inhibitionoffattyacidsynthaseattenuatescd44-associated ...per 100 ml of km20 or ht29 with...

Therapeutics, Targets, and Chemical Biology

InhibitionofFattyAcidSynthaseAttenuatesCD44-AssociatedSignaling and Reduces Metastasis in Colorectal Cancer

Yekaterina Y. Zaytseva1,2, Piotr G. Rychahou1,2, Pat Gulhati1,2, Victoria A. Elliott1, William C. Mustain1,2,Kathleen O'Connor1, Andrew J. Morris4,5, Manjula Sunkara4,5, Heidi L. Weiss1,2,Eun Y. Lee1,2,3, and B. Mark Evers1,2

AbstractFatty acid synthase (FASN) and ATP-citrate lyase, key enzymes of de novo lipogenesis, are significantly

upregulated and activated in many cancers and portend poor prognosis. Even though the role of lipogenesisin providing proliferative and survival advantages to cancer cells has been described, the impact of aberrantactivation of lipogenic enzymes on cancer progression remains unknown. In this study, we found thatelevated expression of FASN is associated with advanced stages of colorectal cancer (CRC) and livermetastasis, suggesting that it may play a role in progression of CRC to metastatic disease. Targetedinhibition of lipogenic enzymes abolished expression of CD44, a transmembrane protein associated withmetastases in several cancers including CRC. In addition, inhibition of lipogenic enzymes and reducedexpression of CD44 attenuated the activation of MET, Akt, FAK, and paxillin, which are known to regulateadhesion, migration, and invasion. These changes were consistent with an observed decrease in migrationand adhesion of CRC cells in functional assays and with reorganization of actin cytoskeleton upon FASNinhibition. Despite the modest effect of FASN inhibition on tumor growth in xenografts, attenuation oflipogenesis completely abolished establishment of hepatic metastasis and formation of secondary metastasis.Together, our findings suggest that targeting de novo lipogenesis may be a potential treatment strategy foradvanced CRC. Cancer Res; 72(6); 1504–17. �2012 AACR.

IntroductionColorectal cancer (CRC) is the second leading cause of

cancer-related deaths in the United States (1). The prognosisand life expectancy of patients with CRC are primarily deter-mined by presence or absence of metastases, not by growth ofthe primary tumor (2). Treatment options for metastatic CRCare limited and more selective therapeutic targets are neededto improve survival.

The signaling networks that determine progression ofcancer to a metastatic phenotype include multiple altera-tions in the oncogenic pathways and significant changes incellular metabolism. The fact that, in contrast to normalcells, the majority of fatty acids in malignant cells arederived from de novo lipogenesis regardless of the availabil-ity of extracellular lipids, suggests the importance of upre-gulation of endogenous lipid biosynthesis in malignant

transformation (3). ATP-citrate lyase (ACLY) and fatty acidsynthase (FASN), the key enzymes of de novo lipogenesis, aresignificantly upregulated in many cancers including CRC (3).Indeed, expression of FASN was increased in 86% of aberrantcrypt foci (ACF) compared with that of adjacent normalcolonic mucosa (4). Furthermore, metabolic profiling of CRChas shown an overall increase in the lipid content of polypsand tumors (5).

Neoplastic lipogenesis provides a selective proliferativeand survival advantage and contributes to drug resistancein cancer cells (6–8). However, the impact of aberrantactivation of lipogenic enzymes on metastases remainsunknown. Expression of FASN is highest in metastatictumors and correlates with decreased survival and diseaserecurrence in several tumor types (9, 10). Interestingly,proteomic characterization of CRC cell lines indicatesthat an increased expression of lipogenic enzymes is asso-ciated with a more aggressive metastatic phenotype (11).Furthermore, pharmacologic inhibition of FASN providesindirect evidence of a possible connection between activa-tion of lipogenesis and metastatic behavior of cancer cells(12–14).

Progression to a metastatic phenotype is associated withdifferential expression of proteins on the cell surface (11, 15).CD44, a transmembrane glycoprotein with multiple iso-forms, is implicated in tumor progression and metastasis(16). Expression of CD44 is increased in CRC and correlateswith poor clinical outcome (17, 18). The role of CD44 in

Authors' Affiliations: 1Markey Cancer Center, Departments of 2Surgeryand 3Pathology and Laboratory Medicine, 4Division of CardiovascularMedicine, and 5Gill Heart Institute, University of Kentucky, Lexington,Kentucky

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: B. Mark Evers, Markey Cancer Center, Universityof Kentucky, 800 Rose Street, CC140, Lexington, KY 40536. Phone: 859-323-6556; Fax: 859-323-2074; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-4057

�2012 American Association for Cancer Research.

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metastases might be linked to its interaction with receptortyrosine kinases such as c-MET, a proto-oncogene involvedin tumor growth, invasion, and metastasis (19). Associationof c-MET with CD44 isoforms at the plasma membraneseems to be essential for activation of c-MET and down-stream signaling in CRC (20).In this study, we determined the role of lipogenic enzymes

in metastatic CRC. We show that in human tissue arrays,FASN is progressively increased with advancing stages ofCRC. For the first time, this study establishes the linkbetween expression of lipogenic enzymes and CD44. Weshow that inhibition of ACLY and FASN dramaticallyreduces expression of CD44 and attenuates CD44-associatedsignaling. We further show that suppressed expression ofFASN decreases the tumorigenic and metastatic potential ofCRC cells in vitro and in vivo. Collectively, our data suggestthat upregulation of de novo lipogenesis is a critical step inCRC progression to metastases and that a better under-standing of the link between metabolic changes in cancercells and development of metastasis may lead to novelstrategies to prevent and/or control advanced CRC.

