EphB/EphrinB Receptors and Wnt Signaling in Colorectal Cancer

Hans Clevers1and Eduard Batlle

2

1Hubrecht Laboratory and Centre for Biomedical Genetics, Utrecht, the Netherlands, and 2Institucio Catalanade Recerca i Estudis Avancats and Barcelona Science Park, Barcelona, Spain

Abstract

Eph receptors and their ephrin ligands mediate cell repulsionduring embryonic development. In the intestinal epithelium,EphB receptors are Wnt signaling target genes that controlcell compartmentalization along the crypt axis. Recentfindings have shown that this family of receptors are keyplayers during colorectal cancer progression. Here, we reviewthe current knowledge of the EphB/ephrinB system in theintestinal epithelium and we discuss their tumor suppressorrole in the context of the multistep progression of colorectalcancer. (Cancer Res 2006; 66(1): 2-5)

Genetic Program Driven by B-Catenin and Tcf inColorectal Cancer

Around 70% of all colorectal cancers show homozygousinactivation of the adenomatous polyposi coli (APC) tumorsuppressor gene (1, 2). This genetic alteration results in theactivation of the Wnt signaling pathway and the constitutivetranscription by the h-catenin/Tcf complex (3, 4). Loss of APCfunction is present throughout the sequence of intestinalcarcinogenesis (i.e., from benign adenomas to fully malignantcolorectal cancer and metastasis). Even the earliest precursors ofcolorectal tumors, the so-called dysplastic crypts, show mutationalactivation of the Wnt pathway (5). In mice, activating mutations inWnt signaling pathway components lead to the formation ofdysplastic crypts and benign adenomas similar to the preneoplasticlesions developed by humans (6, 7). Together, these observationshave led to the notion that constitutive transcription by the h-catenin/Tcf complex is the key step for initiation of colorectalcancer.About 5 years ago, we undertook the identification of the genetic

program driven by h-catenin and Tcf in colorectal cancer. Incolorectal cancer cell lines bearing activating mutations of the Wntpathway, we engineered ways to block the constitutive h-catenin/Tcf–mediated transcription in an inducible manner. Gene expres-sion profile analysis of these tumor cells before and after blockageof h-catenin/Tcf activity revealed a set of target genes consistentlyexpressed in dysplastic crypts and adenomas (8). Strikingly, weobserved that virtually all the target genes identified in our screenwere also expressed in the progenitor compartment of the normal,nontransformed intestinal epithelium of mice and humans. Thisobservation was initially unexpected because the acceptedhypothesis was that the novo activation of the Wnt signalingpathway in the intestinal epithelium initiated colorectal cancer.The explanation for our observations was obvious upon study-

ing h-catenin localization in the intestinal epithelium; whereasmost epithelial cells showed only membranous h-catenin, a fewcells localized at the bottom of the crypts, presumably the stemcells and early progenitors, accumulated h-catenin in the nucleus.Thus, crypt progenitor cells receive Wnt signals as part of theirnormal physiology. The similarities between the genetic programdriven by h-catenin/Tcf in crypt progenitors and early colorectalcancer lesions lead us to propose that the first step towardsmalignancy consists in the acquisition of a crypt progenitor-likephenotype.

Modular B-Catenin and Tcf Program RegulatesIntestinal Crypt Cell Biology

Given the essential role of Wnt signaling in colorectal cancer,we and others have started to decipher the instructions given byh-catenin/Tcf to intestinal progenitors and colorectal cancercells. The genetic program driven by h-catenin/Tcf seems todictate three different sets of instructions that collectively regu-late the biology of the crypt cells. The core module enforcesthe undifferentiated-proliferative phenotype of progenitor cryptcells. Mice genetically manipulated to lack h-catenin/Tcf activityin the intestine lack proliferative progenitors (9–11). Conversely,APC deficiency in the intestinal epithelium leads to an enor-mous amplification of the progenitor compartment at the expenseof the differentiated compartment (12). This core set ofinstructions also determines the proliferative undifferentiatedphenotype of colorectal cancer cells. Blockage of h-catenin/Tcf–mediated transcription in colorectal cancer cell lines resultsin cell cycle arrest and differentiation even in the presenceof multiple alterations in other tumor suppressors and onco-genes (8).The second module of the h-catenin/Tcf program is necessary

for Paneth cell maturation (13, 14). Paneth cells represent asecretory cell type localized close to the bottom of the crypts in thesmall intestine. These cells display prominent nuclear h-cateninlocalization owing to the expression of the Wnt receptor Frizzled-5(Frz5). Several specific Paneth cell differentiation markers, such ascryptdins, are bona fide h-catenin/Tcf target genes, whoseexpression depends on Frz5 and canonical Wnt signaling.Consequently, constitutive activation of the Wnt signaling pathwayin intestinal adenomas and carcinomas results in the expression ofsome Paneth markers in tumors (14, 15). Although a role intumorigenesis has been proposed for at least one h-catenin/TcfPaneth cell target gene (i.e., the matrix metalloproteinase-7/matrylisin; ref. 16), it remains unclear if any other genes withinthis module might play a role during colorectal cancer progression.Of note, Paneth cells remain postmitotic and differentiated despitethe activation of the Wnt signaling pathway, suggesting that theyhold mechanisms to switch off the mitogenic instructions providedby h-catenin and Tcf.The third module of the h-catenin/Tcf program controls the

compartmentalization of epithelial cells along the crypt axis and

Requests for reprints: Eduard Batlle, Barcelona Science Park, Josep Samitier 1-5,08028 Barcelona, Spain. E-mail: ebatlle@pcb.ub.es.

