Lipoprotein Lipase Links Dietary Fat to Solid Tumor Cell Proliferation · over, blocking de novo...

Therapeutic Discovery

Lipoprotein Lipase Links Dietary Fat to Solid TumorCell Proliferation

Nancy B. Kuemmerle1,2, Evelien Rysman3, Portia S. Lombardo2,4, Alison J. Flanagan2,4, Brea C. Lipe1,2,Wendy A. Wells2,5, Jason R. Pettus5, Heather M. Froehlich5, Vincent A. Memoli2,5, Peter M. Morganelli2,6,Johannes V. Swinnen3, Luika A. Timmerman7, Leila Chaychi4, Catherine J. Fricano2,4, Burton L. Eisenberg2,8,William B. Coleman9, and William B. Kinlaw2,4

AbstractMany types of cancer cells require a supply of fatty acids (FA) for growth and survival, and interrupting de

novo FA synthesis in model systems causes potent anticancer effects. We hypothesized that, in addition to

synthesis, cancer cells may obtain preformed, diet-derived FA by uptake from the bloodstream. This would

require hydrolytic release of FA from triglyceride in circulating lipoprotein particles by the secreted enzyme

lipoprotein lipase (LPL), and the expression of CD36, the channel for cellular FA uptake. We find that selected

breast cancer and sarcoma cells express and secrete active LPL, and all express CD36. We further show that

LPL, in the presence of triglyceride-rich lipoproteins, accelerates the growth of these cells. Providing LPL to

prostate cancer cells, which express low levels of the enzyme, did not augment growth, but did prevent the

cytotoxic effect of FA synthesis inhibition. Moreover, LPL knockdown inhibited HeLa cell growth. In contrast

to the cell lines, immunohistochemical analysis confirmed the presence of LPL and CD36 in the majority of

breast, liposarcoma, and prostate tumor tissues examined (n¼ 181). These findings suggest that, in addition to

de novo lipogenesis, cancer cells can use LPL and CD36 to acquire FA from the circulation by lipolysis, and this

can fuel their growth. Interfering with dietary fat intake, lipolysis, and/or FA uptake will be necessary to

target the requirement of cancer cells for FA. Mol Cancer Ther; 10(3); 427–36. �2011 AACR.

Introduction

Many tumors, including those arising in breast, colon,ovary, and prostate, exhibit a lipogenic phenotype. Thisfeatures brisk rates of saturated long-chain fatty acid (FA)synthesis driven by enhanced expression of genes coding

for the 3 enzymes required to produce palmitic acid fromcytosolic citrate [ATP citrate-lyase, acetyl CoA-carboxy-lase, and fatty acid synthase (FASN)]. Importantly, lipo-genic tumor cell growth is slowed in vitro and survival isreduced by FA synthesis inhibitors, whereas nontrans-formed cells are unaffected (reviewed in refs. 1, 2). More-over, blocking de novo lipogenesis with FASN inhibitorsin vivo exerts potent antitumor effects in rodent models ofbreast (3) and prostate (4) cancer. These observations,coupled with the low rates of FA synthesis in mostnormal human tissues (5), have spurred efforts to developanticancer therapies based on inhibiting lipogenicenzyme activities or silencing the corresponding genes.

Attempts to exploit the metabolic requirements oflipogenic cancers have thus far focused solely on disrupt-ing de novo FA synthesis. Cytotoxicity following inhibi-tion of lipid synthesis, however, may be obviated by theprovision of exogenous FA (6–8). This observation, andthe improved outcome of breast cancer patients ingestinga low fat diet (9), led us to hypothesize that triglyceride incirculating lipoprotein particles could provide an addi-tional, exogenous source of FA for tumors. This wouldrequire triglyceride-rich chylomicrons or very low den-sity lipoproteins (VLDL) as substrate, extracellular lipo-protein lipase (LPL) for hydrolysis, and FA translocase(CD36) for cellular uptake of the free FA (reviewed in ref.10). As LPL is a secreted enzyme that is bound to theluminal surface of capillary endothelial cells, it could

Authors' Affiliations: 1Section of Hematology and Oncology, Departmentof Medicine, Dartmouth-Hitchcock Medical Center, and Dartmouth Med-ical School; 2Norris Cotton Cancer Center, Dartmouth-Hitchcock MedicalCenter, Lebanon, New Hampshire; 3Laboratory for Experimental Medicineand Endocrinology, Katholieke Universiteit Leuven, Leuven, Belgium;4Section of Endocrinology and Metabolism, Department of Medicine,and 5Department of Pathology, Dartmouth-Hitchcock Medical Center,and Dartmouth Medical School; 6Department of Immunology and Micro-biology, V. A. Medical Center, White River Junction, Vermont and Dart-mouth Medical School, Lebanon, New Hampshire; 7Cancer ResearchInstitute, UCSF/Helen Diller Comprehensive Cancer Center, Universityof California at San Francisco – San Francisco, California; 8Departmentof Surgery, Dartmouth Medical School, Lebanon, New Hampshire; and9Department of Pathology and Laboratory Medicine, Lineberger Compre-hensive Cancer Center, University of North Carolina School of Medicine,Chapel Hill, North Carolina

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

N.B. Kuemmerle and E. Rysman contributed equally to this work.