Materials and MethodsCell lines, lentiviral transduction, siRNAHuman CRC lines KM20 and HCT116 were used as

described previously and their identity was authenticatedat the Johns Hopkins Genetic Resources Core Facility inOctober 2010, as previously reported (21). HT29 cellswere purchased from American Type Culture Collection.For generation of stable knockdown KM20, HT29, andHCT116 cell lines, the lentiviral transduction particles con-taining short hairpin RNA (shRNA) for ACLY (SHCLNV-NM_001096), FASN (SHCLNV-NM_004104), or nontargetshRNA (CHC002V) in pLKO.1-puro plasmid were purchasedfrom Sigma. Cells were transduced with virus in the pres-ence of polybrene (10 mg/mL) for 24 hours and thenselected on puromycin (10 mg/mL). ON-TARGET plus CD44siRNAs (LU-00999907 and LU-00999908) and control siRNA(D-001810-10) were purchased from Dharmacon and usedin a concentration of 100 mmol/L.

AntibodiesAntibodies for Western blot and immunofluorescent stain-

ing were purchased from Cell Signaling: FASN (#3180), ACLY(#4332), pACLY (#4331), CD44 (#3570), pMET (#3129), MET(#3148), pSrc (#2101), Src (#2109), pAkt (#4058L), Akt (#4691L),p-paxillin (#2541), paxillin (#2542), pFAK (#3283), FAK (#3285),and RhoA (#2117).

Human tissue arraysThe CO702 tissue array (US Biomax), A203 (IV) tissue array

(AccuMax), and FASN antibody (Cell Signaling) were pur-chased. Scoring was carried out blindly by a pathologistaccording to a semiquantitative method (21). The extent scorewas assessed on a scale of 0 to 3 (no positive cells¼ 0,<10%¼ 1,10%–50% ¼ 2, positive staining of >50% ¼ 3); the intensityscore was alsomeasured on a scale of 0 to 3 (negative¼ 0, weak

¼ 1, moderate¼ 2, strong¼ 3). The immunoreactive score wasobtained by summing these 2 scores.

Analysis of de novo lipogenesisDe novo palmitate synthesis was analyzed by stable iso-

tope labeling. Cells were plated in normal growth medium.On day 2, medium was replaced with medium containingsodium acetate (2–13C, 99%, Cambridge Isotope Laborato-ries) for 20 hours at a concentration of 10 mmol/L. Extrac-tion of the lipids was carried out as described previously(22) in the presence of an internal standard. Fatty acidswere converted to 3-acyloxymethyl-1-methylpyridiniumiodide (AMPP) derivatives and the AMPP derivative ofpalmitic acid was quantified using a Shimadzu HPLC cou-pled to ABI 4000 Q-Trap hybrid linear ion trap triplequadrupole mass spectrometer operated in positive elec-trospray ionization (ESI) mode as previously described (23).The instrument was operated in multiple reaction moni-toring mode and the precursor product ion pairs monitoredwere 362.2/124.5 which represent the [MþH]þ mass of theAMPP derivative of palmitic acid and a fragment ioncontaining the N-pyridyl moiety. Enrichment of 13C wasdetermined from the ratio of integrated peak areas corre-sponding to the mz/363.2/124.5 and the Mþ1 isotopomer362.2/124.5 precursor/product ion pairs.

Quantitative real-time PCRTotal RNA was isolated using an RNeasy mini kit (QIAGEN).

cDNA was synthesized using a high capacity cDNA reversetranscription kit (Applied Biosystems). Quantitative real-timePCR (qRT-PCR) was carried out using a TaqMan Gene Expres-sion Master Mix (#4369016) according to manufacture proto-col and TaqMan probes for human CD44 (ID Hs1075862_m1)and human GAPDH (# 4333764F; Applied Biosystems).

Flow cytometry analysis and cell sortingCells were labeled with 2 mg/mL CD44–FITC antibody

(Abcam) per 5 � 106 cells. For cell sorting, cells were washedand resuspended in basic sorting buffer. For analysis of CD44expression on cell surface, cells were resuspended in PBS, 10%FCS, 1% sodium azide. Cell analysis and cell sorting wereconducted by the UK Flow Cytometry Service Facility.

Transwell migration assayA Boyden chamber migration assay with collagen-coated

Transwells was conducted with KM20 and HT29 cells for 20hours as described previously (21). FBS (10%) was used as achemoattractant. Cells were counted in 4 different fields withan inverted microscope.

Endothelial cell adhesion assayCRC cells were labeled with Calcein AM (2.5 mg/mL final

concentration) at 37�C, added atop a monolayer of humanmicrovascular endothelial cells from lungs (HMVEC-L) for 30minutes. Before the experiment, HMVEC-L cells were activatedwith 10 ng/mL of TGFa for 4 hours. Unattached cells wereremoved bywashingwith PBS (5�). Three images perwell weretaken to count attached cells.

