c-kit is suppressed in human colon cancer tissue and...

Molecular and Cellular Pathobiology

c-Kit Is Suppressed in Human Colon Cancer Tissue andContributes to L1-Mediated Metastasis

Nancy Gavert1, Anna Shvab1, Michal Sheffer2, Amir Ben-Shmuel1, Gal Haase1, Eszter Bakos1,Eytan Domany2, and Avri Ben-Ze'ev1

AbstractThe transmembrane neural cell adhesion receptor L1 is aWnt/b-catenin target gene expressed in many tumor

types. In human colorectal cancer, L1 localizes preferentially to the invasive front of tumors and whenoverexpressed in colorectal cancer cells, it facilitates their metastasis to the liver. In this study, we investigatedgenes that are regulated in human colorectal cancer and by the L1-NF-kB pathway that has been implicated inliver metastasis. c-Kit was the most highly suppressed gene in both colorectal cancer tissue and the L1-NF-kBpathway. c-Kit suppression that resulted from L1-mediated signaling relied upon NF-kB, which directly inhibitedthe transcription of SP1, a major activator of the c-Kit gene promoter. Reconstituting c-Kit expression in L1-transfected cells blocked the biological effects conferred by L1 overexpression in driving motility and livermetastasis. We found that c-Kit expression in colorectal cancer cells is associated with a more pronouncedepithelial morphology, along with increased expression of E-cadherin and decreased expression of Slug. Althoughc-Kit overexpression inhibited the motility and metastasis of L1-expressing colorectal cancer cells, it enhancedcolorectal cancer cell proliferation and tumorigenesis, arguing that separate pathways mediate tumorigenicityand metastasis by c-Kit. Our findings provide insights into how colorectal cancer metastasizes to the liver, themost common site of dissemination in this cancer. Cancer Res; 73(18); 5754–63. �2013 AACR.

IntroductionAberrant activation ofWnt/b-catenin signaling is a hallmark

in the majority of human colorectal cancers (1–3). A centralquestion in human colorectal cancer development is theidentification and role of downstream b-catenin-T-cell factor(TCF) target genes. We identified members of the neuralimmunoglobulin-like cell adhesion receptors L1 and Nr-CAM(4, 5) as b-catenin-TCF target genes in colorectal cancer cellsand showed that L1 is exclusively expressed in cells at theinvasive front of human colorectal cancer tissue (5). Over-expression of L1 in human colorectal cancer cells conferredenhanced motility and metastasis to the liver in a mousemetastasis model (6). We further showed that themechanismswhereby L1 confers such metastatic capacities involve anassociation of the L1 juxtamembrane cytoplasmic domainwith the cytoskeletal linker protein ezrin andwith the inhibitorof kB (IkB) component of the NF-kB signaling pathway (7).This association results in ezrin phosphorylation/activation by

Rho-associated protein kinase and an enhanced phosphoryla-tion and proteasomal degradation of IkB leading to the release,nuclear localization, and increased activation of NF-kB-medi-ated transcription (8). In this studywewished to determine thetarget genes of the L1-NF-kBpathway that are also regulated ina large set of human colorectal cancer tissue samples com-pared with normal colon tissue. Surprisingly, we found that theexpression of the tyrosine kinase growth factor receptor c-Kit issuppressed in human colorectal cancer tissue when comparedwith normal colon tissue, as well as in L1-mediated NF-kBsignaling that promotes colorectal cancer cell metastasis. Wereport on the mechanisms whereby the L1-NF-kB pathwayinhibits c-Kit expression and the means by which the metas-tasis suppressive effects are conferred by c-Kit in colorectalcancer cells.

Materials and MethodsCell culture, proliferation, artificial wound closure, andtransfections

HEK 293T, Ls174T, and DLD-1 cells were grown as described(6). Ls174T-L1, Ls174T-p65, and Ls174T-control cells weremaintained in medium containing neomycin (800 mg/mL),Ls174T-L1þshp65, Ls174-L1þIkB-SR, and Ls174T-L1þc-Kitcells in medium with both neomycin (800 mg/mL) and puro-mycin (10 mg/mL). For cell growth assays, 104 cells/well wereseeded in 96-well dishes and cell number determined intriplicates for 5 days. An artificial wound was introduced intoconfluent cell cultures using a micropipette. The medium wasreplaced with fresh medium containing 0.35 mg/mL mitomy-cin-C to inhibit cell proliferation. Pictures were taken at 0 and

Authors' Affiliations: Departments of 1Molecular Cell Biology; and 2Phys-ics and Complex Systems, The Weizmann Institute of Science Rehovot,Israel

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

Corresponding Author: Avri Ben-Ze'ev, Department of Molecular CellBiology, TheWeizmann Institute of Science, Rehovot 76100, Israel. Phone:972-8-9342422; Fax: 972-8-9465261; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-13-0576

�2013 American Association for Cancer Research.

