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 α2β1 integrin with a direct effect on β1 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 α2β1 integrin signaling in cell adhesion and proliferation in colon cancer cells for liver metastasis.
Tumor metastasis depends on different factors, such as growth factors, receptors, proteases, chemokines and cellular adhesion molecules.1 These adhesion molecules include cadherins, integrins and immunoglobulin superfamily molecules, which have been implicated in many steps of the metastatic cascade.2 Adhesion molecules can mediate adhesion between cells (that is, cadherins) or to components of the extracellular matrix such as laminins, collagens or fibronectin (that is, integrins).3 During metastasis, cell–cell adhesion suffers different changes in signaling, loss of contact inhibition, cell migration and stromal interactions.4 However, how adhesion molecules regulate cell phenotype or proliferation in cancer metastasis is not fully understood.
In a previous proteomic analysis of the highly metastatic derivative human colorectal cancer cell line KM12SM and its parental, poorly-metastatic, KM12C cell line,5 we found cadherin-17 (CDH17) as one of the top upregulated proteins in KM12SM cells, suggesting that CDH17 might have an important role in late metastatic events.6 We also found α2 integrin overexpressed in KM12SM cells.6 Integrin α2 has been reported to mediate selective metastasis to the liver.7
CDH17, also known as liver-intestine-cadherin, is a distinctive member of the cadherin superfamily.8 It closely resembles kidney-specific-cadherin (cadherin-16), and they form a subgroup termed 7D-cadherin, because of their seven extracellular domains.9, 10 Together with E-cadherin, CDH17 is specifically expressed in basolateral plasma membrane of enterocytes and goblet cells,11, 12 and seems to be localized exclusively in cholesterol-rich fractions, where it is supposed to keep tissue integrity. Interestingly, transgenic mice showed that enterocytes remained attached despite the loss of E-cadherin, demonstrating that intercellular adhesion does not depend exclusively on E-cadherin.13 Weak homotypic interaction mediated by other molecules (that is, CDH17) might explain cell clustering. Classical cadherins like E-cadherin bind catenin proteins through their cytoplasmic domains to initiate the signaling cascade. Intriguingly, CDH17 has a very short cytoplasmic domain of only 24 amino acids, which seems not to interact with catenins or cytoskeleton proteins,14 raising questions about its mechanism of action.
CDH17 is mainly expressed in fetal liver and gastrointestinal tract during embryogenesis,15 being overexpressed in several types of cancer: hepatocellular carcinoma,16, 17 gastric cancer,18 ductal pancreatic cancer19 and colorectal cancer,20, 21 where 96% of tumor samples show expression of this molecule.22 CDH17 has been proposed as an oncogene and as a disease marker for hepatic and gastrointestinal malignancies.16 The precise involvement of CDH17 in colorectal cancer metastasis, as well as the mechanisms for pathway signaling, remains unclear and contradictory. Some studies reported a reduced expression of CDH17 associated only with lymph node metastasis but not to liver metastasis, based on immunohistochemical analysis,23 or with an increase in metastatic behavior of LoVo cancer cells.24 However, in the first study, the number of analyzed samples was relatively low (<30), with only 11 liver metastasis.23 In the case of LoVo cells, these cells derived from a metastatic nodule in the supraclavicular region, which might imply different colonization capacities that metastatic cells isolated from liver. In another immunohistochemical study, Kwak et al.25 reported that reduced expression of CDH17 was associated with tumor dedifferentiation and poor overall survival, but not with lymph node or distant metastasis. However, in this case, the number of stage IV cases was only 6%. In contrast, another recent study described a clear association of high expression of CDH17 with either primary tumors or lung metastasis in colon cancer.21 Then, the association of CDH17 expression with liver metastasis still requires further clarification.
In this manuscript, we observed a clear association of high CDH17 expression to liver metastasis in colorectal cancer. At the molecular level, a strong reduction in cell adhesion, proliferation and liver metastasis was noticed after CDH17 knockdown in KM12 metastatic colorectal cancer cell lines. These effects were mediated by an interaction between CDH17 and α2β1 integrin, through the regulation of β1 integrin activation. The modulation by CDH17 of the integrin-triggered signaling pathway led to the activation of the focal adhesion kinase (FAK) and the Ras pathway, including mitogen-activated protein kinases and cyclin D1. In summary, our results demonstrated the capacity of CDH17 to regulate α2β1integrin activity for cell proliferation and adhesion in colorectal cancer cells with high metastatic capacity to liver.
