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Cancer Biology and Signal Transduction

Lipid Catabolism via CPT1 as a Therapeutic Target forProstate Cancer

Isabel R. Schlaepfer1, Leah Rider1, Lindsey Ulkus Rodrigues1, Miguel A. Gij�on1, Colton T. Pac1, Lina Romero1,Adela Cimic2, S. Joseph Sirintrapun2, L. Michael Glod�e3, Robert H. Eckel4, and Scott D. Cramer1

AbstractProstate cancer is the most commonly diagnosed malignancy among Western men and accounts for the

second leading cause of cancer-related deaths. Prostate cancer tends to grow slowly and recent studies

suggest that it relies on lipid fuel more than on aerobic glycolysis. However, the biochemical mechanisms

governing the relationships between lipid synthesis, lipid utilization, and cancer growth remain unknown.

To address the role of lipid metabolism in prostate cancer, we have used etomoxir and orlistat, clinically

safe drugs that block lipid oxidation and lipid synthesis/lipolysis, respectively. Etomoxir is an irreversible

inhibitor of the carnitine palmitoyltransferase (CPT1) enzyme that decreases b oxidation in the mitochon-

dria. Combinatorial treatments using etomoxir and orlistat resulted in synergistic decreased viability in

LNCaP, VCaP, and patient-derived benign and prostate cancer cells. These effects were associated with

decreased androgen receptor expression, decreased mTOR signaling, and increased caspase-3 activation.

Knockdown of CPT1A enzyme in LNCaP cells resulted in decreased palmitate oxidation but increased

sensitivity to etomoxir, with inactivation of AKT kinase and activation of caspase-3. Systemic treatment

with etomoxir in nude mice resulted in decreased xenograft growth over 21 days, underscoring the

therapeutic potential of blocking lipid catabolism to decrease prostate cancer tumor growth. Mol Cancer

Ther; 13(10); 2361–71. �2014 AACR.

IntroductionProstate cancer is themost commonly diagnosedmalig-

nancy and the second highest contributor to cancer deathsin men in the United States (1). Currently, the standardsystemic treatment for advanced prostate cancer is basedon androgen deprivation with initial positive responses,but prostate cancer tumors eventually become resistantand restore androgen receptor (AR) signaling (2). Afterprostate cancer becomes castration resistant, no curativetreatments exist, making the identification of novel ther-apies imperative.

The mechanisms by which prostate cancer cells uselipids to their benefit are poorly understood. De novofatty acid synthesis can occur in cancer cells fromglucose, in a pathway largely controlled by the enzymefatty acid synthase (FASN), and is associated with cellgrowth, survival, and drug resistance (3, 4). However,the biochemical mechanisms governing the relation-ships between lipid synthesis, lipid utilization, andcancer growth are still largely unknown.

Overexpression of key enzymes in lipid synthesis inprostate cancer is characteristic of both primary andadvanced disease (5), suggesting that targeting lipidmetabolism enzymes in prostate cancer may offer newavenues for therapeutic approaches. Recent researchhas focused on the development of small FASN inhibi-tors for prostate cancer therapeutics (6). The lipase andFASN inhibitor orlistat has been used in several pre-clinical studies to decrease tumor growth (7–9). How-ever, much less attention is being focused on theoxidation of newly synthesized lipid in prostate cancercells. The lipid utilization pathways in these cells areinferred from indirect evidence, but they are not wellstudied or understood (10, 11).

Several lines of evidence indicate that intracellular lipidturnover (not just lipid synthesis) is important in cancercell survival: monoacylglycerol lipase, which catalyzesthe release of fatty acids from intracellular lipid stores,promotes tumor growth and survival (12); blocking fat

1Department of Pharmacology, University of Colorado Denver, Aurora,Colorado. 2Department of Pathology, Wake Forest University School ofMedicine Winston-Salem, North Carolina. 3Division of Medical Oncology,Department of Medicine, University of Colorado Denver, Aurora, Colorado.4Division of Endocrinology Metabolism and Diabetes, Department of Med-icine, University of Colorado Denver, Aurora, Colorado.

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

Current address for S.J. Sirintrapun: Memorial Sloan-Kettering CancerCenter, New York, NY.

Corresponding Author: Isabel R. Schlaepfer, Department of Pharmacol-ogy, University of Colorado Denver, Anschutz Medical Campus, 12801 E17th Ave., Room L18-6213, Mail Stop 8303, Aurora, CO 80045. Phone:303-724-3365; Fax: 303-724-3663; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-14-0183

�2014 American Association for Cancer Research.

