The Receptor Tyrosine Kinase EPHB4 Has Tumor Suppressor Activities in Intestinal Tumorigenesis

Published OnlineFirst September 8, 2009; DOI: 10.1158/0008-5472.CAN-09-0706

Molecular Biology, Pathobiology, and Genetics

Higinio Dopeso,1,5 Silvia Mateo-Lozano,1 Rocco Mazzolini,1 Paulo Rodrigues,1 Laura Lagares-Tena,1 Julian Ceron,2 Jordi Romero,1,5 Marielle Esteves,1 Stefania Landolfi,4 Javier Herna´ndez-Losa,4 Julio Castan˜o,3 Andrew J. Wilson,6 Santiago Ramon y Cajal,4 John M. Mariadason,7 Simo Schwartz, Jr.,3,5 and Diego Arango1,5

Groups of 1Molecular Oncology, 2Functional Genomics and Genetics, and 3Drug Delivery and Targeting, Molecular Biology and Biochemistry Research Center (CIBBIM-Nanomedicine) and 4Department of Pathology, Vall d’Hebron Hospital; 5CIBER de Bioingenierı´a, Biomateriales y Nanomedicina, Barcelona, Spain; 6Department of Obstetrics and Gynecology, Vanderbilt University Medical Center, Nashville, Tennessee; and 7Ludwig Institute for Cancer Research, Melbourne Centre for Clinical Sciences, Austin Health, Heidelberg, Victoria, Australia

Abstract Introduction Colorectal cancer is the second cause of cancer-related death Colorectal cancer is one of the leading causes of cancer-related in the western world, and although the genetic and molecular death in the western world and accounts for f1 million new mechanisms involved in the initiation and progression of these cases and f500,000 deaths every year worldwide. At the molecular tumors are among the best characterized, there are significant level, the constitutive activation of Wnt signaling is one of the gaps in our understanding of this disease. The role of EPHB hallmarks of colorectal cancer. Wnt activation most frequently signaling in colorectal cancer has only recently been realized. occurs through Apc mutations, preventing the phosphorylation of Here, we use animal models to investigate the role of EphB4 the key coactivator h-catenin, which accumulates in the nucleus, in intestinal tumorigenesis. Modulation of EPHB4levels in binds to TCF/LEF transcriptions factors, and activates the colon cancer cell lines resulted in significant differences in promoter of a large number of mitogenic genes, such as c-MYC, tumor growth in a xenograft model, with low levels of EPHB4 cyclin D1, and several EPHB receptors (1–4). Cell proliferation and associated with faster growth. In addition, using a genetic positioning in both normal and transformed intestinal epithelium model of intestinal tumorigenesis where adenomatous poly- are largely controlled by Wnt signaling through EPH signaling posis coli (Apc) mutations lead to initiation of the tumorigenic cascades. EPH receptors constitute the largest receptor tyrosine process (Apcmin mice), we show that inactivation of a single kinase family with at least 16 different receptors identified, and allele of EphB4results in higher proliferation in both the EPHs are grouped into A and B subtypes according to sequence normal epithelium and intestinal tumors, significantly larger homology and ligand binding specificity (5). EPHB2 and EPHB3 tumors in the small intestine, and a 10-fold increase in the have been shown to regulate both proliferation in the intestinal number of tumors in the large intestine. This was associated crypts and positioning of cells of different lineage within the min with a 25% reduction in the lifespan of Apc mice (P < normal intestinal epithelium (3, 6). Although EPHB2/3 knockout 0.0001). Gene expression analysis showed that EphB4muta- mice do not develop tumors, the oncogenic process is substantially tions result in a profound transcriptional reprogramming, accelerated upon tumor initiation by adenomatous polyposis coli affecting genes involved in cell proliferation, remodeling (Apc) inactivation (4). EPHB4 expression has been shown to be of the extracellular matrix, and cell attachment to the base- frequently lost in advanced human colorectal tumors and we have ment membrane among other functional groups of genes. recently reported that low tumor EPHB4 levels are associated with Importantly, in agreement with the expression profiling expe- poor prognosis of colorectal cancer patients, and modulation of in vitro riments, using an assay, we show that loss of EPHB4 EPHB4 levels in colorectal cancer cell lines interfered with the long- in colon cancer cells results in a significantly increased po- term clonogenic potential of these cells (4, 7). However, EPHB4 tential to invade through a complex extracellular matrix. has oncogenic activities in a variety of tumor types, including pro- Collectively, these results indicate that EphB4has tumor state, breast, endometrium, mesothelioma, head and neck, bladder, suppressor activities and that regulation of cell prolifera- and ovarian cancer (8–14), and after a recent study reported tion, extracellular matrix remodeling, and invasive potential a possible oncogenic effect of EPHB4 (15), the functional role of are important mechanisms of tumor suppression. [Cancer Res EPHB4 in colorectal tumors remains to be elucidated. 2009;69(18):7430–8] In this study, we investigate the role of EPHB4 in colorectal tumorigenesis using animal models. We used a xenograft approach to show that restoring EPHB4 levels in deficient colon cancer cells leads to significantly smaller tumors, whereas inactivation of EPHB4 signaling in cell lines with high levels results in faster Note: Supplementary data for this article are available at Cancer Research Online tumor growth. In addition, loss of a single allele of EphB4 was (http://cancerres.aacrjournals.org/). found to result in a 25% reduction of the lifespan of Apcmin mice Requests for reprints: Diego Arango, Group of Molecular Oncology, Molecular Biology and Biochemistry Research Center (CIBBIM-Nanomedicine), Vall d’Hebron where intestinal tumorigenesis is initiated by Apc inactivation. Hospital Research Institute, Passeig Vall d’Hebron 119-129, Barcelona 08035, Spain. A 2-fold reduction in EphB4 expression was observed in the normal Phone: 34-93-274-6739; Fax: 34-93-489-3893; E-mail: [email protected]. I2009 American Association for Cancer Research. epithelial crypts and intestinal tumors. The reduced lifespan of +/À +/+ doi:10.1158/0008-5472.CAN-09-0706 EphB4 mice compared with EphB4 animals was associated