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24 hours after introducing the wound and percent woundclosure was determined. Transient transfection of HEK 293Tcells was conducted using the calcium-phosphate method.Ls174T and DLD-1 cells were transfected using Lipofecta-mine 2000 (Invitrogen). To measure the phosphorylation ofAKT, the cells were starved overnight and then medium with10% FCS was added for serum activation. Inhibition of phos-phoinositide 3-kinase (PI3K) was achieved using 50 mmol/LLY294002 (Sigma-Aldrich) added 12 hours after platingcells and further incubation of the cells for 36 hours beforeharvesting. The cell lines were obtained from American TypeCulture Collection and passaged for less than 6 months afterreceipt.

PlasmidsThe wt L1, p65, and IkB-SR cDNA were previously described

(7). c-Kit cDNA was obtained from Dr. Francoise Moreau-Gachelin (Institute Curie, Paris, France). The c-Kit responsivepromoter reporter plasmid was provided by Dr. MenasheBar-Eli (University of Texas MD Anderson Cancer Center,Houston, TX). SP1 cDNA was provided by Dr. Jonathon M.Horowitz (North Carolina State University, Chapel Hill, NC)and Dr. Grace Gill (Tufts University Medical School, Boston,MA). p65 shRNA was prepared in pSUPER.puro accordingto the manufacturer's instructions (pSUPER.puro RNAi Sys-tem; OligoEngine) using the target sequences shown in Sup-plementary Table S1.

Luciferase reporter assaysHEK 293T cells were transiently cotransfected in triplicate

plates with 0.1 mg L1, p65, or pcDNA3 expression vector,together with 0.25 mg/mL b-galactosidase plasmid and 0.25mg c-Kit promoter reporter plasmids in pGL3, or with 0.25mg/mL of empty pGL3. Cells were lysed 24 hours after trans-fection, and luciferase and b-galactosidase levels were deter-mined by the luciferase assay system (Promega). Luciferaseactivity was normalized to b-galactosidase activity for trans-fection efficiency.

RT-PCRRNA was isolated using the EZ-RNA kit (Biological Indus-

tries). PCR was conducted using the sequences shown inSupplementary Table S1. Relative gene expression was calcu-lated by quantitative real-time PCR (qRT-PCR) with primersdesigned for glyceraldehyde-3-phosphate dehydrogenase(GAPDH), E-cadherin, AP-2, and SP1 (see SupplementaryTable S1). qRT-PCR was conducted on the ABgene thermo-cycler with the ABsolute SYBR Green ROX Mix (ABgene).Triplicates of 1 ng cDNA template and 500 nmol/L gene-specific primers were used. The GAPDH gene served to nor-malize for RNA levels. Primers were examined for efficiency,displaying an amplification slope of �3.33 � 0.3 and r2 > 0.98.qRT-PCR was started by incubating the samples at 95�C for 10minutes followed by PCR amplification cycles (95�C for 20seconds and 60�C for 1minute for 40 cycles). Data analysis wasconducted with the DDCT method with the ABgene thermo-cycler software. For semiquantitative RT-PCR, the productswere analyzed by agarose gel electrophoresis.

Chromatin immunoprecipitation assaysRabbit anti-p65 (sc-109; Santa Cruz Biotechnology, Inc.) and

SP1 (pep2; sc-59; Santa Cruz Biotechnology, Inc.) were used forthe immunoprecipitation and rabbit anti-immunoglobulin G(IgG; Jackson ImmunoResearch Laboratories, Inc.) as controlantibody. Chromatin immunoprecipitation (ChIP) was carriedout as described (8), with the exception that the DNA waspurified using the PCR Purification Kit (Promega) and sub-jected to PCR with the specific primers shown in Supplemen-tary Table S1.

Immunoblotting and immunofluorescenceImmunoblotting was carried out as described (7) using

the following antibodies: mouse anti-IkBa/MAD-3 (BD Bio-sciences at 1:1,000), goat Ab against NF-kB p65 (sc-109;Santa Cruz Biotechnology, Inc. at 1:1,000), rabbit anti-c-Kit(Cell Signaling Technologies, Inc. at 1:1000), mouse anti-E-cadherin (BD Biosciences at 1:1000), rabbit anti-L1 (gift fromDr. Vance Lemmon, University of Miami, Miami, FL at1:5,000), rabbit anti-total AKT (Sigma-Aldrich at 1:10,000),mouse anti-phospho-AKT (Ser473; Cell Signaling Technolo-gies, Inc. at 1:5,000), mouse anti-SP1 (pep2; Santa CruzBiotechnology, Inc. at 1:10,000), rabbit anti-histone H2B(Merck-Millipore at 1:5,000), and mouse anti-a-tubulin (Sig-ma-Aldrich at 1:200,000). Western blots were developedusing the ECL method (Amersham Biosciences). For immu-nofluorescence, cells cultured on glass coverslips were per-meabilized with 0.5% Triton X-100 and fixed with 3% PFA.Immunostaining for E-cadherin was conducted with cellsfixed in methanol for 5 minutes at 4�C. The secondaryantibodies were Alexa-488-conjugated goat antimouse,anti-rabbit IgG (Invitrogen), and Cy3-labeled goat antimouseor anti-rabbit IgG (Jackson ImmunoResearch Laboratories).Images were acquired by using the Zeiss LSM710 confocalmicroscope with objective 60�/1.4 NA.

