Oncogene (2014) 33, 1658–1669 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

ORIGINAL ARTICLE -17 interacts with a2b1 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 . 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 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 , 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, 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 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 metastatic capacity to liver. SW480 and SW620. The silencing of CDH17 by small interfering RNAs in these cell lines (Supplementary Figure S3) caused a significant reduction in their proliferative (Figure 2g) and adhesive RESULTS properties (Figure 2h). These results suggest that knocking down CDH17 overexpression in human patients is associated to late CDH17 significantly reduce the tumorigenic properties of color- stages and lower overall survival ectal cancer cells, being more evident for the highly metastatic CDH17 was overexpressed in highly metastatic KM12SM cells.6 So, KM12SM cells. we tested other colorectal cancer cell lines for CDH17 expression (Figure 1a). Significant expression levels were observed in all tested cell lines, except in the poorly differentiated RKO cells. Very CDH17 modulates cell adhesion through interaction with a2b1 integrin low CDH17 expression was detected in the cell lines with lower metastatic capacity (SW48 and SW480). To study the role of CDH17 in cell adhesion, we investigated the To investigate the relevance of our results in human colon potential interactions between CDH17 and other proteins to cancer, we analyzed the expression of CDH17 in tumor tissue regulate cellular adhesion. Integrins are particularly relevant cell samples. We used a tissue microarray containing 119 patients adhesion molecules implicated in metastasis within the liver vasculature. Previously, we had observed that a2 integrin was diagnosed and treated for colorectal adenocarcinoma that were 6 followed for more than 5 years and retrospectively selected overexpressed in KM12SM cells. As a2 integrin combines with b1 (Supplementary Table S1). CDH17 expression was moderate in integrin to form a heterodimeric complex called VLA2, a2b1 54.6% of cases and absent or weak in 7.6% of cases. These cases integrin was our initial target to check for direct interactions. We were classified as ‘CDH17 low’ (Figure 1b). The remaining cases carried out coimmunoprecipitation experiments using anti-CDH17 (37.8%) showed a strong membrane staining of CDH17 in tumors and anti-a2 integrin antibodies followed by western blot analysis compared with normal mucosa and were labeled as ‘CDH17 high’ using antibodies against integrin subunits a2 and b1, talin and (Figure 1b). There was a significant correlation between CDH17 CDH17 (Figure 3a). In each case, we observed a coimmunopre- expression and presence of liver metastasis at the moment of cipitation of CDH17 with a2 and b1 integrin subunits, suggesting diagnosis (P ¼ 0.026) (Figure 1c). In addition, overall survival an interaction between these proteins. This interaction was analysis showed a clear association of high CDH17 expression with confirmed by the presence of talin in the immunoprecipitates. Talin binds b1 integrin cytoplasmic domain and is recruited by poor prognosis (P ¼ 0.029) (Figure 1d). However, we did not found 26 a statistically significant association between CDH17 expression FAK to nascent focal adhesions. To prove this interaction was and lymph node involvement. Collectively, these results indicated not particular only to KM12 cells, we tested also SW480 and an association between CDH17 expression and liver metastasis in SW620 colon cancer cells, differing in metastatic properties. Effects colon cancer. were similar to KM12 cells, confirming the interaction between a2b1 integrin and CDH17 (Figure 3b). To explore the molecular mechanisms of the interaction, we Effect of CDH17 on KM12 cell proliferation and adhesion carried out a transient silencing of b1 and a2 integrins in KM12SM To examine the effects of CDH17 on colorectal cancer metastasis cells, followed by immunoprecipitation with anti-CDH17 at the molecular level, CDH17 expression was silenced on KM12SM (Figure 3c). Neither b1 nor a2 integrins were recovered after and KM12C cells by preparing stable short hairpin RNA (shRNA) immunoprecipitation with anti-CDH17 of b1or a2 integrin-silenced transfectants. The lack of expression of CDH17 was confirmed by cells. Thus, both subunits were necessary to interact with CDH17. western blot (Figure 2a) and flow cytometry (Figure 2b). These To analyze whether the effect of CDH17 was mainly through b1

& 2014 Macmillan Publishers Limited Oncogene (2014) 1658 – 1669 CDH17 interacts with a2b1integrin in colon cancer RA Bartolome´ et al 1660 integrin activation, we determined the levels of activated b1 of activated b1 integrin. As before, these effects were more integrin using an antibody that recognizes an epitope expressed prominent for silenced KM12SM cells (Figure 3d). Next, we tested only after its activation.27 Silencing of CDH17 decreased the levels the effect of blocking KM12 cell adhesion using antibodies for