Cancer Res 2009; 69: (18). September 15, 2009 7430 www.aacrjournals.org

EPHB4 in Colorectal Cancer with higher proliferation in both the normal epithelium and intest- Systems) as described previously (19, 20). A 1:1,000 dilution was used to inal tumors as well as a significantly increased tumor burden. detect active caspase-3 (R&D Systems) following treatment for 20 min with j This was linked with the transcriptional reprogramming of citrate buffer (10 mmol/L, pH 6) at 95 C. multiple growth factors and genes involved in extracellular matrix RNA extraction and quantitative reverse transcription-PCR. Total RNA of small intestinal tumors was extracted with the RNeasy Micro Kit remodeling. Importantly, the role of EPHB4 in the capacity of colon (Qiagen) and reverse transcribed to cDNA using the cDNA Archive Kit cancer cells to invade through the extracellular matrix was directly (Applied Biosystems). EphB4, IGFBP5, MMP2, and Col5a2 levels were in vitro shown using an assay. Collectively, these results indicate assessed by real-time PCR using SYBR Green Master Mix (Applied that EPHB4 has tumor suppressor activities in intestinal tumor- Biosystems) and 18S rRNA was used as a standardization control. Primer igenesis and show that tumor suppression is associated with the sequence is available in Supplementary Fig. S3. Relative mRNA levels were ÀDD control of cell proliferation, extracellular matrix remodeling, and assessed using the 2 Ct method as described before (21, 22). invasive potential. Microarray experiments. Fresh tumors from the jejunum were dissected and total RNA was extracted as described above. Equal amounts of RNA from three different tumors of 18-week-old mice were pooled and Materials and Methods processed for hybridization on Affymetrix Mouse 430 2.0 chips (21). Tumor min/+ +/+ min/+ +/À Cell lines. We have previously assessed EPHB4 levels in a panel of pools from two Apc ;EphB4 and two Apc ;EphB4 mice were colorectal cancer cell lines and found that SW837 and HT29 have low and hybridized independently. The expression values obtained from all the high relative levels of EPHB4, respectively (7). SW837 cells were stably hybridizations were normalized using dChip software (23). Functional transfected with a construct expressing full-length EPHB4 (16) or the group enrichment analysis was used to compare the number of genes corresponding empty vector control (pcDNA3.1; Invitrogen). HT29 cells present in the Affymetrix 430 2.0 chip in each one of the Gene Ontology (24) were stably transfected with pEGFP-N2 (Clontech) or pEPHB4DC-EGFP, a categories (cellular component, molecular function, and biological process) and the number of genes in each one of these categories in the list of 183 construct coding for a chimeric EPHB4 protein where the kinase domain min/+ has been substituted by enhanced green fluorescent protein (EGFP). This genes differentially expressed in tumors from Apc mice that are wild- protein has been shown before to function as a dominant negative (17). type or heterozygous for EphB4. This analysis was implemented using L2L P À4 Western blot. Extracts of total protein (50 Ag) in radioimmunopreci- software (25) and a level of significance of <10 (binomial test) was used. pitation assay buffer were loaded on a 10% acrylamide gel. After gel We performed a Functional Network Analysis of the 183 differentially electrophoresis, proteins were transferred to a nitrocellulose membrane and expressed genes found in the microarray experiments using the Web- probed with anti-EPHB4 (1:200; clone 3D7G8; Zymed Laboratories), anti- interface MouseNet version 1 (26). The resulting network was exported to EGFP (1:1,000; Clontech), or anti-h-actin (1:1,000; clone AC74; Sigma) as Cytoscape 2.6.0 for graphical representation (27). described previously (7). Matrigel invasion assay. Cell culture inserts for 24-well plates with an A Xenograft model. Six athymic nude-Foxn1^un mice (Harlan) were 8 m pore size were used (Becton Dickinson). Cells were trypsinized and Â 6 injected s.c. with 1.5 Â 106 SW837-pcDNA3.1 (left flank) and SW837- resuspended at a density of 1 10 /mL in DMEM with 1 mg/mL Matrigel pcDNA3.1-EPHB4 (right flank) cells resuspended on growth medium. The (Becton Dickinson). One hundred microliters were placed in the top j same experimental setup was carried out for the HT29 pEPHB4DC-EGFP chamber of the insert. After incubation for 1 h at 37 C, the bottom derivative line and the corresponding empty vector control. Tumor size compartment was filled with DMEM with 10% fetal bovine serum and the A was measured using a caliper three times per week for a total of 6 weeks. cells were covered with 100 L fresh medium. After 72 h, cells adhering to Tumor volume was calculated with the formula: V =(L Â W2) Â 0.5, where the bottom surface were fixed with gluteraldehyde 5% in PBS, stained with L is the length and W is the width of a xenograft. crystal violet, and counted. Twelve random fields were counted at 400-fold Mouse knockout strains. The C57BL/6J-Apcmin/J strain was obtained magnification. Each sample was assayed in triplicate, and all experiments from The Jackson Laboratory. These mice carry a heterozygous mutation were repeated three times. in Apc. EPHB4/LacZ mice carry an EphB4 allele in which the signal sequence has been deleted and a tau-lacZ indicator gene has been fused in- Results frame with the initiation codon, preventing membrane insertion of the receptor (18). Male Apcmin/+;EphB4+/+ mice were crossed with female EPHB4regulates the growth of colon cancer cells. We have À Apc+/+;EphB4+/ mice and the resulting Apcmin/+;EphB4+/+ and Apcmin/+; reported previously that EPHB4 levels regulate the clonogenic À EphB4+/ offspring were used in the experiments described. potential of colorectal cancer cells (7). Here, we used a xenograft Immunohistochemistry. Eighteen-week-old mice were sacrificed, the model to evaluate the role of EPHB4 on the growth of human colon small and large intestines were dissected and opened longitudinally, and cancer cells. We stably transfected a colon cancer cell line tumor size and number were scored. The tissue was then fixed with 10% expressing relative high levels of EPHB4 (HT29) with a construct formalin and embedded in paraffin. Antigen retrieval was carried out with containing the human EPHB4 extracellular and transmembrane j preheated EDTA (1 mmol/L, pH 8) for 5 min at 95 C. The following domain and EGFP instead of the kinase domain (pEPHB4DC-EGFP; antibodies were used: anti–proliferating cell nuclear antigen (PCNA)-biotin Fig. 1A). This protein has been shown to function as a dominant- (1:200; Biolegend), anti-EPHB4 (0.1 Ag/mL; R&D Systems), and rat anti- negative form of EPHB4 (EPHB4-DN; ref. 17). We then injected CD34 (1:50; MEC14.7; Biolegend). CD105 levels were assessed in frozen Â 6 sections using a phycoerythrin-conjugated rat monoclonal antibody (1:250; s.c. 1.5 10 cells of the empty vector control HT29-EGFP-EV line eBioscience) and 4¶,6-diamidino-2-phenylindole counterstaining. CD105 (left flank) or HT29-EPHB4-DN cells (right flank) in six athymic levels were quantified blindly using ImageJ software8 in at least three nude mice. Xenograft growth was monitored three times per week different random fields from six different xenografts. The levels of EphB4 for a total of 6 weeks. Overexpression of the dominant-negative were quantified blindly using ImageJ in at least five representative fields EPHB4 was shown to result in significantly larger tumors com- from five animals in each group. pared with control cells (Fig. 1B). In addition, using a cell line with Detection of apoptosis. Apoptotic cells were detected using the low relative levels of EPHB4 (SW837), we engineered an isogenic terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling derivative line that overexpressed this receptor (Fig. 1C). When assay or immunodetection with an anti–active caspase-3 antibody (R&D grown as a xenograft in nude mice, overexpression of EPHB4 resulted in significantly slower tumor growth (Fig. 1D). EPHB4 has been reported previously to modulate angiogenesis in normal and 8 http://rsbweb.nih.gov/ij/ tumor tissues (18, 28). However, immunohistochemical staining of www.aacrjournals.org 7431 Cancer Res 2009; 69: (18). September 15, 2009

