sphingosine kinase 1 overexpression contributes to...

Cancer Therapy: Preclinical

Sphingosine Kinase 1 Overexpression Contributes toCetuximab Resistance in Human Colorectal Cancer Models

Roberta Rosa1, Roberta Marciano1, Umberto Malapelle2, Luigi Formisano1, Lucia Nappi1, Claudia D'Amato1,Valentina D'Amato1, Vincenzo Damiano1, Gabriella Marf�e6, Silvana Del Vecchio3, Antonella Zannetti3,Adelaide Greco2,4, Alfonso De Stefano1, Chiara Carlomagno1, Bianca Maria Veneziani5,Giancarlo Troncone2, Sabino De Placido1, and Roberto Bianco1

AbstractPurpose: Although the anti–EGF receptor (EGFR) monoclonal antibody cetuximab is an effective

strategy in colorectal cancer therapy, its clinical use is limited by intrinsic or acquired resistance. Alterations

in the "sphingolipid rheostat"—the balance between the proapoptotic molecule ceramide and the

mitogenic factor sphingosine-1-phosphate (S1P)—due to sphingosine kinase 1 (SphK1) overactivation

have been involved in resistance to anticancer-targeted agents. Moreover, cross-talks between SphK1 and

EGFR-dependent signaling pathways have been described.

Experimental design:We investigated SphK1 contribution to cetuximab resistance in colorectal cancer,

in preclinical in vitro/in vivo models, and in tumor specimens from patients.

Results: SphK1 was found overexpressed and overactivated in colorectal cancer cells with intrinsic or

acquired resistance to cetuximab. SphK1 contribution to resistancewas supportedby thedemonstration that

SphK1 inhibitionbyN,N-dimethyl-sphingosine or silencing via siRNA in resistant cells restores sensitivity to

cetuximab, whereas exogenous SphK1 overexpression in sensitive cells confers resistance to these agents.

Moreover, treatment of resistant cells with fingolimod (FTY720), a S1P receptor (S1PR) antagonist, resulted

in resensitization to cetuximab both in vitro and in vivo, with inhibition of tumor growth, interference with

signal transduction, induction of cancer cells apoptosis, and prolongation of mice survival. Finally, a

correlation between SphK1 expression and cetuximab response was found in colorectal cancer patients.

Clin Cancer Res; 19(1); 138–47. �2012 AACR.

IntroductionIn the last few years, cetuximab and panitumumab, 2

monoclonal antibodies (mAb) targeting the EGF receptor(EGFR), have proven to be effective in combination withchemotherapy or as single agents for treatment of metastaticcolorectal cancer (1). However, as is common in cancertherapy, intrinsic or acquired resistance to anti-EGFR drugsby different mechanisms has been widely observed (2).Molecular alterations such as mutations in genes codifyingfor EGFR-dependent signal transducers [K-Ras, B-Raf, phos-

phoinositide 3-kinase (PI3K), and PTEN] have been relatedto primary refractoriness to cetuximab in colorectal cancer.Other mechanisms, such as alternative signaling by differenttyrosine kinase receptors or induction of angiogenesis bytumor-derived factors, could be also involved, particularly inthe onset of resistance over prolonged treatment (3). There-fore, there is an urgent need to identify novel predictivemarkers of response to cetuximab as well as to develop noveltherapeutic strategies for colorectal cancer patients withintrinsic or acquired resistance to this agent.

Sphingolipids are a family of molecules enriched in lipidrafts that contribute to their unique biochemical properties.Sphingolipidmetabolites including ceramide, sphingosine,ceramide-1-phosphate (C1P), and sphingosine-1-phos-phate (S1P) have emerged as bioactive signalingmolecules,with ceramide and sphingoid bases serving as activators ofcell death pathways whereas S1P and C1P primarily exertmitogenic effects (4). Altered regulation of the S1P/cer-amide ratio can lead to an imbalance in the "sphingolipidrheostat" through which these sphingolipid metabolitesinfluence cell fate and tissue homeostasis. The balance ofthesemolecules is critically regulated by sphingosine kinase(SphK), which converts sphingosine to S1P by phosphor-ylation (5). Two SphK isoforms have been cloned andcharacterized to date. SphK1, activated by a variety of

Authors' Affiliations: Dipartimenti di 1Endocrinologia ed Oncologia Mole-colare e Clinica and 2Scienze Biomorfologiche e Funzionali, Universit�a diNapoli "Federico II"; 3Institute of Biostructures and Bioimages, NationalResearch Council; 4CEINGE, Biotecnologie Avanzate; 5Dipartimento diBiologia e Patologia Cellulare e Molecolare "L. Califano", Universit�a diNapoli "Federico II", Naples; and 6Dipartimento diMedicina Sperimentale eScienze Biochimiche, Universit�a di Roma "Tor Vergata", Rome, Italy