Figure 1. CDH17 expression is associated to human colon cancer metastasis and poor survival of colorectal cancer patients. (a) Twenty-five micrograms of protein lysates of the indicated colorectal carcinoma cell lines were resolved in SDS–PAGE and subjected to western blot using anti-CDH17. (b) Immunohistochemical analysis of CDH17 expression in tissue microarrays showing representative images of weak, moderate or intense staining of different colon carcinomas. Counterstaining was made with hematoxylin. Pictures were taken at  100 (up) or  200 magnification (down). (c) Percentage of patients with invasion of regional lymph nodes (left) or with metastasis (right) at the time of diagnosis in function of CDH17 expression levels; P-values were calculated using a w2-test (*Po0.05). (d) Significant association of CDH17 overexpression with shorter overall survival was found by using the Mann–Whitney test.

Figure 2. CDH17 silencing led to the inhibition of cell proliferation, colony formation and adhesion. (a) KM12C and KM12SM cells were infected with lentiviral vectors containing different shRNAs targeting CDH17, scrambled shRNA or empty vectors. CDH17 expression was assessed by western blot. Bands were quantified with MultiGauge software, Fujifilm (Kansas, KS, USA). RhoGDI (Rho GDP-dissociation inhibitor) was used as loading control. (b) Flow cytometry analysis showing the surface expression of CDH17 on KM12C and KM12SM cells expressing shRNA no. 60 targeting CDH17 or scrambled. (c) Proliferation was determined by MTT assays after 24 h of culture. Optical density was significantly decreased by CDH17 knockdown (**Po0.01; ***Po0.001). (d) The histogram shows the time to develop a cell cycle in scrambled and CDH17-silenced KM12 cells. Doubling time was calculated by the detection of incorporated bromodeoxyuridine and cell cycle analyses. (e) Colony formation assay in soft agar with the indicated cell lines. Number of colonies was significantly inhibited in CDH17-silenced cells (***Po0.001). (f) Cell adhesion to Matrigel of CDH17-silenced or scrambled KM12 cells. Adhesion was significantly downregulated in CDH17- silenced KM12SM cells compared with control cells (***Po0.001). Data represent the mean±s.d. of three independent experiments. (g) Caco2, SW480 and SW620 cells were transfected with small interfering RNA control or targeting CDH17 and subjected to MTT assays. Optical density was significantly decreased after CDH17 knockdown (***Po0.001). (h) The same transfectants of (g) were subjected to adhesion assays to Matrigel. Adhesion was significantly inhibited after CDH17 silencing (*Po0.05; **Po0.01; ***Po0.001).

Oncogene (2014) 1658 – 1669 & 2014 Macmillan Publishers Limited CDH17 interacts with a2b1integrin in colon cancer RA Bartolome´ et al 1661 a2b1 integrin. Addition of either anti a2 or anti b1 antibodies was to specific substrate ligands (collagen type IV) or unspecific able to block the adhesion of KM12 cells to basal levels (Figure 3e), (fibronectin, collagen type I). KM12SM cells were able to adhere particularly for KM12SM cells. To analyze the ligand specificity of only to collagen type IV. This adhesion was also significantly a2b1 integrin on cell adhesion, we tested KM12SM cell adhesion inhibited by CDH17 silencing (Figure 3f). Flow cytometry analysis

& 2014 Macmillan Publishers Limited Oncogene (2014) 1658 – 1669 CDH17 interacts with a2b1integrin in colon cancer RA Bartolome´ et al 1662