Inhibition of Fatty Acid Synthase

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In vivo studiesMale athymic nudenu/nu mice (5–6 weeks old; Charles River

Laboratories) were housed in the Markey Cancer Center SmallAnimal Facility. All procedures were carried out with protocolsapproved by the U.K. Animal Care and Use Committee. Toestablish CRC xenografts, mice were injected with 1� 106 cellsper 100 mL of KM20 or HT29 with nontargeting control (NTC)shRNA and FASN shRNA (5 mice per group). To assess estab-lishment of hepatic metastasis, HT29-GFP-Luc cells (2 � 106

cells per 100 mL) were injected intrasplenically as previouslydescribed (24). For experimental metastasis models, controland stable knockdown HT29-GFP-Luc cells or KM20-GFP-Luc(2 � 106 cells per 300 mL) were injected into the tail vein ofathymic nude mice (n ¼ 4 and 5 per group, respectively). Tomonitor metastasis, mice were anesthetized with isoflurane,given a single i.p. dose of 150 mg/kg D-luciferin in PBS andimaged 8 minutes after injection (IVIS Spectrum; CaliperSciences). Results were analyzed by Living Image 3.0 software.

Statistical methodsANOVA with test for linear trend and pairwise comparisons

was carried out to compare immunoreactivity scores in normalmucosa and CRC of various stages. ANOVA was also employedfor multiple group comparison of shRNA (ACLY and FASN)with NTC for CD44 levels, whereas 2-sample t test or Wilcoxonrank-sum test was used for 2 group comparisons of migration,number of colonies, and tumor weight between FASN versusNTC. Comparison of log values of tumor volume measured

over time was conducted using a linear mixedmodel. Analyseswere conducted separately for each cell line.

ResultsFASN is highly expressed in primary CRCs and livermetastases

Evaluations of FASN in human ACF and primary CRCs haverevealed elevated expression of this enzyme in early stages ofcolorectal tumorigenesis as compared with normal mucosa(4, 25–27). To extend these studies, we analyzed expression ofFASN in CRC clinical samples by immunohistochemistry(Fig. 1A). Expression of FASN was detected in the cytoplasmin all stages of primary CRC, though some variability wasobserved within different regions of individual tumor cores.Statistical evaluation of immunoreactivity scores showedincreased expression of FASN in stages II to IV CRC ascompared with normal mucosa. Analysis of primary tumors,matched liver metastases, and normal colonic mucosa fromstage IV patients showed significantly higher expression ofFASN in both the primary CRC and liver metastasis as com-pared with normal colon mucosa (Supplementary Fig. S1).

We also tested expression of ACLY and FASN in a panel ofCRC cell lines. Consistent with results obtained from humantissue arrays, Western blot analysis showed high expression/activation of lipogenic enzymes (Fig. 1B). These data suggestthat enhanced de novo lipid biosynthesis may play a role inprogression of primary CRC to metastatic disease.

Figure 1. Expression of FASN inCRC tissues and cell lines. A,expression of FASN in normalcolon mucosa and stage I to IVprimary CRC (US Biomax tissuearray CO702, total 69 cores, �200magnification). Immunoreactivityscorewasdetermined according toa semiquantitive method asdescribed in Materials andMethods. B, cell lyses wereprepared from a panel of humanCRC cell lines and immunoblotanalysis was conducted for FASN,ACLY, and pACLY expression.b-actin was used as a loadingcontrol.

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Inhibition of lipogenic enzymes decreases expression ofCD44CD44 and c-Met have been implicated in CRCmetastasis via

regulation of tumor cell growth, adhesion, migration, andinvasion (16, 28, 29). Considering that tumor-produced fattyacids preferentially partition into lipid rafts (30), we hypoth-esized that altered expression of lipogenic enzymes may affectCD44 and c-Met because their functions have been attributedto these rafts (31, 32). First, we analyzed expression of CD44and c-Met in a panel of CRC cell lines. CD44 variant isoforms(CD44v), which are primarily implicated in regulation ofmetastasis, are highly expressed in most cell lines (Fig. 2A).

In contrast with other cell lines, the nonmetastatic SW480 cellline expresses a low level of CD44v and a high level of CD44standard isoform (CD44s). Activation of c-Met was observed inKM20, HT29, and HCT116 cell lines, and we used these 3 celllines to establish a stable shRNA-mediated knockdown ofeither ACLY or FASN to investigate the role of de novolipogenesis in metastatic CRC.

To confirm inhibition of lipid biosynthesis when ACLYand FASN are inhibited, cells were exposed to stable isotope-labeled acetate and its incorporation into palmitate wasmeasured by mass spectrometry. Targeted inhibition ofACLY and FASN led to a 60% to 100% decrease in de novo