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

Corresponding Author: Roberto Bianco, Cattedra di Oncologia Medica,Dipartimento di Endocrinologia e Oncologia Molecolare e Clinica, Uni-versit�a di Napoli "Federico II", Via S. Pansini, 5, 80131 Napoli, Italy. Phone:39-081-7462061; Fax: 39-081-7462061; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-1050

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 19(1) January 1, 2013138

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Published OnlineFirst November 19, 2012; DOI: 10.1158/1078-0432.CCR-12-1050

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growth factors, cytokines and mitogens, is upregulated inmany cancers, often correlating with higher clinical gradeand resistance to standard therapy (6, 7). Consistently,interference with SphK1 activity by dominant-negativemutants or competitive inhibitors such as N,N-dimethyl-sphingosine (DMS), as well as inhibition of S1P bymAbs orS1P receptors antagonists such as fingolimod (FTY720,Novartis), blocks tumorigenesis and tumor angiogenesisin cancer models (8). Moreover, recent studies showed thatalterations of ceramide/S1P rheostatmay be involved in theregulation of resistance to both chemotherapeutics andtargeted agents (9–14).On the basis of this evidence, and as several reports

showed cross-talks between SphK1 and EGFR-dependentsignaling pathways (15, 16), we analyzed the contributionof sphingolipid rheostat alterations to cetuximab resistancein human colorectal cancer models. Moreover, we investi-gated the combination of cetuximab and fingolimod as atherapeutic strategy potentially effective in colorectal can-cers resistant to cetuximab.

Materials and MethodsCompoundsDMS was purchased from Sigma. Cetuximab was kindly

provided by ImClone Systems. Fingolimod was kindlyprovidedbyNovartis International AG(Basel, Switzerland).

Cell culturesHuman SW48, GEO, SW480, LS174T, HCT116, HT29,

and LoVo colorectal carcinoma cell lines were obtainedfrom the American Type Culture Collection. GEO-CR(Cetuximab Resistant) cells were established as previouslydescribed (17).

MTT survival assayCells (104 cells/well) were grown in 24-well plates and

exposed to increasing doses of cetuximab, DMS or fingoli-mod, alone or in combination. The percentage of cellsurvival was determined using the MTT.

Apoptosis assayApoptosis was quantified using the Annexin V-FITC apo-

ptosis kit (BD Biosciences).

Immunoprecipitation and Western blot analysisTotal cell lysates from cell cultures or tumor speci-

mens were resolved by 4% to 15% SDS-PAGE and probedwith anti-human, polyclonal pEGFR, EGFR and SphK1,monoclonal phospho mitogen-activated protein kinase(pMAPK), MAPK and S1PR1/EDG-1 (Santa Cruz Biotech-nology), polyclonal pAkt, Akt and SphK1 phospho-Ser225(Cell Signaling Technologies), and monoclonal actin (Sig-ma-Aldrich). Immunoreactive proteins were visualizedby enhanced chemiluminescence (Pierce). Densitometryanalysis was conducted with Image J software (NIH).

Sphingosine kinase assaySphK1 activity was measured by using Sphingosine

Kinase Activity Assay Kit (Echelon Biosciences; refs. 18, 19).

Quantification of S1PQuantification of S1P from cell protein extracts, tumor

lysates, or mice sera by ELISA assay was conducted using avalidated S1P Assay Kit (Echelon Biosciences; refs. 20, 21).

Hairpin siRNA construct for Sphk1Endogenous Sphk1 expression was downregulated with

sequence-specific pSilencer-siSphK1 851 (Clone1) and1118 (Clone2), as previously described (22), as well aswith Silencer Select Validated siRNA s16957 and s16959. Anonsense sequence was used as a negative control.

Transfection of human SphK1 in human cell linesHuman SphK1 (GenBank accession no. AF200328)

cDNA was cloned into pCMV6-AC-GFP vector (OriGeneRockville). Transient transfections were conducted usingthe Lipofectamine 2000 (Invitrogen).

Nude mice cancer xenograft modelsFive-week-old Balb/c athymic (nuþ/nuþ) mice (Charles

River Laboratories) maintained in accordance with institu-tional guidelines of the University of Naples Animal CareCommittee were injected subcutaneously (s.c.) with GEO-CR cells (107 cells/mice) resuspended in 200 mL of Matrigel(CBP). After 7 days, tumors were detected and groups of 10mice were randomized to receive: Cetuximab 10 mg/kgintraperitoneally (i.p.) 3 times a week for 3 weeks, fingo-limod 2.5 mg/kg i.p. 3 times a week for 3 weeks, or thecombination. Tumor volume (cm3)wasmeasuredusing theformula p/6 � larger diameter � (smaller diameter)2 aspreviously reported (23).

