L1-Mediated Colon Cancer Cell Metastasis Does Not Require ... · changes in L1 levels do not affect...

Angiogenesis, Metastasis, and the Cellular Microenvironment

L1-Mediated Colon Cancer Cell Metastasis Does NotRequire Changes in EMT and Cancer Stem Cell Markers

Nancy Gavert1, Alessia Vivanti1, John Hazin1, Thomas Brabletz2, and Avri Ben-Ze’ev1

AbstractAberrant activation of Wnt/b-catenin signaling is common in most sporadic and inherited colorectal cancer

(CRC) cells leading to elevated b-catenin/TCF transactivation. We previously identified the neural cell adhesionmolecule L1 as a target gene of b-catenin/TCF in CRC cells. Forced expression of L1 confers increased cellmotility, invasion, and tumorigenesis, and the induction of human CRC cell metastasis to the liver. In humanCRC tissue, L1 is exclusively localized at the invasive front of such tumors in a subpopulation of cells displayingnuclear b-catenin. We determined whether L1 expression confers metastatic capacities by inducing an epithelial tomesenchymal transition (EMT) and whether L1 cosegregates with cancer stem cell (CSC) markers. We found thatchanges in L1 levels do not affect the organization or expression of E-cadherin in cell lines, or in invading CRCtissue cells, and no changes in other epithelial or mesenchymal markers were detected after L1 transfection. Theintroduction of major EMT regulators (Slug and Twist) into CRC cell lines reduced the levels of E-cadherin andinduced fibronectin and vimentin, but unlike L1, Slug and Twist expression was insufficient for conferringmetastasis. In CRC cells L1 did not specifically cosegregate with CSC markers including CD133, CD44, andEpCAM. L1-mediated metastasis required NF-kB signaling in cells harboring either high or low levels ofendogenous E-cadherin. The results suggest that L1-mediated metastasis of CRC cells does not require changes inEMT and CSCmarkers and operates by activating NF-kb signaling.Mol Cancer Res; 9(1); 14–24.�2010 AACR.

Introduction

Aberrant activation of Wnt/b-catenin target genes playsa key role both during early and later phases of humancolorectal cancer (CRC) development (1, 2). Members ofthe immunoglobulin-like cell adhesion receptors (Nr-CAM and L1), mostly known for their presence in nervecells (3, 4) but also in many cancer cell types (5), wererecently identified as target genes of b-catenin/TCF sig-naling in CRC cells (6, 7). L1 was detected in a smallsubpopulation of cells at the invasive front of CRC tissueshowing nuclear b-catenin (7). Moreover, the expressionof L1 in human CRC cells lacking L1 confers enhancedmotility and metastasis to the liver (8). A large number ofgenes induced by L1 in such cultured cells are also sharedby human CRC tissue samples, as indicated by geneexpression microarray profiles (8). Analysis of the signal-ing pathways that are involved in L1-mediated CRC cell

metastasis indicated that the NF-kB pathway and ezrin areboth required for conferring metastatic capacities in thesecells (9). A more motile phenotype consistent with anepithelial to mesenchymal transition (EMT)-like processwas suggested to be induced by L1 in MCF7 breast cancercells transfected with L1 (10). EMT was suggested to actin breast cancer progression, by producing cells with CRCstem cell (CSC) characteristics (11, 12). In a recent studywhere CSCs were isolated from human glioma tissue,based on CD133 expression, L1 was identified as acosegregating molecule that was highly enriched inCD133þ cells (13). Moreover, suppression of L1 levelsin such cells inhibited glioma stem cell proliferation andblocked their metastatic capacities suggesting that L1 canrepresent a CSC therapeutic target (13). The presence ofCD133 on the membrane of CRC cells also enabled theisolation of human CSCs (14, 15). We investigated thepossibility that L1 confers its metastatic capacity in CRCcells by inducing EMT-like properties and by possiblycosegregating with cells expressing CSC markers. WhenL1 levels were increased or suppressed in CRC cells inwhich the levels of endogenous L1 and/or E-cadherin weredramatically different, no detectable changes in E-cad-herin, or in other EMT markers were detected. Moreover,when the expression of Slug and Twist (major EMTregulators; ref. 16) were elevated in CRC cells, this wasinsufficient to induce metastatic capacity in these cells. Onisolation of CD133þ and CD133� CRC cell populations

Authors' Affiliations: 1Department of Molecular Cell Biology, WeizmannInstitute of Science, Rehovot, Israel and 2Department of Visceral Surgery,University of Freiburg, Freiburg, Germany

Corresponding Author: Avri Ben-Ze’ev, Department of Molecular CellBiology, Weizmann Institute of Science, P. O. Box 26, Rehovot 76100,Israel. Phone: 972-8-9342422; Fax: 972-8-9465261.E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-10-0406

�2010 American Association for Cancer Research.