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Therapeutics

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oxidation results in significant death of leukemia cellsexposed toproapoptotic agents (13); fatty acid oxidation isassociated with increased resistance to radiation andchemotherapeutic agents (14); finally, fatty acid oxidationfuels the production of metabolites needed to synthesizelipids and to protect cells from oxidative stress (15).Altogether, lipid oxidation is an important component ofcancer metabolism together with aerobic glycolysis andlipogenesis, but remains ill-defined in prostate cancermetabolism.

One way to study the role of lipid oxidation in atranslatable manner is through the use of safe metabolicinhibitors that can be used both in the laboratory andthe clinic. Etomoxir is a safe irreversible inhibitor of thelong-chain fatty acid transporter and has been used inthe treatment of heart failure (16). Etomoxir works byinhibiting carnitine palmitoyltransferase 1 (CPT1) andblocking the entry of long-chain fatty acids into mito-chondria for oxidation, forcing cells to use the oxidationof glucose for energy (17). Only a few studies describethe effect of etomoxir on cancer survival (13, 18), butthere are no studies of its effects on prostate cancertumor metabolism.

In this report, we examined the effects of pharmaco-logically blocking lipid synthesis and oxidation in pros-tate cancer cell viability, AR content, molecular signaling,and tumor growth. Our results suggest that prostatecancer cells are dependent on lipid oxidation for theirsurvival and this may represent a novel avenue to inves-tigate new nontoxic therapeutic approaches to prostatecancer treatment.

Materials and MethodsCell culture and drug treatment

Cell lines were obtained from the University of Color-ado Cancer Center Tissue Culture Core (Aurora, CO;year 2011) and were authenticated by single tandemrepeat analysis. Cells were used at low passage numberand grown in RPMI or DMEM (for VCaP cells) contain-ing 5% FBS supplemented with amino acids and Insulin(Hyclone). CSS was used for androgen-deprived con-ditions. Human prostate-derived cells were isolatedfrom deidentified surgical specimen at Wake ForestUniversity (Winston-Salem, NC) using our previouslydescribed protocol (19). The histologic origin of thesample was determined by analysis of the tissue sur-rounding the plug used for culture. Etomoxir-HCl (Sig-ma) was dissolved as a 15 mmol/L stock solution;orlistat (Sigma) was dissolved as a 50 mmol/L stockin DMSO.

Cell viability and proliferation analysisCell proliferation was analyzed using the Beckman

Coulter Vi-Cell Automated Cell Viability Analyzer. MTSproliferation assays were carried out using CellTiter 96AQueous One solution from Promega according to manu-facturer’s protocols.

qRT-PCRTotal RNA was isolated from cells (RNeasy, Qiagen),

and cDNAwas synthesized (high-capacity cDNA reversetranscription kit; Applied Biosystems) and quantified byRT-PCR using SYBR green (Applied Biosystems) detec-tion. Results were normalized to the housekeeping geneb-2-microglobulin for mRNA and expressed as arbitraryunits of 2�DDCt relative to the control group. X-box bindingprotein-1 was amplified with Taq polymerase and pro-ducts were resolved on 2% Tris-acetate-EDTA agarosegels and imaged on AlphaImager HP. SupplementaryTable S1 contains the primer sequences used for the qPCRstudies.

Immunoblot analysesProtein extracts (20 mg)were separated on 7.5% or 4% to

20% SDS-PAGE gels and transferred to polyvinylidenedifluoride (General Electric) as described (20). All anti-bodieswere fromCell Signaling Technology (Supplemen-tary Table S2). Band signals were visualized with ECLsubstrate (Pierce). Cell lysates were run on different blotsto avoid stripping and reprobing except for phospho-antibody blots.

Glucose uptakeBasal glucose uptakes were determined as previously

reported for human cells in vitro (20). Briefly, cells (105/well) in 6-well plateswere incubatedwith 2-deoxyglucose(0.5mmol/L; Sigma-Aldrich) and [1,2-3H]2-deoxy-D-glu-cose (GE). Counts were converted to moles of glucosetaken-up and normalized to the protein concentration ofthe lysates.

Lipid oxidationCells were plated in 12-well plates and grown to 70%

confluence in their respective growth media conditionsand with 150 mmol/L etomoxir at the indicated times. Atthe time of the assay, 1 mmol/L carnitine, 100 mmol/LBSA-conjugated fatty acids (Sigma) and C14-labeled fattyacids (1 uCi/mL; PerkinElmer), and fresh medium wereadded to the cells for 3 hours. Entrapment of the generated14CO2 was done by injecting perchloric acid as described(21). Radioactivity was measured by scintillation (Beck-man) and normalized to protein.