Morphologic and immunohistochemical analysis ofmouse and human tumor samples

The morphologic evaluation of necrosis grade was doneon hematoxilin/eosin stained 5-mm slides by a semiquan-titative score (0: absence; 1: low level; 2: intermediate level;and 3: high level). The presence of apoptotic cells was

Translational RelevanceIn this study, we investigated the contribution of

sphingosine kinase 1 (SphK1) overexpression to cetux-imab resistance, both intrinsic or acquired, in colorectalcancer. In preclinical models with intrinsic or acquiredresistance, SphK1 inhibition through different appro-aches partially restored sensitivity to cetuximab. Theeffect of fingolimod, a clinically available antagonist ofsphingosine-1-phosphate receptor, in cetuximab-resis-tant colorectal cancer models was also shown, bothin vitro and in vivo. Moreover, we observed a correlationbetween SphK1 expression and cetuximab response inmetastatic colorectal cancer patients. Therefore, our datasupport cetuximab plus fingolimod as a novel therapeu-tic combination to be tested in the clinical setting forcetuximab-resistant colorectal cancer patients.

SphK1 and Cetuximab Resistance in Colorectal Cancer

www.aacrjournals.org Clin Cancer Res; 19(1) January 1, 2013 139




determined immunohistochemically on formalin fixedparaffin embedded (FFPE) 5-mm tissue slides by analyzingthe expression of annexin V by the alkaline phosphatasesystem (EnVision, DAKO).

SphK1 immunohistochemistry was carried out on FFPE5-mm tumor tissue sections. Validation of antibodies (24, 25)and analysis of SphK1 expression on FFPE colorectal cancertissues is described in the Supplementary Methods section.

Statistical analysisThe results of in vitro experiments were analyzed by

Student’s t test and expressed as means and SDs for at least3 independent experiments conducted in triplicates. Thestatistical significance was determined by 1-way ANOVAand Dunnett’s multiple comparison posttest about tumorgrowth, by log-rank test concerning mice survival. Allreported P values were 2-sided. Analyses were conductedwith the BioMeDical Package (BMDP) New System statis-tical package version 1.0 for Microsoft Windows (BMDPStatistical Software).

The results of immunohistochemical analysis on colo-rectal cancer tissue specimens and the clinical parameterswere evaluated for statistical significance. SphK1 expressionpatternwas analyzed as following: Level 3 (high expression)versus levels 0 (no expression), 1 (low expression) and 2(intermediate expression). A multivariate analysis was con-ducted to evaluate the correlation of SphK1 levels and otherclinical/pathologic variables with response rate to cetuxi-mab-based therapy (responders vs. nonresponders).Patients were classified as "responders" in case of completeor partial response, and as "nonresponders" in case of stableor progressive disease. k2 test and logistic regression wereused. Estimation of likelihood events for disease progres-sion or death was calculated according to Kaplan–Meier.Statistical differences between curves were calculated usingthe log-rank test. HR and OR were assessed by multivariateanalysis. A P value of 0.05 or less was considered statisticallysignificant. All the analyses were conducted using IBM SPSSStatistics 18 package software (SPSS Inc.).

ResultsSphK1 is overexpressed and overactivated in humancolorectal cancer cell lines resistant to cetuximab

On the basis of the suggested correlation between SphK1and resistance to anticancer-targeted agents (9–14), weanalyzed SphK1 expression in a panel of human colorectalcancer cell lines, both sensitive or resistant to cetuximab.They include SW48 cells, harboring a wild-type K-Ras gene;GEO cells, positive for a K-Ras mutation (Gly12Ala);the cetuximab-resistant derivative GEO-CR cells; SW480,LS174T, HCT116, and LoVo cells, harboring K-Ras muta-tions (Gly12Val for SW480; Gly12Asp for LS174T, andGly13Asp for HCT116 and LoVo); and HT29 cells, positivefor a B-Raf mutation (Val600Glu). As shown in Supple-mentary Fig. S1A and S1B, cetuximab inhibited survival andinduced apoptosis more efficiently in SW48 and GEO thanin the other cell lines. These data are consistent with the K-Ras or B-Raf status of the cell lines, except for GEO cells

which are sensitive to cetuximab despite the K-Ras muta-tion, as reported by our and other groups (19, 26).

