dependent palmitate supplementation (SupplementaryFig. S2A–S2D). Specifically, the MTT absorbance for PC-3 cellswas nearly zero after 2 and 4 days of 250 mmol/L and 500 mmol/Lpalmitate supplementation (Supplementary Fig. S2A), whereas250 mmol/L palmitate supplementation attenuated MTT absor-bance of C4-2B cells (D2: 120%, D4: 223% of day 0; Supple-mentary Fig. S2B) and 500 mmol/L palmitate supplementationreduced MTT absorbance (D2: 77%, D4: 63% of day 0; Supple-

mentary Fig. S2B). The bluntedMTT signal was due to reduced cellnumber after 4 days exposure to FCS-containing media supple-mented with 250 mmol/L palmitate relative to control, but thenumber of cells were still greater than the number of cells at thestart of the experiment (Fig. 3E). Similar to PC-3 cells, C4-2B cellspretreated with either oleate or FA mix were protected frompalmitate-induced reduction in viable cells (Fig. 3D; Supplemen-tary Fig. S1D) and cell number (Fig. 3E). Collectively, this dem-onstrated that PC-3 cells were highly sensitive to palmitate-induced apoptosis, whereas C4-2B cells were much less sensitivewhile lipid-loading both cell lines protected from this palmitateinsult.

Differences in palmitate handling explain the differentialresponse to palmitate-induced apoptosis in C4-2B andPC-3 cells

Altered intracellular palmitate metabolism is one potentialexplanation for the striking differences in the sensitivity to pal-mitate-induced lipotoxicity between PC-3 and C4-2B cells, bothin the basal state and after lipid loading. To test this, PC-3 and

Figure 3.

PC-3 cells are sensitive to palmitate-induced apoptosis, but C4-2B cells are not, and lipid-loading protects PC-3 cells from palmitate-induced apoptosis. MTTassays (A) and cell number of PC-3 cells incubated in FCS-containing media supplemented with 250 mmol/L palmitate for 4 dayswith or without prior overnightincubation with oleate or 1:2:1 mixture of palmitate:oleate:linoleate (FA Mix; B). MTT results are presented as percentages of MTT absorbance at indicated timepoints relative to that at day 0 for each group. The dashed line represents the number of cells present at day 0 (MTT: five independent experiments performed inquadruplicate; cell count: three independent experiments performed in duplicate). C, Representative immunoblots of cPARP and ATF4 levels of PC-3 cellsincubated in FCS-containing media supplemented with 250 mmol/L palmitate for 1 day with or without prior overnight incubationwith oleate (representative ofthree independent experiments performed in triplicate). MTT assays (D) and cell number (E) of C4-2B cells incubated in FCS-containing media supplementedwith 250 mmol/L palmitate for 4 dayswith or without prior overnight incubationwith oleate. MTT results are presented as percentages of MTT absorbance atindicated time points relative to that at day 0 for each group. The dashed line represents the number of cells present at day 0 (MTT, three independentexperiments performed in quadruplicate; cell count, three independent experiments performed in duplicate). Data are presented as mean� SEM. � , P� 0.05versus palmitate; #, P� 0.05 versus control by two-way ANOVA (A and D) or one-way ANOVA (B and E) followed by Tukey multiple comparisons test.






C4-2B cells were incubated in 14C-palmitate, in the basal state orfollowing overnight exposure to oleate, and the fate of radiola-beled FAs determined. Despite being more sensitive to palmitate-induced apoptosis, PC-3 cells had lower total palmitate uptakecompared with C4-2B cells and there was no effect of overnightoleate exposure on palmitate uptake (Fig. 4A). Interestingly, C4-2B cells had significantly greater rates of palmitate oxidationcompared with PC-3 cells (Fig. 4B), which was likely due toincreased CPT1 protein levels (Fig. 4C). CPT1 catalyzes therate-limiting step in FA oxidation (42). Overnight treatment witholeate induced a modest increase in CPT1 levels in both C4-2Band PC-3 cells (Fig. 4C), but this did not change the rate ofpalmitate oxidation (Fig. 4B).