CPT1A shRNA transductionsTRCN0000036279 (CPTsh1) and TRCN0000036281

(CPTsh2) CPT1A shRNAs and the nontargeting controlSHC002were purchased fromFunctional Genomics Core.Lentiviral transduction and selection were performedaccording to Sigma’s MISSION protocol but using lenti-viral packing plasmids pCMV-R8.74psPAX2 andVSV-G/pMD2.G and transfection reagent TransIT-LT1 (Mirius).

Lipid fractionation and analysis by gaschromatography coupled to mass spectrometry

LNCaP cells were grown in 60-mm plates and treatedwith etomoxir or vehicle for 24 hours. Incubation was

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Mol Cancer Ther; 13(10) October 2014 Molecular Cancer Therapeutics2362

stopped by adding 1 volume of methanol. To an aliquotrepresenting 25% of the total sample, amixture of internalstandards was added: 20 mL of a solution containing 0.5nmol each of 12:0-ceramide, 12:0-sphingomyelin, glucosyl(b)-C12-ceramide and Lactosyl(b)-C12-ceramide (Cer/SphMixture I;Avanti Polar Lipids).After extracting lipidsusing Bligh & Dyer method (22), sphingolipids wereanalyzed by liquid chromatography/tandem mass spec-trometry essentially as described (23).Datawere analyzedusing MultiQuant software from AB Sciex, and are pre-sented as the ratios between the integrated area of theintensity peak of each analyte and the intensity peak of thecorresponding internal standard.

Xenograft production in nude miceMale athymic nude mice, 4- to 6-week-old were pur-

chased fromHarlan Laboratories. Tumor xenografts weregenerated by injecting human VCaP cells in the flank ofnude male mice as described (24). Approximately 2� 106

cells were used for each injection. When tumors werepalpable the mice were randomized into two groups:vehicle or etomoxir. Treatment was carried out withintraperitoneal injection of etomoxir (40 mg/kg) or vehi-cle (water) every other day for 3 weeks. All procedureswere carried out under a protocol approved by theInstitutional Animal Care and Use Committee of theUniversity of Colorado. After treatment, xenografts werecollected and processed for IHC studies using standardprotocols at the UC Denver Pathology core.

Statistical analysisANOVA tests were used to compare between groups

followed by posthoc Tukey tests when appropriate.Analysis of in vivo tumor growth was done with ANOVAfollowed by t tests with SPSS v20 software. All tests weretwo sided. P < 0.05 was considered significant. Datarepresent mean � SD except for qPCR that is mean �SEM. Synergism was analyzed using CalcuSyn 2.0 (Bio-soft) as described (25).

ResultsLipid metabolic inhibitors reduce the viability ofprostate cancer cell linesBenign (BPH-1, epithelial and WPMY-1, stroma) and

prostate cancer (VCaP, LNCaP, and PC3) cell lines weretreated with etomoxir (75 mmol/L) for 48 hours andsubjected to viability analysis using Trypan blueexclusion. Figure 1A shows that prostate cancer cells havedecreased viability in response to etomoxir when com-pared with normal BPH-1 (epithelial) andWPMY-1 (stro-ma) prostate-derived cell lines. VCaP cells showed thehighest sensitivity to etomoxir treatment, with a 60%reduction in viability (P < 0.01), followed by LNCaP(50% reduction, P < 0.01) and PC3 (40% reduction, P <0.01).Because LNCaP cells were sensitive to etomoxir and

they are also known to be sensitive to orlistat (7), we usedetomoxir (75 mmol/L) and orlistat (20 mmol/L) to study

the viability and proliferation of LNCaP and VCaP cellsexposed to both inhibitors. Treatments were done in FBSand androgen-deprived CSS conditions. Figure 1B and Cshow the effects of the drug combination on cell viabilityfor LNCaP and VCaP cells, respectively, indicating astrong effect (P < 0.001) of both inhibitors compared withcontrol.