CDH17 overexpression in human patients is associated to late stages and lower overall survival
CDH17 was overexpressed in highly metastatic KM12SM cells.6 So, 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 low CDH17 expression was detected in the cell lines with lower metastatic capacity (SW48 and SW480).
To investigate the relevance of our results in human colon cancer, we analyzed the expression of CDH17 in tumor tissue samples. We used a tissue microarray containing 119 patients diagnosed and treated for colorectal adenocarcinoma that were followed for more than 5 years and retrospectively selected (Supplementary Table S1). CDH17 expression was moderate in 54.6% of cases and absent or weak in 7.6% of cases. These cases were classified as ‘CDH17 low’ (Figure 1b). The remaining cases (37.8%) showed a strong membrane staining of CDH17 in tumors compared with normal mucosa and were labeled as ‘CDH17 high’ (Figure 1b). There was a significant correlation between CDH17 expression and presence of liver metastasis at the moment of diagnosis (P=0.026) (Figure 1c). In addition, overall survival analysis showed a clear association of high CDH17 expression with poor prognosis (P=0.029) (Figure 1d). However, we did not found a statistically significant association between CDH17 expression and lymph node involvement. Collectively, these results indicated an association between CDH17 expression and liver metastasis in colon cancer.
Effect of CDH17 on KM12 cell proliferation and adhesion
To examine the effects of CDH17 on colorectal cancer metastasis at the molecular level, CDH17 expression was silenced on KM12SM and KM12C cells by preparing stable short hairpin RNA (shRNA) transfectants. The lack of expression of CDH17 was confirmed by western blot (Figure 2a) and flow cytometry (Figure 2b). These analyses showed that less than 10% of the original protein expression levels were detected after knockdown with shRNA no. 60 and shRNA no. 58 in both cell lines. Both shRNAs were used for further experiments to avoid off-target effects of the shRNAs. Given the reported coexpression of CDH17 and E-cadherin in enterocytes, we tested for the presence of E-cadherin in KM12 cells using flow cytometry. No E-cadherin surface expression was detected in parental or in silenced cells (Supplementary Figure S1). So, CDH17 expression appears to be independent of E-cadherin levels in metastatic cancer cells.
We assessed the tumorigenic (proliferation and colony formation) and metastatic (adhesion, migration and invasion) properties of the KM12 cells transfected with vectors containing CDH17 shRNAs compared with cells transfected with scrambled shRNA. CDH17-silenced cells showed a clear reduction in cell proliferation. This reduction was more significant for KM12SM cells and was proportional to the silencing of CDH17 (Figure 2c). In KM12SM cells, the doubling time increased from 32 to 58 h (Figure 2d). In addition, colony-forming ability of KM12-silenced cells was nearly completely abolished (>90%) after CDH17 silencing (Figure 2e).
Then, we analyzed the adhesive properties of the cell lines using Matrigel assays. Adhesion capacity of scramble KM12SM overexpressing CDH17 was three fold higher than that of KM12C cells. After CDH17 silencing, there was a striking decrease in the adhesion capacity for KM12SM cells (50%) with both shRNAs and negligible for KM12C cells (Figure 2f). In contrast, no significant effect was observed on migration (Supplementary Figure S2A) and invasion (Supplementary Figure S2B) after CDH17 silencing. To confirm these results we used other colorectal cell lines: Caco2, 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 properties (Figure 2h). These results suggest that knocking down CDH17 significantly reduce the tumorigenic properties of colorectal cancer cells, being more evident for the highly metastatic KM12SM cells.
CDH17 modulates cell adhesion through interaction with α2β1 integrin
To study the role of CDH17 in cell adhesion, we investigated the potential interactions between CDH17 and other proteins to regulate cellular adhesion. Integrins are particularly relevant cell adhesion molecules implicated in metastasis within the liver vasculature. Previously, we had observed that α2 integrin was overexpressed in KM12SM cells.6 As α2 integrin combines with β1 integrin to form a heterodimeric complex called VLA2, α2β1 integrin was our initial target to check for direct interactions. We carried out coimmunoprecipitation experiments using anti-CDH17 and anti-α2 integrin antibodies followed by western blot analysis using antibodies against integrin subunits α2 and β1, talin and CDH17 (Figure 3a). In each case, we observed a coimmunoprecipitation of CDH17 with α2 and β1 integrin subunits, suggesting an interaction between these proteins. This interaction was confirmed by the presence of talin in the immunoprecipitates. Talin binds β1 integrin cytoplasmic domain and is recruited by FAK to nascent focal adhesions.26 To prove this interaction was not particular only to KM12 cells, we tested also SW480 and SW620 colon cancer cells, differing in metastatic properties. Effects were similar to KM12 cells, confirming the interaction between α2β1 integrin and CDH17 (Figure 3b).