MolecularCancer

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from some CRC cell lines, no cosegregation between L1and CD133 and other CSC markers (CD44 and EpCAM)was observed. In invasive human CRC tissue, both L1 andE-cadherin were expressed in the same cells. Takentogether, our results suggest that the L1-mediated induc-tion of CRC cell metastasis does not require an EMT and/or changes in CSC markers.

Materials and Methods

Cell lines, cell culture, and transfections293T, MDCK, NIH3T3, HCT116, SW480, and

SW620 cells were maintained in DME with 10% bovinecalf serum. Ls174T cells were grown in RPMI with 10%fetal calf serum. Transient transfection of 293T cells wasperformed using calcium phosphate. Ls174T, SW480,SW620, and HCT116 cells were transfected using Lipo-fectamineTM 2000 (Invitrogen). Ls174T or HCT116cells stably expressing human L1 were established byselection to neomycin resistance (500 mg/mL), asdescribed (7). Ls174T cells expressing L1 were cotrans-fected with shRNA targeted against p65 followed byselection with puromycin (10 mg/mL). In SW620 cells,shRNA targeted against L1 was transfected followed byselection to neomycin resistance (500 mg/mL). Cellsinducibly expressing Twist or L1 were obtained by trans-fecting Ls174TR4 cells (expressing the tetracycline recep-tor) with pcDNA4/TO Twist (or pcDNA4/TO L1), orwith the pcDNA4/TO empty vector followed by selectionwith 800 mg/mL zeocin. HCT116 cells stably expressinginducible Slug were obtained in a similar manner. Intransactivation assays, a b-galactosidase plasmid wascotransfected with the reporter plasmid containing 3copies of an NF-kB–responsive promoter sequence linkedto luciferase (3xkB.luc). Cells were plated in triplicates,lysed after 48 hours, and luciferase and b-galactosidaselevels were determined by enzyme assay kits from Pro-mega. For transfection control, luciferase activity wasnormalized to b-galactosidase activity. Fold inductionof the NF-kB reporter plasmid was calculated using anempty reporter plasmid (pGL3).

PlasmidsThe L1 and Twist cDNA constructs were described (7,

9). Drs. D. Wallach and A. Kovalenko (Weizmann Instituteof Science, Rehovot, Israel) provided the NF-kB–respon-sive reporter plasmid (3xkB.luc). shRNA against p65 wasprepared in pSuper-puro according to the manufacturer'sinstructions (pSuper RNAi-System; OligoEngine) using thefollowing target sequences: 5'-CAAGATCAATGGCTA-CACA-30, 50-CTCAAGATCTGCCGAGTGA-30, 50-GATGAGATCTTCCTACTGT-30, 5'-GGATTGAGGA-GAAACGTAA-30. L1 shRNA was prepared in pSuperusing the following target sequences: 50-GGATGGTGTC-CACTTCAAA-30, 50-GAGAAGGGTGGGCTTCCC-30,50-AGACCAGAAGTACCGGATA-30, and the scrambledsequence 50-AGCGCGCTTTGTAGG.

ImmunofluorescenceCells cultured on glass coverslips were permeabilized with

0.5% Triton X-100 and fixed with 3% PFA in PBS. Thecoverslips were incubated with pAb and mAb against L1(provided by V. Lemmon, University of Miami, Miami, FL)and against E-cadherin, mAb HECD-1 (Invitrogen). Thesecondary antibodies were Alexa 488-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen), and Cy3-labeled goatanti-mouse, or anti-rabbit IgG (Jackson ImmunoResearchLaboratories). Images were acquired using Eclipse E1000;Nikon; objectives �60/1.4 NA equipped with a camera(ORCA-ER; Hamamatsu) and using Volocity acquisitionsoftware (Improvision) and figures were mounted usingPhotoshop CS3 software.

Western blottingTheWestern blots were developed using the ECLmethod

(Amersham Biosciences) using the antibodies described ear-lier in the text, and a mAb against CD133 (Miltenyi Biotec),mAb against CD44v6 (provided by M. Zoeller, DKFZ,Heidelberg, Germany), mAb against EpCAM (provided byG. Moldenhauer, DKFZ, Heidelberg, Germany), mAbagainst E-cadherin (Transductions Laboratories, Sigma-Israel), and mAb against tubulin (Sigma-Israel).

PCRRNA was extracted from cells using EZ-RNA Total RNA

Isolation Kit (Biological Industries) according to the man-ufacturer's protocol. After obtaining cDNA, PCR was per-formed using Red Load TaqMaster (Larova) according to themanufacturer's protocol. Primers for L1 were ACGGGCAA-CAACAGCAAC and CGGCTTCCTGTCAATCATG,and for CD133 CTGGGGCTGCTGTTTATTATTCTGand ACGCCTTGTCCTTGGTAGTGTTG.

