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EphB–ephrin-B interactions suppress colorectal cancer progression by compartmentalizing tumor cells

Abstract

The genes encoding tyrosine kinase receptors EphB2 and EphB3 are β-catenin and Tcf4 target genes in colorectal cancer (CRC) and in normal intestinal cells1,2. In the intestinal epithelium, EphB signaling controls the positioning of cell types along the crypt-villus axis1. In CRC, EphB activity suppresses tumor progression beyond the earliest stages3,4. Here we show that EphB receptors compartmentalize the expansion of CRC cells through a mechanism dependent on E-cadherin–mediated adhesion. We demonstrate that EphB-mediated compartmentalization restricts the spreading of EphB-expressing tumor cells into ephrin-B1–positive territories in vitro and in vivo. Our results indicate that CRC cells must silence EphB expression to avoid repulsive interactions imposed by normal ephrin-B1–expressing intestinal cells at the onset of tumorigenesis.

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Figure 1: EphB activity induces cell sorting and compartmentalization of CRC cells in vitro.
Figure 2: E-cadherin is required for EphB-mediated compartmentalization in CRC cells.
Figure 3: EphB–ephrin-B interactions compartmentalize intestinal adenomas in vivo.
Figure 4: Phenotype of mice with conditional knockout of ephrin-B1 in the intestine.
Figure 5: Colorectal tumorigenesis is accelerated in compound Efnb1Int-KO ApcMin/+ mice.
Figure 6: Defects in cell adhesion in colorectal tumors of ApcMin/+ lacking ephrin-B1.

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Acknowledgements

We thank P. Soriano for providing ephrin-B1 conditional knockout mice, H. Clevers for reagents and support, L. Bardia for technical help with microscopy, N. Malats for help with statistics and D. Dominguez and the rest of the laboratory members for discussions and support. We thank D. Baltimore (Caltech) for the gift of the FUW vector backbone. This study was supported by a grant awarded to E.B. by the Ministerio de Educación y Ciencia (SAF2005-4891). C.C. and J.L.F.-M. were recipients of predoctoral fellowships from the Department de Educació i Universitats of the Catalonian government and A.V. holds a Juan de la Cierva Fellowship.

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Authors and Affiliations

Authors

Contributions

C.C. generated and analyzed in vitro models of tumor cell compartmentalization. S.P.-P. generated and analyzed mice lacking ephrin-B1 in the intestine and tumor phenotypes and performed a wide range of stainings. M.I. performed the quantification and histopathological assessment of mice tumor phenotypes. J.L.F.-M. performed analysis of EphB3 activation, and A.V. worked on the analysis of E-cadherin in cell models. G.W. performed Wnt reporter and qRT-PCR assays. M.H., N.P., L.G. and S.J. provided technical assistance with histology and mice work. A.D. generated ephrin-B1 conditional mice. J.L. performed transmission electron microscopy analysis, E.S. provided project supervision and contributed to the writing, and E.B. provided the general concept, design of study, supervision and manuscript writing.

Corresponding author

Correspondence to Eduard Batlle.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1-10, Supplementary Table 1, Supplementary Discussion, Supplementary Methods (PDF 5077 kb)

Supplementary Video 1

Co115 cells infected with a control Ubq-GFP lentivirus (A) or with lentivirus bearing EphB2 (B) or EphB3 (C) cDNAs downstream of the ubiquitin promoter were recorded during 15 minutes (timer from -15 to 0) before the addition of recombinant crosslinked ephrin-B1/Fc protein to the medium. Cells were then filmed for 60 minutes. Movies show DIC images taken every 30 seconds. (MOV 4501 kb)

Supplementary Video 2

Ubq-EphB3 Co115 cells expressing a non-targeting control shRNA (A) or a shRNA targeting E-cadherin (B) were recorded during 15 minutes (timer from -15 to 0) before the addition of recombinant crosslinked ephrin-B1/Fc protein to the medium. Cells were then filmed for 190 minutes. Movies show DIC images taken every 30 seconds. (MOV 3249 kb)

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Cortina, C., Palomo-Ponce, S., Iglesias, M. et al. EphB–ephrin-B interactions suppress colorectal cancer progression by compartmentalizing tumor cells. Nat Genet 39, 1376–1383 (2007). https://doi.org/10.1038/ng.2007.11

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