Cancer Research

Figure 1. EPHB4 levels regulate tumor growth in a xenograft model. HT29 cells (1.5 Â 106) stably transfected with a dominant-negative form of EPHB4 (EPHB4-DC-EGFP) or the corresponding empty vector control (A) were s.c. injected in the flanks of immunocompromised nude mice. Introduction of the dominant-negative EPHB4 (dashed line) resulted in significantly increased tumor growth (B). SW837 cells were stably transfected with a vector expressing full-length EPHB4 or the corresponding empty vector control (C). Reintroduction of EPHB4 in SW837 cells (dashed line) resulted in significantly slower tumor growth (D).

the endothelial markers CD34 and CD105 (endoglin) did not reveal (median survival of 149.5; P < 0.0001, log-rank test; see Fig. 2), significant differences in the vasculature of the xenografts of the suggesting that EphB4 inactivation accelerates the tumorigenic isogenic cell lines where EPHB4 levels were altered (Supplementary process in these animals. Fig. S1A-D). Together, these results suggest that the lower levels EphB4inactivation results in larger tumors in the small of EPHB4 frequently observed in colorectal tumors (4, 7) may intestine and higher tumor number in the large intestine. contribute to tumor progression. To understand the underlying cause of the shorter survival À EphB4inactivation results in shorter survival of Apc min/+ observed in Apcmin/+;EphB4+/ animals compared with Apcmin/+; mice. To further investigate the role of EphB4 on intestinal tumori- EphB4+/+, we investigated the number, size, and location of the genesis, we used a genetic mouse model carrying an inactivated tumors found in animals sacrificed at age 18 weeks. There were EphB4 allele (18). Homozygous mutations in this receptor cause no significant differences in the number of tumors found in the embryonic lethality, whereas heterozygous animals are viable and small intestine (22.1 F 11.8 and 26.1 F 12.7 in EphB4+/+ and À have no gross abnormalities (18). Because no tumors have been EphB4+/ , respectively; P = 0.48; Fig. 3A). However, the average size À À reported in the gastrointestinal tract of EphB4+/ mice, we decided of the tumors found in Apcmin/+;EphB4+/ mice was >50% larger to initiate the tumorigenic process introducing a mutant allele of than the tumors found in Apcmin/+;EphB4+/+ mice (1.59 F 0.44 and Apc. Heterozygous Apc mutant animals (Apcmin/+) developed 1.06 F 0.18 mm, respectively; P = 0.0002; Fig. 3B). The majority multiple tumors within the gastrointestinal tract and had a median of the tumors observed in Apcmin/+ mice were located in the lifespan of 199 days (Fig. 2). Introduction of a single mutant allele jejunum. However, mutation of one allele of EphB4 in Apcmin/+ of EphB4 on the Apcmin/+ background shortened the lifespan animals resulted in a shift toward more proximally located tumors of these animals by f25% compared with EphB4+/+ littermates (P = 0.0007, Fisher’s test). Whereas only 20% of the tumors in