DNA microarraysLs174T clones expressing L1 were compared with Ls174T

cells expressing L1þIkB-SR, p65, or the empty vector. Twoindividually isolated clones were used in each group. Micro-array analysis with RNA extracted from these cells was con-ducted at the Weizmann Institute Microarray Facility onAffymetrix 1.0st GeneChips. The chips were processed andanalyzed as follows: the data were preprocessed using robustmultiarray average (RMA) followed by cyclic Lowess correctionalgorithm (9). Intensity-dependent variance was estimated foreach probe set, using the distribution of the differencesbetween repeats of probe sets having similar mean intensity(10). To look for differentially expressed probe sets between thedifferent conditions, 3 comparisons were made: L1 versuscontrol (denoted C1), p65 versus control (C2), and L1þIkB-SR versus L1 (C3). Each comparison was made between 2corresponding pairs of replicates using a filtering step thatincluded probe sets that were above threshold (t¼ 6) in at leastone of the conditions of each comparison. Probe sets withunknown gene symbol were removed from the analysis. Mod-ified Fisher inverse c2 approach (11) was used to calculate ameta P-value from the comparisons described earlier. For each

Suppression of c-Kit in CRC Metastasis

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comparison, the genes were ranked according to their P-valuesand assigned a new uniform distribution based on their rankdivided by the total number of genes Rij ¼ rank(P-valueij)/N,for gene i in comparison to j. The values of Rij were used forcalculating Si ¼ �2log(P jRij), which follows a c2 distribution.The meta P-values were assigned according to the c2 distri-bution and 25% false discovery rate (FDR) was used to controlthe FDR. From this list we chose the genes that were over-expressed in both C1, C2 and underexpressed in C3, or under-expressed in C1, C2 and overexpressed in C3. Expression datafrom a large set of colorectal cancer and normal tissue (12) wasused to determine the differential expression of the chosengenes, between 52 samples of normal tissue versus the union of182 primary tumors and 30 metastasis samples (Mann–Whit-ney, 1% FDR). Data were normalized using MAS5 and cyclicLowess correction algorithm (9) followed by log2 transforma-tion. The expression level for each gene was determined as theaverage expression of its probe sets. The reduction in c-Kit RNAwas similar in primary tumors and metastases as comparedwith normal colonic tissue.

Tumor growth and metastasis assaysTumor growth was induced by injecting s.c. 2.5� 106 cells in

200 mL PBS into the flanks of nude mice (5 mice per group).Control cells were injected into the opposite flank of the samemice and tumors were removed and compared after 2 to 4weeks. For metastasis assays, groups of 10 to 15 mice wereinjected with 1� 106 cells in 20 mL PBS into the distal tip of thespleen. After 6 to 7 weeks the spleens and livers were removedas described (6).

StatisticsStatistical significance was determined by the Fisher exact

test for mouse metastasis experiments. Tumor mass wascompared and significance determined by ANOVA. The sig-nificance of qRT-PCR comparisons for RNA levels was deter-mined by ANOVA. In wound closure and luciferase reporterassay studies the significance was determined by ANOVA. A Pvalue of < 0.05 was considered significant. Standard deviationfor phosphorylated AKT as a percentage of total AKT wascalculated from the results of 3 experiments. The rate ofincrease in phosphorylated AKTwas calculated after assigninga linear function to each curve: L1þc-Kit; y¼ 15.19x þ 49.213;R2 ¼ 0.99 and L1; y ¼ 8.81x þ 31.748; R2 ¼ 0.98.

ResultsL1 suppresses c-Kit expression in colorectal cancer cellsby activating NF-kB signaling

In recent studies, we showed that the neural transmembraneadhesion receptor L1 promotes the metastasis of humancolorectal cancer cells by activating NF-kB signaling (7). Wewished to identify genes that are involved in L1-NF-kB-medi-ated promotion of colorectal cancer metastasis in humancolorectal cancer tissue. For this, gene expression profiles ofhuman colorectal cancer cells overexpressing L1 were com-pared with those of cells overexpressing L1 together with theIkB-super repressor (IkB-SR) inhibiting NF-kB activation (Fig.1A). To identify only those genes that are L1 and NF-kB