Figure 2. Inhibition of ACLY andFASN attenuates expression ofCD44. A, immunoblot analysis forCD44, p-MET, and total MET in apanel of CRC cell lines. b-Actin wasused as a loading control. B, de novolipogenesis was analyzed by stableisotope labeling. Abundance of 13Cacetate in palmitic acid wasdetermined in CRC cell lines withNTC shRNA, ACLY shRNA, andFASN shRNAbymass spectrometry.Data shown as the ratio of integratedpeak areas (means � SD of triplicatedeterminations). No13C is anabundance of 13C in unlabeled cells(a baseline isotope ratio).�, P < 0.05 versus control. C,expression of CD44 in KM20, HT29,and HCT116 CRC cell lines with NTCshRNA, ACLY shRNA, and FASNshRNA. D, expression of CD44 inHT29 cell line treated with variousconcentrations of C-75 (mmol/L), aninhibitor of FASN, for 24 hours. E,expression of FASN and CD44 inHT29 cell line transiently transfectedwithmyr-Akt1, myr-Akt2, or a controlplasmid. F, immunoblot analysis forFASN and CD44 in HT29 FASNshRNA cells labeled with CD44-FITCantibodyand sorted for expression ofCD44. Cells with the lowest (10%)and the highest (10%) expression ofCD44 were used for analysis. G,relative CD44 mRNA levels in KM20,HT29, and HCT116 cell lines withstable knockdown of ACLY or FASNassessed by real-time PCR. CD44mRNA expression was calculated bythe DDCt method areas (means� SDof triplicate determinations; �, P <0.05 vs. control).






palmitate synthesis (Fig. 2B). Interestingly, reduced expres-sion of ACLY and FASN was also associated with changes incellular morphology (Supplementary Fig. S2). We show thatinhibition of ACLY and FASN significantly decreases expres-sion of the CD44 protein. A more pronounced inhibitionof CD44 was observed in cells with knockdown of FASNcompared with that of ACLY (Fig. 2C). Treatment of HT29cells with C-75, a synthetic inhibitor of enzymatic activity ofFASN, also led to a dose-dependent decrease in expression of

CD44 (Fig. 2D). Interestingly, upregulation of FASN expres-sion by transient transfection of myristoylated, constitutive-ly active form of Akt1 (myr-Akt1) or Akt2 (myr-Akt2) had anopposite effect and increased expression of CD44 in HT29cells (Fig. 2E).

To further evaluate the correlation of CD44 expression andFASN, we carried out direct labeling of FASN knockdownHT29cells with the CD44-FITC antibody. These cells were culturedfor 3 weeks before the experiment and showed a mixed

Figure 3. Inhibition of ACLY andFASN attenuates c-MET signaling.A, immunoblot analysis for p-METand MET in KM20 and HT29 celllines with NTC shRNA, ACLYshRNA, and FASN shRNA. B, NTCshRNA, ACLY shRNA, and FASNshRNA KM20 or HT29 cells werestarved for 24 hours, thenstimulated with HGF (10 ng/mL) for20 minutes, lysed, andimmunoblotted for FASN, ACLY,p-MET, and MET. C, immunoblotanalysis for p-MET and METexpression in KM20 and HT29 celllines transfected with 2 differentCD44 siRNAs or scrambled siRNAas a control.

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population of cells with differential expression of FASN asconfirmed by immunofluorescent staining (data not shown).CD44-FITC–labeled cells were sorted based on the level ofCD44 expression. Cells with low expression of CD44 showedconcomitant reduction of FASN expression (Fig. 2F).To elucidate themechanism for the downregulation of CD44

in ACLY and FASN knockdown cells, we assessed the level ofCD44 mRNA in KM20, HT29, and HCT116 cell lines each withstable knockdown of either ACLY or FASN. We observed aslight decrease in CD44mRNA inHCT116 andKM20, but not inthe HT29 cell line (Fig. 2G). It has been previously shown thatexpression of CD44 can be regulated by posttranscriptionalmodifications such as palmitoylation (32). To test the possibleinvolvement of this mechanism in the regulation of CD44expression in CRC cells, we used 2-bromopalmitate (2-BP), anonmetabolizable palmitate analog, to block the incorporationof palmitate into proteins. HT29 NTC and FASN knockdown

cells were treatedwith either dimethyl sulfoxide (DMSO) or 200mmol/L of 2-BP, labeled with CD44-FITC antibody, and ana-lyzed by flow cytometry. The analysis conducted a 2-folddecrease in signal intensity on the cell surface in FASN knock-down cells and 2-BP–treated cells versus control HT29 cells(Supplementary Fig. S3A). Consistent with these data, subcel-lular fractionation revealed that expression of CD44 in themembrane fraction is significantly lower in FASN knockdownand 2-BP–treated cells compared with control HT29 cells(Supplementary Fig. S3B). Together, these data suggest thatde novo lipogenesis regulates expression of CD44 at a post-transcriptional level, possibly by altering its palmitoylation.

Inhibition of lipogenic enzymes attenuates c-Metsignaling in CRC cells

Recent studies have shown a functional link between expres-sion of FASN and activation of c-Met (33). To test whether a

Figure 4. De novo lipid biosynthesis promotes tumorigenic potential of CRC cells. A, KM20 and HT29 cells with NTC shRNA and FASN shRNA were plated insoft agar, and the number of colonies formed in each well was counted (day 20) using Alpha Innotech Imaging system and AlphaEase software. Datashown as difference in number of colonies formed in agar by control and FASN knockdown cells. Data are representative of 3 independent experiments.�, P < 0.05 versus control. Images of representative wells are shown. B, KM20 and HT29 cells with NTC shRNA and FASN shRNA were added ontop of an HMVEC-Lmonolayer, and cell adhesionwas assessed as described inMaterials andMethods. Data shown asmean fold changes in number of CRCcells with knockdown of FASN attached to HMVEC-L cells versus control cells (�, P < 0.05). Data are representative of 3 independent experiments.Representative images show adhesion of control and FASN knockdown cells to HMVEC-L cells. C, Transwell migration assaywas conductedwith KM20 andHT29 cells with NTC shRNA and FASN shRNA. Data shown as mean of cell number migrated per hpf; �, P < 0.05 versus control. Data are representativeof 3 independent experiments.






decrease in expression of lipogenic enzymes affects activity ofc-Met in CRC, we first analyzed the expression and activationof c-Met in KM20 and HT29 with stable knockdown of ACLYand FASN. As shown in Fig. 3A, phosphorylation of Tyr 1234/1235 in the c-Met kinase domain is inhibited by knockdown ofFASN inKM20 cells and by knockdown of bothACLY and FASNin HT29 cells.