The incorporation of palmitate into TAG for storage was alsogreater in C4-2B cells compared with PC-3 cells (Fig. 4D). Thisdifference was not explained by differences in the amount ofDGAT-1 or DGAT-2 (Fig. 4E), which catalyze the final reactionin TAG synthesis (43, 44). Pretreatment with oleate increasedTAG synthesis in PC-3 cells up to rates equivalent to C4-2Bcells, which did not change in response to oleate (Fig. 4D). Inaddition, ATGL protein levels were greater in C4-2B cellscompared with PC-3 cells, and ATGL expression was increasedin both cell lines with overnight oleate treatment (Fig. 4E),consistent with increased expression in oleate-treated PDEs(Fig. 1B and C) and enhanced lipolysis seen in oleate-treatedcells (Fig. 2J and K). Overall, the partition of FAs betweenmitochondrial oxidation and storage was similar in C4-2B andPC-3 cells, but this intracellular partitioning of FA was shifted

toward storage relative to oxidation in PC-3 cells followingpretreatment with oleate (Fig. 4F).

Palmitate is a critical substrate for de novo ceramide synthe-sis (45) and one hypothesis to explain the enhanced sensitivity topalmitate in PC-3 cells compared with C4-2B cells was enhancedceramide synthesis (46). However, the rate of palmitate incorpo-ration into ceramidewas lower in PC-3 cells comparedwithC4-2Bcells (Fig. 4G). Interestingly, overnight oleate exposure reducedceramide synthesis inC4-2B cells, but not in PC-3 cells. Therewereno differences in the rate of palmitate incorporation in thephospholipid pool (Fig. 4H).

From these observations, the most striking differences in pal-mitate metabolism between C4-2B and PC-3 cells were higherpalmitate oxidation rates in C4-2B cells and the increase inpalmitate incorporation into TAG following overnight oleatetreatment in PC-3 cells. These differences in intracellular palmi-tate handlingmay explain the differential sensitivity to palmitate-induced apoptosis in C4-2B and PC-3 cells (Fig. 3).

Inhibition ofmitochondrial FA oxidation sensitizes C4-2B cellsto palmitate-induced apoptosis

Another explanation for the observed resistance of C4-2B cellsto palmitate-induced apoptosis compared with PC-3 cells may behigher palmitate oxidation and higher CPT1A protein levels(Fig. 4B and C). Therefore, we tested whether inhibiting CPT1-mediated palmitate oxidation sensitized C4-2B cells to palmitate-induced apoptosis. Treating cells with 100 mmol/L of the CPT1inhibitor, etomoxir, lowered palmitate oxidation (Fig. 5A). The

Figure 4.

PC-3 and C4-2B cells metabolize palmitate differently and this is selectively altered by pretreatment with oleate. 14C-palmitate uptake (A) and oxidation (B) inC4-2B and PC-3 cells with or without prior overnight incubation with 150 mmol/L oleate. C, Representative immunoblots and densitometric quantitation of CPT1Ain C4-2B and PC-3 cells with or without prior overnight incubation with oleate. D, 14C-palmitate incorporation into TAG in C4-2B and PC-3 cells with or withoutprior overnight incubation with oleate. E, Representative immunoblots of DGAT1, DGAT2, and ATGL, and densitometric quantitation of ATGL in C4-2B and PC-3cells with or without prior overnight incubation with oleate. F, Intracellular partitioning of 14C-palmitate expressed as the ratio of 14C-palmitate incorporation intotriacylglycerol (storage) versus 14C-palmitate oxidation in C4-2B and PC-3 cells with or without prior overnight incubationwith oleate. 14C-palmitateincorporation into ceramide (G) and phospholipid (H) in C4-2B and PC-3 cells with or without prior overnight incubation with oleate. Data are presented asmean� SEM of three independent experiments performed in triplicate. †, P� 0.05 main effect for cells; � , P� 0.05 versus - Oleate; #, P� 0.05 versus C4-2Bcells - Oleate by two-way ANOVA followed by Tukey multiple comparisons test.