To explore the molecular mechanisms of the interaction, we carried out a transient silencing of β1 and α2 integrins in KM12SM cells, followed by immunoprecipitation with anti-CDH17 (Figure 3c). Neither β1 nor α2 integrins were recovered after immunoprecipitation with anti-CDH17 of β1or α2 integrin-silenced cells. Thus, both subunits were necessary to interact with CDH17. To analyze whether the effect of CDH17 was mainly through β1 integrin activation, we determined the levels of activated β1 integrin using an antibody that recognizes an epitope expressed only after its activation.27 Silencing of CDH17 decreased the levels of activated β1 integrin. As before, these effects were more prominent for silenced KM12SM cells (Figure 3d). Next, we tested the effect of blocking KM12 cell adhesion using antibodies for α2β1 integrin. Addition of either anti α2 or anti β1 antibodies was able to block the adhesion of KM12 cells to basal levels (Figure 3e), particularly for KM12SM cells. To analyze the ligand specificity of α2β1 integrin on cell adhesion, we tested KM12SM cell adhesion to specific substrate ligands (collagen type IV) or unspecific (fibronectin, collagen type I). KM12SM cells were able to adhere only to collagen type IV. This adhesion was also significantly inhibited by CDH17 silencing (Figure 3f). Flow cytometry analysis showed that the levels of α2β1integrin were similar between scrambled and silenced cells, indicating that α2β1 integrin surface expression was independent of CDH17 (Supplementary Figure S4). Finally, we did not observe differences in β-catenin expression after CDH17 targeting in colorectal cancer cells (Supplementary Figure S5). Collectively, these results indicate an association between CDH17 and α2β1 integrin in colon cancer cells with specific binding to collagen type IV. This interaction requires the presence of both subunits and provokes β1 integrin activation.
Identification of protein networks after immunoprecipitation with CDH17 and α2 integrin antibodies
To characterize the interaction network of CDH17, we carried out a proteomic approach. KM12SM cell lysates were immunoprecipitated using anti-CDH17 or anti-α2 integrin antibody coupled to sepharose beads. As a control, we incubated the lysates with an unrelated immunoglobulin G coupled to sepharose beads to discard unspecific proteins. Precipitated proteins were fractionated by SDS–PAGE (polyacrylamide gel electrophoresis) followed by mass spectrometry analysis. We identified a total of 389 proteins coimmunoprecipitated with CDH17 with at least two different peptides identified with high confidence, using a q-value threshold of 0.01. Proteins identified in control beads and involved in common biological processes, such as ribosomal, nuclear or mitochondrial proteins, chaperons, transport or degradation were skipped from further analysis. Thus, the remaining 78 proteins were considered as specifically associated with CDH17 (Supplementary Table S2, Figure 4a). For α2 integrin, we followed a similar approach that gave us a final list of 132 specific proteins (Supplementary Table S2, Figure 4b). The identified proteins were similar for both proteins: 65 out of 78 proteins immunoprecipitated with anti-CDH17 antibodies were also present in α2 integrin immunoprecipitates (Figure 5a). β1 integrin and talin were among the proteins present in the CDH17-immunoprecipitates, confirming our previous results.
Using STRING software and data mining, proteins were classified into seven clusters: focal adhesion, actin cytoskeleton, microtubule-associated proteins, Rho GTPase modulators, mitogen-activated protein kinase pathway, Wnt signaling pathway and CD44-associated proteins. Interestingly, CD44-related proteins were present only in CDH17-associated proteins (Figure 4a). CDH17 was associated to several proteins involved in focal adhesion that were undetected by proteomics approach, as FAK/protein tyrosine kinase2, paxillin, RhoA, Rac and Ras. For α2-integrin, we also found association to Rac and Ras, but not RhoA. Western blot analysis confirmed the identification of all tested proteins in immunoprecipitates from KM12SM and SW620 cell lines (Figure 5b). In summary, proteomic analysis confirmed previous interaction data and revealed a complex network of interacting proteins for CDH17 and α2 integrin.