Cancer Res 2009; 69: (18). September 15, 2009 7432 www.aacrjournals.org

EPHB4 in Colorectal Cancer

The histology of a total of 101 intestinal tumors was micro- scopically examined. They presented as sessile or plaque-like lesions. The vast majority of these tumors were adenomas with low-grade (55 of 101) and high-grade (46 of 101) dysplasia. Six of the examined tumors were intramucosal adenocarcinomas with lamina propia invasion and one of them showed evidence of vascular invasion. No significant differences were observed in the type of tumors in Apcmin/+ mice that were EphB4 wild-type or heterozygous. As reported previously (4), the high levels of EphB4 expression observed in the proliferating cells within the normal crypts were maintained in the adenomatous lesions of Apcmin/+;EphB4+/+ mice (Fig. 4C). Here, we show that the tumors À of Apcmin/+;EphB4+/ mice have reduced EphB4 protein levels (Fig. 4C and D). In good agreement, a 51% reduction in EphB4 mRNA levels (P = 0.0019, Student’s t test) was also observed in min/+ Figure 2. Inactivation of a single allele of EphB4 in Apc mice shortens tumors from EphB4 heterozygous mice as assessed by quantitative animal survival. Heterozygous inactivation of EphB4 in animals carrying Apc mutations (dashed line; n = 26) results in a 25% reduction of the lifespan of these reverse transcription-PCR. To study the underlying mechanisms animals compared with EphB4 wild-type animals (n = 24; median survival of leading to larger small intestinal tumors, we investigated the effects 149.5 and 199 d, respectively; P < 0.0001, log-rank test). of the reduced EphB4 levels on apoptosis, vascularization, and proliferation. No changes were observed in the number of EphB4+/+ mice were located in the duodenum, 35% of the tumors in apoptotic cells (terminal deoxynucleotidyl transferase–mediated À EphB4+/ were found in the duodenum. In addition, whereas the dUTP nick end labeling or active caspase-3 positive) in the tumors À large intestine of Apcmin/+;EphB4+/+ animals had only an average of of Apcmin/+;EphB4+/ mice compared with tumors in Apcmin/+; À 0.25 macroscopically visible tumors per mouse, Apcmin/+;EphB4+/ EphB4+/+ animals (data not shown). In addition, immunostaining mice had 2.4 tumors on average (9.6-fold increase; Fig. 3C and D). The larger tumors found in the small intestine and the higher tumor frequency in the large intestine are likely to contribute to À the shorter survival of the Apcmin/+;EphB4+/ compared with the Apcmin/+;EphB4+/+ counterpart. EphB4levels, proliferation, apoptosis, and differentiation. We used immunohistochemical staining to evaluate the effects of EphB4 mutations in the levels of expression of EphB4 in the normal and transformed intestinal epithelium of Apcmin mice. Rigorous quantification of EphB4 immunostaining showed that targeted deletion of one allele of EphB4 led to a 52% reduction in the levels of expression of EphB4 in the proliferative compartment of the normal small intestine (P = 0.02, Student’s t test; Fig. 4A and B). To further investigate the underlying mechanisms responsible for the shorter survival and higher tumor burden in À Apcmin/+;EphB4+/ mice relative to the Apcmin/+;EphB4+/+ counterpart, we studied the effects on differentiation, apoptosis, and proliferation in the normal intestinal epithelium. Although other EphB receptors have been reported to control lineage-specific cell sorting within the intestinal crypts (3), no differences in the number or localization of goblet cells (Alcian blue positive; Sup- plementary Fig. S2A and B), Paneth cells (lysozyme positive; Sup- plementary Fig. S2C and D), or enteroendocrine cells (Grimelius staining; Supplementary Fig. S2E and F) were observed in the in- À testinal epithelium of Apcmin/+;EphB4+/ and Apcmin/+;EphB4+/+ mice. In addition, no differences were observed in the number of apoptotic cells that were positive for active caspase-3 or terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining (data not shown). In the epithelial lining of the normal intestine, cell division is confined to the crypt compartment. We assessed proliferation in the small intestinal crypts by scor- ing the number of cells that were positive for immunostaining Figure 3. Tumor number and size. A, number of tumors in Apcmin/+;EphB4+/À with PCNA, a well-established proliferative marker (Fig. 5). mice (n = 18) was not significantly different from Apcmin/+;EphB4+/+ animals The number of proliferating cells was significantly higher in (n = 7). However, the average size of the tumors observed was 50% larger in the min/+ Apcmin/+;EphB4+/À mice compared with Apcmin/+;EphB4+/+ animals (B). The Apc mice that are heterozygous for EphB4 compared with min/+ +/À P t number of tumors in the Apc ;EphB4 was f9.6-fold higher than in EphB4 wild-type animals (18.3% increase; = 0.03, Student’s test; EphB4 wild-type mice (C), although these tumors were smaller than in Fig. 5A and C). Apcmin/+;EphB4+/+ mice (D). www.aacrjournals.org 7433 Cancer Res 2009; 69: (18). September 15, 2009