To further confirm that attenuation of c-Met signaling is dueto inhibition of lipogenic enzymes, we serum starvedHT29 and

KM20 cells (control, ACLY, and FASN knockdown) for 24 hoursand then treated them with 10 ng/mL of hepatocyte growthfactor (HGF), a c-Met receptor ligand, for 20 minutes. Consis-tent with data from the previous experiment, activation ofc-Met by HGF was prevented by knockdown of FASN in bothKM20 and HT29 cell lines (Fig. 3B).

To test whether changes in activity of c-MET are CD44dependent, KM20 and HT29 cells were transiently transfectedwith 2 different CD44 siRNAs or control siRNA. As shown

Figure 5. Inhibition of de novolipogenesis affects expression ofproteins involved in regulation ofcell adhesion, motility, andinvasion and inducesrearrangement of the actincytoskeleton. A, schematicillustration of regulation of celladhesion, migration, and invasionby the CD44/c-MET complex. B,immunoblot analysis for FASN,ACLY, pSrc, and total Src in KM20,HT29 cells, and HCT116 cell lineswith NTC shRNA, ACLY shRNA,and FASN shRNA. C, immunoblotanalysis for FASN, ACLY, pAkt,total Akt, p-paxillin, and totalpaxillin in KM20 and HT29 cellswith NTC shRNA, ACLY shRNA,and FASN shRNA. D, immunoblotfor pAkt, total FAK, and RhoA inKM20 and HT29 cells with NTCshRNA, ACLY shRNA, and FASNshRNA. E, immunoblot analysis forpAkt, RhoA, pSrc, p-paxillin, andp-FAK in KM20 and HT29 cell linestransfected with 2 different CD44siRNAs or scrambled siRNA as acontrol for 48 or 72 hours. F, totalinternal reflection fluorescenceimaging of control and FASNknockdown KM20 and HT29 cellsstained for F-actin (green) andeither p-paxillin (red) or pFAK (red).

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in Fig. 3C, inhibition of CD44 decreases c-MET activity inHT29,but not in KM20 cells.Together, these findings suggest that upregulation of lipo-

genesis during cancer progression regulates c-Met signaling.

Expression of FASN regulates CRC cell growth, adhesion,and migrationAn important prometastatic characteristic of cancer cells is

their ability to grow in an anchorage-independent manner.KM20 and HT29 cells (control and FASN knockdown) weretested for their ability to grow without attaching to a substrateusing soft agar assay. In both cell lines, we observed a signif-

icant decrease in the number of colonies formed when expres-sion of FASN was inhibited (Fig. 4A).

Tumor cell intravasation and extravasation are importantsteps in metastasis and require cancer cells to interact andadhere to endothelial cells. In CRC, CD44 is implicated inregulation of adherence of cancer cells to endothelial cells andtheir subsequent transendothelial migration (34). To testwhether inhibition of FASN can affect the ability of CRC cellsto attach to endothelial cells, we used control and FASNknockdown KM20 and HT29 cells labeled with Calcein AM.Cells were added atop a monolayer of HMVEC-L preactivatedwith TGFa and incubated for 30 minutes. Unattached cells

Figure 6. Inhibition of FASNsuppresses primary CRC tumorgrowth and establishment of livermetastasis. A, KM20 and HT29 cells(1 � 106; NTC or FASN shRNA) wereinjected subcutaneously intoathymic nude mice. Immunoblotanalysis for FASN andCD44 in tumorcells before injections shown withtumor volume for days 1 to 27 (5miceper group). B, tumor weights incontrol and FASN shRNA groups ofKM20 and HT29 xenografts. C,immunoblot analysis for FASN andCD44 in tumor tissues from KM20and HT29 xenograft (day 27). D,images of GFP-positive cancer cellsin athymic nude mice injectedintrasplenically with HT29-NTC-Luc/GFP or HT29-FASNshRNA-Luc/GFPcells (abdominal view). Arrow headsindicate primary tumors. E, images ofGFP-positive cells in liver. F,representative images of histologicanalysis (H&E staining) of livermetastasis. L, normal liver; M,metastasis.






were removed by multiple washes. Data showed that down-regulation of FASN inKM20 andHT29 cells impairs their abilityto attach to endothelial cells (Fig. 4B).

Enhanced cell migration is associated with a metastaticphenotype. The results from the Transwell migration assayshowed that inhibition of FASN impairs migration in bothKM20 and HT29 cell lines (Fig. 4C). Collectively, these findingssuggest that de novo lipogenesis plays a significant functionalrole in progression of CRC.