Figure 3. CDH17 interacts with a2b1 integrin and regulates integrin activation. (a) Transfectants of KM12 cells were lysed and the extracts subjected to immunoprecipitation with anti-a2 integrin or anti-CDH17 antibodies. Immunoprecipitates were analyzed by western blot using antibodies to a2 integrin, b1 integrin, talin and CDH17. (b) SW480 and SW620 cells were examined for CDH17–a2b1 integrin interaction. Lysates from both cell lines were subjected to immunoprecipitation assays as in (a). (c) KM12SM cells were transfected with small interfering RNAs control or targeting to a2 integrin or b1 integrin, after 50 h cells were lysed, and the extracts subjected to immunoprecipitation as in (a). Aliquots of lysates were analyzed by western blot to confirm integrin knockdown. RhoGDI was used as loading control. (d) To determine the expression of b1 integrin in high-affinity conformation, the same transfectants were subjected to flow cytometry using a specific antibody for activated b1 integrin. Mean fluorescence intensities are showed inside the panels. (e) Blocking of cell adhesion to Matrigel by preincubation with the indicated antibodies (**Po0.01; ***Po0.001). (f) Inhibition of KM12SM cell adhesion to the indicated ligands after preincubation with BB BBB the indicated antibodies (***Po0.001) or CDH17 knockdown ( Po0.01; Po0.001).

showed that the levels of a2b1integrin were similar between discard unspecific proteins. Precipitated proteins were fractio- scrambled and silenced cells, indicating that a2b1 integrin surface nated by SDS–PAGE (polyacrylamide gel electrophoresis) followed expression was independent of CDH17 (Supplementary Figure S4). by mass spectrometry analysis. We identified a total of 389 Finally, we did not observe differences in b-catenin expression proteins coimmunoprecipitated with CDH17 with at least two after CDH17 targeting in colorectal cancer cells (Supplementary different peptides identified with high confidence, using a q-value Figure S5). Collectively, these results indicate an association threshold of 0.01. Proteins identified in control beads and involved between CDH17 and a2b1 integrin in colon cancer cells with in common biological processes, such as ribosomal, nuclear or specific binding to collagen type IV. This interaction requires the mitochondrial proteins, chaperons, transport or degradation were presence of both subunits and provokes b1 integrin activation. skipped from further analysis. Thus, the remaining 78 proteins were considered as specifically associated with CDH17 (Supplementary Table S2, Figure 4a). For a2 integrin, we followed Identification of protein networks after immunoprecipitation with a similar approach that gave us a final list of 132 specific proteins CDH17 and a2 integrin antibodies (Supplementary Table S2, Figure 4b). The identified proteins were To characterize the interaction network of CDH17, we carried out a similar for both proteins: 65 out of 78 proteins immunopre- proteomic approach. KM12SM cell lysates were immunoprecipi- cipitated with anti-CDH17 antibodies were also present in a2 tated using anti-CDH17 or anti-a2 integrin antibody coupled to integrin immunoprecipitates (Figure 5a). b1 integrin and talin sepharose beads. As a control, we incubated the lysates with an were among the proteins present in the CDH17-immunoprecipi- unrelated immunoglobulin G coupled to sepharose beads to tates, confirming our previous results.

Oncogene (2014) 1658 – 1669 & 2014 Macmillan Publishers Limited CDH17 interacts with a2b1integrin in colon cancer RA Bartolome´ et al 1663

Figure 4. Protein networks identified by proteomic analyses after immunoprecipitation with CDH17 or a2 integrin antibodies. KM12SM lysates were immunoprecipitated using anti-CDH17 (a) or anti-a2-integrin (b) antibodies. Immunoprecipitates were resolved by a short SDS–PAGE, divided in three fractions and each fraction was digested with trypsin. The resulting peptides were analyzed by nanoLC-MS/MS. The cartoon displays the detected proteins according to known interactions bioinformatics analysis using STRING and data mining.

& 2014 Macmillan Publishers Limited Oncogene (2014) 1658 – 1669 CDH17 interacts with a2b1integrin in colon cancer RA Bartolome´ et al 1664 Using STRING software and data mining, proteins were CDH17-promoted proliferation through a2b1 integrin, FAK and classified into seven clusters: focal adhesion, actin cytoskeleton, Ras activation microtubule-associated proteins, Rho GTPase modulators, mito- Integrins cluster to form focal adhesions, which are implicated gen-activated protein kinase pathway, Wnt signaling pathway and in cytoskeletal reorganization28 and cell cycle progression.29 a2 CD44-associated proteins. Interestingly, CD44-related proteins integrin mediates collagen type IV-dependent activation of FAK.7 were present only in CDH17-associated proteins (Figure 4a). We examined (i) the effect of CDH17 on proliferation signaling CDH17 was associated to several proteins involved in focal pathways mediated through integrins, talin and focal adhesion adhesion that were undetected by proteomics approach, as FAK/ proteins and (ii) the effect of FAK inhibitor 14 (FAK I-14), which protein tyrosine kinase2, paxillin, RhoA, Rac and Ras. For prevents FAK autophosphorylation at Tyr397, on mitogen- a2-integrin, we also found association to Rac and Ras, but not activated protein kinase pathway activation in the presence or RhoA. Western blot analysis confirmed the identification of all absence of Matrigel (Figure 6a). We observed a clear over- tested proteins in immunoprecipitates from KM12SM and SW620 expression of activated and total FAK in scrambled KM12SM cell lines (Figure 5b). In summary, proteomic analysis confirmed respect to KM12C. CDH17 silencing suppressed FAK activation in previous interaction data and revealed a complex network of KM12SM cells at a similar level that FAK I-14. Regarding MAP interacting proteins for CDH17 and a2 integrin. kinase pathways, activated and total jun N-terminal kinase