CDH17-promoted proliferation through α2β1 integrin, FAK and Ras activation
Integrins cluster to form focal adhesions, which are implicated in cytoskeletal reorganization28 and cell cycle progression.29 α2 integrin mediates collagen type IV-dependent activation of FAK.7 We examined (i) the effect of CDH17 on proliferation signaling pathways mediated through integrins, talin and focal adhesion proteins and (ii) the effect of FAK inhibitor 14 (FAK I-14), which prevents FAK autophosphorylation at Tyr397, on mitogen-activated protein kinase pathway activation in the presence or absence of Matrigel (Figure 6a). We observed a clear overexpression of activated and total FAK in scrambled KM12SM respect to KM12C. CDH17 silencing suppressed FAK activation in KM12SM cells at a similar level that FAK I-14. Regarding MAP kinase pathways, activated and total jun N-terminal kinase (JNK) and extracellular signal-regulated kinase were also more abundant in KM12SM cells. Silencing of CDH17 and FAK I-14 also reduced the activation of JNK and extracellular signal-regulated kinase. Consequently, cyclin D1 was increased in KM12SM cells and reduced by silencing of CDH17 and the use of FAK inhibitor.
To determine whether FAK signaling was mediated through Ras, we studied the presence of active Ras in KM12 cells (Figure 6b). After FAK inhibition, we observed a significant decrease of active Ras in scrambled KM12SM cells. In CDH17-silenced KM12 cells, there was a clear decline in active Ras, which was not affected by FAK inhibition. These results suggested that Ras activation was modulated by CDH17 signaling. Biochemical data were confirmed by confocal microscopy. KM12SM 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 showed staining and colocalization of CDH17 with paxillin and anti-phospho-FAK. In contrast, CDH17-silenced cells showed ‘spindle-like’ cell morphology, without cell spreading and/or focal contacts, with a strong reduction in phospho-FAK expression (Figure 6c).
In addition, we analyzed the effect of the different antibodies and inhibitors on CDH17-induced proliferation (Figure 6d). The most significant reduction of proliferation was associated to FAK I-14, followed by UO126, a MEK1/2 inhibitor, and JNK I-II, a JNK inhibitor. Although at a lower extent, antibodies against CDH17 or the integrin subunits α2 β1 were also effective in the reduction of proliferation. In contrast, a SRC inhibitor (PP2) was ineffective, probably as a consequence of the lack of effect of CDH17 on cell migration. Together these results indicate that CDH17-promoted adhesion and proliferation are mediated through α2β1 integrin, followed by FAK and Ras activation via MAP kinase pathway, and can be abolished by FAK, MEK and JNK inhibitors.
Targeting CDH17 abolishes in vivo tumor growth and increases mice survival
Finally, to verify the role of CDH17 targeting in colorectal cancer metastasis, we carried out subcutaneous and intrasplenic inoculations of scrambled and silenced KM12 cells in nude mice to determine tumor growth and mice survival. CDH17-silenced cells did not develop measurable tumors after subcutaneous inoculation, whereas scrambled KM12 cells showed large size tumors as expected (Figure 7a). In addition, mice inoculated intrasplenically with CDH17-silenced KM12SM cells showed longer survival than those with highly metastatic scrambled KM12SM cells (Figure 7b). This prolonged survival was probably due to the lower capacity for liver colonization and the impaired growth of tumor cells observed when some animals developed tumors in other places (that is, injection site). No metastatic nodes were detected in knockdown CDH17 KM12SM cells, whereas the number of macroscopic metastasis as well as the liver surface covered by metastatic foci was highly significant in scrambled KM12SM cells (Figure 7c). Collectively, these results indicate that differences in metastasis formation and colonization should be attributed to advantages in cell adhesion and proliferation.
We report a new role for the atypical cadherin-17 in adhesion and proliferation of metastatic colorectal cancer cells. Our conclusions were based on the following observations: (i) CDH17 high expression was associated to liver metastasis and overall survival, (ii) CDH17 silencing decreased cell adhesion, colony formation and proliferation in KM12 cells, particularly in highly metastatic KM12SM cells, (iii) CDH17 formed large complexes with α2β1 integrin and other proteins in colon cancer cells, (iv) cadherin-integrin signaling was mediated through FAK, Ras and MAP kinase activation (Figure 7d) and (v) CDH17 silencing decreased in vivo tumor growth and increased mice survival. Together, these data confirm that CDH17 have a major role in cell proliferation, tumor growth and liver metastasis colonization in colon cancer.