Metastasis assaysGroups of 5 mice were anesthetized by peritoneal injec-

tion of xylazine (20 mg/mL) and ketamine (100 mg/mL).Through a 1-cm incision in the upper left lateral abdomen,the spleen was delivered into the wound, and 106 cells in 20mL of PBS were injected, using a Hamilton syringe, into thedistal tip of the spleen that was replaced in the abdomen andthe incision closed with staples. After 5 to 6 weeks, theanimals were sacrificed and the spleen and liver removed forexamination.

Artificial wound healingA round "wound" was introduced into a confluent mono-

layer of cells, in 24-well dishes, with the tip of a micropipetteusing suction to remove the cells. The culture medium wasreplacedwith freshmedium, and 0.1mg/mLofMitomycinCwas added to inhibit cell proliferation. Wounds (in quad-ruplicate) were photographed every hour for 18 to 24 hoursand the percentage of wound closure was calculated from thephotographs taken at the start of the experiment and after 18to 24 hours by using the PhotoshopCS3 analyzer tomeasurethe wound area not closed compared with the area of thewound at the beginning of the experiment.

L1 Induces Colon Cancer Metastasis but not EMT and CSC Markers

www.aacrjournals.org Mol Cancer Res; 9(1) January 2011 15




Fluorescence-activated cell sorting and analysisHuman CRC cell lines were subjected to fluorescence-

activated cell sorting (FACS). Human-specific anti-CD133(293C3) conjugated to phycoerythrin (PE; Milyteni Biotec)was used for FACS analysis. The cells were pelleted andresuspended in FACS buffer (PBS pH 7.2, 0.5% bovineserum albumin, 2 mmol/L EDTA) containing the anti-CD133 antibody (1:100). After incubation for 20 minutesat 4�C, the cells were washed and the samples analyzedusing a Becton Dickinson (BD) FACSVantage SE flowcytometer. The BD Cell Quest software was used for gatingand analysis.

ImmunohistochemistryImmunohistochemistry was carried out on 25 paraffin-

embedded human colorectal adenocarcinomas as described(17). In brief, antigen retrieval included pretreatment incitrate buffer pH 6.0 in a pressure cooker for 30 minutes.For L1 and E-cadherin detection, polyclonal rabbit anti-L1antiserum (18), diluted 1:1,000, and a monoclonal anti-body against E-cadherin (clone 36; BD Transduction;diluted 1:200) were used for overnight staining at 4�C.The streptomycin/AB system (Dako) was used for detectionaccording to the manufacturer's protocol. Sections werecounterstained with hemalaun (Merck).

StatisticsStatistical significance was determined by Fisher's exact

test (19) for mouse metastasis experiments. In woundclosure, significance was determined using non-pairedStudent's t-test. P < 0.05 was considered significant.

Results

Overexpression of L1 in CRC cells containing high orlow levels of E-cadherin increases cell motility andinduces liver metastasisThe expression of L1 in Ls174T CRC cells that lack L1

(7), and only possess very low levels of E-cadherin (Fig. 1A,lane 2), confers enhanced motility and the injection of suchcells into the spleen of nude mice causes liver metastasis (8,9). To examine whether overexpression of L1 in CRC cellsdisplaying high levels of E-cadherin will cause similarresponses and whether L1 will affect the organizationand levels of E-cadherin, L1 was transfected intoHCT116 CRC cells that display low levels of endogenousL1 and high levels of E-cadherin (Fig. 1A, lane 3). Twoindependent cell clones overexpressing L1 were isolated(Fig. 1B, lanes 2 and 3) and these CRC cells displayed amore than 2-fold increase in motility as measured by theirability to close an artificial wound in a monolayer (Fig. 1C,left and right). Moreover, on injection of these HCT116cell clones overexpressing L1 into the spleen of nude mice,they formed liver metastases (Fig. 1D, Cl1 and Cl2, arrows).HCT116 cells transfected with the empty vector onlyformed primary tumors at the site of injection after 6 weeks(P < 0.05; Fig. 1D, control, arrowheads). As with Ls174Tcells, there was no correlation between the sizes of tumors

A

C

D

B

Figure 1. Overexpression of L1 in CRC cells expressing either high orlow E-cadherin levels increases cell motility and induces livermetastasis. A, the levels of L1 and E-cadherin were determined byWestern blotting in human CRC cell lines Ls174T, HCT116, and SW620.NIH3T3 cells served as a negative control and MDCK as a positivecontrol for E-cadherin expression. Tubulin served as loading control.B, L1 was stably transfected into HCT116 cells and the level of L1 in2 individual cell clones (L1/Cl1 and L1/Cl2) was determined. C, thecapacity of HCT116-L1 cells (described in B) to close an artificial woundwas compared with that of control HCT116 cells after 24 hours. C, left,the motility of HCT116-L1/Cl1 and Cl2 was compared with that of controlHCT116 cells by determining simultaneously the closure of 4 wounds foreach cell line. The same areas were photographed immediately afterwounding (0 hours) and 24 hours later (C, right). D, the cell clonesdescribed in B were injected into the spleen of nude mice and tumorgrowth at the site of injection (spleen) and formation of metastases (liver)were determined. The arrowheads point to tumors formed in the spleenand the arrows to large macrometastases in the liver.