Corresponding Author: William B. Kinlaw, 606 Rubin Building, Dart-mouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon,NH 03756. Phone: 603-653-9961; Fax: 603-653-9952. Email: [email protected]

doi: 10.1158/1535-7163.MCT-10-0802

�2011 American Association for Cancer Research.

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potentially be supplied by tumor cells or by nonmalig-nant cells in the tumor microenvironment.

Materials and Methods

cDNA microarray analysisProduction of the expression dataset has been pre-

viously described in detail, as have culture conditionsfor cell lines (ref. 11; http://cancer.lbl.gov/breastcancer/data.php). RNA from 45 human breast cancer cell lines(ICBP45) grown at subconfluence was harvested, reversetranscribed, and hybridized to Affymetrix U133A genechips. Resulting Affymetrix image files were normalized(RMA; ref. 12). Unsupervised average linkage clusteranalysis of log2 signal intensities was done using approxi-mately 14,000 probeset IDs of highest variance, using theCluster Software package, and the resulting dendrogramimage produced with Treeview (ref. 13; http://rana.lbl.gov/eisen/?page_id¼7). Probeset IDs identifying CD36,LPL, and FASN were identified, median centered, nor-malized, and a heat map produced indicating the relativehybridization intensity for each sample.

RT-PCRRNA was isolated by the RNeasy Mini Kit (Quiagen).

One microgram RNAwas reverse transcribed by randomhexamer primers with M-MULV reverse transcription(New England Biolabs). PCR was done as described(14). Primers used are described in SupplementaryTable S1. Different primers were used for real-time RT-PCR.

Quantitative real-time RT-PCRRNA was prepared by the PureLink Total RNA pur-

ification system (Invitrogen). The purity and concentra-tion of RNA were assessed by a NanoDrop DM-1000spectrophotometer (NanoDrop Technologies). RNA wasconverted to cDNA by Superscript II RT and randomhexamer primers, according to the manufacturer’s pro-tocol (Invitrogen). Primer sequences for LPL were 50-TATCCGCGTGATTGCAGAGA-30 (forward) and 50-GCCTTACTTGGATTTTCTTCATTCA-30 (reverse). SYBRgreen was used for detection and 18S rRNA was used asan internal control. Primer sequences for 18S were 50-CGCCGCTAGAGGTGAAATTC-30 (forward) and 50-TTGGCAAATGCTTTCGCTC-30 (reverse). PCR was inthe 7500 Fast Real-Time PCR System (Applied Biosys-tems). The program used included 2 minutes at 50�C,1 minute at 95�C, and 40 cycles of 3 seconds at 95�C and30 seconds at 60�C. The average of the Ct values foreach triplicate reaction was expressed relative to theamount of 18S rRNA in the sample.

Tissue cultureLiSa-2 liposarcoma cells were from Martin Wabitsch

(University of Ulm, Germany) and we confirmed theiridentity by the ability to produce lipid droplets thatstained with oil red-O on confluence. All other lines

were from the American Type Culture Collectionexcept VCaP, which was from ECACC, and these lineswere acquired recently and were of low passage num-ber (<10). Cells were grown in DMEM:F12 supplemen-ted with 10% fetal calf serum (FCS; Atlanta Biologicals),1% penicillin/streptomycin, and 2 mmol/L L-gluta-mine, in 5% CO2 at 37�C. Cell growth was monitoredin an MTT assay (15). Lipoprotein-deficient FCS wasprepared by the method of Goldstein and coworkers(16).

Knockdown of LPLHeLa cells were transfected with 10 nmol/L siRNA

targeting LPL (siRNA A: 50-GGUAGAUAUUGGAGAA-CUA; siRNA B: 50-GGAUGGAGGAGGAGUUUAA;Dharmacon) with the use of Lipofectamine RNAiMAX(Invitrogen). A siRNA targeting luciferase (50-CGUACGCGGAAUACUUCGA) was used as control.RNA was harvested 48 hours later, and cell viabilitywas assessed 96 hours after transfection.

Fluorescent labeling of VLDL particlesDialyzed VLDL (0.5 mg; Kalen Biomedical) were incu-

bated at 37�C for 15 hours with 25 mL diI D282 (Invitro-gen; 3mg/mL in dimethyl sulfoxide). Free dye wasremoved by dialysis against PBS. Prior to observationby confocal microscopy, 25 mL labeled VLDLs wereadded to slide chambers (Tissue Tek #177402) containing106 cells grown for 3 days in delipidated Dulbecco’sModified Eagle’s Medium. Excitation was at 549 nm,and fluorescence was detected at 565 nm.