Our data agree with a recent histopathological study showing that CDH17 expression in colon cancer was associated to lung and lymph node metastasis.21 In addition to an increased expression in liver metastasis, we have observed a significant correlation of CDH17 overexpression with poor survival in colorectal cancer. The divergent results found in previous literature regarding CDH17 expression in colorectal cancer might be explained, at least partially, by technical reasons due to different treatment conditions as, for instance, sample fixation time, archival storage on the long-term, quality of antibodies, and so on. In our study, the initial management of the samples was standardized following a protocol that should minimize the heterogeneity of our results.
Although our results indicate that CDH17 interaction requires the presence of both α2β1 integrin subunits, only changes in β1 integrin conformation were visible. Therefore, the regulation should involve a conformational change induced by cadherin interaction to induce β1 integrin activation, as the HUTS21 antibody used in our study only recognizes a conformational epitope present in the high-affinity conformation of the β1 integrin.27 This might explain also why despite similar levels of total α2β1 integrin expression, scrambled and silenced cells present different metastatic potential. The observed macromolecular complexes for CDH17 and α2β1 integrin link extracellular matrix with actin cytoskeleton dynamics, and constitute signal transduction centers at sites of integrin clustering. The modulation of α2β1 integrin activity by CDH17, followed by FAK and Ras activation, is a novel mechanism that creates a link between non-conventional cadherins like CDH17 and the focal adhesion complex to promote an adhesive phenotype, and to increase cell proliferation in metastatic cells (Figure 7d).
Integrin regulation by CDH17 can promote cell adhesion to collagen type IV, which is highly enriched in the liver, and migration within the liver vasculature. We have observed some differences between lymph node and liver metastasis regarding the expression of CDH17. Collagen type IV is found primarily in the basement membrane of epithelial tissues, but it is almost absent in lymph nodes or other lymphatic tissues. Therefore, activation of α2β1 integrin through CDH17 for increased adhesion to collagen type IV might explain differences between liver and lymph node metastasis. For lymph nodes, the presence of a critical mass of tumor cells in the subcapsular sinuses is likely to promote metastasis at the regional level. This obviates the need for metastasizing cells to acquire properties for extravasation and increasing the probabilities of metastasis.30 In distant organs, the tumor cells require an appropriate microenvironment (‘soil’) to bind, grow and proliferate.
In colon cancer metastasis, the presence of CDH17 might facilitate cell clustering through homotypic cadherin interactions and cell adhesion through integrin activation to initiate micrometastasis formation, and further growth by increased proliferation. 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 the canonical epithelial E-cadherin and some integrins as, for instance, the mucosal lymphocyte integrin αEβ7, which might have implications in adhesion and activation.31 In addition, E-cadherin forms a ternary complex with insulin-like growth factor 1 receptor and αv integrin at cell–cell contact sites that correlates with an increase in cell migration of colon cancer cells.32 Another link between α6β4 integrin with E-cadherin through cdc42 has been reported.33 The effector cdc42 was also present in our CDH17 complex. More recently, Marchio et al.34 described the formation of E-cadherin and α6 integrin complex on the surface of colorectal cancer cells. This complex together with angiopoietin-like 6 seems to have a role in liver homing and colonization of colon cancer cells. Some authors have proposed that cadherin domains might adopt a tertiary structure rather, like immunoglobulin domains, resembling that of cellular integrin ligands. Still, no description of interactions between other cadherins, different from E-cadherin, and integrins was described to our knowledge. E-cadherin was expressed in some metastatic lesions in colon cancer, although membrane expression was significantly higher in primary tumors compared with their metastases.35 There are some other tumors, particularly prostate cancer, where there is a clear re-expression of E-cadherin in liver metastasis.36 Relationship between E-cadherin and CDH17 in metastatic cells will require further studies.
In summary, we have demonstrated that CDH17 interacts with α2β1 integrin and is a key determinant in the regulation of α2β1 integrin activity on cell adhesion and proliferation and, consequently, in the acquisition of liver metastatic capacity in colorectal cancer cells.