Cancer Research with the endothelial marker CD34 did not reveal differences in the genes were represented by more than one probe set showing vascularization of the small intestinal tumors from Apcmin/+ mice excellent agreement between independent expression measure- that are wild-type or heterozygous for EphB4 (Supplementary ments for the same gene (Pearson’s correlation R = 0.94; P < Fig. S1E and F). However, when assessed as the number of PCNA- 0.0001). Of the 183 genes differentially expressed, 21 had reduced positive cells, the loss of a single allele of EphB4 led to a significant expression and 162 elevated expression in tumors from Apcmin/+ increase (33%; P = 0.02, Student’s t test) in the number of pro- mice that were EphB4 heterozygous compared with Apcmin/ liferating cells (Fig. 5B and D). The higher proliferative rates +;EphB4+/+ animals (Supplementary Table S1), indicating a global observed in the tumors from Apcmin/+ mice that are EphB4 inhibitory role imposed by EphB4 in intestinal tumors. Quantitative heterozygous are in good agreement with the 50% increase in real-time reverse transcription-PCR was used to confirm the tumor size observed. expression differences observed in the microarray experiments for Transcriptional reprogramming imposed by EphB4inacti- four genes (EphB4, Igfbp5, Mmp2, and Col5a2). An excellent vation. To gain further insight into the molecular mechanisms correlation was observed between microarray and quantitative underlying the tumor suppressor activity observed for EphB4 in the reverse transcription-PCR assessments of gene expression (R = intestine of Apcmin/+ mice, we used oligonucleotide microarray 0.99; P = 0.003; Supplementary Fig. S3). analysis to assess differences in the expression levels of >39,000 Activation of the Wnt signaling pathway is one of the hallmarks transcripts in tumors from Apcmin/+ mice that are EphB4 wild-type of intestinal tumorigenesis. Loss of EphB4 in the normal epithelium or heterozygous for EphB4. To assess the level of background and small intestinal tumors did not result in differences in Wnt variability between hybridizations, we examined the mean fold signaling as assessed by the absence of expression differences in difference of 78 housekeeping genes (29) in tumors from EphB4+/+ a list of 20 well-characterized h-catenin/TCF4 target genes (fold À and EphB4+/ animals and was found to be 1.1 F 0.1 (mean F SD). difference <2; Supplementary Table S2), h-catenin immunostaining The 183 genes with >2-fold expression difference in tumors from (Supplementary Fig. S4A-D), and direct assessment of h-catenin/ EphB4 wild-type and heterozygous mice were considered to be TCF4 transcriptional activity using transient transfection of a differentially expressed. This cutoff value is >20 times the SD reporter construct in colon cancer cells with different levels observed in the set of 78 housekeeping genes used to define the of EphB4 (Supplementary Fig. S4E). Previous studies have in- baseline variability. Seventeen of the 183 differentially expressed vestigated the transcriptional changes leading to the transition

Figure 4. EphB4 levels in the intestine. Levels of expression of EphB4 in the intestinal crypts (A and B)and tumors (C and D)ofApcmin/+;EphB4+/À mice were significantly reduced compared with Apcmin/+;EphB4+/+ animals when assessed by immunohistochemistry.

Cancer Res 2009; 69: (18). September 15, 2009 7434 www.aacrjournals.org

EPHB4 in Colorectal Cancer

Figure 5. Proliferation in the normal intestine and tumors of Apcmin/+;EphB4+/À animals. A and B, presence of PCNA-positive cells in the normal small intestinal epithelium and small intestinal tumors, respectively, of Apcmin/+;EphB4+/+ (left) and Apcmin/+;EphB4+/À (right). C, quantification of PCNA-positive cells in at least 30crypt columns from 6 Apcmin/+;EphB4+/+ mice and 12 Apcmin/+;EphB4+/À animals showed a significant increase in the number of proliferating cells in the normal small intestine of EphB4 heterozygous mice. D, quantification of PCNA-positive cells in two independent fields from three different tumors from the same animals showed a 33% increase in the number of proliferating cells in the small intestinal tumors from EphB4 heterozygous animals. N, normal; T, tumor.

from adenoma to carcinoma in intestinal tumors of Apcmin/+ mice single copy of EphB4 leads to transcriptional changes that favor and identified differences in nine key genes (30). Remarkably, tumor progression. an excellent correlation (R = 0.96; P < 0.003) was observed between Functional group analysis and interactome analysis identified a the fold expression difference observed for these genes in the subset of genes involved in extracellular matrix reorganization, adenoma-to-carcinoma transition and in tumors from Apcmin/+ accounting for >28% (52 of 183) of the genes differentially expressed mice that are wild-type or heterozygous for EphB4 (Supplementary (Supplementary Fig. S6; Supplementary Tables S4-S6). Among these Fig. S5; Supplementary Table S3). In addition, there were a sig- genes, there were 12 different types of collagen (Col15a1, Col18a1, nificant number of growth factors up-regulated in tumors from Col1a1, Col1a2, Col3a1, Col4a1, Col4a2, Col5a1, Col5a2, Col5a3, Col6a2, À EphB4+/ mice compared with EphB4+/+ animals, including and Col6a3), 3 matrix metalloproteinases (MMP2, MMP10, and insulin-like growth factor-I, connective tissue growth factor, MMP13), as well as other proteins involved in remodeling of the fibroblast growth factor-7, amphiregulin, epiregulin, discoidin extracellular matrix such as decorin, biglycan, lysyl oxidase–like 2, domain receptor tyrosine kinase 2, and platelet-derived growth lumican, and Sparc (see Supplementary Table S1). These results factor receptor (see Supplementary Table S1). The transcrip- strongly suggested the possibility that EphB4 may regulate the capa- tional changes observed in tumors from Apcmin/+;EphB4+/+ mice city of intestinal tumor cells to invade through the extracellular matrix. are likely to be responsible for the increased proliferation found EPHB4regulates the migration/invasion of colon cancer in these tumors. Overall, these results indicate that inactivation of a cells. To test the hypothesis that reduced EPHB4 levels favor www.aacrjournals.org 7435 Cancer Res 2009; 69: (18). September 15, 2009