Production of anti-human LPL antibodiesMice were immunized with a peptide (Sigma) repre-

senting human LPL residues 21–36 (CASRGG-VAAAQRRDFID) coupled to keyhole limpethemocyanin. After fusion of splenocytes to mouse multi-ple myeloma cells, media from candidate clones werescreened for reactivity to bacterially expressed LPL in anenzyme-linked immunosorbent assay. Positive cloneswere further screened by Western blot of skeletal musclefrom transgenic mice expressing a human muscle-speci-fic LPL transgene (MCK-LPL, kindly supplied by IraGoldberg, Columbia School of Medicine, New York,NY) and against human breast milk. Mouse tissues werehomogenized in immunoprecipitation assay buffer con-taining 10 mg/mL phenylmethylsulfonylfluoride. Sam-ples were centrifuged at 10,000 � g for 10 minutes � 2.Protein content was determined by the BCA assay(Pierce). Samples were boiled in 2X sample buffer andfractionated through 15% acrylamide. Following transferto polyvinylidene difluoride (PVDF) membranes (Immo-bilon–FL; Millipore), blocking was with SuperBlock(Pierce). Incubation with 1:200 dilution of the primaryantibody in TBS-Tween was overnight at 4�C, followedby 2 TBS washes. Recombinant protein A/G conjugatedwith horseradish peroxidase (Pierce) was applied fordetection at 1:5,000 in TBS-Tween for 1 hour at RT. After

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4 TBS washes, membranes were developed with NBT-BCIP (Pierce).

Bacterial expression of human LPLThe 2.3 kb EcoRI–HindIII fragment coding LPL was

excised from pCMV-SPORT6-LPL (Open Biosystems)and inserted into the pProEx-HTa His-tag vector. Mostof the fusion protein could not be solubilized, but thoserecovered showed reactivity with the anti-His4 antibody(Invitrogen). For immunodot assays 10 ng protein fromcleared lysates of Escherichia coli DH5a transformed withempty or LPL plasmid were spotted onto PVDF mem-branes, blocked, and incubated with antibody asdescribed earlier in the text.

Affinity isolation of LPLHuman milk and conditioned cell culture media were

fractionated over heparin sepharose (Sigma) by a proce-dure modified from Hata and colleagues (17).

LPL activityWe used the radiochemical assay of Nilsson-Ehle and

Schotz (18) or a colorimetric assay based on determina-tion of glycerol production (BioVision). We used aprotocol based on that of Cruz and colleagues (19) fordetermination of heparin-releasable LPL. Briefly, 5 � 106

cells from 75 cm2 flasks were cultured for 72 hours, andscraped pellets were washed 3 times in PBS with orwithout 100 U/mL heparin. Media and lysed cell pelletswere assayed in triplicate for residual LPL activity.

ImmunohistochemistryImmunohistochemistry was done as previously

described (20). Anti-LPL monoclonal antibody clone 43was used at a dilution of 1:10, with Citra Plus antigenretrieval (Biogenix). CD36 was assessed by an affinity-purified rabbit polyclonal antibody (Thermo Scientific)according to the supplier’s protocol. The InstitutionalReview Board (IRB)-approved the use of breast cancertissue and the tissue microarray containing 147 primarybreast cancers from postmenopausal women, diagnosedbetween 2000 and 2007 at Dartmouth-Hitchcock MedicalCenter, Lebanon, NH. Each case was represented by onetissue core 1.0 mm in diameter. The liposarcoma tissuemicroarray, also IRB approved, contained 26 liposarco-mas diagnosed between 1995 and 2008 at Dartmouth-Hitchcock Medical Center. Each case was representedby two to four 1.0-mm tissue cores. Prostate cancerspecimens were acquired at the Katholieke UniversiteitLeuven, Belgium, with IRB approval.

Results

We used a cDNA microarray to screen 45 breast can-cer–derived cell lines from the dataset of Neve andcolleagues (11) for LPL gene expression, and for FASNmRNA as amarker for de novo FA synthesis. We analyzedcell lines because breast tumor samples may contain

adipocytes, which express high levels of LPL and FASN.We also sorted the breast cancer lines by their global geneexpression signatures (21). These signatures include theluminal type (estrogen receptor–positive; ERþ), the basal,or triple-negative type that lacks receptors for estrogen,progestin, and trastuzumab (22), and the typewithHer2/neu amplification. Only 6 breast cancer cell lines(HCC2157, HCC1008, HCC1599, Du4475, SUM149, andSUM190) expressed high levels of LPL mRNA, and eachof these exhibited the aggressive basal gene expressionsignature (Supplementary Fig. S1). Expression of LPLmRNA by selected cell lines was verified by RT-PCR(Fig. 1A), as was expression of CD36 mRNA (Fig. 1B).LiSa-2 liposarcoma cells, which we previously showed to