Gavert et al.

Mol Cancer Res; 9(1) January 2011 Molecular Cancer Research16




formed at the site of injection and the metastatic capacity(8, 9). This indicates that the low level of endogenous L1 inHCT116 cells is insufficient to cause liver metastasis withinthis time period (6 weeks), and only at much later times(3.5–4 months) liver metastases are observed when controlHCT116 cells were injected (data not shown).

Overexpression of L1 induces NF-kB activation inHCT116 cells and suppression of p65 levels blocks thecapacity to confer liver metastasisThe transfection of L1 into Ls174T CRC cells was

recently reported to induce NF-kB signaling, and theactivated NF-kB subunit p65-P was shown to colocalizein cells at the invasive CRC tissue front together with L1(9). We found that L1 transfection into HCT116 cellssimilarly induced the transcriptional activity of an NF-kBreporter plasmid (Fig. 2A), and the stable suppression ofendogenous p65 levels by shRNA to p65 (Fig. 2B) resultedin the suppression of NF-kB activity (Fig. 2C) and blockedthe metastatic capacity of these cells (P < 0.05; Fig. 2D).This suggests that L1 overexpression in both Ls174T andHCT116 CRC cells confers liver metastasis by a mechanismrequiring the activation of NF-kB signaling.

Overexpression of L1 in CRC cells does not affect thelevels of E-cadherin or other EMT markersL1 was detected in a small subpopulation of CRC tissue

cells at the invasive tumor front (7) that often lose E-cadherin expression (20), indicating the possibility thatL1 may induce cellular properties compatible with anEMT. Indeed, a recent study suggested that in MCF7breast cancer cells the transfection of L1 enhances invasivecell motility by affecting the organization of E-cadherin,reminiscent of an EMT-like process (10). We thereforeexamined the levels of E-cadherin and other epithelial andmesenchymal markers in CRC cell lines in which the levelsof L1 were either increased or suppressed. As shown inFigure 3A, the stable transfection of L1 into Ls174T cells(Fig. 3A, lane 3) did not influence the level of endogenousE-cadherin, which remained low as in control Ls174T cells(Fig. 3A, compare lane 3 with lane 2). The high levels of E-cadherin also remained unaffected in HCT116 cells stablytransfected with L1 (Fig. 3A, compare lane 5 with lane 4).In SW620 CRC cells that possess endogenous L1 and highlevels of E-cadherin (Fig. 1A, lane 4; Fig. 3A, lane 6),suppression of L1 levels by shRNA to L1 had no significanteffect on E-cadherin levels (Fig. 3A, compare lane 7 withlane 6). It is noteworthy that the suppression of L1 inSW620 reduced their tumorigenic capacities in nude mice(9). In addition to E-cadherin, no changes were observed inthe epithelial cell specific cytokeratin 18, and an increase inthe levels of the mesenchymal markers N-cadherin orvimentin was not apparent when L1 levels were manipu-lated (Fig. 3A).Because EMT is considered to be a very transient process,

both during embryogenesis and tumor progression (21), weprepared Ls174T CRC cells expressing a doxycycline-indu-cible L1 and followed the expression of L1 and E-cadherin

at various times after treating the cells with doxycycline(Fig. 3B). L1 was already partially induced after 6 hours(Fig. 3B, compare lane 6 with lane 1) and continued toaccumulate reaching much higher levels between 12 and72 hours after doxycycline treatment (Fig. 3B, lanes 7–10). As previously shown (22), the levels of E-cadherinincrease in CRC cell lines as cell density increases, andaccordingly an increase in E-cadherin at later times of cellsin culture, especially after 72 hours, was noted (Fig. 3B,lanes 5 and 10). However, this density dependent increasein E-cadherin was the same in cells lacking L1 and in cellswhere high levels of L1 were induced (Fig. 3B, lanes 5and 10).Because Ls174T CRC cells are among the few CRC cell

lines that express wt p53, we also examined the possibilitythat induction of L1 will elicit a response from the p53pathway. As shown in Figure 3B, the levels of p53 remainedlargely unchanged during the 3 days of L1 induction,whereas L1 levels increased dramatically. These resultsimply that when L1 levels are extensively modulated inCRC cells by stable shRNA-mediated suppression, over-expression, or induction by a doxycycline-responsive sys-tem, there were no detectable changes in the expression ofkey EMT markers in such cells.