Cancer Research migration/invasion, we used isogenic HT29 colon cancer cells cancer genes in the tumorigenic process in vivo. Here, we show that expressing high levels of EPHB4 and a derivative line stably inactivation of a single allele of EhpB4 is sufficient to cause a 25% transfected with a dominant-negative form of EPHB4 (EPHB4DC- reduction in the lifespan of mice where the tumorigenic process is EGFP; see Fig. 1C). This system models the reduced EPHB4 expres- initiated by heterozygous Apc mutations. We show that the reduced À À sion observed in tumors from Apcmin/+;EphB4+/ mice compared lifespan of EphB4+/ animals is associated with increased prolifer- with tumors from Apcmin/+;EphB4+/+ animals (see Fig. 4C-E) and in ation, larger tumors in the small intestine, and a 9.6-fold increase in human tumors. We used a Matrigel invasion assay to show that the number of tumors in the large intestine of these mice, indicating reduced EPHB4 signaling resulted in a 5.6-fold increase in the a tumor suppressor function for EphB4 in this organ. number of invading cells (Fig. 6A). Moreover, overexpression of Several EPHB receptors, including EPHB2, EPHB3, and EPHB4, EPHB4 in SW837 cells led to a 2.5-fold reduction of the invasive have been reported to be direct transcriptional targets of h-catenin/ potential of these cells (Fig. 6B). These results suggest that the TCF and EphB2 and EphB3 signaling has been shown to be profound expression reprogramming observed in intestinal tumors important for maintaining active proliferation in the crypts of the À from EphB4+/ mice compared with the wild-type counterpart intestinal epithelium (3, 6). Abrogation of EphB2/EphB3 signaling by leads to an increased capacity of the tumor cells to invade/migrate targeted inactivation in murine models results in f50% reduction through the extracellular matrix surrounding the tumor. of the number of proliferating cells in normal intestinal crypts and adenomas observed in Apcmin/+ mice (6). In this study, we observed significantly increased proliferation in the normal epithelium and Discussion small intestinal adenomas of Apcmin/+ mice that are heterozygous EPHB4 plays important roles during embryogenesis and mainte- for EphB4 compared with EphB4 wild-type animals. Therefore, nance of normal homeostasis of different adult tissues such as the unlike other EphB receptors, disruption of EphB4 signaling seems brain and the vasculature (5). In addition, the expression to promote tumorigenesis by increasing proliferation. of EPHB4 has been reported to be deregulated during tumori- Previous studies investigating transcriptional changes associated genesis in different tissues. Most of the reports point at EPHB4 being with tumor progression using the Apcmin/+ mouse model identified overexpressed in several tumor types such as prostate, mesotheli- a small set of genes associated with the transition from adenoma to oma, head and neck, bladder, and ovarian cancer, and elevated levels carcinoma (30). The expression changes induced by the inactiva- are linked to a protumorigenic role of this receptor (8, 10–13). tion of a copy of EphB4 in Apcmin/+ mice, which are associated with However, conflicting results have been reported for EPHB4 in breast larger tumor size and shorter animal lifespan, closely resembled tumors, where it may have tumor suppressor activity (9, 31). the expression changes observed in the adenoma-to-carcinoma Although the tumor suppressor effects of other EPHB receptors have transition (see Supplementary Fig. S5; R = 0.96; P < 0.003). The recently been shown (4, 6, 32), the possible role of EPHB4 in expression changes induced by EphB4 inactivation in Apcmin/+ intestinal tumorigenesis remains unclear. Some studies have mice and in the adenoma-to-carcinoma transition include the up- reported EPHB4 overexpression in colorectal tumors (33, 34) and regulation of insulin-like growth factor binding protein 5, which the elevated EPHB4 levels have been suggested to confer a growth may potentiate signaling through the proliferatory insulin-like advantage to colon tumor cells (15). Moreover, EPHB4 targeting has growth factor signaling pathway, and the serine peptidase inhibitor been suggested to be of therapeutic value in colorectal tumors (15). Serpine2. In addition, there was a reduction in the expression However, this is in disagreement with the results from other studies of S100g (calbindin D9K), which regulates the intracellular levels showing that EPHB4 expression is frequently lost in colorectal of calcium, and the transcriptional regulator Sox17, which is a tumors (4, 7). Moreover, the loss of EPHB4 expression is associated negative regulator of Wnt signaling and is silenced by promoter with tumor dissemination and reduced patient survival (4, 7). hypermethylation in most colorectal tumors (35, 36). Also, deletion Animal models constitute a powerful tool to elucidate the role of of a copy of EphB4 leads to a significant shift in the balance

Figure 6. EPHB4 regulates cell invasion. A Matrigel invasion assay revealed that interfering with EPHB4 signaling in HT29 cells using a dominant-negative form of EPHB4 resulted in a 5.6-fold higher number of invading cells (A). Conversely, reintroduction of EPHB4 into SW837 cells led to a 2.5-fold decrease of the invasive potential (B). *, P < 0.01 (Student’s t test).