Figure 1. LPL, CD36, and FASN gene expression in cancer cells. A–C,ethidium-stained gel electrophoresis of RT-PCR products. Cell linesanalyzed are listed above each lane. stds, electrophoretic size standards;LiSa-2, liposarcoma line; Du4475, breast cancer cells lacking receptors forsex steroids and trastuzumab; T47D, breast cancer cells with receptors forestrogen and progesterone, but not trastuzumab; BT474, breast cancercells with receptors for sex steroids and trastuzumab; PC3, LNCaP, andVCaP, prostate cancer lines; fibro, human fibroblasts. A, primerscorresponded to cyclophilin (cyc) or LPLmRNAs. B, primers correspondedto the FA translocase CD36. C, primers detected FASNmRNA. D, real-timeRT-PCR quantitation of LPL mRNA (normalized to 18S rRNA, mean� SEM, n¼ 3 wells/cell line). HeLa adenocarcinoma cells are included as apositive control, as we previously reported expression of LPL mRNA bythis cell line (25).

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exhibit the lipogenic phenotype (23), also expressed LPLand CD36, as expected for a tumor cell derived from anadipocytic lineage. All of the cell types expressed sub-stantial FASN mRNA (Fig. 1C), and in the breast cancercell lines this did not vary among the gene expressionsignatures (Supplementary Fig. S1). Quantitative real-time RT-PCR of representative lines confirmed thatLiSa-2 liposarcoma and triple-negative Du4475 breastcancer cells expressed the highest levels of LPL mRNA(Fig. 1D). In contrast, prostate cancer cells, which arehighly lipogenic (24), expressed relatively low levels ofLPL mRNA, and ERþ T47D and BT474 breast cancer cellsexpressed essentially none.

We examined conditioned tissue culture media for LPLenzyme activity, and it paralleled the levels of LPLmRNA (Fig. 2A). LPL activity accumulated over timein culturemedia of LiSa-2 liposarcoma andDu4475 breastcancer cells (Fig. 2B). In contrast, ERþ T47D, ERþ Her2/

neuþ BT474 breast cancer cells, and fibroblasts did notsecrete detectable lipase activity. Prostate cancer cellsproduced low levels of the enzyme. LPL activities inbreast milk and murine striated muscle were substan-tially greater than those observed in any of the condi-tioned (72 hours) media.

We found that available antibodies were not suffi-ciently specific to analyze LPL protein by immunohisto-chemistry. We therefore raised a mouse monoclonalantibody using a peptide representing residues 20 to36 of the human enzyme as antigen. This antibody ishighly specific (Supplementary Fig. S2), and permitteddetection of heparin sepharose–purified LPL from tissueculture media conditioned by Du4475 breast cancer andLiSa-2 liposarcoma cells (Fig. 2C, top). The band recog-nized by this antibody in Western analysis of milk wasverified to represent LPL by mass spectrometry. Wecould not detect LPL protein in media from ERþ breast

Figure 2. Production of LPL activity by breast cancer, liposarcoma, and prostate cancer cells and in a breast cancer tissue sample. A, lipase activity[mean � SEM, 4 samples/group, corrected for cellular protein content and normalized to the value observed in milk (9 � 103 cpm/2h)]. Human breastmilk (50 mL), mouse gastrocnemius muscle (50 mg protein, 45 � 103 cpm/2h), or tissue culture media conditioned by the indicated cell lines for 3 days wereassessed for lipase activity (mean � SEM, 4 samples/group, corrected for cellular protein content and activity observed in unconditioned media). The dottedline denotes the LPL activity found in unconditioned culture medium. B, time course of accumulation of lipase activity in conditioned culture media. Media(50 mL) were removed from cultures at the indicated intervals (mean cpm/mg protein� SEM, n¼ 4 wells/timepoint). C, top, identification of LPL in conditionedcell culture media. LPL was heparin-sepharose affinity purified from 10 mL fresh culture medium, 1.0 mL human breast milk, or 10 mL culture mediaconditioned (72 hours) by LiSa-2 liposarcoma or DU4475 triple-negative breast cancer cells, eluted with 0.6 to 0.8 mol/L NaCl, and analyzed by Western blotusing anti-human LPL clone 43 (1:200). The band from milk was verified to contain LPL by mass spectrometry. Bottom, Western analysis of a breast tumorhomogenate (50 mg protein without affinity purification) and breast milk (10 mL) for LPL. A band of the appropriate size is apparent in the tumor sample. D,estimation of the heparin-releasable LPL pool in breast cancer tissue and HeLa cells. Left, tumor associated LPL activity is significantly reduced by heparintreatment (P ¼ 0.0001). Right, heparin reduced LPL activity residing in HeLa cell pellets by 29% (P < 0.04). HR, heparin releasable.