Inhibition of lipogenic enzymes alters the signalingnetwork that regulates adhesion and motility in CRC

The cooperation between CD44 and c-Met at the plasmamembrane and their dynamic interactions with multipledownstream molecules leads to activation of the signalingnetwork that regulates cell growth, adhesion, and migration(16). Activation of tyrosine kinase Src by c-Met contributes tothe metastatic potential of CRC cells via regulation of the actincytoskeleton and modulation of the formation of adhesivestructures (35). Furthermore, expression of CD44 and activa-tion of c-MET promote phosphorylation of focal adhesionkinase (FAK) and promote cell motility (36). Interestingly,cooperation of Src and FAK kinases leads to phosphorylationof paxillin and also contributes to stimulation of cell motility(37; Fig. 5A). Our data show that inhibition of expression ofACLY and FASN inKM20 andHT29 decreases phosphorylationof Src (Fig. 5B). Because phosphorylation of Src is associatedwith downstream activation of FAK and paxillin, we examinedexpression of these proteins in HT29 and KM20 with stableknockdown of either ACLY or FASN. Phosphorylation of pax-illin on Tyr 118 was inhibited only by knockdown of FASN inboth KM20 and HT29 cell lines (Fig. 5C). Interestingly, theeffect of FASN knockdown on phosphorylation of FAK wasmore prominent than the effect of ACLY knockdown (Fig. 5C).Consistent with several studies showing the connectionbetween upregulation of lipogenesis and activation of Akt(7, 8), we observed a decrease in phosphorylation of Akt inbothKM20 andHT29 cell lineswith stable knockdownof eitherACLY or FASN (Fig. 5D). Furthermore, CD44-dependentexpression of RhoA induces formation of stress fibers and isrequired for focal adhesion formation (38). Our data show asignificant decrease in expression of RhoA in ACLY and FASNknockdown KM20 and HT29 cell lines (Fig. 5D).

To test whether these changes are CD44 dependent, KM20and HT29 cells were transiently transfected with CD44 siRNAsor control siRNA and expression/activity of aforementionedproteins were assessed. As shown in Fig. 5E, inhibition of CD44significantly decreases expression of RhoA and activity of Aktin both cell lines. Interestingly, knockdown of CD44 decreasedactivity of Src and FAK only in HT29 cells and did not affectactivity of paxillin in either cell line (Fig. 5E).

Actin cytoskeleton plays an important role in regulation ofcell growth, adhesion, motility, and invasion. Considering thatactivation of CD44/c-Met and Src affects cell motility byregulating actin polymerization, depolymerization, and stressfiber formation, we carried out immunofluorescence stainingfor F-actin. Phalloidin staining showed significant changes inorganization of actin cytoskeleton in cells with FASN knock-

down compared with control cells (Fig. 5E). Furthermore, inagreement with Western blot analysis, immunofluorescentstaining of control and FASN knockdown cells for phospho-FAK and phospho-paxillin showed a significant decrease innumber of focal adhesions in FASN knockdown KM20 andHT29 cells. Our findings suggest that inhibition of lipogenesisin CRC cells induces molecular changes and reorganization ofactin cytoskeleton, which are consistent with a less motile andinvasive phenotype of cancer cells.

Inhibition of FASN decreases tumor growth andattenuates CRC liver metastasis

It has been previously shown that CRC xenografts treatedwith C-75 exhibit inhibition of tumor growth (7). To testwhether molecular inhibition of FASN expression affectstumor growth in vivo, we injected control or FASN knockdowncells subcutaneously into athymic nude mice. Tumor growthwas monitored by measurement of tumor volume (Fig. 6A).Mice were sacrificed at day 27 after the inoculation, and tumorweight was recorded (Fig. 6B). We observed a slower rate oftumor growth and smaller tumor size in the FASN-treatedmiceversus control (P ¼ 0.002 for KM20; P ¼ NS for HT29).Expression of FASN and CD44 in tumors from control andFASN knockdown groups was assessed by Western blot andshowed partial recovery of expression of both proteins at theend of the experiment (Fig. 6C).

The liver is the most common site for CRC metastasis, andintrasplenic injections recapitulate the process of liver metas-tasis. We injected GFP-labeled control or FASN knockdownHT29 cells into the spleens of athymic nude mice and assessedthe presence of liver metastasis by GFP imaging and immu-nohistochemical analysis at 6 weeks after injection. Primarytumors were observed in 100% of mice in the control groupversus 40% of mice in the FASN knockdown group (Fig. 6D).Liver metastases were observed in 80% of the mice injectedwith control HT29 cells, whereas complete absence of livermetastasis was observed in mice injected with FASN knock-down cells (Fig. 6D–E). Presence or absence of liver metastasiswas confirmed by hematoxylin and eosin (H&E) staining (Fig.6F). Data from these experiments provide support for the roleof lipogenesis in tumor growth and potentially in the metas-tasis of CRC.