Cancer Res 2009; 69: (18). September 15, 2009 7436 www.aacrjournals.org

EPHB4 in Colorectal Cancer maintaining the proliferative state of the tumor cells with several We have reported previously that EPHB4 expression is reduced or mitogenic genes being up-regulated, such as Ptgs2/Cox2, Wnt5a, lost in primary human colorectal tumors compared with the normal and Igf1, and some counterbalancing antiproliferative genes, such proliferating cells in intestinal crypts (7). Moreover, EPHB4 levels are as secreted frizzled-related sequence protein 1. Collectively, the further reduced in lymph node metastases compared with primary expression reprogramming observed is likely to underlie the in- tumors and there is frequent EPHB4 promoter hypermethylation in creased proliferation, the larger tumor size, and ultimately the colorectal tumors (7). In addition, we observed that low EPHB4 À shorter animal survival observed in the EphB4+/ mice. Despite the tumor levels are significantly associated with shorter survival of larger adenoma size, we did not observe a shift toward a more colorectal cancer patients (7). Here, we report that targeted inac- À aggressive phenotype in tumors from EphB4+/ mice. This is in tivation of a single allele of EphB4 in Apcmin/+ mice leads to a 25% good agreement with the observation that the loss of EPHB4 is shortening of animal survival and this is associated with increased specifically associated with the transition from small to large proliferation and larger tumor size in the small intestine and a adenoma in human lesions (4). Whereas >70% of the human small 9.6-fold increase in the number of tumors in the large intestine. adenomas (<5 mm) retain high levels of EPHB4, 90% of the large Also, we show that reduced EPHB4 expression results in changes in adenomas (>5 mm) show reduced EPHB4 levels compared with the expression of multiple genes involved in extracellular matrix the normal proliferating epithelial cells (P = 0.001, Fisher’s test). remodeling, strongly suggesting that tumor cells expressing low A large proportion of the genes differentially expressed in the EPHB4 levels have increased migration/invasion potential. We intestinal tumors from Apcmin/+ mice that are wild-type or hetero- directly tested this hypothesis and showed that low EPHB4 levels zygous for EphB4 are involved in the synthesis and remodeling are associated with increased invasion of colon cancer cells through of the extracellular matrix and regulation of cell attachment to a complex extracellular matrix. Collectively, in this study, we provide the basement membrane. The profound reprogramming caused mechanistic insight showing that the loss of EphB4 results in by heterozygous EphB4 inactivation included the coordinate up- increased proliferation and migration/invasion potential, contribut- regulation of 12 different types of collagen as well as multiple ing to the progression from small to large intestinal adenomas. In enzymes involved in remodeling of the extracellular matrix such as conclusion, our findings support the idea that EPHB4 has tumor several matrix metalloproteinases (MMP2, MMP10, and MMP13), suppressor activities in intestinal tumorigenesis. decorin, biglycan, lysyl oxidase–like 2, lumican, and Sparc (see Sup- plementary Table S1). Here, we show that EPHB4 modulates the capacity of colon cancer cells to invade through a complex extra- Disclosure of Potential Conflicts of Interest cellular matrix. HT29 cells transfected with a dominant-negative D. Arango, J.M. Mariadason, and S. Schwartz, Jr. are co-inventors of a related form of EPHB4 showed significantly increased invasive potential, patent: PCT/EP2007/056222 ‘prognostic method in colorectal cancer’. The other authors disclosed no potential conflicts of interest. whereas overexpression of wild-type EPHB4 in SW837 cells significantly reduced the number of Matrigel invading cells. These results are in good agreement with the changes observed in EphB4 Acknowledgments heterozygous mice compared with the wild-type counterpart and Received 2/23/09; revised 6/17/09; accepted 7/6/09; published OnlineFirst 9/8/09. may contribute to explain both the transition from small to large Grant support: Spanish Ministry of Science and Innovation grants FIS051394, adenoma associated with the loss of EPHB4 (4) and the poor CP05/00256, and SAF2008-00789 and Fundacio´n Me´dica Mutua Madrilen˜a (D. Arango). The costs of publication of this article were defrayed in part by the payment of page prognosis of patients with low tumor levels of EPHB4 (7). There- charges. This article must therefore be hereby marked advertisement in accordance fore, although up-regulation of this receptor is part of the with 18 U.S.C. Section 1734 solely to indicate this fact. transcriptional reprogramming observed in intestinal adenomas We thank Dr. David J. Anderson (Howard Hughes Medical Institute, Caltech) for kindly providing us with the EphB4/LacZ mice, Dr. Elena B. Pasquale (The Burnham triggered by Wnt activation, disruption of EPHB4 signaling is Institute) for the EPHB4-#C-EGFP vector, and Dr. Jens Glienke (Schering) for the important for tumor progression beyond this stage. pcDNA3.1-EPHB4 plasmid.

References receptor tyrosine kinase EphB4 is overexpressed in in colorectal cancer progression. Cancer Res 2009;69: ovarian cancer, provides survival signals and predicts 3736–45. 1. He TC, Sparks AB, Rago C, et al. Identification of poor outcome. Br J Cancer 2007;96:1083–91. 16. Sturz A, Bader B, Thierauch KH, Glienke J. EphB4 c-MYCas a target of the APCpathway. Science 1998;281: 9. Kumar SR, Singh J, Xia G, et al. Receptor tyrosine signaling is capable of mediating ephrinB2-induced 1509–12. kinase EphB4 is a survival factor in breast cancer. Am J inhibition of cell migration. Biochem Biophys Res 2. Tetsu O, McCormick F. h-Catenin regulates expression Pathol 2006;169:279–93. Commun 2004;313:80–8. of cyclin D1 in colon carcinoma cells. Nature 1999;398: 10. Xia G, Kumar SR, Stein JP, et al. EphB4 receptor tyro- 17. Noren NK, Lu M, Freeman AL, Koolpe M, Pasquale 422–6. sine kinase is expressed in bladder cancer and provides EB. Interplay between EphB4 on tumor cells and 3. Batlle E, Henderson JT, Beghtel H, et al. h-Catenin and signals for cell survival. Oncogene 2006;25:769–80. vascular ephrin-B2 regulates tumor growth. Proc Natl TCF mediate cell positioning in the intestinal epitheli- 11. Xia G, Kumar SR, Masood R, et al. Up-regulation of Acad Sci U S A 2004;101:5583–8. um by controlling the expression of EphB/ephrinB. Cell EphB4 in mesothelioma and its biological significance. 18. Gerety SS, Wang HU, Chen ZF, Anderson DJ. 2002;111:251–63. Clin Cancer Res 2005;11:4305–15. Symmetrical mutant phenotypes of the receptor EphB4 4. Batlle E, Bacani J, Begthel H, et al. EphB receptor acti- 12. Xia G, Kumar SR, Masood R, et al. EphB4 expression and its specific transmembrane ligand ephrin-B2 in vity suppresses colorectal cancer progression. Nature and biological significance in prostate cancer. Cancer cardiovascular development. Mol Cell 1999;4:403–14. 2005;435:1126–30. Res 2005;65:4623–32. 19. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of 5. Castano J, Davalos V, Schwartz S, Jr., Arango D. EPH 13. Sinha UK, Kundra A, Scalia P, et al. Expression of programmed cell death in situ via specific labeling of receptors in cancer. Histol Histopathol 2008;23:1011–23. EphB4 in head and neck squamous cell carcinoma. Ear nuclear DNA fragmentation. J Cell Biol 1992;119:493–501. 6. Holmberg J, Genander M, Halford MM, et al. EphB Nose Throat J 2003;82:866, 869–70, 887. 20. Marshman E, Ottewell PD, Potten CS, Watson AJ. receptors coordinate migration and proliferation in the 14. Berclaz G, Karamitopoulou E, Mazzucchelli L, et al. Caspase activation during spontaneous and radiation- intestinal stem cell niche. Cell 2006;125:1151–63. Activation of the receptor protein tyrosine kinase EphB4 induced apoptosis in the murine intestine. J Pathol 2001; 7. Davalos V, Dopeso H, Castano J, et al. EPHB4 and in endometrial hyperplasia and endometrial carcinoma. 195:285–92. survival of colorectal cancer patients. Cancer Res 2006; Ann Oncol 2003;14:220–6. 21. Arango D, Laiho P, Kokko A, et al. Gene-expression 66:8943–8. 15. Kumar SR, Scehnet JS, Ley EJ, et al. Preferential profiling predicts recurrence in Dukes’ Ccolorectal 8. Kumar SR, Masood R, Spannuth WA, et al. The induction of EphB4 over EphB2 and its implication cancer. Gastroenterology 2005;129:874–84. www.aacrjournals.org 7437 Cancer Res 2009; 69: (18). September 15, 2009