Figure 1D and E show the proliferation of LNCaP cellstreated with drugs in the presence of FBS or CSS media,respectively. A dose-dependent effect and a strong inhi-bition of proliferation were observed with the combina-tion of drugs (P < 0.001 comparedwith either drug alone).The combinatorial index (CI) for the drugs is indicated atthe bottom of figures. A number less than 1 indicatessynergism between both drugs, whereas 1 or greaterreflects additive or antagonistic effects, respectively.These results indicate that lower doses were needed toobtain a synergistic effect on proliferation. CSS mediaincreased the sensitivity of LNCaP cells to the drugs,especially etomoxir. Figure 1F and G shows the sameparadigm for VCaP cells. Treatments in the presence ofCSS media resulted in increased sensitivity to the combi-nation of drugs compared with treatments with FBS-containing media. This was reflected in the strong syner-gistic CI scores.

We also examined the effect of etomoxir and orlistaton the proliferation of patient-derived primary humanprostate epithelial cells. Purity of the epithelial cultureswas assessed by E-cadherin and vimentin expression(Supplementary Fig. S1). Figure 1H and I shows the effectof inhibitors on benign and cancer primary cells, respec-tively. Increasing drug concentrations decreased prolif-eration (P < 0.001) for both cell types. However, the cancercells were more sensitive to each drug than the benigncells at the lower drug concentrations, andmore sensitiveto the combination of drugs at the higher concentrations(P < 0.05). Strong synergism was observed in the cancercell lines (CI ¼ 0.5) compared with the benign cells(CI ¼ 0.75), suggesting a higher sensitivity of the cancercells to the inhibitors, especially etomoxir. Additionalpatient-derived primary prostate cell lines are shown inSupplementary Fig. S2.

Etomoxir and orlistat decrease AR isoformexpression and modify lipid oxidation and glucoseuptake

Because AR activity is associated with lipid metabo-lism, we examined the expression ofARmRNA aswell asits downstream targets PSA andNKX3.1. Figure 2A and Bshows a significant decrease of transcripts in the presenceof inhibitors, regardless of the presence of androgens (P�0.05). Similar results were obtained for VCaP cells but to alesser extent (Fig. 2C and D). Examination of the effect ofinhibitors on lipid oxidation in prostate cancer and BPH-1cells was done by trapping the CO2 produced by the cellsafter treatment. Figure 2E shows increased lipid oxidationin prostate cancer cells compared with the benign BPH-1cells (�5-fold, P < 0.01). Orlistat incubation did not affect

Lipid Catabolism Blockade Effects in Prostate Cancer

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oxidation rate significantly, except for VCaP cells. How-ever, addition of etomoxir decreased the oxidation rate by50% in LNCaP and VCaP cells (P � 0.05). These resultsdemonstrate that prostate cancer cells are lipolytic, andsuggest that their survival strongly depends onAR actionand lipid utilization.Because etomoxir is known to increase glucose uptake

in heart cells (26), we also examined the effect of inhibitorson glucose uptake (Fig. 2F). Interestingly, the drug com-bination produced a significant increase in glucose uptakein all the cells but was greater in BPH-1 cells, suggesting

that different metabolic pathways operate in benign andcancer cells.

Lipid catabolism blockade results in decreasedmTOR signaling and increased apoptosis

To study the molecular mechanisms of etomoxir andorlistat on prostate cancer cells, we examined the phos-phorylation status of the proapoptotic BAD protein,which has been shown to be associated with the met-abolic status of the cell and is necessary to protectprostate cancer cells from apoptosis, likely mediated

Figure 2. Etomoxir and orlistatdecrease AR isoform expressionand modify lipid oxidation andglucose uptake. Expression of full-length AR (ARfl), variant 7 (ARv7),total AR,PSA, andNKX3.1genes inLNCaP (A and B) and VCaP (C andD) cells treated with etomoxir (75mmol/L) and/or orlistat (20 mmol/L).Posthoc tests compared withvehicle: A, �, P � 0.004; B, �, P �0.05; C, �, P � 0.03; and D, �, P �0.05. E, rate of 14C-palmitateoxidation in LNCaP (ANOVA, P ¼0.008), VCaP (ANOVA P ¼ 0.02),and BPH-1 cells exposed toinhibitors for 6 hours. �, P < 0.01; ^,P� 0.02 compared with vehicle. $,P < 0.02 prostate cancer cellscompared with BPH-1. F, glucoseuptake of LNCaP (ANOVA, P <0.0001), VCaP (ANOVA P < 0.001),and BPH-1(ANOVA, P ¼ 0.002)cells exposed to inhibitors for6 hours. Posthoc tests:comparisons with vehicletreatment: #, P < 0.05; �, P < 0.05;^, P � 0.007. VCaP and BPH-1comparedwith LNCaP treatedwithetomoxir, a, P < 0.05.

