Oncogene (2014) 33, 1658–1669 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE Cadherin-17 interacts with a2b1 integrin to regulate cell proliferation and adhesion in colorectal cancer cells causing liver metastasis RA Bartolome´ 1, R Barderas1,5, S Torres1, MJ Fernandez-Acen˜ ero2, M Mendes1, J Garcı´a-Foncillas3, M Lopez-Lucendo4 and JI Casal1 Liver metastasis is the major cause of death associated to colorectal cancer. Cadherin-17 (CDH17) is a non-classical, seven domain, cadherin lacking the conserved cytoplasmic domain of classical cadherins. CDH17 was overexpressed in highly metastatic human KM12SM and present in many other colorectal cancer cells. Using tissue microarrays, we observed a significant association between high expression of CDH17 with liver metastasis and poor survival of the patients. On the basis of these findings, we decided to study cellular functions and signaling mechanisms mediated by CDH17 in cancer cells. In this report, loss-of-function experiments demonstrated that CDH17 caused a significant increase in KM12SM cell adhesion and proliferation. Coimmunoprecipitation experiments demonstrated an interaction between CDH17 and a2b1 integrin with a direct effect on b1 integrin activation and talin recruitment. The formation of this complex, together with other proteins, was confirmed by mass spectrometry analysis. CDH17 modulated integrin activation and signaling to induce specific focal adhesion kinase and Ras activation, which led to the activation of extracellular signal-regulated kinase and Jun N-terminal kinase and the increase in cyclin D1 and proliferation. In vivo experiments showed that CDH17 silencing in KM12 cells suppressed tumor growth and liver metastasis after subcutaneous or intrasplenic inoculation in nude mice. Collectively, our data reveal a new function for CDH17, which is to regulate a2b1 integrin signaling in cell adhesion and proliferation in colon cancer cells for liver metastasis. Oncogene (2014) 33, 1658–1669; doi:10.1038/onc.2013.117; published online 22 April 2013 Keywords: Cadherin-17; a2b1 integrin; adhesion; proliferation; liver metastasis; colorectal cancer INTRODUCTION Tumor metastasis depends on different factors, such as growth Together with E-cadherin, CDH17 is specifically expressed in factors, receptors, proteases, chemokines and cellular adhesion basolateral plasma membrane of enterocytes and goblet cells,11,12 molecules.1 These adhesion molecules include cadherins, integrins and seems to be localized exclusively in cholesterol-rich fractions, and immunoglobulin superfamily molecules, which have been where it is supposed to keep tissue integrity. Interestingly, implicated in many steps of the metastatic cascade.2 Adhesion transgenic mice showed that enterocytes remained attached molecules can mediate adhesion between cells (that is, cadherins) despite the loss of E-cadherin, demonstrating that intercellular or to components of the extracellular matrix such as laminins, adhesion does not depend exclusively on E-cadherin.13 Weak collagens or fibronectin (that is, integrins).3 During metastasis, homotypic interaction mediated by other molecules (that is, cell–cell adhesion suffers different changes in signaling, loss of CDH17) might explain cell clustering. Classical cadherins like contact inhibition, cell migration and stromal interactions.4 E-cadherin bind catenin proteins through their cytoplasmic However, how adhesion molecules regulate cell phenotype or domains to initiate the signaling cascade. Intriguingly, CDH17 proliferation in cancer metastasis is not fully understood. has a very short cytoplasmic domain of only 24 amino acids, which In a previous proteomic analysis of the highly metastatic seems not to interact with catenins or cytoskeleton proteins,14 derivative human colorectal cancer cell line KM12SM and its raising questions about its mechanism of action. parental, poorly-metastatic, KM12C cell line,5 we found CDH17 is mainly expressed in fetal liver and gastrointestinal cadherin-17 (CDH17) as one of the top upregulated proteins in tract during embryogenesis,15 being overexpressed in several KM12SM cells, suggesting that CDH17 might have an important types of cancer: hepatocellular carcinoma,16,17 gastric cancer,18 role in late metastatic events.6 We also found a2 integrin ductal pancreatic cancer19 and colorectal cancer,20,21 where 96% overexpressed in KM12SM cells.6 Integrin a2 has been reported of tumor samples show expression of this molecule.22 CDH17 has to mediate selective metastasis to the liver.7 been proposed as an oncogene and as a disease marker for CDH17, also known as liver-intestine-cadherin, is a distinctive hepatic and gastrointestinal malignancies.16 The precise member of the cadherin superfamily.8 