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-3849

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regulates their ordered migration (17). The main effectors of thisfunction are the h-catenin/Tcf targets EphB2 and EphB3, twomembers of the Eph family of receptors. Eph receptors comprisethe largest subgroup of receptor tyrosine kinases characterized forbinding to membrane-bound ligands known as ephrins (18). With afew exceptions, there is a ligand subclass specificity in the Eph/ephrin family; that is, B-type Eph receptors bind preferentiallytransmembrane ephrins (B-type ephrins), whereas A-type Ephreceptors bind Gpi-membrane anchored ephrins (A-type ephrins).Eph receptors are well-known mediators of cell compartmentali-zation and ordered migration during embryonic development(18, 19). This function has been associated with their ability toprovoke cell repulsion upon activation by ephrin ligands. There arenumerous examples in embryonic development where Eph-mediated repulsion plays a key role, including the path finding ofsome axonal tracts or the formation of boundaries betweenadjacent cell populations in segmented structures, such as thehindbrain or the somites.In the small intestine, EphB3 expression is driven by h-catenin

and Tcf in epithelial cells that reside at the bottom-most posi-

tions of the crypts; that is, Paneth Cells and progenitor cells

intermingled between Paneth Cells. EphB2 is expressed by all

proliferative crypt cells in a decreasing gradient from the bottom

to the top of the crypts. In the large intestine, the expression of

both receptors is restricted to progenitor cells localized at the

crypt base, but the extent of overlapping between their expres-

sion domains has not been clearly characterized yet. The EphB

ligands, ephrinB1 and ephrinB2, are highly expressed by differ-

entiating surface and villus epithelial cells. Thus, EphB and

EphrinB genes are expressed in counter gradients along the crypt-

villus axis.We described three phenotypes in the small intestine of

EphB2/EphB3–deficient mice (17). In adult EphB3�/� mice, Panethcells are no longer sorted to the crypt base but are rather scat-tered along the crypt and the villus. Double deficient EphB2/EphB3 mice lose the boundary between the differentiated andproliferative cell compartments, a phenotype that is evident inthe intestine of juvenile mice where the crypts are not yet formed.In double EphB3�/�;EphB2�/� adult mice, progenitor cells do notmigrate towards the lumen unidirectionally but rather seem torandomly intermingle with their neighbors. Overall, these resultsindicate that EphB2 and EphB3 control cell positioning and

Figure 1. A model for tumor suppression by EphB receptors in the intestinal epithelium. A, initial tumor founder cells receive a mutation in APC or h-catenin(blue arrow ) that triggers constitutive activation of the h-catenin/Tcf complex. Consequently, the tumor founder cell remains locked into a progenitor phenotypeand repopulates the crypt with their mutant descendants (B and C). These initial tumor cells express EphB receptors as a consequence of active Wnt signaling,yet they will only encounter ephrinB bearing cells when they reach the surface epithelium. The activation of EphB receptors in tumor cells, upon interaction withephrinB–positive surface epithelial cells (D ), compartmentalizes the tumor (black arrows ) and impairs its ability to repopulate the adjacent epithelium. When tumorcells learn to silence EphB expression (E), they resume their expansion and repopulate the adjacent crypts in a top-down fashion (adapted from ref. 25).

EphB/ephrinB Receptors, Wnt, and Colorectal Cancer

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ordered migration in the intestinal epithelium. It is worth notingthat we recently found EphB4, a close homologue of EphB2 andEphB3, expressed in the intestinal epithelium with a similarpattern to that of EphB2 (20). Thus, EphB-mediated repulsionin the intestine may not be entirely abolished in EphB2-EphB3double-mutant mice. Unfortunately, EphB4 knock-out mice dieearly in embryonic development precluding the analysis of EphB4function in the intestine (21).

EphB Receptors and Colorectal Cancer Progression

The role of EphB receptors as suppressors of colorectal cancerprogression was initially suspected after analyzing the h-catenin/Tcf target gene program in a collection of human colorectal cancersamples at different stages of malignancy (20). Dysplastic cryptsand small adenomas retained expression of most h-catenin/Tcftargets present in crypt progenitors pinpointing a common tumorinitiation mechanism through mutational activation of the Wntsignaling pathway. These initial lesions showed homogenousEphB2, EphB3, and EphB4 expression in all cells at equivalentlevels to that of normal crypt progenitors. Strikingly, the majority ofcolorectal carcinomas contained >50% EphB receptor–negativecells despite evident nuclear h-catenin localization. Although wedid not find a correlation between the frequency of EphB positive(EphB+) and EphB negative (EphB�) cells and tumor staging, theirdistribution was not equivalent. Low- and medium-grade tumorareas were enriched in EphB+ cells, whereas clusters of EphB� cellscorresponded mostly to high-grade areas. As adenomas representthe benign precursors of carcinomas and tumors of higher gradeoften behave more aggressively than low-grade ones, our obser-vations implied that silencing of EphB expression has occurredin a subset of tumor cells concomitantly with the acquisition ofmalignancy.Does loss of EphB expression confer any advantages to