Figure 1. Lipidmetabolic inhibitors reduce the viability of prostate cancer cell lines. A, relative cell viability of prostate-derived cell lines exposed to etomoxir (75mmol/L) for 48hours, �,P<0.001 comparedwith vehicle. B, viability of LNCaPcells exposed to inhibitors etomoxir (75mmol/L), orlistat (20mmol/L): ^,P�0.001,compared with vehicle, #, P � 0.016, compared with single drug. C, viability of VCaP cells: �, P � 0.001, compared with vehicle, #, P � 0.001,comparedwith single drug. D,MTSproliferation assay of LNCaP cells in FBSmedia. �,P < 0.02; ��,P¼ 0.001 combination versus single drug. E, CSSmedia �,P < 0.001 combination versus single drug. F, MTS assay of VCaP cells in FBS: �, P < 0.001 combination versus single drug. G, CSS media �, P � 0.003;^, P ¼ 0.026 combination versus single drug. H and I, MTS assay of patient-matched prostate-derived benign (H) and cancer (I) cells exposed to inhibitorsfor 48 hours. Two-tailed t tests: a, P < 0.01 compared with orlistat treatment in benign cells; b, P < 0.05 compared with etomoxir treatment in benign cells;c, P < 0.05 compared with combinatorial treatment in benign cells. Combinatorial index is shown at bottom of the graph, where CI < 1.0 indicates synergy.



by AKT activation (27). Figure 3A and B shows blots ofetomoxir and/or orlistat-treated LNCaP and VCaPlysates, respectively. Decreased BAD S112 phosphory-lation was observed in both cells lines with etomoxirtreatment at 6 hours.

mTOR and AMPK are also involved in nutrientsensing and integrate fuel homeostasis and cell surviv-al. Etomoxir treatment resulted in decreased activationof mTORC1 and its downstream substrates S6Kand 4EBP1, which are involved in protein synthesisand survival, especially the S6K-BAD signaling axis(ref. 28; Fig. 3C). Interestingly, less caspase-3 activationwas observed in the BPH-1 cells (SupplementaryFig. S3). In addition, strong suppression of ACC, anenzyme involved in fat synthesis, was also observedwith treatment, indicating AMPK activation. Figure 3Dshows AMPK activation after 16 hours of treatment inLNCaP cells. Figure 3E shows a putative diagram of

these molecular players. Figure 3F and G shows blotsfor VCaP lysates. Decreased mTOR signaling axis andincreased caspase-3 activation was observed for thedrug combination. Orlistat also increased caspase-3,suggesting increased sensitivity to orlistat in VCaPcells.

ERstress andapoptotic ceramides are increased afterlipid metabolism blockade in LNCaP cells

Because mTOR inactivation is associated with survivalsignals like endoplasmic reticulum (ER) stress and autop-hagy,we examined the expression of canonicalmarkers inLNCaP and VCaP lysates. Figure 4A shows activation ofthe ER stress transcription factor XBP-1 by splicing inresponse to etomoxir. Interestingly, addition of palmitateto the treatments resulted in less XBP-1 splicing, under-scoring the role of lipogenesis/lipolysis cycle in cellhomeostasis (Supplementary Fig. S4). ER stress-related

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Figure 3. Lipid catabolism blockade results in decreasedmTOR signaling and increased apoptosis. AKT and BAD phosphorylation of LNCaP (A) and VCaP (B)lysates treatedwith etomoxir (75 mmol/L) and/or orlistat (20 mmol/L) for 6 hours. C, expression ofmTOR-S6K-BAD-Caspase-3 axis aftermetabolic treatmentsfor 16 hours in LNCaP cells. D, blot of AMPK activation and ACC2 inactivation of LNCaP lysates E, diagram ofmolecular pathway likely involved in the LNCaPcells. F, expression of mTOR-S6K-BAD-Caspase-3 axis after 16-hour treatments in VCaP cells. G, AMPK and phospho-ACC2 in VCaP lysates.

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factors ATF4, CHOP, GADD34, and GRP78 were alsoincreased after etomoxir treatment (Fig. 4B). The mostdramatic changes were observed in the expression ofCHOP (C/EBP homologous protein) and GADD34(growth arrest and DNA damage 34) expression, both ofwhich have been associated with the proapoptotic side ofthe ER stress response (29). No changes in the mRNAexpression of ER stress-related factors were observed inVCaP cells (not shown).Because ER stress also leads to a block in protein

translation, we examined phospho-eIF2a in the sameLNCaP samples that had XBP1 activation (Fig. 4C). Aslight increase in p-eIF2a was observed with orlistat asshown before (30), but stronger signals were observed inthe etomoxir-treated samples. The weaker p-eIF2a signalin the combination treatmentwas parallel to the increasedexpression of the GADD34 phosphatase regulator (Fig.