Inhibition of FASN attenuates establishment of CRCmetastasis

To further investigate the role de novo lipogenesis plays inmetastasis, we examined the effect of FASN inhibition in anexperimental metastasis model in vivo. Luciferase and GFP-labeled HT29 and KM20 cells with stable knockdown of FASNwere injected i.v. into athymic nude mice, and formation ofsystemic metastasis was assessed by bioluminescent andfluorescent imaging. All mice in the HT29 control groupshowedmetastatic nodules (Fig. 7A). The distribution of valuesfor total luciferase signal per animal is shown in Fig. 7B.Luciferase signal was associated with metastatic nodules onthe backs of the animals and metastasis to the mesentericlymph nodes. In contrast, none of the metastatic nodules wereformed in the HT29 FASN knockdown group. However,

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histologic examination of lungs showed the presence of pul-monary micrometastasis in both the control and FASN knock-down groups (Fig. 7C).Similar results were obtained when we used another met-

astatic CRC cell line, KM20. Systemicmetastases were noted in80% of mice from the control group; however, knockdown ofFASN completely abolished establishment of metastasis asdepicted by luciferase imaging and quantification of totalluciferase signal per mouse (Fig. 7D and E). Surprisingly, incontrast to HT29-inoculated mice, histologic examinationshowed the presence of pulmonary micrometastasis in 60%of mice in the KM20 control group and complete absence ofmicrometastasis in the group injected with FASN knockdownKM20 cells (Fig. 7F). The presence of pulmonary metastasis inthe control group was also visualized with GFP imaging(Supplementary Fig. S4). Taken together, these findings sug-gest that overexpression of FASNmay play an important role inestablishment of CRC metastasis in vivo.

DiscussionIn this study, we investigated the role of de novo lipogenesis

in CRCmetastases. Although, multiple studies have focused onthe contribution of aberrant expression of lipogenic enzymesto growth and survival of cancer cells, their role in metastasisremains unknown (39, 40). Here, we show an increase inexpression of FASN with advancing stages of CRC, suggestingthat FASN may play an important role in the progression ofCRC to metastatic disease. Furthermore, this study shows thatinhibition of lipogenic enzymes decreases expression of CD44and attenuates the CD44-associated signaling, thus establish-ing the novel link between lipid biosynthesis and metastasis.Finally, we show that targeted inhibition of FASNdecreases thetumorigenic potential of CRC cells in vitro and attenuatesestablishment of metastasis in 2 experimental metastasismodels in vivo.Increased expression of CD44 correlates with metastasis in

many cancers (16). We showed that changes in CD44 in CRCcells with inhibited expression of lipogenic enzymes are notdue to transcriptional regulation, but possibly to posttran-scriptional modifications of protein. CD44 undergoes asequential proteolytic cleavage and releases CD44 intracellulardomain, which acts as a transcriptional factor and regulatesexpression of its own gene (36). This mechanism can explain aslight decrease in CD44 mRNA noted in KM20 and HCT116cellswhen lipogenic enzymes are inhibited. Expression of CD44is dramatically higher in HT29 cells, so it is possible that CD44is not dependent on self-regulation.c-Met is implicated in promotion of metastasis (19). Con-

sistent with studies of diffuse large B-cell lymphoma andprostate cancer, showing the link between inhibition of FASNanddecreased phosphorylation of c-Met (33, 41), our data showthat inhibition of lipogenic enzymes decreases endogenousactivation of c-Met and prevents its activation by HGF in CRCcells. Interestingly, CD44 is required for c-Met activation andsignaling in several cancers (20, 42). Consistently, we showedthat decreased phosphorylation of c-Met in FASN-depletedcells is CD44 dependent in HT29 cells. The differential effect ofCD44 inhibition on c-Met activity in KM20 and HT29 cells can

be explained by a significantly higher level of CD44 expressionand c-MET activation in HT29 cells.

Overexpression of FASN in breast cancer cells increasedthe size and number of colonies in soft agar (43). In agree-ment with this study, we show that inhibition of FASN leadsto a significant decrease in the ability of CRC cells to grow inan anchorage-independent manner. In addition, we showthat altered lipogenesis regulates attachment of CRC cells toendothelial cells. Suppressed migration is associated withreduced activity of FASN in ovarian carcinoma cells (14).Consistently, we also observed a significant decrease inmigration of both KM20 and HT29 cell lines when expressionof FASN is inhibited. Considering that suppression of lipo-genic enzymes inhibits expression of CD44 and decreasesactivation of c-Met, these functional changes can beexplained by alterations in the signaling network down-stream of these 2 proteins. Our data show that KM20 andHT29 cells, with inhibited expression of FASN, exhibit sig-nificant suppression of Src, FAK, and paxillin activation, theproteins which are downstream of the CD44/c-Met complexand implicated in regulation of adhesion dynamics, migra-tion, and invasion of cancer cells (44). Indeed, the level ofFAK receptors and their phosphorylation has been shown toincrease during the adenoma to carcinoma transition ofcolonic epithelia (45). Furthermore, activation of Src con-tributes to anoikis resistance in CRC cells through activationof phosphatidylinositol 3-kinase (PI3K)/Akt pathway andphosphorylation of Akt (46). In addition, overexpression ofFASN has been associated with activation of Akt in humanCRC tissue samples (7). Consistent with these findings, wealso observed a CD44-dependent decrease in activation ofAkt in HT29 and KM20 cells with inhibited expression oflipogenic enzymes. Interestingly, inhibition of CD44 attenu-ates activation of Src and FAK in HT29, but not in the KM20cell line, suggesting that the effect of inhibition of lipogenicenzymes on downstream signaling may be mediated byaltered composition of lipid rafts within the plasma mem-brane and impairment of activation/function of membrane-associated receptors or oncoproteins other than CD44 and c-MET (47).