Figure 5. Proteins coimmunoprecipitated with CDH17 or a2 integrin. (a) Venn diagram corresponding to proteins identified in both immunoprecipitations. (b) KM12SM and SW620 lysates were immunoprecipitated using anti-CDH17 or anti-a2-integrin antibodies, resolved by SDS–PAGE and analyzed by western blot using the indicated antibodies.

Oncogene (2014) 1658 – 1669 & 2014 Macmillan Publishers Limited CDH17 interacts with a2b1integrin in colon cancer RA Bartolome´ et al 1665

Figure 6. CDH17 silencing downregulates FAK activation, Ras signaling and pathways involved in cell proliferation. (a) KM12 transfectants were added to empty (control) or Matrigel-coated plates in the presence or absence of FAK inhibitor 14, and subsequently lysed. Protein extracts were resolved by SDS–PAGE and subjected to western blot using the indicated antibodies. (b) KM12 cells were treated as in (a) and the extracts were subjected to a GTPase activity assay to determine the presence of active Ras. Aliquots of the extracts were used to assess total Ras protein content with specific antibodies. (c) KM12SM stable transfectants were subjected to confocal microscopy using anti-CDH17, anti-paxillin or phospho-Y397-FAK, followed by Alexa-555 or Alexa-647-conjugated secondary antibodies. Cells were also stained with 4,6- diamidino-2-phenylindole and Phalloidin-fluorescein isothiocyanate. (d) Cell proliferation and viability was evaluated by MTT assay in the presence of the indicated antibodies or inhibitors (*Po0.05; **Po0.01; ***Po0.001).

& 2014 Macmillan Publishers Limited Oncogene (2014) 1658 – 1669 CDH17 interacts with a2b1integrin in colon cancer RA Bartolome´ et al 1666 (JNK) and extracellular signal-regulated kinase were also more contacts, with a strong reduction in phospho-FAK expression abundant in KM12SM cells. Silencing of CDH17 and FAK I-14 (Figure 6c). also reduced the activation of JNK and extracellular signal- In addition, we analyzed the effect of the different antibodies regulated kinase. Consequently, cyclin D1 was increased in and inhibitors on CDH17-induced proliferation (Figure 6d). The KM12SM cells and reduced by silencing of CDH17 and the use most significant reduction of proliferation was associated to FAK of FAK inhibitor. I-14, followed by UO126, a MEK1/2 inhibitor, and JNK I-II, a JNK To determine whether FAK signaling was mediated through inhibitor. Although at a lower extent, antibodies against CDH17 or Ras, we studied the presence of active Ras in KM12 cells the integrin subunits a2 b1 were also effective in the reduction of (Figure 6b). After FAK inhibition, we observed a significant proliferation. In contrast, a SRC inhibitor (PP2) was ineffective, decrease of active Ras in scrambled KM12SM cells. In CDH17- probably as a consequence of the lack of effect of CDH17 on cell silenced KM12 cells, there was a clear decline in active Ras, which migration. Together these results indicate that CDH17-promoted was not affected by FAK inhibition. These results suggested adhesion and proliferation are mediated through a2b1 integrin, that Ras activation was modulated by CDH17 signaling. Bio- followed by FAK and Ras activation via MAP kinase pathway, and chemical data were confirmed by confocal microscopy. KM12SM can be abolished by FAK, MEK and JNK inhibitors. scrambled cells showed major spreading and presence of focal contacts. Besides a membrane distribution, CDH17 was localized in peripheral protrusions of spreading cells. Focal contacts Targeting CDH17 abolishes in vivo tumor growth and increases showed staining and colocalization of CDH17 with paxillin and mice survival anti-phospho-FAK. In contrast, CDH17-silenced cells showed Finally, to verify the role of CDH17 targeting in colorectal cancer ‘spindle-like’ cell morphology, without cell spreading and/or focal metastasis, we carried out subcutaneous and intrasplenic