4B), potentially leading to suppression of the unfoldedprotein response and induction of apoptosis (31). Finally,becauseunresolvedERstress activates autophagy (32),weexamined the conversion of LC3-II (17 KDa), which wasevident in the etomoxir-treated samples (Fig. 4C). VCaPcells did not show increased phospho-eIF2a with eto-moxir, but changes in autophagy were noticeable withorlistat (Fig. 4D).

Because ceramides containing 16- and 18-carbon fattyacids are also associated with decreased mTOR activityand autophagy (33), we examined the levels of differentceramide species present in etomoxir-treated LNCaP cellsafter 24 hours. Figure 4E shows a significant increase inceramide species containing palmitic (16:0) and stearic(18:0) acyl chains. Interestingly, 16C and 18C containing-ceramides seem to be most important for intrinsic apo-ptosis induction (34).

Figure 4. ER stress and apoptoticceramides are increased after lipidmetabolism blockade in LNCaPcells. A, orlistat and etomoxirtreatments induce strong activationof the XBP-1 transcription factorafter 16 hours of treatment. B,relative qPCR analysis of the ERstress-related factors after16 hours. For each gene examined,ANOVA < 0.001 across treatments.Posthoc tests: �, P < 0.01; and a,P ¼ 0.002 compared with vehicle.C and D, Western blot analyses forphospho-eIF2a and LC3 fragmentsof LNCaP and VCaP lysates,respectively, treated with inhibitors.Total eIF2a bands were used asloading controls. E, ceramidespecies in LNCaPcellswere treatedwith etomoxir for 24 hours andharvested for lipid extraction andceramide analysis. Two-sidedt test: �, P � 0.05. Fatty acidcomposition of the ceramidemolecules are indicated in thex-axis.



Downregulation of CPT1A decreases fat oxidationand leads to apoptosis

To verify the role of CPT1A as the target of etomoxiraction, we used two different shRNAs to knock down(KD) CPT1A expression in LNCaP cells. Control cellswere transduced with a nontargeting shRNA construct.Clones were treated with vehicle (V, H2O/DMSO), orli-stat (O, 20 mmol/L), orlistat/etomoxir (OE, 20 mmol/L/75mmol/L) or etomoxir alone (E, 75 mmol/L) for 24hours. Figure 5A shows thedecrease inCPT1Aexpressionin theKD clones comparedwith control cells (V lanes). Anunexpected increase in CPT1A expression with etomoxirwas observed in all clones, suggesting a compensatoryfeedback effect. A lack of S112 and S155 phosphorylationof BAD was observed in the combinatorial treatment,suggesting an activation of BAD proapoptotic activity(27, 28). Decreases in pAKT and mTOR action (via pS6K)were also observed, concomitant with cleaved caspase-3signal, suggesting apoptosis induction. VCaP cells werenot viable after CPT1AKD selection but they also showeda slight increase of CPT1A protein expression with eto-moxir (Fig. 5B).

Analysis of cell viability and sensitivity of clones toetomoxir was also examined (Fig. 5C). CPT1A KD clones

were sensitive to 50 mmol/L etomoxir (reduced by 60%,P < 0.001) while the control cells showed a 20% decreasein viability. This effect was dose dependent as the 75 and100 mmol/L doses decreased viability in all the clones.Because the total AKT expressionwas strongly reduced inthe CPT1A KD clones with the etomoxir treatments, thissuggests a lackof compensatory survivalpathway leadingto decreased viability. Concomitant with decreasedCPT1A expression, we also observed lower palmitateoxidation in the KD clones (Fig. 5D). Clone CPTsh1showed a stronger decrease in fat oxidation (60%decrease, P < 0.01) followed by CPTsh2 (25% decrease,P ¼ 0.01).