Materials and methods
Cell lines, culture and reagents
KM12C and KM12SM human colon cancer cells5 were obtained directly from Dr Fidler’s lab (MD Anderson Cancer Center, Houston, TX, USA). SW480, SW620, Caco2, RKO, SW48, Colo320, HT29 were purchased directly from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (Invitrogen) and antibiotics at 37 °C in a 5% CO2-humidified atmosphere.
Inhibitor UO126 was from Calbiochem (Darmstadt, Germany), PP2 from Sigma-Aldrich (St Louis, MO, USA), JNK inhibitor II from Merck Chemicals (Darmstadt, Germany) and FAK inhibitor 14 from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies used in this work are listed in Supplementary Table S3. The monoclonal antibody specific for CDH17 was a kind gift of Dr John M Luk (Hong Kong University).
Transfections of KM12 cells
pLKO.1 vectors containing shRNAs for CDH17 were purchased from Open Biosystems (Lafayette, CO, USA), clone IDs TRCN0000055658-TRCN00000555662 (shRNAs no. 58–62). As shRNA control vectors, we used a scrambled shRNA and an empty pLKO.1 vector obtained from AddGene (Cambridge, MA, USA); clone IDs 1864 and 8453, respectively.37, 38 Stably transfected cells were obtained by lentiviral infection, as previously described.39 Briefly, HEK293T cells were transfected with pLKO.1 vectors and the packaging vectors pCMV-rev, pMDLg-pRRE and pNGVL-VSVG using jetPRIME Transfection Reagent (Polyplus, Illkirch, France). After incubating the cells for 12–15 h in serum-free medium, it was replaced by fresh Dulbecco’s modified Eagle medium containing 10% fetal bovine serum and antibiotics. The day after, media containing lentiviral particles was added to KM12C and KM12SM cells. After 3 days of incubation, infected cells were selected using 1 μg/ml puromycin (Sigma-Aldrich) for 2–3 weeks. Then, cells were maintained in media containing 0.5 μg/ml puromycin.
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 described.40
Cell adhesion and proliferation
Cell proliferation, adhesion and invasion using Matrigel assays were carried out according to previously published procedures.41 For other ligands, cells were incubated 30 min on plates coated with fibronectin (10 μg/ml), collagen type I (5 μg/ml) or collagen type IV (5 μg/ml).
Western blot, inmunoprecipitation and GTPase activity assays
Cells were starved for 4 h and incubated in the presence or absence of inhibitors, and/or allowed to adhere to empty or Matrigel-coated plates for 45 min. Then, cells were detached, washed and lysed with protease and phosphatase inhibitors in lysis buffer (1% Igepal, 100 mM NaCl, 2 mM MgCl2, 10% glycerol in 50 mM Tris–HCl). Protein extracts were separated in SDS–PAGE and transferred to nitrocellulose membranes as previously described.39 For immunoprecipitation, cells were lysed and 500 μg of cell lysate were incubated with the indicated antibodies and the immunocomplex captured with 100 μl of Protein G-Sepharose beads (Sigma-Aldrich). After washing, samples were loaded on SDS–PAGE for western blot analysis.
For GTPase activity assays, cells were starved for 4 h, treated with inhibitors and/or exposed to Matrigel in previously coated plates and lysed. Aliquots of the extracts were kept for total lysate controls and the rest was incubated for 16 h at 4 °C with glutathione-S-transferase (GST)-Raf fused protein (a kind gift from Dr JM Rojas, ISCIII, Spain) and glutathione-agarose beads (Sigma-Aldrich). The beads were centrifuged, washed and bound proteins were eluted in Laemmli buffer, resolved in SDS–PAGE and analyzed by western blot using anti-Ras antibodies.