Overexpression of Slug or Twist in CRC cells reducesE-cadherin levels but does not induce metastasis bythese cellsNext, we addressed the possibility that the induction of

an EMT-like process in CRC cells by the overexpression ofmajor EMT transcription regulators, such as Twist andSlug, will be sufficient to cause liver metastasis by these cells.Two Ls174T cell clones overexpressing Twist were isolatedand the level of E-cadherin in such cells was determinedwhen the cells were either grown as sparse or dense culturesfor 3 days (Fig. 4A). As shown previously (22), E-cadherinlevels in control cells are higher in dense cultures whereextensive cell–cell contacts are established (Fig. 3B; Fig. 4A,compare lane 5 with lane 6). The transfection of Twist intoLs174T cells dramatically reduced the levels of E-cadherineven in dense cell cultures (Fig. 4A, compare lanes 1 and 3with control, lane 5). Interestingly, a dramatic shift infibronectin expression (a mesenchymal marker) from a veryhigh level in sparse cultures to almost undetectable levels indense cultures could be observed (Fig. 4A, compare lanes 1,3, and 5 with lanes 2, 4, and 6). However, this shift infibronectin expression by cell culture density was notaffected by Twist expression. In addition, Ls174T cellsexpressing a doxycycline-inducible Twist construct wereisolated (8) and the cells were injected into the spleen ofnude mice and the expression of Twist in these mice waskept under either uninduced or induced conditions (withdoxycycline). The expression of Twist in the injected cells,however, was insufficient to confer metastatic capacity inthese cells (Table 1; Fig. 4D, top and bottom). Slug isanother major regulator of EMT whose expression ismodulated in CRC cells (22). The overexpression of Slugby stable transfection into Ls174T cells (Fig. 4B, left) or






under a doxycycline-inducible promoter in HCT116 cells(Fig. 4B, right) reduced the levels of E-cadherin in bothsparse and dense cell cultures compared with untransfected,or uninduced cells (Fig. 4B, left, compare lane 1 with lane 3;Fig. 4B, right, compare lane 1 with 3 and 2 with 4). Slugalso induced the expression of mesenchymal markers(vimentin and fibronectin; Fig. 4C, left and right).HCT116 cells expressing inducible Slug were injected intothe spleen of nude mice, and while forming tumors at thesite of injection, Slug expression was insufficient to confermetastatic capacities in these cells (Table 2). These resultsimply that whereas the major regulators of EMT, Twist andSlug, reduced the levels of E-cadherin and inducedmesenchymal markers in CRC cells, their overexpression(unlike that of L1) is insufficient to confer metastasis bythese cells in nude mice.

Changes in L1 levels do not result in a differentorganization of E-cadherin in CRC cells and tumortissueIn MCF7 breast cancer cells, the transfection of L1 was

suggested to result in a dramatic loss of E-cadherin from

adherens junctions and its replacement by L1 and theinduction of a motile, EMT-like phenotype in such cells(10). We therefore examined the organization of L1 and E-cadherin in CRC cells in which the levels of L1 weremodulated. The organization of endogenous L1 and E-cadherin in SW620 CRC cells was mostly detected at cell–cell contact sites, but with rather little overlap (Fig. 5A–C).The complete suppression of L1 levels in these cells byshRNA to L1 (Fig. 5E) did not result in a detectable changein E-cadherin organization at cell–cell contacts (Fig. 5D).The overexpression of L1 in HCT116 cells (Fig. 1B)produced cells expressing L1 at cell–cell contact sites(Fig. 5H) and E-cadherin localized in adherens junctions(Fig. 5G). In such cells, there was also rather little overlapbetween the localization of these 2 molecules in the cellmembrane (Fig. 5I). Note that the single untransfected cellmarked in the image shown in Figure 5G–I (arrowheads)displayed a similar organization of E-cadherin like theneighboring HCT116 cells that overexpressed the trans-fected L1.We have also studied the organization of L1 and E-

cadherin in cells at the invasive front of CRC tissue (in 25

A B C

D

Figure 2. Overexpression of L1 inCRC cells induces NF-kBactivation and suppression of p65expression blocks the capacity toconfer liver metastasis. A, the NF-kB–responsive reporter plasmid3xkB.luc, or a control vector, wastransfected together with ab-galactosidase control vector (fortransfection efficiencynormalization) into humanHCT116 CRC cells stablyoverexpressing L1 (L1/Cl1 and L1/Cl2). Fold NF-kB activation wasdetermined after dividingluciferase activity by the valuesobtained with the empty reporterplasmid. B, Ls174T CRC cellclones stably overexpressing L1and shRNA to p65 (L1 þ shp65/Cl1 and Cl2) or a control shRNA(L1þ control) were analyzed for L1and p65 expression. Ponceaustaining determined equal proteinloading. C, the activity of NF-kB inthe cell clones described in B wasdetermined as described in A. D,the CRC cell clones (described inB) were injected into the spleen ofnude mice, and tumor growth andmetastasis were determined asdescribed in Figure 1D.