Inhibition of lipogenic enzymes decreases cell growth inseveral types of cancers (40). Furthermore, FASN inhibitionwith C-75 slows growth of CRC xenografts (7). Our findings areconsistent with these studies and show that inhibition oflipogenesis attenuates the rate of tumor growth and decreasestumor size in both KM20 and HT29 xenografts. Interestingly,we observed the partial recovery of FASN expression and asubsequent increase in expression of CD44 in xenografts,supporting the importance of lipogenesis for cancer cell sur-vival and growth.

Implantation of cancer cells into the spleens of nude miceresults in primary tumor growth and establishment ofhepatic metastases (24). The liver metastasis model primar-ily evaluates late stages of metastasis and recapitulates thepattern of colorectal metastasis in humans (24). Using thismodel, we showed that despite detection of primary tumorsin both control and FASN knockdown groups, inhibition ofFASN prevented formation of liver metastasis. Furthermore,






results from the experimental metastasis model, carried outwith both KM20 and HT29 cell lines, showed that inhibitionof FASN expression blocked establishment of metastaticnodules in nude mice. The control mice developed multiplemetastatic nodules, predominantly located on the upperback and mesenteric lymph nodes. This pattern of tumorformation is similar to what was reported in a previouspublication by our laboratory (21). Surprisingly, gross exam-ination of the lungs showed a different effect of the down-regulation of FASN on establishment of microscopicnodules. Knockdown of FASN in KM20 cells preventedformation of micrometastasis in the lungs; however, itsinhibition in the HT29 cells did not affect formation oftumor nodules. This discrepancy can likely be explained bythe different genetic backgrounds of HT29 and KM20 celllines. It is possible that downregulation of FASN has differ-ent consequences on modulation of signaling pathways andmetastatic potential in these cell lines. Furthermore, in the

experimental metastasis model, colonization of cells in thelungs does not recapitulate all steps of the metastaticcascade, and formation of secondary metastatic lesions,which were depicted by the bioluminescent imaging in ourexperiment, provides a better model for evaluation of themetastatic potential of cancer cells. Taken together, datafrom our in vivo experiments suggest that downregulation oflipogenesis in cancer cells has a more prominent effect onmetastasis than on the growth of primary tumors.

Due to the strong correlation between de novo lipid biosyn-thesis and the malignant phenotype, lipogenic enzymes havebecome an attractive target for therapeutic intervention. Inparticular, FASN, due to its structure and tumor-specificexpression, represents a unique target for cancer drug discov-ery (48). Despite, multiple publications clearly showing the roleof FASN in cancer cell growth and survival, our study is the firstto provide insight into the contribution of aberrant de novolipogenesis progression andmetastasis of CRC suggesting that

Figure 7. Inhibition of FASNprevents establishment of CRCmetastasis. A, in vivo total bodybioluminescence images ofathymic nude mice in dorsal andventral positions (IVIS@ ImagingSystem) injected i.v. with HT29-NTC-Luc or HT29-FASNshRNA-Luccells. B, quantitative analysis ofmetastasis (estimated by totalluciferase counts per animal) inathymicnudemice injected i.v.withHT29-NTC-Luc or HT29-FASNshRNA-Luc cells. C,representative images of histologicanalysis of lung micrometastasis inmice injected i.v. with HT29-NTC-Luc or HT29-FASNshRNA-Luccells.

Zaytseva et al.





inhibition of FASN may be a potential therapeutic strategy foradvanced stages of CRC. We also show the link betweenexpression of lipogenic enzymes and expression of CD44, theprotein implicated in establishing of metastasis in manycancers including CRC. Humanized CD44 antibodies haveshown promising results in clinical trials, but those trials wereterminated due to skin-related toxicities (49, 50). The findingspresented in this study open the possibility of targeting CD44through inhibition of lipogenesis, thus avoiding antibody-associated toxicities.

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

AcknowledgmentsThe authors thank Donna Gilbreath, Jennifer Rogers, and Nathan Vanderford

for help with manuscript preparation and Greg Bauman and Jennifer Strange forassistance with FACS.

Grant SupportThis work was supported by P20CA1530343 (GI SPORE; B.M. Evers) and

GM50388 and P20RR021954 (A.J. Morris).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received December 16, 2011; revised January 12, 2012; accepted January 13,2012; published OnlineFirst January 19, 2012.

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Figure 7. (Continued ) D, in vivo totalbody bioluminescence images ofathymic nude mice in dorsal andventral positions (IVIS@ ImagingSystem) injected i.v. with KM20-NTC-Luc/GFP or KM20-FASNshRNA-Luc/GFP cells. E,quantitative analysis of metastasis(estimated by total luciferase countsper animal) in athymic nude miceinjected i.v. with KM20-NTC-Luc/GFP or KM20-FASNshRNA-Luc/GFP cells. F, representative imagesof histologic analysis (H&E staining)of lung micrometastasis in athymicnude mice injected i.v. with KM20-NTC-Luc/GFP or KM20-FASNshRNA-Luc/GFP cells.






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2012;72:1504-1517. Published OnlineFirst January 19, 2012.Cancer Res Yekaterina Y. Zaytseva, Piotr G. Rychahou, Pat Gulhati, et al. Signaling and Reduces Metastasis in Colorectal CancerInhibition of Fatty Acid Synthase Attenuates CD44-Associated

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