It closely resembles kidney- involvement of CDH17 in colorectal cancer metastasis, as well as specific-cadherin (cadherin-16), and they form a subgroup termed the mechanisms for pathway signaling, remains unclear and 7D-cadherin, because of their seven extracellular domains.9,10 contradictory. Some studies reported a reduced expression of 1Department of Cellular and Molecular Medicine, Centro de Investigaciones Biolo´ gicas (CIB-CSIC), Madrid, Spain; 2Pathology Department, Fundacio´n Jime´nez Dı´az, Madrid, Spain; 3Oncology Department, Fundacio´n Jime´nez Dı´az, Madrid, Spain and 4Proteomics Core Facility, CIB-CSIC, Madrid, Spain. Correspondence: Dr JI Casal, Department of Cellular and Molecular Medicine, Centro de Investigaciones Biolo´ gicas (CIB-CSIC), Ramiro de Maeztu, 9, Madrid 28040, Spain. E-mail: [email protected] 5Present address: Department of Biochemistry and Molecular Biology I, Universidad Complutense, Madrid, Spain. Received 20 November 2012; revised 19 February 2013; accepted 25 February 2013; published online 22 April 2013 CDH17 interacts with a2b1integrin in colon cancer RA Bartolome´ et al 1659 CDH17 associated only with lymph node metastasis but not to analyses showed that less than 10% of the original protein liver metastasis, based on immunohistochemical analysis,23 or expression levels were detected after knockdown with shRNA no. with an increase in metastatic behavior of LoVo cancer cells.24 60 and shRNA no. 58 in both cell lines. Both shRNAs were used for However, in the first study, the number of analyzed samples was further experiments to avoid off-target effects of the shRNAs. relatively low (o30), with only 11 liver metastasis.23 In the case of Given the reported coexpression of CDH17 and E-cadherin in LoVo cells, these cells derived from a metastatic nodule in the enterocytes, we tested for the presence of E-cadherin in KM12 supraclavicular region, which might imply different colonization cells using flow cytometry. No E-cadherin surface expression was capacities that metastatic cells isolated from liver. In another detected in parental or in silenced cells (Supplementary Figure S1). immunohistochemical study, Kwak et al.25 reported that reduced So, CDH17 expression appears to be independent of E-cadherin expression of CDH17 was associated with tumor dedifferentiation levels in metastatic cancer cells. and poor overall survival, but not with lymph node or distant We assessed the tumorigenic (proliferation and colony forma- metastasis. However, in this case, the number of stage IV cases tion) and metastatic (adhesion, migration and invasion) properties was only 6%. In contrast, another recent study described a clear of the KM12 cells transfected with vectors containing CDH17 association of high expression of CDH17 with either primary shRNAs compared with cells transfected with scrambled shRNA. tumors or lung metastasis in colon cancer.21 Then, the association CDH17-silenced cells showed a clear reduction in cell proliferation. of CDH17 expression with liver metastasis still requires further This reduction was more significant for KM12SM cells and was clarification. proportional to the silencing of CDH17 (Figure 2c). In KM12SM In this manuscript, we observed a clear association of high cells, the doubling time increased from 32 to 58 h (Figure 2d). In CDH17 expression to liver metastasis in colorectal cancer. At the addition, colony-forming ability of KM12-silenced cells was nearly molecular level, a strong reduction in cell adhesion, proliferation completely abolished (490%) after CDH17 silencing (Figure 2e). and liver metastasis was noticed after CDH17 knockdown in KM12 Then, we analyzed the adhesive properties of the cell lines metastatic colorectal cancer cell lines. These effects were using Matrigel assays. Adhesion capacity of scramble KM12SM mediated by an interaction between CDH17 and a2b1 integrin, overexpressing CDH17 was three fold higher than that of KM12C through the regulation of b1 integrin activation. The modulation cells. After CDH17 silencing, there was a striking decrease in the by CDH17 of the integrin-triggered signaling pathway led to the adhesion capacity for KM12SM cells (50%) with both shRNAs and activation of the focal adhesion kinase (FAK) and the Ras pathway, negligible for KM12C cells (Figure 2f). In contrast, no significant including mitogen-activated protein kinases and cyclin D1. In effect was observed on migration (Supplementary Figure S2A) and summary, our results demonstrated the capacity of CDH17 to invasion (Supplementary Figure S2B) after CDH17 silencing. To regulate a2b1integrin activity for cell proliferation and adhesion in confirm these results we used other colorectal cell lines: Caco2, colorectal cancer cells with high