When analyzed by Western blot analysis, levels of SphK1protein, as well as of its activated/phosphorylated form(SphK1 phospho-Ser225), were observed to be higher incetuximab-resistant cells compared with cetuximab-sensitivecells. Among the resistant cell lines, low levels of SphK1expression/activation were detected only in HCT116 cells(Fig. 1A), as previously reported (27). Consistently, SphK1enzymatic activity (Fig. 1B), as well as levels of S1Pmeasuredin cell lysates (Fig. 1C), were higher in cells with intrinsic oracquired resistance to cetuximab. Lower SphK1 activity andS1P levels were detected in HCT116 cells (Fig. 1B and C).

We then tested sensitivity of colorectal cancer cell lines toDMS, a potent competitive inhibitor of SphK1.Consistentlywith the previous finding, higher doses ofDMSwere neededto achieve complete enzyme saturation and survival inhi-bition in resistant cells (Fig. 1D). Moreover, the proapop-totic molecule ceramide, a precursor of sphingosine,induced apoptosis less efficiently in resistant than in sen-sitive cells, consistently with the idea that increased SphK1levels mediate S1P synthesis by ceramide in resistant cells.Also in this case, the only exception was the HCT116 cellline (Fig. 1E). As a confirm of SphK1 overactivity, SphK1inhibition by DMS potentiated the effects of ceramide inresistant GEO-CR and SW480 cells, with a 2-fold increase inapoptosis after combined treatment compared with cer-amide alone (Fig. 1F).

SphK1 inhibition partially restores sensitivity tocetuximab in resistant colorectal cancer cell lines

On the basis of the overexpression of SphK1 in resistantcells, we investigated the involvement of this kinase incetuximab resistance by conducting combined treatmentof GEO-CR, SW480, LS174T, HT29 and LoVo cells withDMS and cetuximab. DMS was able to significantly restoresensitivity to cetuximab in resistant cells (SupplementaryFig. S2A). Most interestingly, whereas neither DMS orcetuximabwere able to produce significant apoptosis induc-tion in resistant cells, the combination of these agents waseffective; this result was comparable to that obtained withcetuximab alone in sensitive cells (Supplementary Fig. S2B).These data show that SphK1 blockade may restore cetux-imab activity in resistant cancer cells.

We also studied the effects of modulating SphK1 expres-sion in resistant cancer cell lines. SphK1 gene silencing viasiRNA in GEO-CR, SW480, HT29, and LoVo SphK1-over-expressing resistant cells caused a marked decrease inSphK1 protein expression (Fig. 2A and Supplementary Fig.S3A). SphK1 silencing was not achieved in LS174T cells dueto low transfection efficiency (data not shown). In all thetested cells, treatment with SphK1 siRNA led to reducedAkt phosphorylation/activation (Fig. 2A and Supplemen-tary Fig. S3A), SphK1 enzyme activity (Fig. 2B) and S1Pproduction (Supplementary Fig. S3B). Moreover, as shownin Fig. 2C, SphK1 silencing increased sensitivity to cetux-imab-induced apoptosis in resistant cells. Conversely,SphK1 overexpression in GEO and SW48 sensitive cells

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Clin Cancer Res; 19(1) January 1, 2013 Clinical Cancer Research140




through a full-length expression vector increased SphK1and SphK1 phospho-Ser225 protein levels (Fig. 2D andSupplementary Fig. S3C), enzyme activity (Fig. 2E), and S1Pproduction (Supplementary Fig. S3D). As a further confirm,SphK1 overexpression significantly prevented cell death inresponse to cetuximab in GEO/SphK1 and SW48/SphK1cells compared with controls. Moreover, in these cells,SphK1 inhibition by DMS significantly restored sensitivityto cetuximab-induced apoptosis (Fig. 2F).

The S1PR antagonist fingolimod restores sensitivity tocetuximab in colorectal cancer cell linesTo further assess the role of SphK1 in cetuximab resis-

tance,we used fingolimod (FTY720), a S1PR antagonist thatcould also act as a SphK1 inhibitor. This drug is currentlyavailable for the treatment ofmultiple sclerosis (28) andhasshowed anticancer properties in different models of humancancers (8, 29, 30). This agent moderately inhibits survivalof all cell lines, with an IC50 � 5 mmol/L (data not shown).Then we evaluated fingolimod capability to restore cetux-imab activity in resistant cells. As shown in Fig. 3A and B,fingolimod significantly potentiated survival inhibition andapoptosis induction by cetuximab in GEO-CR, SW480,HT29, and Lovo cells. Therefore, we analyzed the effect ofthe combination cetuximab plus fingolimod on SphK1