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or prostate cancer cells. Western analysis of a clinicalbreast tumor homogenate (50 mg protein) without affinitypurification revealed a single band exhibiting the samemigration as that observed in milk (Fig. 2C, bottom).It seemed possible that expression of heparanase

could inactivate LPL, and thus could vitiate the meta-bolic relevance of LPL expression by tumors. Weassessed expression of the heparanase gene (HPSE) usingcDNAmicroarray data from 45 human breast cancer celllines. This showed that the cells generally express verylow levels of heparanase mRNA, as was the general casefor LPL mRNA. We were intrigued to note that thesubgroup of triple-negative cell lines exhibiting sub-stantial LPL expression also expressed the lowest levelsof heparanase mRNA. Indeed, linear regression of therelationship between LPL and heparanase mRNAs inlines with the basal A signature revealed a statisticallysignificant inverse correlation (P ¼ 1.27 � 10�5,, R2 ¼0.38). Thus, the coupling of high LPL with low hepar-anase expression seems to provide an advantage to thesubset of cells that produce substantial LPL. Our exam-ination of total and heparin-releasable LPL activity in afreshly prepared breast tumor homogenate also reflectson this question, as heparin-releasable activity wasreadily detectable, arguing against depletion of a cellsurface–bound LPL pool in breast tumors (see in thefollowing text).We carried out 2 experiments to determine whether

cancer-associated LPL is bound to tumor cells by non-covalent interactions with cell surface heparan sulfateproteoglycans, using a protocol based on that of Cruz andcolleagues (19). First, we homogenized freshly resectedinvasive breast cancer tissue shown to contain LPLimmunoreactivity (Fig. 2C, bottom), and extracted equalaliquots with buffer containing or not containing heparin.LPL activity in the control sample was 1,032 � 8 withoutheparin, 768 � 4 with heparin treatment (mean � SE,nmol/L glycerol produced/g tumor/h, measured in tri-plicate; P < 0.0001). This represented a heparin-releasablefraction of 26% of the total tumor-associated LPL activity(represented by the portion of the bar labeledHR, Fig. 2D,left).Second, we determined the heparin-releasable fraction

of LPL in HeLa cells, and calculated turnover rates forcellular LPL pools (Fig. 2D, right). Residual LPL activityin cell pellets was 13,260� 1,080 without, and 9,360� 820with heparin exposure (units are nmol/L glycerol pro-duced/flask/h, mean � SEM, n ¼ triplicate measure-ments/group; P < 0.04). We thus estimate that 29% of theHeLa cell–associated pool of LPL is heparin-releasable(indicated by HR on the graph), a fraction similar to thatobserved in the breast tumor sample. Measurement ofLPL activity in culture media indicated that 36,000 �4,000 units of LPL activity were secreted per 24 hours. Wetherefore estimate that the total cellular LPL pool turnsover more than 2.7 times/d, whereas the heparin-labilepool (3,900 units/well) turns, presumably by secretion,more than 9.2 times/d.

Our FCS contained 660 mg triglyceride/mL. LPLsecreted by cells is removed when culture media arereplaced, so the enzyme content in tissue culture neverapproaches that observed in tissues. We thereforeassessed the functional significance of LPL by addingthe enzyme to media containing 10% FCS and measuringcell accumulation. LPL activity under these culture con-ditions approximated that observed in mouse muscle.LPL enhanced the growth of T47D breast cancer cells,which do not express LPL, and of LiSa-2 liposarcomacells, which express LPL (Fig. 3A and B). This effect ofLPL was greatly reduced in media containing FCS thatwas nearly depleted of trigyceride (20 mg/mL).

LNCaP prostate cancer cell growth was not acceleratedby LPL addition. The ability of these cells to use exogen-ous triglyceride-derived FA to maintain growth wasrevealed, however, in the presence of soraphen A, apotent inhibitor of the lipogenic enzyme acetyl CoA-carboxylase (7). The cells were rescued from SoraphenA–induced cytotoxicity by provision of LPL in the pre-sence, but not in the absence, of lipoproteins (Fig. 3C).Experiments using PC3 prostate cancer cells yieldedsimilar results (Fig. 3D).

In complementary studies we assessed the impact ofsiRNA-mediated knockdown of LPL mRNA on thegrowth of HeLa cells, which we previously reported toexpress the LPL gene (25), and its interaction with inhibi-tion of lipogenesis by soraphen A. Two different siRNAseach caused greater than 90% disappearance of LPLmRNA, whereas a nonspecific siRNA was without effect(Fig. 3E). Soraphen A caused a major inhibition of HeLacell accumulation, and this effect was prevented by pro-vision of LPL to the cultures (Fig. 3F). Transfection of LPLsiRNA A or B, but not of the nonspecific siRNA, sig-nificantly inhibited HeLa cell growth, and the anticancereffects of the 2 LPL siRNAs were further enhanced byexposure to soraphen A.

We used immunohistochemistry to assess the rele-vance of our findings in cultured cells to human tumors.We assessed the expression of markers of de novo lipo-genesis [FASN, THRSP (Spot 14, S14)], lipolysis (LPL),and exogenous FA uptake (CD36) in a panel of 147 breast,24 liposarcoma, and 10 prostate tumor tissues (examplesin Fig. 4). FASN was cytosolic, in agreement with pre-vious studies. S14, which promotes expression of theFASN gene (26, 27), was primarily nuclear, as reported(20).