Mass spectrometry analysis of immunoprecipitated proteins
For proteomic analysis, 10 mg of cell lysates were immunoprecipitated as before, and the proteins were loaded in SDS–PAGE, which were divided in three slices for in-gel digestion as previously described.6 Peptides were loaded onto a C18-A1 ASY 2 cm precolumn (ThermoScientific, Waltham, 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 B (Buffer A: 0.1% formic acid/2% ACN; Buffer B: 0.1% formic acid in ACN) on a nanoEasy HPLC (Proxeon, Odense, Denmark) coupled to a nanoelectrospay ion source (Proxeon). Mass spectra were acquired in a linear trap quadrupole (LTQ) Orbitrap Velos in a data-dependent mode with an automatic switch between mass spectrometry and MS/MS scans using a 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-induced dissociation in the LTQ using normalized collision energy of 35% 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 22.214.171.1249, ThermoScientific) against the Uniprot Database (2012_02). Search parameters included precursor and fragment mass tolerances of 10 p.p.m. and 0.8 Da, respectively, a maximum of two missed cleavages allowed, a fixed modification of carbamidomethyl cysteine and a variable modification of methionine oxidation. Identified peptides were validated using Percolator algorithm with a q-value threshold of 0.01.
Cells directly attached onto Matrigel-coated coverslips, were fixed with 1% paraformaldehyde (Sigma-Aldrich) in phosphate-buffered saline containing 0.1% Triton X-100. Cells were washed three times with phosphate-buffered saline and incubated with primary antibodies, washed again and incubated with secondary antibodies labeled with Alexa-555 or Alexa-647, and with fluorescein isothiocyanate-phalloidin (Invitrogen) and 4,6-diamidino-2-phenylindole. After incubation with fluorochrome-conjugated secondary antibodies, samples were mounted with Mounting Fluorescence Medium (Dako, Copenhagen, Denmark) and images were captured using a TCS-SP2-AOBS-UV confocal microscope (LEICA, Wetzlar, Germany) with × 63 oil immersion objective. Displayed images were captured at the same sections in the different samples.
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 survival between years 2005 and 2007 in the Hospital Fundación Jimenez Díaz (Madrid). All surgical specimens were received at the Pathology Department in less than 1 h after resection, following protocols approved by the Ethical Committee of the hospital. Then, samples were fixed for 24 h 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 the risk of expression heterogeneity. Each sample was subjected to deparaffinization and antigen retrieval with a PT Link Module (Dako) at a high pH, for 20 min and subsequent incubation with the primary antibody against CDH17 (1 μg/ml). The reaction was revealed with DAB (3,3'-diaminobenzidine) as a chromogen and counterstained with hematoxylin and observed in an Olympus microscope. The intensity of CDH17-staining on membrane was graded as high (if intense) or low (either negative or moderate staining similar to control areas of normal colonic mucosa). In all cases, sections from normal colonic mucosa distant from the tumor site were used as negative controls.
In vivo animal experiments
The Ethical Committee of the Consejo Superior de Investigaciones Científicas (Spain) approved the protocols used for experimental work with mouse. For metastasis experiments, 106 KM12 cells expressing shRNA in 0.1 ml phosphate-buffered saline were injected intrasplenic in Swiss nude mice (Charles River). Mice were daily inspected for signs of disease, such as abdominal distension, locomotive deficit or tumor detectable by palpation. When symptoms were noted, mice were euthanized and 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 measured with external caliper, and volume was calculated as (4π/3) × (width/2)2 × (length/2). When tumors had reached an average size of 1200 mm3, animals were euthanized and tumors were excised.
Data were analyzed by one-way analysis of variance followed by Tukey-Kramer multiple comparison test. In both analyses the minimum acceptable level of significance was P<0.05. For discrete variable data, as presence or absence of metastasis or lymph node invasion, we used χ2 test. For continuous variable data with not Gaussian distribution, as overall survival, we performed a Mann–Whitney test.
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We thank Prof John M Luk (Hong Kong University) by the kind supply of CDH17 antibody. RA Bartolomé was supported by a grant to established research groups of the Asociación Española Contra el Cáncer (AECC). R Barderas was a recipient of a JAE-DOC/FSE Contract (CSIC) and is currently a fellow of the Ramón y Cajal program. S Torres was a recipient of a ProteoRed contract and Juan de la Cierva program. This research was supported by grant BIO2009-08818 from the Spanish Ministry of Science and Innovation, grant to established research groups (AECC) and grant S2010/BMD-2344/ Colomics2 from Comunidad de Madrid.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Oncogene website
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Bartolomé, R., Barderas, R., Torres, S. et al. Cadherin-17 interacts with α2β1 integrin to regulate cell proliferation and adhesion in colorectal cancer cells causing liver metastasis. Oncogene 33, 1658–1669 (2014). https://doi.org/10.1038/onc.2013.117
- α2β1 integrin
- liver metastasis
- colorectal cancer
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