Figure 7. CDH17 silencing suppresses tumor growth and liver metastasis in KM12 cells. (a) KM12 transfectants were inoculated subcutaneously in nude mice. Tumor size was measured every day for 2 weeks. (b) Kaplan–Meier survival assay of nude mice inoculated intrasplenic with the indicated KM12 cell transfectants. (c) Mice were examined for macroscopic metastases in liver. (Left) number of macroscopic metastases in the liver was significantly reduced in mice inoculated intra-spleen with CDH17-silenced KM12SM cells (**Po0.01). B (Diamonds): number of metastases in each mouse. Average and s.d. are shown. (Right) liver surface covered by metastatic foci. (d) Model for CDH17-dependent regulation of cell proliferation. CDH17 interacts with and increases a2b1 integrin affinity for their ligands in the extracellular matrix leading to talin binding and enhanced activation of FAK and Ras, which in turn activates MAP kinase pathways for an increment in the expression of cyclin D1 and, consequently, in cell proliferation.

Oncogene (2014) 1658 – 1669 & 2014 Macmillan Publishers Limited CDH17 interacts with a2b1integrin in colon cancer RA Bartolome´ et al 1667 inoculations of scrambled and silenced KM12 cells in nude mice to the expression of CDH17. Collagen type IV is found primarily in the determine tumor growth and mice survival. CDH17-silenced cells basement membrane of epithelial tissues, but it is almost absent did not develop measurable tumors after subcutaneous inocula- in lymph nodes or other lymphatic tissues. Therefore, activation of tion, whereas scrambled KM12 cells showed large size tumors as a2b1 integrin through CDH17 for increased adhesion to collagen expected (Figure 7a). In addition, mice inoculated intrasplenically type IV might explain differences between liver and lymph node with CDH17-silenced KM12SM cells showed longer survival than metastasis. For lymph nodes, the presence of a critical mass of those with highly metastatic scrambled KM12SM cells (Figure 7b). tumor cells in the subcapsular sinuses is likely to promote This prolonged survival was probably due to the lower capacity for metastasis at the regional level. This obviates the need for liver colonization and the impaired growth of tumor cells metastasizing cells to acquire properties for extravasation and observed when some animals developed tumors in other places increasing the probabilities of metastasis.30 In distant organs, the (that is, injection site). No metastatic nodes were detected in tumor cells require an appropriate microenvironment (‘soil’) to knockdown CDH17 KM12SM cells, whereas the number of bind, grow and proliferate. macroscopic metastasis as well as the liver surface covered by In colon cancer metastasis, the presence of CDH17 might metastatic foci was highly significant in scrambled KM12SM cells facilitate cell clustering through homotypic cadherin interactions (Figure 7c). Collectively, these results indicate that differences in and cell adhesion through integrin activation to initiate micro- metastasis formation and colonization should be attributed to metastasis formation, and further growth by increased prolifera- advantages in cell adhesion and proliferation. tion. Regarding the association between cadherins and integrins there were only a few reports involving E-cadherin. Previous reports demonstrated a direct and regulated interaction between DISCUSSION the canonical epithelial E-cadherin and some integrins as, for We report a new role for the atypical cadherin-17 in adhesion and instance, the mucosal lymphocyte integrin aEb7, which might have proliferation of metastatic colorectal cancer cells. Our conclusions implications in adhesion and activation.31 In addition, E-cadherin were based on the following observations: (i) CDH17 high forms a ternary complex with insulin-like growth factor 1 receptor expression was associated to liver metastasis and overall survival, and av integrin at cell–cell contact sites that correlates with an (ii) CDH17 silencing decreased cell adhesion, colony formation and increase in cell migration of colon cancer cells.32 Another link proliferation in KM12 cells, particularly in highly metastatic between a6b4 integrin with E-cadherin through cdc42 has been KM12SM cells, (iii) CDH17 formed large complexes with a2b1 reported.33 The effector cdc42 was also present in our CDH17 integrin and other proteins in colon cancer cells, (iv) cadherin- complex. More recently, Marchio et al.34 described the formation integrin signaling was mediated through FAK, Ras and MAP kinase of E-cadherin and a6 integrin complex on the