In contrast to our findings in breast cancer cell lines,LPL immunoreactivity was observed in all of the breasttumors examined, and, also in contrast to the cell lines,expression was not limited to triple-negative tumors.Similarly, all liposarcoma and prostate tumors examinedexpressed readily detectable LPL by immunohistochem-istry. Intracellular LPL showed an asymmetric, perinuc-lear distribution suggestive of localization to the Golgiapparatus, as predicted for a glycosylated and secretedprotein (Fig. 4C, insets). As expected, extracellularLPL was found on the luminal surfaces of capillaries






(Supplementary Fig. S2C, left). We stained tonsil tissue asa negative control, based on previous work showingundetectable LPL mRNA in lymphoid cells (25). The

lymphoid stroma indeed showed no staining except forscattered isolated monocytes, whereas the highly prolif-erative basal (stem cell) layer of the mucosal epithelium

Figure 3. LPL stimulates tumor cell growth in the presence of lipoproteins. A, T47D breast cancer cells were grown for 72 hours in media containingcomplete or lipoprotein-depleted FCS (triglyceride content 660 and 20 mg/mL, respectively) plus the indicated concentrations of LPL. Media were replacedat 24-hour intervals. Data in this and other panels are mean � SEM, normalized to the control group (seeded in 24-well plates at 20,000 cells/well,n ¼ 6 wells/group). *, P < 0.05 compared with control. B, LiSa-2 liposarcoma cells were grown for 72 hours in media containing complete or lipoprotein-depleted FCS plus the indicated concentrations of LPL were replaced at 24-hour intervals. *, P < 0.05 compared with control. C, LnCaP prostatecancer cells were treated with the indicated concentrations of LPL with or without 100 nmol/L soraphen A to inhibit lipid synthesis. Comparisons are within thelipoprotein plus and minus groups. *, P < 0.05 compared with no LPL or soraphen A; #, P < 0.05 compared with no LPL, þ soraphen A. D, PC3 prostatecancer cells were treated as in C. *, P < 0.05 compared with no LPL or soraphen A; #, P < 0.05 compared with no LPL, þ soraphen A. E, 2 LPL siRNAs(A, B), but not a nonspecific siRNA (NS), cause a substantial decline in LPL mRNA. Data are mean LPL mRNA signal normalized to 18S RNA � SEM,4 wells/group. RNA was harvested 48 hours after transfection. *, P < 0.05 compared with the nonspecific siRNA. F, LPL siRNA impairs the growth of HeLacells and augments the antiproliferative effect of soraphen A. Data are viable cells/well, mean � SEM (n ¼ 4 wells/group). Cell growth was assessed 96 hoursafter siRNA transfection. *, P < 0.05 compared with the no siRNA, no soraphen A, no LPL group; #, P < 0.05 compared with the control siRNA, no soraphen Agroup; @, P < 0.05 compared with the respective siRNA groups (A, B) without soraphen A.

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overlying the tonsil unexpectedly showed a strong signal(Supplementary Fig. S2C, right).The majority of tumors also stained for CD36 (Fig. 4D).

Interestingly, 2 distinct staining patterns were observedin breast cancer tissue. Of the 144 evaluable cores, 42exhibited diffuse cytoplasmic staining without accentua-tion at the plasma membrane (Fig. 4D, left, top), whereas100 also showed a strong cell surface signal (Fig. 4D, left,bottom). Only two breast cancer cases were devoid ofstaining. A statistically significant difference in the pre-valence of the membranous staining pattern between thetriple-negative and ERþ breast cancers was shown by c2

analysis (42% vs. 76%, P < 0.02).Of the 25 liposarcoma cases, 21 stained for CD36,

almost all in a mixed cytoplasmic plus plasmamembranepattern (Fig. 4D, middle), including all 9 cases of well-differentiated liposarcoma. Of the 9 evaluable prostatecancers, 4 showed focally positive staining in a mixedcytoplasmic and plasma membrane pattern (Fig. 4D,right), whereas 5 cases scored negative for CD36.Expression of LPL by breast cancer cells suggested the

possibility that the cells could use the enzyme not only tohydrolyze extracellular triglyceride, but also for receptor-mediated endocytosis of triglyceride-rich lipoproteins.This process uses LPL as a bridge between the cell surface

receptor syndecan-1 and the lipoprotein (28). RT-PCRrevealed readily detectable syndecan-1 mRNA fromDU4475 breast cancer cells, whereas LiSa-2 and T47Dexhibited a faint signal (Supplementary Fig. S3A). Weincubated fibroblasts andDU4475 cells with fluorescentlylabeled VLDL particles, and assessed for cellular uptakeby confocal microscopy. Abundant uptake was observedin fibroblasts (Supplementary Fig. S3B) but not in DU4475cells (Supplementary Fig. S3C). Occasional fluorescencewas detected on the cell surface (Supplementary Fig. S3D)but never within the breast cancer cells.