enzymatic activity and S1P production. In all cell lines,cetuximab did not affect SphK1 activity and S1P levels,while fingolimod reduced them. Statistically significantreductions of SphK1 enzymatic activity and S1P levels weredetected with the combined treatment (Fig. 3C, D). Finally,we investigated the activation of EGFR signal transducersmainly involved in the onset of resistance to cetuximab. In4 different resistant cells, cetuximab was ineffective ininhibiting Akt and/or MAPK phosphorylation. Fingolimodproduced a variable effect, whereas the combined treat-ment strongly interfered with both Akt and MAPK phos-phorylation/activation in all cell lines (Fig. 3E).

Fingolimod restores sensitivity to cetuximab incolorectal cancer xenografts in nude mice

To confirm the antitumor effect of the combinationfingolimod plus cetuximab also in in vivo models of cetux-imab resistance,we xenograftedGEO-CR cells in nudemice.

On day 70 (10 weeks after tumor cells injection) all themice in the control group reached the maximum allowedtumor size of about 2 cm3. GEO-CR tumors treated withcetuximab initially responded to this agent, but thenresumed an exponential growth rate, reaching 2 cm3 onday 91. Fingolimod inhibited growth of tumors that did notreach the size of 2 cm3 until the end of the experiment, on

Figure 1. Overexpression and overactivation of SphK1 in human colorectal cancer cell lines resistant to cetuximab. A, Western blot analysis of SphK1 andSphK1 phospho-Ser225 expression. Bottom shows the relative optical density of pSphK1 (Ser225) and SphK1 normalized to the actin level. B, SphK1activity was measured by using SphK1 Activity Assay Kit. C, levels of S1P production (mmol/L), as measured by ELISA assays on cell lysates. D, percentageof survival of cells treated with increasing doses of DMS (0.1–5 mmol/L), as measured by the MTT assay. �, 2-sided P < 0.005 versus control. Bars,SDs. E, percentage of apoptosis of cells treated with ceramide (10 mmol/L), as measured by the annexin V assay. �, 2-sided P < 0.005 versus control.F, percentage of apoptosis of GEO-CR and SW480 cells treated with ceramide (10 mmol/L), DMS (1 mmol/L), or the combination, as measuredby the annexin V assay. �, 2-sided P < 0.005 versus ceramide alone. Data represent the mean (�SD) of 3 independent experiments, each conductedin triplicate. Bars, SDs.






day 105. The combination of cetuximab and fingolimodcaused a potent and long-lasting cooperative antitumoractivity, with about 70% of growth inhibition (tumor sizeof 0.6 cm3) until day 105, 11 weeks after treatment with-drawal. Comparison of tumor sizes among different treat-ment groups was statistically significant (Fig. 4A). Consis-tently, as shown in Fig. 4B, mice treated with the combi-nation showed a statistically significantly prolonged medi-an survival duration compared with the controls [mediansurvival 102.5 vs. 37.50 days; HR, 0.07552; 95% confidenceinterval (CI), 0.02130–0.2677; P < 0.0001] or to micetreated with cetuximab as single agent (median survival102.5 vs. 57; HR, 0.1570; 95% CI, 0.04962–0.4966; P ¼0.0016). Treatments were well tolerated; no weight loss orother signs of acute or delayed toxicity were observed(Supplementary Table S1).

Western blot analysis on tumor samples frommice sacri-ficed on day 25 showed that fingolimod not only inhibitsphosphorylation of SphK1 on Ser225, but efficiently inter-feres also with EGFR-dependent signal transduction, byreducing EGFR-, Akt-, andMAPK-activated forms. The com-bination was more efficient than fingolimod alone, pro-ducing an almost total suppression of EGFR and Akt phos-phorylation/activation. No alterations in expression ofSphK1 and S1PR1 were detected (Fig. 4C). ELISA assays ontumor lysates and mice sera revealed that fingolimod sig-nificantly reduces S1P production by tumor cells, but thecombined treatment was much more effective (Fig. 4D).