Balaban et al.





addition of 250 mmol/L palmitate to growth media reduced cellviability (Fig. 5B); however, the combination of etomoxir andpalmitate completely abolished MTTmetabolic activity (Fig. 5B),cell number (Fig. 5C), and activated PARP signaling (Fig. 5D).Similar, but less striking, patterns were observed in siRNA-medi-ated CPT1A knockdown in C4-2B cells. Knockdown of CPT1A(Fig. 5E) lowered palmitate oxidation (Fig. 5F), but this wasassociated with a mild reduction in MTT metabolic activity(Fig. 5G) and no effect on cell number (Fig. 5H). The additionof 250 mmol/L palmitate to growthmedia attenuated cell viability(Fig. 5G); however, the combination of CPT1A knockdown andpalmitate further lowered MTT metabolic activity (Fig. 5G) andcell number (Fig. 5H). Collectively, these results indicate thatinhibition of FA oxidation sensitized C4-2B cells to palmitate-induced apoptosis.

Inhibition of oleate-stimulated TAG synthesis restoressensitivity to palmitate-induced apoptosis in lipid-loaded PC-3cells

Pretreatment with either oleate alone or the FA mixture pro-tected PC-3 cells from palmitate-induced and serum starvation–

induced apoptosis (Fig. 3; Supplementary Fig. S1). Radiometricanalysis of palmitate metabolism pointed to an increase in TAGsynthesis to shunt palmitate into lipid droplets (Fig. 4D) as apotential mechanism by which pretreatment with FAs protectedPC-3 cells from palmitate-induced apoptosis. We directly testedthis by inhibiting TAG synthesis through the addition of a DGAT-1 inhibitor only during the oleate preincubation of PC-3 cellsprior to palmitate treatment. As expected, DGAT-1 inhibitionblunted oleate-induced increase in PC-3 TAG content (Fig. 6A)due to reduced incorporation of radiolabeled oleate into TAG(Fig. 6B). As previously observed, palmitate treatment inducedapoptosis, as determined by reduced MTT (Fig. 6C), PARP acti-vation (Fig. 6D), and reduced cellular protein content (Fig. 6E).Preincubation with oleate blunted this effect (Fig. 6C–E); how-ever, lowering intracellular TAG levels by DGAT inhibition in thepresence of oleate restored PC-3 cell sensitivity to palmitate(Fig. 6C–E). Importantly, preincubation with DGAT-1 inhibitordid not affect subsequent PC-3 (Supplementary Fig. S3) andC4-2B (data not shown) cell growth in FCS-containing media.Therefore, TAG synthesis is required for the protective effects ofpretreating PC-3 cells with FAs to palmitate-induced apoptosis.

Figure 5.

Inhibition of FA oxidation in C4-2B cells results in sensitization to palmitate-induced apoptosis. A, 14C-palmitate oxidation in C4-2B cells that were treated with orwithout 100 mmol/L Etomoxir (Eto; three independent experiments performed in triplicate). MTT assays (B) and cell number (C) of C4-2B cells incubated in FCS-containing media supplemented with 250 mmol/L palmitate (Palm), etomoxir (Eto), or a combination for 4 days. MTT results are presented as percentages ofMTT absorbance at indicated time points relative to that at day 0 for each group. The dashed line represents the number of cells present at day 0 (MTT: fourindependent experiments performed in quadruplicate; Cell count: three independent experiments performed in duplicate). D, Representative immunoblots ofcPARP levels of C4-2B cells incubated in FCS-containing media supplemented with 250 mmol/L palmitate, etomoxir or a combination for 1 days (representativeof three independent experiments performed in triplicate). E, Representative immunoblots of CPT1A of C4-2B cells treated with control or CPT1A siRNA for 2days (representative of three independent experiments performed in triplicate). F, 14C-palmitate oxidation in C4-2B cells treated with or without CPT1A siRNA for4 days (three independent experiments performed in triplicate). MTT assays (G) and cell number (H) of C4-2B cells treated with control or CPT1A siRNA for 2days then incubated in FCS-containing media with or without supplementation with 250 mmol/L palmitate for 4 days. MTT results are presented as percentagesof MTT absorbance at indicated time points relative to that at day 0 for each group. The dashed line represents the number of cells present at day 0 (MTT, fourindependent experiments performed in quadruplicate; cell count, three independent experiments performed in duplicate). Data are presented as mean� SEM.A, � , P� 0.05 versus control by unpaired Student t test. B and C, � , P� 0.05 versus palmitate; #, P� 0.05 versus etomoxir by two-way ANOVA followed byTukey multiple comparisons test. F, � , P� 0.05 versus control by unpaired Student t test. G and H, � , P� 0.05 versus palmitate; #, P� 0.05 versus CPT1A KD bytwo-way ANOVA followed by Tukeymultiple comparisons test.