dependent, we further compared this gene expression patternwith that of cells overexpressing the p65 NF-kB subunit (Fig.1A, relevant genes). We detected both genes that were upre-gulated and genes that were downregulated in these compar-isons (Supplementary Tables S2 and S3). To select only thosegenes that have in vivo biological significance, we comparedthe gene expression patterns of Supplementary Tables S2 andS3 with genes that were regulated in 212 human colorectalcancer tissue samples and compared metastases with 52normal colon tissue samples. About 20% of the genes thatwere downregulated by L1 and NF-kB were also expressed atlower levels in human tumors as compared with normal colontissue (Fig. 1C and Supplementary Table S4). Surprisingly,among these genes, c-Kit was on top of the list of genes whoseexpression was most robustly reduced by both L1, NF-kB andin human colorectal cancer tissue samples (Fig. 1C). c-Kit ismost commonly associated with oncogenesis (13), but accord-ing to the data in Fig. 1C c-Kit seemed to be among the genesthat are suppressed during tumorigenesis. We were thereforeinterested in investigating how, in L1-NF-kB colorectal cancercell signaling, c-Kit may act to suppress tumor progression.

We used Ls174T human colon cancer cell lines in whicheither L1, NF-kB (p65), or a combination of L1 and IkB-SRwereoverexpressed (7) to examine the levels of c-Kit. RT-PCRanalysis revealed that Ls174T cells overexpressing L1 (Fig.2A, lane 2) displayed a marked reduction in c-Kit RNA (Fig.2A, lane 2 compare to lane 1) and protein levels as comparedwith control (Fig. 2B, lanes 3 and 4 compare to lanes 1 and 2).When NF-kB signaling was inhibited in L1-transfected cells bythe stable expression of IkB-SR (Fig. 2A, lanes 3 and 4), or withshRNA to the p65 NF-kB subunit (Fig. 2A, lanes 5 and 6), c-KitRNA levels were partially restored in 2 independently isolatedcell clones in each case (Fig. 2A, lanes 3–6). Furthermore, 2clones of colorectal cancer cells overexpressing the p65 subunitof NF-kB (but not L1) also downregulated c-Kit RNA (Fig. 2A,lanes 7 and 8 compare to lane 1) and protein levels (Fig. 2B,lanes 5 and 6 compare to lanes 1 and 2), similar to L1 (Fig. 2A,lane 2; 2B, lanes 3 and 4). This ability of L1 to suppress c-Kitexpression was not limited to Ls174T colorectal cancer cellsand was also observed in clones of DLD-1 colorectal cancercells overexpressing L1 (Fig. 2C). The reduction in c-Kit RNAlevels resulted from a decrease in c-Kit transcription, becauseboth L1 and the p65 subunit of NF-kB inhibited the activationof the c-Kit gene promoter reporter plasmid (Fig. 2D). More-over, the inhibition of NF-kB activity, by IkB-SR in L1-expres-sing cells, restored the activity of the c-Kit promoter (Fig. 2D,compare pcDNA3 to L1þIkB-SR). Together, these resultsindicate that L1 reduces the transcription of c-Kit through theactivation of NF-kB signaling.

L1 andNF-kB are suppressing c-Kit transcription via SP1regulation

We next wished to determine the mechanism whereby L1,via NF-kB activation, downregulates c-Kit transcription.Because previous reports indicated that c-Kit gene expressionis regulated by the AP-2 transcription factor (14), we deter-mined the levels of AP-2 in Ls174T cells stably expressing L1,the p65 NF-kB subunit, or the empty vector, but did not find

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Cancer Res; 73(18) September 15, 2013 Cancer Research5756




significant differences in AP-2 RNA levels in such cells (Sup-plementary Fig. S1). Next, we identified 3 putative NF-kBbinding sites within the promoter region of c-Kit (Supplemen-tary Fig. 2A) and conducted ChIP analyses to examine whetherNF-kB directly binds to these sequences to inhibit c-Kit tran-scription. However, we did not detect a binding of the p65NF-kB subunit to these sequences in the c-Kit promoter(Supplementary Fig. S2B). Another known transcriptionalregulator of c-Kit expression is the transcription factor SP1(15). Quantitative RT-PCR analysis detected a significantdecrease in SP1 RNA levels in both L1 and even more in p65overexpressing colorectal cancer cell clones compared withcontrol (Fig. 3A), in accordance with the reduction in c-Kitprotein level by L1 and p65 (Fig. 2B). A marked decrease in SP1levels was also detected in p65 overexpressing cell clones(Fig. 3B, lanes 7 and 8). Moreover, we found that L1-expressingcells displayed a dramatic reduction in the activity of the c-Kitgene promoter as compared with control cells and re-intro-

duction of SP1 into these cells (with or without L1) activatedthe c-Kit promoter (Fig. 3C). By ChIP experiments we alsoshowed that SP1 binds to the c-Kit promoter in Ls174Tcolorectal cancer cells (Fig. 3D). Because a previous studyshowed that NF-kB can reduce SP1 transcription directly byits binding to SP1 promoter sequences (16), we conductedChIP experiments to examine whether in Ls174T colorectalcancer cells NF-kB binds to the SP1 promoter and found thatthis is indeed the case (Fig. 3E). We therefore conclude thatchanges in SP1 levels are involved in reducing c-Kit transcrip-tion, via an NF-kB-mediated inhibition of the SP1 promoter,suggesting that by this mechanism NF-kB downregulatesc-Kit transcription in L1-expressing cells (Fig. 3F).