On the basis of the proapoptotic effect observed in vitrowith the combination of cetuximab and fingolimod, weconducted imaging of apoptosis in GEO-CR tumor-bearingnude mice, 72 hours after the first treatment. As shown in

Figure 2. Effects of SphK1 modulation on cetuximab sensitivity in resistant colorectal cancer cell lines. A, Western blot analysis of protein expression inGEO-CR, SW480, HT29, and LoVo cells transfected with an SphK1-specific siRNA or with a negative control. B, SphK1 activity was measured in cellstransfected with a SphK1 specific siRNA or with a negative control by using SphK1 Activity Assay Kit. �, 2-sided P < 0.005 versus cells transfected with thenegative control. C, percentage of apoptosis of cells transfected with a SphK1 specific siRNA or a negative control and treated with cetuximab (140 nmol/L),as measured by the annexin V assay. �, 2-sided P < 0.005 versus cells transfected with the SphK1 specific siRNA, but untreated with cetuximab. D,Western blot analysis of protein expression inGEOandSW48 cells transfectedwith humanSphK1 cDNA or an empty vector. E, SphK1 activity wasmeasuredin cells transfected with human SphK1 cDNA or an empty vector by using SphK1 Activity Assay Kit. �, 2-sided P < 0.005 versus cells transfected withthe empty vector. F, percentage of apoptosis of cells transfectedwith human SphK1 cDNA or an empty vector and treated with cetuximab (140 nmol/L), DMS(1 mmol/L), or the combination, as measured by the annexin V assay. �, 2-sided P < 0.005 versus cetuximab alone. Data represent the mean (�SD) of 3independent experiments, each conducted in triplicate. Bars, SDs.

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Supplementary Fig. S4, a relevant apoptosis induction wasdetected in mice treated with the combination comparedwith mice treated with cetuximab or fingolimod as singleagents. We also investigated the long-term induction ofapoptosis by conducting immunohistochemical analysisof annexin V on tumor samples from mice sacrificed onday 25: a relevant staining was found only in tumors treatedwith both cetuximab and fingolimod (Fig. 5A). Moreover,the necrosis grade was low in the control, intermediate inthe cetuximab- or fingolimod-treated tumors, and high inthe combination-treated tumors (Fig. 5B).

SphK1 expression is related to cetuximab response incolorectal cancer patientsOn the basis of this body of data, and as alterations of

ceramide/S1P rheostat mediated by SphK1 overexpressionhave been involved in reduced response to therapy inhuman cancers (9–12, 14), we evaluated SphK1 expressionin paraffin-embedded, archived clinical tumor tissue speci-mens obtained from 50 cases of K-Ras wild-type colorectalcancer patients enrolled in controlled clinical trials and

treated with cetuximab-containing regimens. To evaluateSphK1 expression, 3 different Abs were tested, but only oneyielded a strong signal intensity (Supplementary Fig. S5A).This polyclonal Ab was validated for specificity and repro-ducibility by using a previously described algorithm (25).Briefly, it showed a band of the expected molecular weightin Western blot analysis, produced a specific and localizedstaining when titered on tissue microarray containing con-trol tissues, and was reproducible between different runsand lots (Supplementary Fig. S5B and S5C).

Representative pictures of samples with different SphK1expression levels are shown in Supplementary Fig. S6A. Asreported in Table 1, 22 of the 50 tumor samples (44%)expressed high levels of SphK1. Among them, 19 (87%)derived from patients who did not respond to cetuximab-based therapy. Statistical analysis showed that the correla-tion between high levels of SphK1 expression and poorresponse to cetuximabwas significant (P¼0.03).Moreover,as shown in Supplementary Fig. S6B, progression freesurvival (PFS) seemed in favor of patients with low levelsof SphK1 expression, with an advantage of 2.4 months

Figure 3. Effects of the S1PR antagonist fingolimod on cetuximab sensitivity in colorectal cancer cell lines. A, percentage of survival of cells treated withincreasing doses of cetuximab (7–350 nmol/L), in presence or not of fingolimod (1 mmol/L), as measured by the MTT assay. �, 2-sided P < 0.005 versus cellstreated with cetuximab alone. B, percentage of apoptosis of cells treated with cetuximab (140 nmol/L), fingolimod (5 mmol/L), or the combination, asmeasured by the annexin V assay. �, 2-sided P < 0.005 versus control. C, SphK1 activity was measured by using SphK1 Activity Assay Kit. ��, 2-sidedP < 0.05 and �, 2-sided P < 0.005 versus untreated control cells. D, percentage of S1P production, as measured by ELISA assays on cell lysates. ��, 2-sidedP < 0.05 versus control. E, Western blot analysis of protein expression on GEO-CR, SW480, HT29, and LoVo cells treated for 24 hours with cetuximab(140nmol/L), fingolimod (1mmol/L), or the combination. Data represent themean (�SD) of 3 independent experiments, eachconducted in triplicate. Bars, SDs.