surface of colorectal activation (Figure 7d) and (v) CDH17 silencing decreased in vivo cancer cells. This complex together with angiopoietin-like 6 seems tumor growth and increased mice survival. Together, these data to have a role in liver homing and colonization of colon cancer confirm that CDH17 have a major role in cell proliferation, tumor cells. Some authors have proposed that cadherin domains might growth and liver metastasis colonization in colon cancer. adopt a tertiary structure rather, like immunoglobulin domains, Our data agree with a recent histopathological study showing resembling that of cellular integrin ligands. Still, no description of that CDH17 expression in colon cancer was associated to lung and interactions between other cadherins, different from E-cadherin, lymph node metastasis.21 In addition to an increased expression in and integrins was described to our knowledge. E-cadherin was liver metastasis, we have observed a significant correlation of expressed in some metastatic lesions in colon cancer, although CDH17 overexpression with poor survival in colorectal cancer. The membrane expression was significantly higher in primary tumors divergent results found in previous literature regarding CDH17 compared with their metastases.35 There are some other tumors, expression in colorectal cancer might be explained, at least particularly prostate cancer, where there is a clear re-expression of partially, by technical reasons due to different treatment E-cadherin in liver metastasis.36 Relationship between E-cadherin conditions as, for instance, sample fixation time, archival storage and CDH17 in metastatic cells will require further studies. on the long-term, quality of antibodies, and so on. In our study, In summary, we have demonstrated that CDH17 interacts with the initial management of the samples was standardized following a2b1 integrin and is a key determinant in the regulation of a2b1 a protocol that should minimize the heterogeneity of our results. integrin activity on cell adhesion and proliferation and, conse- Although our results indicate that CDH17 interaction requires quently, in the acquisition of liver metastatic capacity in colorectal the presence of both a2b1 integrin subunits, only changes in b1 cancer cells. integrin conformation were visible. Therefore, the regulation should involve a conformational change induced by cadherin interaction to induce b1 integrin activation, as the HUTS21 MATERIALS AND METHODS antibody used in our study only recognizes a conformational Cell lines, culture and reagents epitope present in the high-affinity conformation of the b1 KM12C and KM12SM human colon cancer cells5 were obtained directly integrin.27 This might explain also why despite similar levels of from Dr Fidler’s lab (MD Anderson Cancer Center, Houston, TX, USA). total a2b1 integrin expression, scrambled and silenced cells SW480, SW620, Caco2, RKO, SW48, Colo320, HT29 were purchased directly present different metastatic potential. The observed from the American Type Culture Collection (ATCC) and cultured in macromolecular complexes for CDH17 and a2b1 integrin link Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA, USA) 1 extracellular matrix with actin cytoskeleton dynamics, and containing 10% fetal calf serum (Invitrogen) and antibiotics at 37 Cina 5% CO2-humidified atmosphere. constitute signal transduction centers at sites of integrin Inhibitor UO126 was from Calbiochem (Darmstadt, Germany), PP2 from clustering. The modulation of a2b1 integrin activity by CDH17, Sigma-Aldrich (St Louis, MO, USA), JNK inhibitor II from Merck Chemicals followed by FAK and Ras activation, is a novel mechanism that (Darmstadt, Germany) and FAK inhibitor 14 from Santa Cruz Biotechnology creates a link between non-conventional cadherins like CDH17 (Santa Cruz, CA, USA). Antibodies used in this work are listed in and the focal adhesion complex to promote an adhesive Supplementary Table S3. The monoclonal antibody specific for CDH17 phenotype, and to increase cell proliferation in metastatic cells was a kind gift of Dr John M Luk (Hong Kong University). (Figure 7d). Integrin regulation by CDH17 can promote cell adhesion to Transfections of KM12 cells collagen type IV, which is highly enriched in the liver, and pLKO.1 vectors containing shRNAs for CDH17 were purchased from migration within the liver vasculature. We have observed some Open Biosystems (Lafayette, CO, USA), clone IDs TRCN0000055658- differences between lymph node and liver metastasis regarding TRCN00000555662 (shRNAs no. 58–62). As shRNA control vectors, we

