Published OnlineFirst September 8, 2009; DOI: 10.1158/0008-5472.CAN-09-0706 Molecular Biology, Pathobiology, and Genetics The Receptor Tyrosine Kinase EPHB4 Has Tumor Suppressor Activities in Intestinal Tumorigenesis 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 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst September 8, 2009; DOI: 10.1158/0008-5472.CAN-09-0706 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