(median PFS 23.6 vs. 14.0 weeks, P¼ 0.05). Despite a trendin favor of patients with low SphK1 levels, the difference interms of overall survival (OS) did not reach the statisticalsignificance (median OS 144.9 vs. 82.7 weeks, P¼ 0.5; datanot shown). To exactly delineate the role of SphK1 expres-sion as a predictive/prognostic marker in the cohort ofpatients presented, a multivariate analysis was conductedincluding other potential confounding factors, such aschemotherapy line, type of cetuximab-containing regimen,tumor stage at the time of diagnosis. As reported in Sup-plementary Table S2, SphK1 expression was the only factorshowing a statistically significant correlation with responseto cetuximab (P ¼ 0.03).

Overall, these data support the hypothesis that SphK1expression may correlate with resistance to EGFR targetedtherapy in colorectal cancer patients.

DiscussionAlthough a number of molecular alterations have been

identified as responsible for resistance to EGFR inhibitorsavailable in clinical practice, such as cetuximab for colo-rectal cancer, each of these mechanisms only partiallyjustifies the lack of response in patients. Therefore, the

search for further determinants of resistance may help tobetter select patients potentially responsive to cetuximaband to develop novel therapeutic strategies for resistantcancers (1, 2).

On the basis of the evidence that alterations of ceramide/S1P rheostat may be involved in resistance to biologicagents (9, 13, 14) and as some reports showed cross-talksbetween SphK1 and EGFR-dependent pathways (15,16, 31), we investigated the role of SphK1 in the onset ofresistance to cetuximab in colorectal cancers. To this aim,weanalyzed SphK1 expression and activation in preclinicalmodels of colorectal cancer, both sensitive orwith intrinsic/acquired resistance to cetuximab. We found that SphK1 isoverexpressed and overactivated in colorectal cancer celllines resistant to cetuximab. In fact, high expression of theactivated form of SphK1, produced by MAPK-mediatedphosphorylation on Ser225 (32), as well as high levels ofenzyme activity and S1P production, were detected inresistant cells. Then, we investigated whether SphK1 inhi-bition could restore cetuximab sensitivity in resistant cancercells. To this aim, we used different approaches, includingDMS, a potent, even if not specific (33), competitive inhib-itor of SphK1, the S1PRs antagonist fingolimod, and siRNAs

Figure 4. Effects of the combination cetuximab plus fingolimod on tumor growth, survival, and signal transduction ofmice xenografted with GEO-CR resistanttumors. A, after 7 days following subcutaneous injection of GEO-CR cells, mice were randomized (10/group) to receive cetuximab, fingolimod, or theircombination, as described in the Materials and Methods section. The 1-way ANOVA test was used to compare tumor sizes among treatment groups at themedian survival time of the control group (35 days). The results were statistically significant for the combination versus single agents (P < 0.0001). B, mediansurvival was statistically significant for the combination versus control or cetuximab (log-rank test). C, Western blot analysis was conductedon total lysates from tumor specimens of 2 mice sacrificed on day 25. D, quantification of S1P (mmol/L) on tumor lysates and mice sera by ELISA assays.��, 2-sided P < 0.05 and �, 2-sided P < 0.005 versus control. Data represent the mean (�SD) of 3 independent experiments, each conducted in triplicate,and are presented relative to control. Bars, SDs.

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specific for SphK1. By combining each of these tools withcetuximab on resistant cells, we showed that SphK1 inhi-bition was able to partially restore cetuximab capability toaffect cell survival, apoptosis and EGFR-dependent signaltransduction. We particularly focused on signal transducersclassically involved in the development of resistance toEGFR inhibitors, such as the PI3K/Akt/mTOR and theRas/mitogen-activated protein–extracellular signal-regulat-ed kinase/MAPK pathways: The combination of cetuximabwith fingolimod produced a strong suppression of Akt andMAPK phosphorylation/activation in resistant cells. These

results are consistent with other studies showing the capa-bility of S1P to induce cell proliferation and survivalthrough activation of the Akt pathway (34–36). Mostimportantly, the resensitization to cetuximab induced bySphK1 inhibition was observed in different models ofresistance: SW480, HT29, and LoVo cells, whose intrinsicresistance to cetuximab is related to K-Ras or B-Raf muta-tions and consequent overactivation of the Ras/MAPKpathway, and GEO-CR cells, whose acquired resistance isdue to PI3K/Akt overactivation (17).