& 2014 Macmillan Publishers Limited Oncogene (2014) 1658 – 1669 CDH17 interacts with a2b1integrin in colon cancer RA Bartolome´ et al 1668 used a scrambled shRNA and an empty pLKO.1 vector obtained from Confocal microscopy 37,38 AddGene (Cambridge, MA, USA); clone IDs 1864 and 8453, respectively. Cells directly attached onto Matrigel-coated coverslips, were fixed with 1% Stably transfected cells were obtained by lentiviral infection, as previously paraformaldehyde (Sigma-Aldrich) in phosphate-buffered saline contain- 39 described. Briefly, HEK293T cells were transfected with pLKO.1 vectors ing 0.1% Triton X-100. Cells were washed three times with phosphate- and the packaging vectors pCMV-rev, pMDLg-pRRE and pNGVL-VSVG buffered saline and incubated with primary antibodies, washed again and using jetPRIME Transfection Reagent (Polyplus, Illkirch, France). After incubated with secondary antibodies labeled with Alexa-555 or Alexa-647, incubating the cells for 12–15 h in serum-free medium, it was replaced by and with fluorescein isothiocyanate-phalloidin (Invitrogen) and 4,6- fresh Dulbecco’s modified Eagle medium containing 10% fetal bovine diamidino-2-phenylindole. After incubation with fluorochrome-conjugated serum and antibiotics. The day after, media containing lentiviral particles secondary antibodies, samples were mounted with Mounting Fluorescence was added to KM12C and KM12SM cells. After 3 days of incubation, Medium (Dako, Copenhagen, Denmark) and images were captured using a infected cells were selected using 1 mg/ml puromycin (Sigma-Aldrich) for TCS-SP2-AOBS-UV confocal microscope (LEICA, Wetzlar, Germany) with 2–3 weeks. Then, cells were maintained in media containing 0.5 mg/ml  63 oil immersion objective. Displayed images were captured at the same puromycin. sections in the different samples. For transient transfections, small interfering RNAs targeting specifically CDH17 (SASI-Hs01-00166354, Sigma-Aldrich) or control small interfering RNAs were transfected with JetPrime (Polyplus Transfection) as previously Immunohistochemistry analysis 40 described. Immuhistochemistry was performed on four tissue microarrays, consisting of a total of 119 patients of colorectal cancer with clinical data about metastasis, lymph node invasion at the time of diagnosis and overall Cell adhesion and proliferation survival between years 2005 and 2007 in the Hospital Fundacio´n Jimenez Cell proliferation, adhesion and invasion using Matrigel assays were carried Dı´az (Madrid). All surgical specimens were received at the Pathology out according to previously published procedures.41 For other ligands, cells Department in less than 1 h after resection, following protocols approved were incubated 30 min on plates coated with fibronectin (10 mg/ml), by the Ethical Committee of the hospital. Then, samples were fixed for 24 h collagen type I (5 mg/ml) or collagen type IV (5 mg/ml). in 10% buffered formalin, sectioned and embedded in paraffin in an automatic inclusion system. Paraffin blocks were stored in the same conditions. This standardized protocol for sample management reduces Western blot, inmunoprecipitation and GTPase activity assays the risk of expression heterogeneity. Each sample was subjected to deparaffinization and antigen retrieval with a PT Link Module (Dako) at a Cells were starved for 4 h and incubated in the presence or absence of high pH, for 20 min and subsequent incubation with the primary antibody inhibitors, and/or allowed to adhere to empty or Matrigel-coated plates for against CDH17 (1 mg/ml). The reaction was revealed with DAB (3,3’- 45 min. Then, cells were detached, washed and lysed with protease and diaminobenzidine) as a chromogen and counterstained with hematoxylin phosphatase inhibitors in lysis buffer (1% Igepal, 100 mM NaCl, 2 mM MgCl2, and observed in an Olympus microscope. The intensity of CDH17-staining 10% glycerol in 50 mM Tris–HCl). Protein extracts were separated in SDS– on membrane was graded as high (if intense) or low (either negative or PAGE and transferred to nitrocellulose membranes as previously 39 moderate staining similar to control areas of normal colonic mucosa). In all described. For immunoprecipitation, cells were lysed and 500 mg of cell cases, sections from normal colonic mucosa distant from the tumor site lysate were incubated with the indicated antibodies and the were used as negative controls. immunocomplex