c-Kit suppresses L1-mediated motility and metastasis incolorectal cancer cells

Because c-Kit was among the genes whose expression issuppressed by L1 during human colorectal cancer progression

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Figure 1. c-Kit RNA levels arereduced by both L1 and NF-kBin human colon cancer tissuesamples. A, Ls174T clonesexpressing L1 were comparedwithcontrol clones (genes regulated byL1, blue circle in diagram). Thisgroup was compared with genesexpressed in Ls174T cellsexpressing L1þIkB-SR ascompared with controls (genesregulated by IkB-SR in L1-expressing cells, yellow circle) andfurther comparedwith Ls174T cellsexpressing p65 or the empty vector(genes regulated by NF-kB, redcircle). Two individually isolatedclones were checked in eachgroup. Genes that were regulatedin all 3 comparisons were chosento identify those upregulated byL1 and p65 and downregulatedby IkB-SR or downregulated byL1 and p65 and upregulated byIkB-SR. Microarray analysis withRNA extracted from these cellswas conducted on AffymetrixGeneChips. B and C, heat maps ofgene expression data from a largeset of colorectal tumor and normalcolon tissue were used to checkfor differential expression of thechosen genes (relevant genes in A),between 52 samples of normaltissue and 212 samples of primarytumors and metastases (Mann–Whitney, 1% FDR). Log2-foldratios of both upregulated (B) anddownregulated (C) genes fromthis comparison are shown.Upregulation, red; downregulation,blue.






(Fig. 1C), we wished to determine whether the reduction inc-Kit contributes to L1-mediated promotion of metastasis (6,7). We reintroduced c-Kit into L1 overexpressing cells by stabletransfection (Fig. 4A) and found that cells expressing both L1and c-Kit are lessmotile than cells expressing L1 alone (Fig. 4B).In fact, these cells were only asmotile as control cells lacking L1(Fig. 4C). Most importantly, the metastasis of L1 transfectedcells in which c-Kit expression was reconstituted was dramat-ically reduced (Fig. 4D and E). Fewer than 10% of micedeveloped liver metastasis after splenic injection comparedwith almost 90% in L1-expressing colorectal cancer cells(Fig. 4E). These cells continued to express L1 in vivo as showedby analysis of the splenic primary tumor and, in the case ofisolated liver metastases, also in the liver metastatic tissue(Fig. 4F). We conclude that without the suppression of c-Kit,L1 expression in colon cancer cells is insufficient to promotemetastasis, and the loss of c-Kit is an essential step in themechanism of L1-mediated metastasis in colorectal cancer.Interestingly, when we analyzed the proliferative and tumor-igenic abilities of the L1þc-Kit expressing colorectal cancercells, we found that c-Kit expression enhanced their prolifer-ation both in medium containing 10% of serum and in 0.5% ofserum (Supplementary Fig. S3A and S3B). Moreover, upon

subcutaneous injection into nude mice, the L1þc-Kit cellsformed larger tumors than cells only expressing L1 (Supple-mentary Fig. S3C–S3E), indicating that they are more tumor-igenic. We conclude that overexpression of the oncogene c-Kit,while promoting the tumorigenic capacity of these cells,inhibits their metastatic spread.

c-Kit promotes an epithelial phenotype by suppressingSlug and increasing E-cadherin via AKT activation

To begin to understand the changes that colorectal cancercells undergo as a result of c-Kit expression, we examinedthe colorectal cancer cell lines described in Fig. 4A for mor-phological and molecular characteristics. L1-expressing colo-rectal cancer cells are characterized by the spontaneousformation of three-dimensional aggregates in culture, evenwhen only moderately dense (Fig. 5A; ref. 5). In sharp contrast,these L1-transfected cells, when reconstituted to also expressc-Kit, formed flat colonies of well-spread cells with cell–cellcontacts and clear margins resembling an epithelial morphol-ogy even at moderate density (Fig. 5B). This phenotype wasassociated with an increase in E-cadherin RNA and proteinlevels in L1þc-Kit expressing cells (Fig. 5C–E). The change inneoplastic properties from metastatic to nonmetastatic, as

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Figure 2. L1 suppresses c-Kit expression in colorectal cancer cells by activating NF-kB signaling. A, RNA was extracted from individual clones isolated fromstably transfected Ls174T cells expressing L1, control plasmid (pcDNA3), L1 together with IkB-SR or shRNA against p65, and p65 alone. SemiquantitativeRT-PCR was conducted using primers for L1, c-Kit, and GAPDH for loading control. B, Western blot analysis for many of the cell lines shown in A.C, theDLD-1 colon cancer cell linewas stably transfectedwith L1, 3 individual cloneswere isolated, and the levels of L1 andc-Kit were determined byWesternblot analysis. D, the c-Kit promoter reporter plasmid was transfected together with pSV b-galactosidase control vector (for transfection efficiencynormalization) into Ls174T colorectal cancer cells stably transfected with L1 or the p65 subunit of NF-kB (p65) or L1 together with the IkB super repressor(L1þIkB-SR) and a control clone (pcDNA3). Fold c-Kit promoter activation was determined after dividing luciferase activity by the values obtained withthe empty reporter plasmid.