DiscussionCancer cells require adaptive alterations in intermediarymetab-

olism to fulfil the energy requirements and biochemical needs oftheir uncontrolled growth capacity (47). Unlike many other solidtumors, prostate cancer exhibits a low rate of glucose utilizationand an increased dependence on lipids as a major energysource (48). While enhanced de novo fatty acid synthesis inprostate cancer has been firmly established (see ref. 23), thecrucial importance of extracellular FAs in prostate cancer progres-sion has been underappreciated and less well-studied. Using arange ofmodels, we demonstrate that prostate cancer cells take upFAs and incorporate them into intracellular TAG for storage in adose-dependent manner. Furthermore, these extracellular FAs area greater contributor to intracellular synthesis compared withglucose and glutamine, which are used as substrates for de novo

fatty acid synthesis. We also show for the first time, strikingheterogeneity in the intracellular handling of FAs in castration-resistant C4-2B and PC-3 prostate cancer cells and in theirresponse to palmitate-induced apoptosis. Specifically, C4-2B cellshave increased FA oxidative capacity that underpins their resis-tance to palmitate-induced apoptosis. On the other hand, PC-3cells are highly sensitive to palmitate-induced apoptosis, whichwas inhibited by prior lipid-loading to stimulate TAG synthesis.These observations highlight the diversity of intracellular FAmetabolism in prostate cancer cells.

Storage of lipids is an evolutionarily conserved phenomenon,which can buffer energy fluctuations and promote survival in allcells and organisms (49). In this scenario, lipid storage predom-inantly refers to the partitioning of excess FAs into TAG for storagein cytosolic lipid droplets. Lipid droplets are closely localizedwith

Figure 6.

Inhibition of TAG synthesis in PC-3 cells blunts the protective effects of prior oleate treatment to palmitate-induced apoptosis. A, PC-3 cell TAG levels in cellstreated with 150 mmol/L oleate (Ol) with or without 60 nmol/L DGAT inhibitor AZD3988 (iDGAT) for 24 hours (three independent experiments performed induplicate). B, 14C-oleate incorporation into TAG in PC-3 cells that were treated with or without DGAT inhibitor (three independent experiments performed induplicate). C,MTT assays of PC-3 cells incubated in FCS-containing media supplemented with 250 mmol/L palmitate (Palm) for 4 days following prior incubationwith 150 mmol/L oleate (Ol) with or without 60 nmol/L DGAT inhibitor AZD3988 (iDGAT). MTT results are presented as percentages of MTT absorbance atindicated time points relative to that at day 0 for each group (three independent experiments performed in quadruplicate). D, Representative immunoblots ofcPARP of PC-3 cells incubated in FCS-containing media supplemented with 250 mmol/L palmitate for 1 day following prior incubationwith oleate with or withoutDGAT inhibitor (three independent experiments performed in triplicate). E, Protein levels in PC-3 cells incubated in FCS-containing media supplemented with250 mmol/L palmitate for 4 days following prior incubation with 150 mmol/L oleate with or without DGAT inhibitor (three independent experiments performed intriplicate). Data are presented as mean� SEM. A, � P� 0.05 versus control; #, P� 0.05 versus oleate by one-way ANOVA followed by Tukeymultiplecomparisons test. B, #, P� 0.05 versus Oleate by Student t test. C, � , P� 0.05 versus palmitate; #, P� 0.05 versus control, $, P� 0.05 versus palmþ oleate bytwo-way ANOVA followed by Tukeymultiple comparisons test. E, � , P� 0.05 versus palmitate; #, P� 0.05 versus control; $, P� 0.05 versus palmþ oleate byone-way ANOVA followed by Tukeymultiple comparisons test.