Systemic treatment with etomoxir decreasesxenograft tumor growth in nude mice

Male nude mice were grafted with VCaP cancer cellssubcutaneously, randomized to four groups (two vehiclesand two treatment doses)when the tumorswere palpable,and treated with etomoxir systemically for 21 days. VCaPcells were used instead of LNCaP because they growwellas xenografts in nude mice (35) and are also sensitive toetomoxir (Fig. 1A). To account for possible toxicity, weused twodifferent doses of etomoxir. Figure 6A shows the

Figure 5. Downregulation of CPT1Adecreases fat oxidation and leadsto apoptosis. A, Western blotanalysesofCPT1AKDcells treatedwith vehicle (V), orlistat (O),orlistatþetomoxir (OE), or etomoxiralone (E) for 24 hours. B, CPT1Aexpression in VCaP cells treatedwith inhibitors. C, Trypan blueviability assay of shRNA clonestreated with three doses ofetomoxir for 2 days; �, P < 0.01comparedwith control cells treatedwith vehicle. D, palmitate oxidationrate in KD clones compared withcontrol, �, P � 0.01 compared withcontrol shRNA clone.

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progressive growth of tumors using a 40 mg/kg doseevery other day. Significant differences were observedin the last week of treatment (ANOVA P < 0.001, posthocP < 0.05) suggesting that systemic inhibition of fat oxida-tion impairs growth of implanted tumors. Animals werehealthy and their body weight remained stable overthe 3 weeks of treatments (Fig. 6B). CPT1A content in thetumors was still strong after 21 days, albeit weaker inthe etomoxir group (Fig. 6C). Phospho-S6 (marker ofmTOR action) did not show any changes. The study using20mg/kg did not produce significant results over 3weeks(data not shown).

DiscussionThe results of our study point to an important role of

b-oxidation of fatty acids in prostate cancer. We haveobserved a significant decrease in viability when prostatecancer cells are grown in the presence of the CPT1 inhib-itor etomoxir, but this effect was not replicated in non-

tumor forming cells, suggesting a possible therapeuticwindow for prostate cancer. Furthermore, because lipo-genesis from sugar carbons is a well-documented obser-vation in prostate cancer (36), the combinatorial approachof inhibiting fat oxidation and fat synthesis simultaneous-ly, with etomoxir and orlistat, respectively, generatedsynergistic results in prostate cancer cell growth assays.Unfortunately, the main problem with targeting FASN isthe low solubility and bioavailability of currentlyapproved drugs (like orlistat), mainly due to the hydro-phobicity of the FASNactive site (37),making it difficult toadvance to clinical trials.

ARs are involved in the activation of lipid metabolism(38) as well as the growth of prostate cancer cells, even incastration-resistant or recurrent prostate cancer (39). Verylittle is known about how lipids regulate the AR and itsvariants in prostate cancer. Our results blocking lipidcatabolism and significantly decreasing AR expression inboth LNCaP and VCaP cells suggest that thwarting theability of prostate cancer cells to utilize lipids, regardlessof the PTEN status, may synergize with current antian-drogen therapies for a more effective AR blockade.Expression of PSA and NKX3.1 was also decreased, sug-gesting a reducedAR-signaling axis. Furthermore, benignBPH-1 cells showed higher glucose uptake than prostatecancer cells when treated with both etomoxir and orlistat,suggesting that theywere able to compensate for the lipidblockade. This lack of metabolic flexibility in prostatecancer cells may contribute to their decreased viabilitywhen challengedwithmetabolic stress, opening doors forcombinatorial treatments to be explored clinically, likeetomoxir and enzalutamide.

The mammalian kinase mTOR is deregulated in nearly100% of advanced human prostate cancers. However,there are not clinically effective drugs that target mTORactivity (40). We have observed decreased mTOR activa-tionwhen cellswere challengedwithmetabolic inhibitors,leading to increased 4EBP1 inhibitor activity (less phos-phorylated) that likely reduces protein translation andgrowth (41). Interestingly, activation of caspase-3 wasdifferent between LNCaP and VCaP cells. Etomoxir wasthe driver for apoptosis in LNCaP cells, whereas VCaPapoptosis seemed dependent on orlistat treatment. Thesecell line differences in response to the inhibitors may restin their genetic differences: LNCaP cells have activation ofsurvival AKT pathways, whereas VCaP likely rely onother pathways. The observation that pAKT wasincreased with orlistat in the CPT1A KD clones wasunexpected, but could reflect a compensatorymechanismto increase FASN activity, as was previously observed inprostate cancer (42).