Gavert et al.





tumor samples), where most of the L1 molecules arelocalized in CRC (7, 9). The loss of differentiation in smallclusters of invasive cells was characterized by reduced orcytoplasmic expression of E-cadherin and nuclear b-cateninlocalization in all cases (Fig. 6B, C, E, and F). Approxi-mately 75% (20 out of 25) of the cases displayed variableexpression of L1 in invading tumor cells (Fig. 6A). Thus,cytoplasmic localization of E-cadherin was not restricted toonly L1-expressing tumor cells (Fig. 6B), but was alsodetectable in L1-negative cells (Fig. 6E). Note the mem-branal localization of E-cadherin in the more central anddifferentiated areas of the tumor that lack L1 (Fig. 6H). Thearrowheads (Fig. 6D) indicate normal L1 expression innerve bundles of the submucosal layer where invadingCRC cells are found (7). Taken together, in both culturedhuman CRC cell lines and in CRC tissue cells the presenceof L1 does not appear to have a detectable effect on theorganization of E-cadherin in these cells.

Expression of CRC stem cell markers and L1 in CRCcellsL1 is most abundant in nerve cells and in tumors of the

nervous system (3). A recent study reported on the isolationof CSCs from human glioma and found that L1 cosegre-gates with glioma stem cells expressing the CD133 marker(13). Suppression of L1 levels in such cells blocked braintumor formation in mice indicating that L1 could functionas a glioma CSC target for therapy. Because CD133

positivity was also suggested to be a key marker for humanCRC stem cells (14, 15), we wished to determine whetherL1 expression in CRC cells cosegregates with CD133 andwith other human CRC stem cell markers. FACS analysiswas employed to separate from the HCT116 cell line, cellpopulations that either express or lack CD133 (Fig. 7A,left). Approximately 94% (in different experiments) of thecells in the HCT116 cell line displayed CD133. CD133þ

and CD133� HCT116 cells were separated by 2 cycles ofpurification using FACS analysis and when equal amountsof protein were analyzed by Western blotting for CD133,an effective separation between CD133þ and CD133� cellscould also be confirmed by this method (Fig. 7A, right,second panel). No enrichment for L1 in CD133þ cells wasobserved compared with CD133� cells and both cellpopulations expressed similar amounts of L1 (Fig. 7A, right,top panel). The analysis of other CRC stem cell markers,including CD44 and EpCAM, did not indicate a specificenrichment in CD133þ cells (Fig. 7A, right). We alsoexamined the possibility of L1 and CD133þ cosegregationin SW620 cells that displayed a more significant amount ofCD133� cells (approximately 65%; Fig. 7B, left). How-ever, in these cells, also there was no enrichment for L1 inthe CD133þ cells compared with CD133� cells (Fig. 7B,right), and the expression of EpCAM was similar in bothcell populations of SW620 cells. We analyzed the levels ofCD133 in individual HCT116 and SW620 cell clones inwhich the levels of L1 were manipulated by either over-

A B

Figure 3. Overexpression of L1 in CRC cells does not affect the levels of endogenous E-cadherin or other EMT markers. A, the human CRC cell lines Ls174Tand HCT116 stably transfected with L1 or with control plasmid, and SW620 stably transfected with shRNA to L1 or with control shRNA, were analyzedby Western blotting for the expression of L1, E-cadherin, N-cadherin, vimentin, and cytokeratin 18 (CK18). NIH3T3 cells were used to displaymesenchymal markers and MDCK cells for epithelial markers. Tubulin served as loading control. B, Ls174T cells stably transfected with a doxycycline-inducible L1 plasmid were induced with doxycycline for up to 72 hours. The expression of L1, E-cadherin and p53 were analyzed by Western blotting inboth induced and uninduced cells. Ponceau staining determined equal protein loading.






expression or shRNA-mediated suppression (Fig. 7C). Hereagain, the level of CD133 RNA (Fig. 7C, left) and protein(Fig. 7C, right) remained unchanged (or did not correlatewith L1) in the individual cell clones in which the level of L1

was dramatically altered. Taken together, these studiesimply that in human CRC cell lines expressing endogenousL1 there is no specific cosegregation of L1 with cellpopulations expressing CRC stem cell markers.