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most intracellular organelles, notably mitochondria and endo-plasmic reticulum (ER) and are highly conserved in yeast throughtomammals (50) but the size and number of lipid droplets variesbetween cell types. Prostate cancer cells have detectable levels ofTAGs and lipid droplets (11, 51) and the amounts of thesecorrelate with disease grade (11). Here, we show that PDEs,LNCaP, and C4-2B spheroids and a range of prostate cancer cellscan respond to changing levels of extracellular FAs and accumu-late these as TAG in a dose-dependentmanner, independent of FAspecies. Collectively, these in vitro observations suggest that thelipid biology of prostate cancer is likely to be influenced by thelocal lipid environment of the host, including local adipose tissuethat may be expanded in obese patients and/or the circulatinglipid profile, which itself is influenced by diet and body compo-sition (52, 53).

Several studies have reported that exogenous FAavailability caninfluence prostate cancer cell growth in vitro (54–60). Monoun-saturated FAs, including oleate (C18:1), stimulate LNCaP cellproliferation (54), but activate apoptosis inDU145 cells (60) andretard growth in PC-3 cells (58, 59). However, another studyreported that oleate stimulates PC-3, but not LNCaP cellgrowth (55), which cannot be explained by differences in con-centration. Similar inconsistent observations have been madeusing other FA species including linoleate (C18:2), arachidonicacid (C20:4), and eicosapentaenoic acid (C20:5; refs. 54, 55, 59,60). Despite these inconsistencies, overall these observationssuggest that FAs can have both pro- or antiproliferative effects.Surprisingly, little is known about the effects of extracellularpalmitate (C16:0), which accounts for approximately 20%–

22% of FA species of complex lipids (i.e. phospholipids, TAGsetc.; ref. 61) or approximately 30% of free FAs (62) in humanplasma, on prostate cancer cells. The saturated FA palmitate caninduce apoptosis in a range of cells including 3T3 fibroblasts (41),peripheral blood mononuclear cells (63), human cardiac pro-genitor cells (64), pancreatic b cells (65, 66), macrophages (67),breast cancer cells (21, 40), and hepatocytes (68). We observedthat palmitate indeed activates apoptosis in PC-3 cells but attenu-atesC4-2B cell growth. This is similar to our recent observations inbreast cancer cells, where palmitate induced apoptosis in triple-negative MDA-MB-231 cells but only attenuated growth in estro-gen receptor a–positive MCF-7 cells (21). The precise mechanismby which palmitate induces apoptosis remains to be elucidatedbut several mechanisms have been proposed. These include ERstress (65), impaired autophagy (63), altered NAD metabo-lism (68), and ceramide synthesis (66).

Prostate cancer is initially sensitive to hormonal manipulation;however, resistance to androgen deprivation therapy ultimatelyoccurs, which results in the development of lethal metastaticcastration-resistant prostate cancer. Here, we identified that thedifferential responsiveness of castration-resistant C4-2B and PC-3cells to palmitate was associated with differences in palmitatehandling. Specifically, attenuation of C4-2B cell growth by pal-mitate treatment, rather than activation of apoptosis, was due toenhanced FA oxidation compared with PC-3 cells. FA oxidationhas been proposed to be a dominant bioenergetic pathway inprostate cancer cells (10). While the relative contribution ofvarious substrates to ATP turnover in prostate cancer cells is yetto be reported, we provide clear evidence that prostate cancer cellshave increased FA oxidation compared with prostate epithelialPNT-1 cells. Our data compliment a previous observation thatLNCaP and VCaP prostate cancer cells have greater palmitate

oxidation compared with the benign prostatic hyperplasia epi-thelial cell line BPH-1 (69). We also show that the differencesbetweenC4-2B andPC-3 cellsmaybe related toCPT1Aexpressionand function. Inhibition of FA oxidation by etomoxir and CPT1Aknockdown in C4-2B cells attenuated growth, consistent with aprevious observation in castration-sensitive LNCaP and VCaPcells and xenografts (69). Interestingly, the attenuation of C4-2B cell growth by the CPT1A inhibitor etomoxir was similar topalmitate treatment, with the combination of FA oxidation inhi-bition and palmitate treatment leading to cell death. Our resultsshed new light on the contribution of FA oxidation in castration-resistant C4-2B cells where pharmacologic and genetic inhibitionof FA oxidation sensitized C4-2B cells to palmitate-inducedapoptosis and in combination with other observations (69) sug-gests that targeting FA oxidation is an attractive therapeuticstrategy in prostate cancer.