Discussion

Our data show that cancer cells may use two differentmechanisms to acquire FA to fuel proliferation. Breastand liposarcoma tumors are equipped for both lipidsynthesis and for LPL-mediated extracellular lipolysisfollowed by FA uptake via CD36. Prostate cancer cells,which have a very high capacity for de novo lipogenesis(24), express very little LPL. The low LPL expressioncould be explained in part by the reported loss of hetero-zygosity at the LPL locus in 47% of prostate tumors,owing to the presence of a nearby tumor suppressorgene (29). These cells, however, can acquire sufficient

Figure 4. Immunohistochemicalanalysis of markers of FAmetabolism in breast,liposarcoma, and prostate tumors.Slides from a representativeinvasive ductal carcinoma of thebreast (left column), liposarcoma(middle column), and prostaticadenocarcinoma (right column)were immunostained for (A) FASN,(B) THRSP (Spot 14, S14), (C) LPL,or (D) CD36. Originalmagnification,�40. Detection waswith peroxidase (brown pigment),and slides were counterstainedwith hematoxylin (blue pigment).FASN staining is cytosolic, andS14 is nuclear. LPL showed anasymmetric, perinucleardistribution (arrows in insets)compatible with localization to theGolgi apparatus. Note that thewell-visualized prostate tumorstroma does not expressdetectable LPL. CD36 exhibited 2distinct patterns of subcellularlocalization in breast tumors. Onlya cytosolic signal was seen in 29%of cases (D, left, top), whereasprominent cell surface stainingwas seen in 69% (D, left, bottom),and only 2% were devoid of CD36immunoreactivity. Staining wasalso seen inmost liposarcoma andprostate cancers. This wasprimarily a cell surface pattern,and was not uniformly presentacross those tumors.






exogenous FA to maintain growth in the face of FAsynthesis inhibition when they are supplied with LPLand triglyceride-rich lipoprotein particles.

LPL expression has been shown to be a marker of poorprognosis in chronic B-cell lymphocytic leukemia (B-CLL; refs. 30, 31). The single reported examination ofthe functional significance of LPL in B-CLL was difficultto interpret because Orlistat, a compound that inhibitsboth LPL and FASN (4), was used to inhibit LPL in thosestudies (32). To our knowledge, these are the first experi-ments to show widespread expression of LPL by solidtumors. We find that, in contrast to cultured breast cancercell lines, where substantial LPL is found only in a subsetwith a triple-negative gene expression signature, theenzyme is a universal component of breast tumors, irre-spective of biomarker status. Moreover, we also find thatall liposarcoma and prostate tumors examined alsoexpress LPL.

Several plausible explanations exist for the discrepancybetween cell lines and tumors with respect to LPL expres-sion. First, the cell lines have been passaged over time inculture systems lacking vascular endothelium, which isthe physiologic site for LPL action, or reliably fixedconcentrations of triglyceride-rich lipoprotein substrate,whereas cell culture media generally contain high con-centrations of glucose. Thus, de novo synthesis, ratherthan lipolysis or receptor-mediated endocytosis, mayhave been selected as the preferred mechanism for FAacquisition in cell culture. Second, it is possible thatinteractions with stroma elicit LPL expression. Third,each of the breast cancer cell lines that we find to expresssubstantial levels of LPL are not only triple-negative, butare also nonadherent to tissue culture plasticware. Inview of reports that cellular detachment provokes majormetabolic adaptations in Her2/neu-expressing breastcancer cells (33), we examined the hypothesis that cellulardetachment (72 hours) would provoke enhanced LPLmRNA expression. This proved, however, not to be thecase (data not shown). Irrespective of the cause of thediscrepancy, it is important to recognize that tissueculture experiments may not faithfully recreate in vivophysiology.

Efficient utilization by cancer cells of FA released byextracellular lipolysis would require the expression ofboth LPL and CD36. It was therefore not surprising tofind CD36 expression in the majority of tumor tissuesexamined. CD36 is known to traffic from cytoplasm to theplasma membrane in response to insulin stimulation ofadipocytes (34). We observed cell surface localization in�70% of breast cancers, whereas �30% exhibited only acytoplasmic signal. On the basis of our observation thatcell surface staining was significantly less frequent intriple-negative tumors, we speculate that CD36 traffick-ing may be driven by cell surface acting growth factorsand/or sex steroids in breast cancers.

Although further experiments are required to delineatethe precise roles of lipogenesis and lipolysis in transfor-mation, proliferation, and metastasis, recent studies have

advanced this area. Previous work established a tightlinkage of enhanced FA synthesis to transformation (35),and recent studies have defined the role of an intracel-lular lipase, monoacyl glycerol lipase in promotingtumorigenesis. Monoacyl glycerol lipase provides, byde-esterification, a stream of intracellular free FA to fuelproliferation, growth, and migration (36). This studyshows a complementary role for LPL, an extracellularlipase, in providing a stream of FA to fuel cancer cellproliferation.