It has been described that S1P produced by SphK1 mayfunction as a second messenger inside the cell or may besecreted to bind to S1PRs on the cell surface. This signaling,involved in several human diseases including cancer, isdefined as "inside-out" S1P signaling (37, 38). Therefore,the resensitization to cetuximab that we observed in our cellmodels may depend on cross-talks of EGFR pathway withboth intracellular S1P and/or extracellular S1P/S1PRs sig-naling pathways. By comparing the effects of SphK1 andS1PRs inhibitors, we attempted to explain this point anddiscriminate between intracellular and extracellular effectsof S1P. Fingolimod has been initially defined as a S1PRsantagonist: its phosphorylated form, produced by SphK2,binds to S1PRs and elicits their polyubiquitination, endo-cytosis, and degradation (39). In this study, we observed noalteration in S1PR1 protein expression after treatment ofGEO-CR xenografts with fingolimod. This result seems torule out that the effect of fingolimod depends on its

Figure 5. Effects of the combination cetuximab plus fingolimod on apoptosis and necrosis of GEO-CR tumor xenografts. A, immunohistochemical analysisof the apoptotic marker annexin V on FFPE 5-mm tissue slides. B, morphologic evaluation of necrosis-grade on hematoxilin/eosin stained 5-mm slides.

Table 1. Correlation of SphK1 expression withresponse to cetuximab-based therapy incolorectal cancer patients

Cross tabulation response versus SphK1

SphK1

Lowexpression

Highexpression Total

Response Responders 11 3 14Nonresponders 17 19a 36

Total 28 22 50

aP ¼ 0.03 vs. responders group.






antagonistic activity on S1PRs. However, it has beendescribed that fingolimod can act also as a SphK1 inhibitor(29). Therefore, the resensitization to cetuximab in our cellmodels may depend on cross-talks of EGFR pathway withintracellular S1P signaling rather than with extracellularS1P/S1PRs signaling pathways. Further investigations areneeded to clarify this point.

As several preclinical studies reported the activity offingolimod in human cancer models (8), the antitumoreffect of this agent in colorectal cancers with acquiredresistance to cetuximab was investigated also in vivo, innude mice xenografted s.c. with GEO-CR cells. The combi-nation of cetuximab and fingolimod caused a potent andlong-lasting cooperative antitumor activity, with inhibitionof tumor growth, interference with signal transduction,induction of apoptosis, and prolongation of mice survival.

As a further confirmation of our preclinical data, weexamined SphK1 expression in tumor specimens fromcolorectal cancer patients. To date, SphK1 overexpressionhas been described in colorectal cancers compared withcorresponding normal tissues (40), but we reported for thefirst time an interesting correlation between SphK1 expres-sion and poor response to cetuximab therapy. This resultshould be interpreted with caution due to the limitednumber of patients included in the analysis and it couldbe used to design dedicated, prospective clinical trials toexplore the potential role of SphK1 as a biologic marker ofresistance to cetuximab.

Taken together, we show for the first time that SphK1inhibition is effective in restoring cetuximab antitumoractivity in colorectal cancers resistant to this agent. More-

over, as fingolimod is a clinically available drug (28), theresults of the present study suggest cetuximab plus fingoli-mod as a novel therapeutic strategy to be tested in theclinical setting for colorectal cancer patients with resistanceto cetuximab.

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

Authors' ContributionsConception and design: R. Rosa, B.M. Veneziani, S. De Placido, R. BiancoDevelopmentofmethodology:R. Rosa, V.D’Amato, V. D’Amato, A.Greco,S. De Placido, R. BiancoAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): R. Rosa, R. Marciano, U.Malapelle, L. Formisano,L. Nappi, C.D’Amato, G.Marfe, S. Del Vecchio, A. Zannetti, A. De Stefano, C.Carlomagno, R. BiancoAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): R. Rosa, R. Marciano, L. Formisano, L.Nappi, C. D’Amato, V. D’Amato, A. De Stefano, G. Troncone, S. De Placido,R. BiancoWriting, review, and/or revision of themanuscript: R. Rosa, V. D’Amato,C. Carlomagno, B.M. Veneziani, R. BiancoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R. Marciano, L. Formisano, L.Nappi, C. D’Amato, A. De Stefano, R. BiancoStudy supervision: S. De Placido, R. Bianco

Grant SupportThis study was supported by Associazione Italiana per la Ricerca sul

Cancro (AIRC) My First Grant 2011-2014 (MFAG-11473) to R. Bianco.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 indicatethis fact.

ReceivedMarch30, 2012; revisedOctober 24, 2012; acceptedNovember 8,2012; published OnlineFirst November 19, 2012.

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2013;19:138-147. Published OnlineFirst November 19, 2012.Clin Cancer Res Roberta Rosa, Roberta Marciano, Umberto Malapelle, et al. Resistance in Human Colorectal Cancer ModelsSphingosine Kinase 1 Overexpression Contributes to Cetuximab

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