colorectal cancer cells? The results of our genetic experimentswere unequivocal. We engineered mice where the APCmin muta-tion was placed in a genetic background with low EphB activity.APCmin/+ mice develop benign intestinal lesions, such as dys-plastic crypts and adenomas, as a result of constitutive acti-vation of the Wnt signaling pathway (6, 7, 22). In the absence ofEphB activity, tumor progression in the large intestine ofAPCmin/+ mice is strongly accelerated resulting in the develop-ment of aggressive colorectal adenocarcinomas. Therefore,whereas constitutive activation of the Wnt signaling pathwayis required for the initiation of tumorigenesis (transition fromnormal epithelium to early adenoma stage), not all the instruc-tions codified within the h-catenin/Tcf crypt progenitor prog-ram promote tumorigenesis. Rather, the module that specifiescell positioning seems to block tumor progression beyond theearlier stages.Our observations have been recently strengthened by two inde-

pendent studies. Jubb et al. have shown that the extent of EphB2silencing in colorectal cancer correlates inversely with patientsurvival (23). Similarly, Lugli et al. also showed that loss of EphB2expression is a strong indicator of poor overall survival in colorectalcancer (24).

Tumor Compartmentalization: A New Mechanism ofTumor Suppression?

At the beginning of tumorigenesis, APC mutant cells areconfined to the epithelium within the so-called dysplastic crypts.

These tumor founder cells expand laterally and repopulate thesurrounding crypts with their mutant descendants (25). It is duringthis initial phase that EphB receptors most likely suppress tumorgrowth as EphB+ tumor cells are continuously in contact withnormal epithelial cells expressing ephrinB ligands. It is temptingto hypothesize that tumor cells are forced to respect the bound-aries imposed by EphB/ephrinB interactions much like normalprogenitors and Paneth cells are compartmentalized in the healthytissue (Fig. 1). Silencing of EphB receptors would generate a subsetof tumor cells with unrestricted capacity for repopulating theepithelium. Indeed, the EphB� population is already noticeable insome small adenomas and is present in most large adenomas,suggesting that silencing initiates at early stages. Alternatively,EphB receptors could also suppress tumor progression in moreadvanced stages, yet it is important to consider that ephrinBligands are membrane-bound proteins and, therefore, EphBactivation can only occur upon interaction of tumor cells withephrinB bearing cells. Unfortunately, the lack of good anti-ephrinBantibodies has hampered the study of their expression patterns incolorectal cancer.But how do EphB/ephrinB signals suppress tumor progression

at the molecular level? The best understood function of EphBreceptors is the control of cell shape by the Rho family of smallGTPases. Colorectal cancer cell lines remodel their actin cytoskel-eton and contract upon ephrinB stimulation, a process which isdependent on inhibition of rap-1 activity and activation of Rho/Rock (17, 26, 27). Although a change in cell shape might benecessary to restrict the migration of normal and transformed cells,it remains unclear whether it is sufficient to inhibit tumorprogression. The study of the crosstalk between EphB receptorsand the signaling pathways that drive intestinal tumorigenesis willbe central to understand the tumor suppressor role of EphBreceptors in colorectal cancer.Finally, the mechanism of silencing of EphB expression in

colorectal cancer also remains uncharacterized. Point mutations inEphB2 have been identify in a small fraction of prostate cancers(28). EphB2 is localized in 1p35-p36.1, a region of frequent allelicloss in colorectal cancer. A study of 50 colorectal cancer samplesidentified loss of heterozygosity (LOH) of EphB2 locus in 33% ofsamples, yet no mutations in the remaining allele were found inany tumor (29). Assessment of the methylation status of the wild-type allele in tumors with EphB2 LOH would be required tocomplement this study. Although genetic alterations or epigeneticsilencing in individual EphB genes will be probably identified in afraction of colorectal cancers, our observations indicate thatEphB2, EphB3, and EphB4 are coordinately silenced in the majorityof colorectal cancer samples (20). Thus, it is unlikely thatmutations, LOH, or methylation account for the coordinatedsilencing of all three EphB genes during colorectal cancerprogression. In the great majority of cancers, down-regulation ofEphB receptors occurs at the mRNA level (20, 23). EphB expressionis fully dependent on h-catenin/Tcf activity, yet many colorectalcancers and cell lines down-regulate EphB levels despite constitu-tive activation of the Wnt signaling pathway. Together, theseobservations point to a common mechanism of transcriptionalsilencing of EphB genes that acts in a dominant fashion overh-catenin/Tcf activation.

Acknowledgments

Received 10/24/2005; accepted 11/4/2005.

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