The role of BAD proteins in sensing mitochondrialmetabolism is well described (43), linking glucose usewith apoptosis. Our studies provide evidence that lipiduse by prostate cancer cells is also connected to theapoptotic machinery because phospho-BAD S112 wasdecreased in both cell lines leading to caspase activationwith the drug combination. The possibility that the

Figure 6. Systemic treatment with etomoxir decreases xenograft tumorgrowth in nude mice. A, tumor growth progression (mean � SD) in micetreated with 40mg/kg Etomoxir injections for 3 weeks. (�,P < 0.05 Tukey,compared with vehicle-treated tumors). B, mouse body weight over thecourse of the experimental treatments. C, representative CPT1A andphospho-S6 stains of tumor xenografts at the end of study.



observeddecreasedmTOR-S6K axis is responsible for thiseffect needs to be further validated.

Additional consequences of blocking lipid turnoverin LNCaP cells are ER stress, autophagy, and ceramideproduction. It is unknown which phenomenon occursfirst but it is possible that accumulated palmitate in theER (that is not oxidized) leads to activation of theunfolded protein response and a survival autophagicresponse, an effect that has been previously reported inyeast (44) and more recently in leukemia cells (45),where phosphorylation of eIF2a by PERK leads to asurvival autophagy response. Ceramide synthesis inprostate cancer is also another potential target for ther-apeutic intervention. We have observed significantincreases in ceramide species in LNCaP cells treatedwith etomoxir, suggesting that the excess fatty acids(mainly C16:0 and C18:0) that could not get oxidized inthe mitochondria due to CPT1 inhibition, were used togenerate proapoptotic ceramides. Indeed, ceramidasesare becoming therapeutic targets for advanced prostatecancer because these degrading enzymes are abundantand contribute to chemoresistance (34).

Themost significant preclinical extension of ourwork isthe result from the prostate cancer xenografts inmice. Theeffect of fat oxidation inhibition in leukemia cells is welldocumented (13, 46), but there are no reports of its effectson prostate cancer cells, which depend on fat metabolismfor survival. Results from injections with etomoxirrevealed a dose-dependent effect on tumor growth with-out affecting the body weight or health of the mice. Theseresults emphasize the dependence of prostate cancer cellson fatty acid availability for oxidation and ATP produc-tion. Because the use of orlistat in our in vitro studiesfurther decreased the viability of the prostate cancer cells,this suggests that de novo lipogenesis and/or lipase activ-ity are likely the sources of fatty acids for b-oxidation inprostate cancer cells. The possibility of using 2-DG (non-usable glucose) and etomoxir in combination is a prom-ising therapeutic avenue for prostate cancer that needs tobe explored.

Several studies have indicated that fat availability totumors (via high-fat diets or obesity) leads to prostate

cancer growth (47, 48). However, lipidmarkers like FASNand insulin-like growth factor-I levels do not fully explainthe association between obesity and poor prostate canceroutcome (48), indicating that the availability of lipids totumors may be an important factor worth exploring indepth. This is relevant in the setting of androgen depri-vation therapy, where the metabolic syndrome withaltered blood lipid profile favors increased fatty acidavailability to the growing prostate cancer tumors. Inaddition, our data suggest that lipid catabolism alsomodulates AR content, likely creating a feed-forwardcycle that sustains prostate cancer growth. In conclusion,systemically targeting lipid use by tumors offers possi-bilities to reduce prostate cancer tumor burden.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: I.R. Schlaepfer, R.H. Eckel, S.D. CramerDevelopment of methodology: R.H. Eckel, S.D. CramerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): I.R. Schlaepfer, L. Rider, L.U. Rodrigues,M.A. Gij�on, C.T. Pac, A. Cimic, S.J. Sirintrapun, S.D. CramerAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): I.R. Schlaepfer, L.U. Rodrigues, M.A. Gij�on,R.H. Eckel, S.D. CramerWriting, review, and/or revision of the manuscript: I.R. Schlaepfer,L. Rider, L.U. Rodrigues, M.A. Gij�on, A. Cimic, S.D. CramerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): C.T. Pac, L. Romero, S.D. CramerStudy supervision: I.R. Schlaepfer, L.M. Glod�e, S.D. Cramer

Grant SupportThis work was supported by NIH (K01CA168934; to I.R. Schlaepfer),

ACS (PF-117219 and IRG-57-001-50; to I.R. Schlaepfer), contributions fromHerbert Crane Endowment, William R. Meyn Foundation, and RobertRifkin Endowment (to L.M. Glod�e), and the Region 4 TransdisciplinaryGeographic Management Program (GMaP) program CA153511 (to I.R.Schlaepfer).

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.

Received March 4, 2014; revised July 11, 2014; accepted July 26, 2014;published OnlineFirst August 13, 2014.

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