Various hypotheses have been proposed to explain thedependence of tumors on lipogenesis, but it is clear thatthe primary metabolic fate of FA in proliferating tumorcells is incorporation into phospholipids destined formembrane biosynthesis (37, 38). As mitochondrial pro-duction and export of citrate are the key steps required tomaintain de novo lipogenesis in the cytosol, this begs thequestion of how such mitochondrial metabolism may bemaintained under the hypoxic (but not anoxic) conditionsthat prevail in tumors. Indeed, hypoxia-induced factor-1,a key mediator of the cellular response to hypoxia,reduces the fractional entry of glucose-derived carbonintomitochondria by downregulating pyruvate dehydro-genase, thus driving the increased lactate production thatis the most well-recognized aspect of intermediary meta-bolism in tumors (39). However, net flux of carbonthrough the glycolytic pathway is substantially elevatedin glucose-avid tumor cells, because of increased uptakeand trapping. The reduced amount of carbon directed tomitochondria is thus sufficient to provide an estimated60–85% of the ATP generated (40). Brisk citrate exportfrom mitochondria seems to be favored by incompletecombustion, as a consequence of the truncated Krebscycle in tumor mitochondria (41), which also may serveto reduce oxygen use by reducing carbon flux throughsteps downstream from citrate in the cycle. Thus, thecompeting oxygen-sparing and anabolic demands ontumors are met by a balanced set of metabolic alterations,the former favored by hypoxia-induced factor-1, and thelatter driven by oncogenes (reviewed in ref. 42). Overall,it seems that the uptake of exogenous FA, for which thisstudy shows most tumors to be equipped, would be anadvantageous response to the metabolic dilemma ofhypoxic, proliferating cancer cells.

Our findings have several implications. First, thera-peutic efforts aimed solely at inhibition of long-chain FAsynthesis may not be effective for tumors that are pro-vided with LPL and express CD36. Such tumors may besensitive to agents that inhibit the enzymes for bothlipogenesis and lipolysis, such as Orlistat (4) or the diet-ary supplement conjugated linoleic acid, which can sup-press the genes required for both pathways (8, 43). Effortsto target LPLwill need to take into account the possibilitythat prolonged systemic suppression of LPL activitycould result in hypertriglyceridemia and consequentpancreatitis, particularly if dietary fat intake is not cur-tailed. Second, the ability of nearby nonmalignant cells toprovide LPL to the tumor microenvironment may favor

Kuemmerle et al.





the ability of tumor cells, particularly those with lowlipogenic potential, to establish metastases in LPL-richtissues such as lung or fatty bonemarrow. To benefit fromLPL provided by tumor stroma, the expression of CD36by the tumor would be required. Third, the presence ofLPL in the tumor vasculature may mediate the reportedeffects of dietary fat intake on outcome (9). In addition tothe well-characterized lipogenic tumor phenotype, ourstudies indicate the expression of a previously unappre-ciated lipolytic pathway active in cancer cells as well.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

MCK-LPL transgenic mice were kindly supplied by Ira Goldberg,Columbia School of Medicine, New York, NY. We thankMartin Wabitsch(University of Ulm, Germany) for the LiSa2 cells. Soraphen A was kindlyprovided by Klaus Gerth and Rolf Jansen, Helmholtz-Zentrum f€ur Infek-

tionsforschung, Braunschweig, Germany. Triglyceride measurementswere kindly provided by Hong K. Lee, Department of Pathology,Dartmouth-Hitchcock Medical Center, Lebanon, NH. We speciallythank Rebecca O’Meara MT (ASCP), Pathology Translational ResearchLaboratory at Dartmouth Medical School, for doing the immunohisto-chemistry.

Grant Support

This work was supported by NIH Grant RO1CA126618 (W.B. Kinlaw),NIH Training Grant DK07508 (N.B. Kuemmerle), a Howard HughesMedical Foundation Fellowship 52005870 (A.J. Flanagan), Norris CottonCancer Center Prouty grants (B.L. Eisenberg, W.B. Kinlaw), GrantG.0590.08 (J.V. Swinnen), a fellowship (E. Rysman) from the ResearchFoundation-Flanders (FWO), the N.C.I. Bay Area Breast Cancer SPOREP50 CA58207 (L.A. Timmerman), and the Program in Experimental andMolecular Medicine at Dartmouth Medical School (C.J. Fricano).

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 August 25, 2010; revised January 11, 2011; accepted January19, 2011; published OnlineFirst January 31, 2011.

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2011;10:427-436. Published OnlineFirst January 31, 2011.Mol Cancer Ther Nancy B. Kuemmerle, Evelien Rysman, Portia S. Lombardo, et al. ProliferationLipoprotein Lipase Links Dietary Fat to Solid Tumor Cell

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