A

C D

B

Figure 4. Overexpression of Slug or Twist in CRC cells reduces E-cadherin and induces vimentin and fibronectin expression. A, clones of Ls174T CRC cellsstably transfected with Twist (Twist Cl1 and Cl2), or with control vector, were grown as sparse (s) or dense (d) cultures for 48 hours. The levels of E-cadherin,fibronectin, and Twist were determined by Western blotting. Tubulin served as loading control. B, left, Ls174T cell clones stably transfected with a Slugplasmid, or an empty vector, were grown in dense and sparse cultures and the levels of E-cadherin, Slug, and tubulin were determined as in A; right, HCT116cells were stably transfected with a doxycycline-inducible Slug plasmid and were either induced (þDOX) or left untreated (�DOX). C, Ls174T cells stablytransfected with Slug were analyzed for the induction of vimentin (left) and fibronectin (right). D, Ls174T expressing inducible Twist (top) were injected into thespleen of nude mice and the expression of Twist was determined in splenic tumors of mice provided, or not, with Dox in their drinking water (bottom).

Table 1. Tumor growth and liver metastasis inTwist-expressing Ls174T cells

Primarysplenic tumor

Livermetastasis

Ls174T CellsL1 4/4 3/4pcDNA3 4/4 0/4

Ls174T-inducible TwistDoxycycline þ 4/4 0/4Doxycycline� 3/4 0/4

NOTE: Half of the mice were fed doxycycline to induceTwist. Tumor growth and liver metastasis were determinedas described in Figure 1D.

Table 2. Tumor growth and liver metastasis inSlug-expressing HCT116 cells

Primarysplenic tumor

Livermetastasis

HCT116 4/TO cellsDoxycyclineþ 4/4 0/4Doxycycline� 4/4 0/4

HCT116-inducible SlugDoxycyclineþ 3/3 0/3Doxycycline� 3/4 0/4

NOTE: Half of themice were fed doxycycline to induce Slug.Tumor growth and liver metastasis were determined asdescribed in Figure 1D.

Gavert et al.





Discussion

EMT is thought to endow cancer cells with migratory,invasive, and stem cell properties by activating a variety ofsignaling pathways including the Wnt/b-catenin and NF-kB pathways (11, 21, 23–25) and their target genes.Because the expression of L1 (a b-catenin/TCF targetgene) in human CRC cells enhances cell motility andinvasion (7), and confers metastatic capacity to the liver innude mice (8) by activating NF-kB signaling (9), in thisstudy we determined whether these properties conferredby L1 are associated with changes in EMT and CSCmarkers.

This study showed that L1 expression induced NF-kBsignaling in CRC cells and is necessary for conferringenhanced motility and liver metastasis in both CRC cellsthat express very low levels (Ls174T) or high levels(HCT116) of endogenous E-cadherin. A recent studyshowed that NF-kB is also induced by L1 in pancreaticductal carcinoma cells (26). The forced expression of L1(in Ls174T and HCT116), or the shRNA-mediatedsuppression of endogenous L1 in SW620 CRC cells thathad profound effects on the tumorigenic capacity of thesecells (9), was not associated with changes in the expressionand organization of E-cadherin. The analysis of E-cad-herin localization in small clusters of invasive CRC cells

Figure 5. Changes in L1 levels donot result in a differentorganization of E-cadherin in CRCcells. A–F, SW620 cell clonesstably transfected with shRNA toL1, or with control shRNA weredoubly immunostained with anti–E-cadherin (A and D) and anti-L1(B and E) antibodies. Nuclei werestained with DAPI (F). C, mergedimage of A and B. G–I, HCT116cells stably transfected with L1were immunostained with anti E-cadherin (G) and anti L1 (H)antibodies. A cell that does notoverexpress L1 is marked byarrowheads (G–I). The scale barrepresents 20 mm.

A B C

D E F

G H I






at the tumor front showed a similar organization andexpression of E-cadherin in tumor cells displaying orlacking L1, implying that the regulation of L1 and E-cadherin levels are unrelated to each other in CRC cells.The levels of other epithelial or mesenchymal markers alsoremained unchanged in CRC cells when L1 expressionwas altered.The reversible plasticity in the expression of EMT mar-

kers by epithelial cells and in the induction of b-catenin/TCF signaling can readily be shown in both nontumori-genic (27, 28) and CRC cells (22) by changing the cellculture density for short time periods, as also shown in thisstudy. Nevertheless, the stable suppression of E-cadherinlevels together with the induction of mesenchymal markersby the major EMT regulators, Twist and Slug, was insuffi-cient to endow these CRC cells with metastatic capacity,suggesting that L1 confers metastatic abilities in these cellsby other mechanisms not involving changes in EMTmarkers.A previous study withMCF7 breast cancer cells transfected