Cancer Research

22. Arango D, Mariadason JM, Wilson AJ, et al. c-Myc over- software environment for integrated models of biomolec- 32. Alazzouzi H, Davalos V, Kokko A, et al. Mechanisms expression sensitises colon cancer cells to camptothecin- ular interaction networks. Genome Res 2003;13:2498–504. of inactivation of the receptor tyrosine kinase EPHB2 in induced apoptosis. Br J Cancer 2003;89:1757–65. 28. Kertesz N, Krasnoperov V, Reddy R, et al. The soluble colorectal tumors. Cancer Res 2005;65:10170–3. 23. Li C, Wong WH. Model-based analysis of oligonucle- extracellular domain of EphB4 (sEphB4) antagonizes 33. Stephenson SA, Slomka S, Douglas EL, Hewett PJ, otide arrays: expression index computation and outlier EphB4-2 interaction, modulates angiogenesis, and Hardingham JE. Receptor protein tyrosine kinase EphB4 detection. Proc Natl Acad Sci U S A 2001;98:31–6. inhibits tumor growth. Blood 2006;107:2330–8. is up-regulated in colon cancer. BMCMol Biol 2001;2:15. 24. Ashburner M, Ball CA, Blake JA, et al. Gene ontology: 29. de Jonge HJ, Fehrmann RS, de Bont ES, et al. 34. Martiny-Baron G, Korff T, Schaffner F, et al. tool for the unification of biology. The Gene Ontology Evidence based selection of housekeeping genes. PLoS Inhibition of tumor growth and angiogenesis by soluble Consortium. Nat Genet 2000;25:25–9. ONE 2007;2:e898. EphB4. Neoplasia 2004;6:248–57. 25. Newman JC, Weiner AM. L2L: a simple tool for 30. Paoni NF, Feldman MW, Gutierrez LS, Ploplis VA, 35. Zhang W, Glockner SC, Guo M, et al. Epigenetic discovering the hidden significance in microarray Castellino FJ. Transcriptional profiling of the transition inactivation of the canonical Wnt antagonist SRY-box expression data. Genome Biol 2005;6:R81. from normal intestinal epithelia to adenomas and containing gene 17 in colorectal cancer. Cancer Res 26. Kim WK, Krumpelman C, Marcotte EM. Inferring carcinomas in the ApcMin/+ mouse. Physiol Genomics 2008;68:2764–72. mouse gene functions from genomic-scale data using a 2003;15:228–35. 36. Sinner D, Kordich JJ, Spence JR, et al. Sox17 and Sox4 combined functional network/classification strategy. 31. Noren NK, Foos G, Hauser CA, Pasquale EB. The EphB4 differentially regulate h-catenin/T-cell factor activity Genome Biol 2008;9 Suppl 1:S5. receptor suppresses breast cancer cell tumorigenicity and proliferation of colon carcinoma cells. Mol Cell Biol 27. Shannon P, Markiel A, Ozier O, et al. Cytoscape: a through an Abl-Crk pathway. Nat Cell Biol 2006;8:815–25. 2007;27:7802–15.

Cancer Res 2009; 69: (18). September 15, 2009 7438 www.aacrjournals.org

The Receptor Tyrosine Kinase EPHB4 Has Tumor Suppressor Activities in Intestinal Tumorigenesis

Higinio Dopeso, Silvia Mateo-Lozano, Rocco Mazzolini, et al.

Cancer Res 2009;69:7430-7438. Published OnlineFirst September 8, 2009.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-09-0706

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2009/09/01/0008-5472.CAN-09-0706.DC1

Cited articles This article cites 36 articles, 13 of which you can access for free at: http://cancerres.aacrjournals.org/content/69/18/7430.full#ref-list-1

Citing articles This article has been cited by 5 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/69/18/7430.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/69/18/7430. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.