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well as the morphological reversion from highly transformedthree-dimensional aggregates to flat epithelial colonies isreminiscent of mesenchymal-to-epithelial transformation(MET). However, further analysis of mesenchymal and epithe-

lial markers did not reveal differences in the expression ofvimentin or cytokeratins 8 and 18, regardless of whether c-Kitwas expressed in these cells (Fig. 5D). In addition, the analysisof several colonic epithelial cell markers (17) did not show

Figure 3. L1 and NF-kB suppress c-Kit expression via SP1 regulation. A, RNA levels of SP1 were determined using qRT-PCR in Ls174T cells stablytransfected with either empty vector (pcDNA3) or L1 or the p65 subunit of NF-kB (2 clones, p65 Cl1 and p65 Cl2); P¼ 6.61E�05. B, Western blot analysis forSp1, L1, p65, histone H2B, and tubulin from equivalent cell volumes of nuclear and cytoplasmic extracts from the colorectal cancer cell clones describedin A. Quantitation of SP1 and p65 in the nuclear fraction was determined by densitometry. C, the c-Kit reporter plasmid was transfected together withpSV b-galactosidase control vector (for transfection efficiency normalization) into Ls174T cells stably expressing L1 or a control clone (pcDNA3). Foldc-Kit promoter activation was determined after dividing luciferase activity by the values obtained with an empty reporter plasmid. For L1, P ¼ 0.0002; forpcDNA3, P ¼ 0.0047. D, ChIP-based PCR analysis using nuclear lysates from Ls174T cells. A primer sequence that amplifies the SP1 binding site(scheme) or a control (NS) irrelevant sequence was used. Rabbit anti-SP1 and nonimmune IgGwere used. E, ChIP-based PCR analysis using nuclear lysatesfromLs174T cells stably transfectedwith L1. Aprimer set that amplifies theNF-kB-binding sequence (scheme) or a control (NS) irrelevant sequencewasused.Binding of p65 to the NF-kB-binding sequence in the IkB gene was used as positive control. DNA fragments conjugated with nuclear proteins wereimmunoprecipitated with rabbit anti-NF-kB or nonimmune goat IgG and amplified by PCR. F, scheme depicting L1 that promotes the phosphorylation ofIkB and the release of NF-kB to act in the cell nucleus (see ref. 7) to inhibit the transcription of SP1 that results in the reduction of c-Kit transcription.






changes in their expression in cells overexpressing c-Kitand/or L1 (Supplementary Fig. S4). To understand how E-cadherin expression is regulated in these cells, we analyzedthe levels of some classic inhibitors of E-cadherin transcrip-tion, Snail, and Slug. Although Snail RNA levels remainedunchanged, Slug RNA was reduced in c-Kit-expressing cellsas compared with cells only expressing L1 (Fig. 5F), provid-ing a likely mechanism whereby c-Kit can enhance E-cad-herin protein levels.

Next, we wished to explore the mechanism/s whereby c-Kitenhances E-cadherin expression. Because c-Kit exerts many ofits effects by activating the PI3K/AKT signaling pathway (18–20), we determined whether the activation of AKT increases inL1þc-Kit overexpressing cells compared with L1 cells. Wefound that after an overnight serum starvation, L1þc-Kit cellsexpressed more phosphorylated AKT (p-AKT) than L1 cells(Fig. 6A, lane 5 compare to lane 1). Furthermore, upon serumactivation, L1þc-Kit cells reacted with increased AKT phos-phorylation as compared with L1-expressing cells (Fig. 6A andB). The LY294002 inhibitor reduced not only the levels of

p-AKT, but also of E-cadherin protein in both L1 and L1þc-Kittransfected cells (Fig. 6C, lane 4 compare to lane 3), indicatingthat E-cadherin expression in these cells is modulated by thePI3K/AKT pathway.