The saturated FA palmitate can activate apoptosis in a range ofcells (21, 40, 41, 63–68, 70), via a range of proposedmechanism,and we show that this also occurs in PC-3 prostate cancer cells.Another common observation is that the addition or presence ofoleate ameliorates the cytotoxic effects of palmitate (67, 68, 71–74). Interestingly, oleate also prevents arachidonic acid andlinoleic acid induced cell death in DU-145 prostate cancercells (60). Several mechanisms have been proposed includingrestoring insulin stimulated protein kinase B (Akt) signaling (73),attenuating palmitate-induced ER stress (71), preventing activa-tion of the unfolded protein response (75), activating prosurvivalpathways of ER stress (72), and activation of AMP-activatedprotein kinase andmTOR signaling (74). Cotreatmentwith oleatecan also modulate palmitate metabolism to increase TAG syn-thesis andprevent diacylglycerol accumulation (73, 76) andblockmitochondrial dysfunction and production of reactive oxygenspecies (76). We observed that pretreatment of PC-3 cells witheither oleate or a FA mixture prevented palmitate-induced apo-ptosis, as well as serum starvation, by increasing palmitate parti-tioning into TAG synthesis for storage in lipid droplets. We alsoobserved that pretreatment of C4-2B cells with either oleate or aFA mixture prevented palmitate-induced attenuation of growth.The ability of FA pretreatment to alter the response to palmitatetreatment by PC-3 and C4-2B cells was not due to changes inpalmitate uptake. We did observe a reduction in palmitate incor-poration into ceramide in C4-2B cells; however, C4-2B cells havehigher rates of ceramide synthesis from palmitate compared withPC-3 cells, despite C4-2B cells being less sensitive to the cytotoxiceffects of palmitate. This suggests that ceramide synthesis does notactivate apoptosis in these cells and that the preventative ability ofFA pretreatment was due to altered TAG synthesis and oxidation.

We and others have demonstrated that TAG synthesis canprotect cells from palmitate-induced lipotoxicity (21, 41, 77).For example, oleate supplementation promotes TAG synthesis inCHOcells that canprevent palmitate-induced apoptosis inmouseembryonic fibroblasts but this protection was not seen inDGAT1�/� mouse embryonic fibroblasts, which lack the abilityto synthesize TAG (78). Recently, we demonstrated that TAGsynthesis was required for oleate to protect MDA-MB-231 breastcancer cells from palmitate-induced apoptosis (21) and here weshow that this also occurs in PC-3 cells. The final step of TAGsynthesis is catalyzed by DGAT1 and DGAT2 and knockdown ofDGAT1 in LNCaP cells reduced cell growth and colony forma-tion (79). Collectively, these observations demonstrate that TAGsynthesis supports cancer cell progression. Interestingly, the






inability to breakdown TAG to liberate FAs also impairs cellgrowth and invasion in LNCaP cells (51, 79). TAG hydrolysis iscatalyzed by ATGL and knockdown of ATGL in LNCaP cellsablated cell invasion and growth (51). We observed that pretreat-ment with oleate increased ATGL protein levels as well as oxida-tion of TAG-derived FAs in both C4-2B and PC-3 cells. Collec-tively, these observations suggest that TAG-derived FAs maysupport prostate cancer cell progression and therefore suggestthat FAs that traverse lipid droplet contained TAG pool play animportant role in prostate cancer biology.