with L1 suggested that L1 disrupts E-cadherin containingadherens junctions and induces cell scattering and an EMT-like conversion (10). However, no changes in E-cadherinlevels and mesenchymal markers or in key EMT regulators(Twist, Slug, Snail) were provided to support this conclusioninMCF7 cells (10). The current studies with CRC cells whilefurther supporting an increase in cell motility induced by L1,

did not observe concomitant changes in E-cadherin in avariety of CRC cell lines, or in the more motile and invasiveCRC cells of the human tumor tissue front. InCaenorhabditiselegans, the L1 homologue SAX-7 and E-cadherin play aredundant role during development and can substitute foreach other (29). These 2 molecules, however, do not com-pletely colocalize and their organization and expression is notaffected when either one of the molecules is geneticallyeliminated (29). Our results are consistent with this inde-pendence in the 2 adhesive systems mediated by L1 and E-cadherin and the lack of influence on their expression whenthe level of either of the molecules is altered.Recent studies have suggested that CSCs can be isolated

from a variety of cancer tissues using the cell surface markerCD133, including CSCs from CRC tissue (14, 15). EMT isbelieved to act in tumor development by producing cells withstem cell characteristics (11, 12) and L1 was found tocosegregate with CD133þ glioma stem cells and determinetheir metastatic capacity (13). We therefore investigatedwhether the L1-mediated increase in motility and metastaticcapacity inCRCcells is associatedwith changes in the levels ofCSC and of other markers associated with the more advancedstages in tumor progression (CD133, CD44, and EpCAM).Our results did not support a specific enrichment for L1, orcosegregation of CD133with L1when employing a variety ofCRC cell lines that expressed mixed populations of CD133þ

and CD133� cells. Recent studies have challenged the use of

A B C

D E F

G H I

Figure 6. The organization andexpression of E-cadherin is similarin cells displaying or lacking L1 intumor cells at the invasive front ofCRC tissue. A–F, adjacent serialsections of human colon cancertissue from the invasive front ofthe tumor were immunostainedwith anti-L1 (A and D), anti-E-cadherin (B and E), and anti-b-catenin (C and F). Nuclearcounterstaining is in blue color. G–I, serial sections from the centralmore differentiated region of theprimary tumor were stained as inA–F. Scale bar, 75 mm (G). Thearrowheads point to nerve cellsthat are positive for L1 (D).

Gavert et al.





CD133 as a specific marker for CSCs and found a wide rangeof epithelial tissues expressing CD133 (30). Another studyshowed that CD133� populations of tumor cells are even

more tumorigenic than their CD133þ counterparts (31).Moreover, the expression of colon CSC properties (includingthe activation of Wnt signaling) are modulated by cells andfactors in the microenvironment that can reverse the moredifferentiated cancer cells to display stem cell properties (25).The analysis of CD133 presence in human CRC tissueindicated that CD133 is not detected in the invasive cellsat the tumor front, but in the more differentiated areas of thetumor in a subpopulation of glandular cells that do not displaynuclear b-catenin (32). Because L1 is only detected in theinvasive front in cells displaying nuclear b-catenin (8), ourresults indicating no L1-CD133 cosegregation are neitherunexpected nor surprising.Although L1 expression in CRC cells did not confer an

EMT signature, overexpression of Ras in CRC cells wasshown to result (by gene expression profiling) in theinduction of an EMT-like signature (33, 34). BecauseL1-mediated metastasis in CRC cells requires NF-kBsignaling in cells expressing different E-cadherin levels (thisstudy and ref. 9), and active NF-kB colocalizes with L1 incells at the invasive tumor front (9), future studies of genearray patterns induced by L1 and NF-kB should providemore information on the mechanisms by which L1 confersenhanced motility and metastatic abilities in CRC cells.Such studies could aid in both CRC diagnosis and ther-apeutics.

Acknowledgments

We thank Dr. Shani Raveh and Dr. Mirjam Nordling for performing some of theexperiments.

Grant Support

This study was supported by grants from Israel Cancer Research Fund(ICRF), the Israel Science Foundation (ISF), the Davis Cancer Research Fund,and from Dr. and Mrs. Arnold Leibowitz. T. Brabletz is funded by the GermanResearch Organisation (DFG, grants BR1399/6-1 and SFB850, B2).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 3, 2010; revised October 20, 2010; accepted November 18,2010; published OnlineFirst December 1, 2010.

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2011;9:14-24. Published OnlineFirst December 1, 2010.Mol Cancer Res Nancy Gavert, Alessia Vivanti, John Hazin, et al. Changes in EMT and Cancer Stem Cell MarkersL1-Mediated Colon Cancer Cell Metastasis Does Not Require

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