DiscussionThe major finding in this study is that a decrease in the

expression of c-Kit is associated with colorectal cancer devel-opment in a large set of human colorectal cancer tissuesamples compared with normal colon tissue, and that thesuppression of c-Kit is necessary for L1-mediated colorectalcancer metastasis to the liver in a mouse model of metastasis.The mechanisms involved in the suppression of c-Kit includethe L1-mediated activation of NF-kB signaling (7, 8) and thesuppression of SP1 transcription by NF-kB, by the directbinding of NF-kB to SP1 promoter sequences. Because SP1 isa major activator of c-Kit transcription (21), the inhibition ofSP1 expression resulted in reduced c-Kit transcription andconsequently, in reduced c-Kit protein levels in L1-expressing

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Figure 4. c-Kit inhibits L1-mediatedmotility andmetastasis. A,Westernblot analysis of Ls174T cells stablytransfected with L1, with a controlvector (pcDNA3), or with L1 andc-Kit (2 individual clones, L1þc-KitCl1 and L1þc-Kit Cl2). B, thecapacity of Ls174T-L1þc-Kit cells(described in A) to close an artificialwound was compared with that ofLs174T-L1 and control cells(pcDNA3) after 24 hours. C, themotility of Ls174T-L1þc-Kit Cl1and Cl2 was compared with thatof control Ls174T cells andLs174T-L1 cells by determiningsimultaneously the closureof 4 wounds for each cell line(P ¼ 3.13762E�06). The sameareas were photographedimmediately after wounding(0 hour) and 24 hours later. D, thecell clones described in A wereinjected into the spleen of nudemice and tumor growth at the siteof injection (spleen) and formationof metastases (liver) wasdetermined. The arrows pointto tumors in the spleen andthe arrowheads to largemacrometastases in the liver.E, table showing the group ofmice described in D (P ¼ 1.7E�05for the reduction in metastasis ofL1þc-Kit cells vs. L1 cells). F,Western blot analysis for L1 intissue samples of spleen tumorsand liver metastases.

Gavert et al.





cells (via NF-kB signaling). This also occurred in the absenceof L1 when NF-kB signaling was enhanced. Although NF-kBis considered mainly as an activator of transcription in can-cer and inflammation (22, 23), previous studies have alreadyreported on the inhibition of SP1 transcription by NF-kB,including in colon cancer cell lines (16). The reconstitutionof c-Kit expression by its transfection into L1-expressingcolorectal cancer cells inhibited all the effects conferred by

L1 in colorectal cancer cells, and included a reduction incell motility and, most importantly, blocked the metastaticcapacity of such cells. In addition, c-Kit expression also altered

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Figure 5. c-Kit promotes an epithelial morphology and increasesE-cadherin expression. Phase contrast images of live Ls174T cells stablyexpressing L1 (A) or L1þc-Kit (B). Scale bar, 75 mm.C, the cells fromA andB were doubly immunostained with anti-E-cadherin and anti-L1antibodies. Scale bar, 20 mm. D, Western blot analysis of Ls174T cellsstably expressing L1 (2 individual clones) or L1þc-Kit (2 individualclones) for L1, c-Kit, E-cadherin, cytokeratins 8 and 18 (CK), vimentin, andtubulin. E, RNA levels of E-cadherin were measured using qRT-PCR inLs174T cells stably transfected as detailed in A–C (P ¼ 0.00234). F,RNA was extracted from individual cell clones as described in Dand semiquantitative RT-PCR was conducted using primers for Slug,Snail, and GAPDH followed by agarose gel electrophoresis.

A

B

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Figure 6. Increased AKT phosphorylation in c-Kit-expressing cellsincreases E-cadherin levels. A, Ls174T cells stably transfected with L1or L1þc-Kit were starved overnight and then serum stimulated forvarious times. Western blot analysis was conducted for the levelsof L1, c-Kit, p-AKT, and total AKT. B, graphic representation of 3separate experiments as described in A. Densitometric measurementsof films for determining the amount of p-AKT as compared with totalAKT in the same sample for each time point were conducted asdescribed in Materials and Methods. C, the cells described in A weretreated with the LY 294002 PI3K inhibitor or with DMSO and collectedafter 48 hours. The levels of L1, c-Kit, E-cadherin, p-AKT, and totalAKT were determined by Western blot analysis.






the cell phenotype to a more epithelial organization intocolonies with clear borders, increased E-cadherin at cell–celljunctions, and a reduction in Slug. Although this phenotype isconsistent with an MET, many other markers of EMT did notshow a change in their levels and this was also the case fordifferentiation markers of colonic epithelial cells.

Our findings seem to be in contrast to numerous reports onthe potential oncogenic role of the tyrosine kinase c-Kitreceptor (13, 24, 25). The fact that loss of c-Kit can promotea more aggressive cancer phenotype was already reported in anumber of cancers, including melanoma (14), where c-Kitexpression in a highly metastatic cell line resulted in suppres-sion of its metastatic capacity (14). In addition, studies inbreast (25–27) and some other types of cancer (28) alsoreported on a decrease in c-Kit at more advanced stages ofthe disease.