captured with 100 ml of Protein G-Sepharose beads (Sigma-Aldrich). After washing, samples were loaded on SDS–PAGE for western blot analysis. In vivo animal experiments For GTPase activity assays, cells were starved for 4 h, treated with The Ethical Committee of the Consejo Superior de Investigaciones inhibitors and/or exposed to Matrigel in previously coated plates and Cientı´ficas (Spain) approved the protocols used for experimental work lysed. Aliquots of the extracts were kept for total lysate controls and the with mouse. For metastasis experiments, 106 KM12 cells expressing shRNA rest was incubated for 16 h at 4 1C with glutathione-S-transferase (GST)-Raf in 0.1 ml phosphate-buffered saline were injected intrasplenic in Swiss fused protein (a kind gift from Dr JM Rojas, ISCIII, Spain) and glutathione- nude mice (Charles River). Mice were daily inspected for signs of disease, agarose beads (Sigma-Aldrich). The beads were centrifuged, washed and such as abdominal distension, locomotive deficit or tumor detectable by bound proteins were eluted in Laemmli buffer, resolved in SDS–PAGE and palpation. When symptoms were noted, mice were euthanized and analyzed by western blot using anti-Ras antibodies. inspected for metastasis in the liver. For xenografts, tumors were induced by subcutaneous injection of 5  106 KM12 cells in phosphate-buffered saline supplemented with 0.1% glucose in nude mice. Tumors were Mass spectrometry analysis of immunoprecipitated proteins measured with external caliper, and volume was calculated as (4p/3)  (width/2)2  (length/2). When tumors had reached an average size of For proteomic analysis, 10 mg of cell lysates were immunoprecipitated as 3 before, and the proteins were loaded in SDS–PAGE, which were divided in 1200 mm , animals were euthanized and tumors were excised. three slices for in-gel digestion as previously described.6 Peptides were loaded onto a C18-A1 ASY 2 cm precolumn (ThermoScientific, Waltham, Statistical analyses MA, USA) and separated on a Biosphere C18 column (NanoSeparations) using a flow rate of 250 nl/min and a 100 min gradient from 0–95% Buffer Data were analyzed by one-way analysis of variance followed by Tukey- B (Buffer A: 0.1% formic acid/2% ACN; Buffer B: 0.1% formic acid in ACN) on Kramer multiple comparison test. In both analyses the minimum acceptable level of significance was Po0.05. For discrete variable data, a nanoEasy HPLC (Proxeon, Odense, Denmark) coupled to a nanoelec- 2 trospay ion source (Proxeon). Mass spectra were acquired in a linear trap as presence or absence of metastasis or lymph node invasion, we used w quadrupole (LTQ) Orbitrap Velos in a data-dependent mode with an test. For continuous variable data with not Gaussian distribution, as overall automatic switch between mass spectrometry and MS/MS scans using a survival, we performed a Mann–Whitney test. top 15 method. Full scans were acquired in the Orbitrap with a mass range of 400 to 1200 Th, a target value of 1 000 000 ions and a resolution of 30 000 (at m/z 400). The 15 most intense ions were submitted to collision- CONFLICT OF INTEREST induced dissociation in the LTQ using normalized collision energy of 35% The authors declare no conflict of interest. and a target value of 10 000 ions. Dynamic exclusion was enabled with a repeat count of one and exclusion duration of 30 s. Mass spectra were searched using SEQUEST search engine with Proteome Discoverer (version 1.3.0.339, ThermoScientific) against the ACKNOWLEDGEMENTS Uniprot Database (2012_02). Search parameters included precursor and We thank Prof John M Luk (Hong Kong University) by the kind supply of CDH17 fragment mass tolerances of 10 p.p.m. and 0.8 Da, respectively, a maximum antibody. RA Bartolome´ was supported by a grant to established research groups of of two missed cleavages allowed, a fixed modification of carbamidomethyl the Asociacio´n Espan˜ola Contra el Ca´ncer (AECC). R Barderas was a recipient of a JAE- cysteine and a variable modification of methionine oxidation. Identified DOC/FSE Contract (CSIC) and is currently a fellow of the Ramo´n y Cajal program. S peptides were validated using Percolator algorithm with a q-value Torres was a recipient of a ProteoRed contract and Juan de la Cierva program. This threshold of 0.01. research was supported by grant BIO2009-08818 from the Spanish Ministry of

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