In conclusion, we report for the first time that prostate cancercells can use extracellular FAs as both fuel for oxidation and theprimary substrates for complex lipid synthesis such as TAG, andthat high extracellular lipid availability further enhances FAflux inthese cells. Furthermore, we identified distinct differences inpalmitate handling and sensitivity between castration-resistantC4-2B and PC-3 cells. The reduced sensitivity of C4-2B cells topalmitate-induced apoptosis was due to the high rates of FAoxidation, thus suggesting a potential therapeutic vulnerabilityfor AR-positive prostate cancer types with high levels of CPT1A.Oleate pretreatment, which stimulated TAG synthesis, preventedpalmitate-induced apoptosis in AR-negative PC-3 cells and addsto a growing body of evidence suggesting that proteins regulatingintracellular TAG homeostasis may be further therapeutic targets.The outcomes from these experiments inform the potential rolethat obesity-associated dyslipidemia (see ref. 23) or the hostcirculating lipidome (24) may play in influencing prostate cancerprogression and therefore, these metabolic traits might be thebasis for novel, targeted treatment interventions in prostatecancer.

Disclosure of Potential Conflicts of InterestA.Y. Zhang has received speakers bureau honoraria from AstraZeneca.

L.G. Horvath reports receiving a commercial research grant from Astellas. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: M. Schreuder, D.N. Saunders, L.G. Horvath,L.M. Butler, A.J. HoyDevelopmentofmethodology:A.Y. Zhang, E.Hosseini-Beheshti,M. Schreuder,R.-A. Hardie, J. Holst, A.J. Hoy

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Z.D. Nassar, A.Y. Zhang, E. Hosseini-Beheshti,M.M. Centenera, M. Schreuder, H.-M. Lin, A. Aishah, B. Varney, L.S. Lee,R.F. Shearer, R.-A. Hardie, M.S. Kakani, L.G. HorvathAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Balaban, Z.D. Nassar, A.Y. Zhang, E. Hosseini-Beheshti, M.M. Centenera, M. Schreuder, H.-M. Lin, F. Liu-Fu, L.S. Lee,R.F. Shearer, R.-A. Hardie, N.L. Raftopulos, D.N. Saunders, J. Holst,L.M. Butler, A.J. HoyWriting, review, and/or revision of the manuscript: A.Y. Zhang, H.-M. Lin,S.R. Nagarajan, R.F. Shearer, R.-A. Hardie, D.N. Saunders, J. Holst, L.G. Horvath,L.M. Butler, A.J. HoyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.Y. Zhang, A. Aishah, S.R. Nagarajan, A.J. HoyStudy supervision: L.G. Horvath, L.M. Butler, A.J. HoyOther (performed experiments with guidance): M.S. Kakani

AcknowledgmentsThe authors thank the Bosch InstituteMolecular Biology Facility for technical

support. L.M. Butler, A.J. Hoy, and J. Holst acknowledge grant support from TheMovember Foundation/Prostate Cancer Foundation of Australia (MRTA3 andMRTA1). A.J. Hoy is supported by a University of Sydney Robinson Fellowshipand was supported by Helen and Robert Ellis Postdoctoral Research Fellowshipfrom the SydneyMedical School Foundation and funding from theUniversity ofSydney. R.-A. Hardie and A.J. Hoy received support from the Sydney MedicalSchool. S. Balaban was a recipient of a University of Sydney Australian Post-graduate Award. Z.D. Nassar is supported by an Early Career Fellowship fromthe National Health and Medical Research Council of Australia and JohnMills Young Investigator Award from the Prostate Cancer Foundation ofAustralia. L.M. Butler is supported by a Principal Cancer Research Fellowshipproduced with the financial and other support of Cancer Council SA's BeatCancer Project on behalf of its donors and the State Government of SouthAustralia through the Department of Health and was supported by a FutureFellowship from the Australian Research Council (FT130101004). D.N. Saunderswas supported by the National Health and Medical Research Council (projectgrant GNT1052963). M. Schreuder was supported by funding from the DutchCancer Institute KWF.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedMay 4, 2018; revised September 22, 2018; accepted January 7, 2019;published first January 15, 2019.

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2019;17:949-962. Published OnlineFirst January 15, 2019.Mol Cancer Res Seher Balaban, Zeyad D. Nassar, Alison Y. Zhang, et al. Synthesis in Prostate CancerExtracellular Fatty Acids Are the Major Contributor to Lipid

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