Several immunohistochemical studies in colorectal canceralso detected a reduction in c-Kit that is associated withlater stages of tumor development (29, 30). However, studieson c-Kit expression in normal human colon and coloncancer tissue showed contradicting results: originally, c-Kitwas detected throughout human adult and fetal tissues andin the colonic mucosa, submucosa, and intestinal smoothmuscle layers (31). More recently, c-Kit was detected in stemcells of colonic crypts (32), whereas in colon cancer, c-Kitwas found in 1.6% to as many as 25% of human colon cancerspecimens (33, 34). Although c-Kit immuno-staining is un-common in primary human colon tumors, c-Kit is expressedat lower levels in more advanced cancer as compared witha higher expression in adenomas. This implies a loss of c-Kitin later stages of colorectal cancer progression in agreementwith our study.

Reports on various colorectal cancer cell lines showedthat c-Kit is expressed in some cell lines including HT29 (35)and DLD-1 (36), but not in many others (37), in agreementwith our studies showing c-Kit in DLD-1 and Ls174T cells,but not in others (such as SW480 and HCT116, data notshown). c-Kit inhibitors were proposed as therapeuticagents in the treatment of colorectal cancer, and ST1571(imatinib mesylate) reduced HT29 colorectal cancer cellproliferation and tumor growth in mice (38). These findingsare in line with our studies showing that c-Kit promotescolorectal cancer cell proliferation both in vitro and in vivo.An additional study (39) with this inhibitor showed a reduc-tion in the proliferation and transwell migration of LS180cells in contrast to our findings on the effect of c-Kit on cellmotility. Because EGF was used to stimulate the migration ofLS180 cells, this could conceivably activate signaling path-ways that are inhibited by imatinib (such as the Brc/Ablpathway), independently of c-Kit activity.

Although the metastasis to the liver mediated by L1 incolorectal cancer cells was suppressed by c-Kit, the prolifer-ation of such cells in culture and their tumorigenic capacityupon subcutaneous injection into mice was enhanced, asexpected from a strong proto-oncogene such as c-Kit. Inter-estingly, a recent study also reported on such dichotomiceffect when the oncogene c-Myc was transfected into breastcancer cell lines resulting in enhanced tumorigenesis, but a

decrease in their metastatic capacity (40). These studies sup-port the notion that the promotion of earlier stages in tumordevelopment, including cell proliferation and tumorigenesis,do not necessarily also promote metastasis and may in somecases interfere with later stages of the disease, includingmetastasis. Recent studies have also shown such dichotomicbehavior for EMT that was amply shown to be associatedwith tumor dissemination (41). These studies (42, 43) show-ed, using transgenic mice expressing an inducible form ofTwist 1 (another EMT transcriptional regulator), that althoughEMT is important during the early stages of tumor develop-ment (tumor cell dissemination), a switch to MET (i.e., areversion of EMT) is required for efficient colonization andmacrometastasis (43) and when EMT continued, no effectivemetastasis occurred.

Our study emphasizes the differential role of proto-onco-genes such as c-Kit in earlier stages of tumor developmentand their inhibitory effect (most probably by promoting aless motile epithelial phenotype) at later stages of colorectalcancer metastasis, as showed here for the L1-NF-kB signalingpathway.

Although some studies have shown that pharmacologicinhibition of c-Kit reduces both cell and tumor proliferation(37, 38) and have recommended their use in the treatmentof colorectal cancer, we believe that a more conservativeapproach is required. Unlike intestinal stromal tumors wherec-Kit overexpression is well documented and primary tumorsshrink upon treatment to operable size, neoadjuvant therapybefore surgery is much less common. Because ours is thefirst study showing a relationship between c-Kit suppressionand metastasis development in colorectal cancer, furtherstudies on the mechanisms governing c-Kit suppressionand the promotion of metastasis are needed before recom-mending changes in the use of c-Kit inhibitors in tumortherapy.

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

Authors' ContributionsConception and design: A. Ben-Ze'ev, N. Gavert, E. BakosDevelopment of methodology: A. Ben-Ze'ev, N. Gavert, E. DomanyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Ben-Ze'ev, N. GavertAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Ben-Ze'ev, N. Gavert, A. Shvab, M. Sheffer, A. Ben-Shmuel, G. Haase, E. DomanyWriting, review, and/or revision of themanuscript: A. Ben-Ze'ev, N. Gavert,A. Shvab, A. Ben-Shmuel, G. HaaseAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A. Ben-Ze'ev, N. GavertStudy supervision: A. Ben-Ze'ev, N. Gavert, A. Shvab

Grant SupportThis study was supported by grants from the Israel Cancer Research Fund

(ICRF) and from the Israel Science Foundation (ISF) and by the Leir CharitableFoundation (E. Domany and M. Sheffer).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 27, 2013; revised July 3, 2013; accepted July 17, 2013;published OnlineFirst September 5, 2013.

Gavert et al.





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2013;73:5754-5763. Published OnlineFirst September 5, 2013.Cancer Res Nancy Gavert, Anna Shvab, Michal Sheffer, et al. Contributes to L1-Mediated Metastasisc-Kit Is Suppressed in Human Colon Cancer Tissue and

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