Loss of Vinculin and Membrane-Bound Β-Catenin

Li et al. Molecular Cancer 2014, 13:263 http://www.molecular-cancer.com/content/13/1/263

RESEARCH Open Access Loss of vinculin and membrane-bound β-catenin promotes metastasis and predicts poor prognosis in colorectal cancer Ting Li1†, Hanqing Guo2†, Ying Song2†, Xiaodi Zhao1, Yongquan Shi1, Yuanyuan Lu1, Sijun Hu1, Yongzhan Nie1, Daiming Fan1 and Kaichun Wu1*

Abstract Background: Loss of cell-cell adhesion is important for the development of cancer invasion and metastasis. Vinculin, a key adhesion-related protein, can affect metastasis and prognosis in several tumours. Here, we determined the biological roles of vinculin in the metastasis of colorectal cancer (CRC) and evaluated its clinical significance as a potential disease biomarker. Methods: The expression level of vinculin in CRC cell lines and tissues was measured using Real-Time PCR and western blotting. Moreover, vinculin function was analysed using Transwell assays and in vivo metastasis assays in gain- and loss-of-function experiments. Furthermore, the impact of vinculin together with membrane-bound β-catenin on the prognosis of 228 CRC patients was investigated by immunohistochemistry. Additionally, the expression of epithelial-mesenchymal transition (EMT) indicators was verified by immunohistochemistry in CRC tissues obtained from these patients. Result: Vinculin expression was found to be significantly downregulated in highly metastatic CRC cell lines and metastatic tissues. Both in vitro and in vivo experiments showed that vinculin suppressed invasion, migration and metastasis in CRC cells and that this suppression could be attenuated by silencing β-catenin. Moreover, the expression of vinculin and membrane-bound β-catenin were positively correlated in CRC tissues, and lack of vinculin expression emerged as an independent prognostic factor in patients with CRC. Finally, the loss of vinculin and membrane-bound β-catenin was associated with node metastasis, organ metastasis and expression of EMT indicators. Conclusion: Our results suggest that vinculin may play specific roles in the EMT and metastasis of CRC and that loss of vinculin could be used as a prognostic factor for CRC. Keywords: Vinculin, β-catenin, Colorectal cancer, Metastasis, Prognosis, EMT

Background deaths have still occurred, making CRC the fourth leading Colorectal cancer (CRC) is the third most commonly diag- cause of cancer deaths in males and the third leading nosed cancer in males and the second most commonly di- cause of cancer deaths in females [1]. The poor prognosis agnosed cancer in females [1]. The CRC death rates have of CRC is associated with tumour invasion and metastasis, been decreasing in several Western countries [2], largely which often leads to therapeutic failure. Recently, it has resulting from improved treatment, increased awareness been reported that some loss of cell-cell adhesion may be and early detection [3]. However, an estimated 608,700 important for the development of CRC invasion and metastasis [4,5]. Vinculin is a ubiquitously expressed, actin-binding protein * Correspondence: [email protected] † that localises to the cytoplasmic face of integrin-mediated Equal contributors 1Department of Gastroenterology & State Key Laboratory of Cancer Biology, cell-extracellular matrix junctions (focal adhesions) and Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, China cadherin-mediated cell-cell junctions [6]. Normally, vinculin Full list of author information is available at the end of the article

© 2014 Li et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. Molecular Cancer 2014, 13:263 Page 2 of 15 http://www.molecular-cancer.com/content/13/1/263

plays a key role in focal adhesion formation [7], cell pro- In this study, we investigated the biological function of liferation [8] and regulation of the actin cytoskeleton vinculin and its prognostic value in CRC. We identified [9,10]. Loss of vinculin has been found in the develop- significant downregulation of vinculin in metastatic CRC ment of many cancers, such as squamous carcinoma cells and tissues. Furthermore, restoration of vinculin sup- [11,12], rhabdomyosarcoma [13] and breast cancer [14], presses CRC metastasis in vitro and in vivo, whereas loss implying that vinculin may have anti-tumour effects. of vinculin promotes CRC invasion and migration. In Recent studies confirmed this finding, as vinculin inhib- addition, we found that vinculin may regulate CRC inva- ited multiple processes associated with malignant tu- sion and migration at least partially through β-catenin. mours, including invasion, metastasis and apoptosis We further verified the positive correlation between the [8,15]. Specifically, overexpression of vinculin caused expression of vinculin and membrane-bound β-catenin reduced cell migration, whereas knockdown of vinculin and their correlation with an EMT indicator. More im- enhanced cell motility [16-18]. Researchers also found portantly, our data provide novel evidence that vinculin that vinculin-null cells had upregulated activity of extra- and membrane-bound β-catenin expression can serve as cellular signal-regulated kinase (ERK), leading to en- predictive biomarkers of poor prognosis in CRC patients. hanced survival and motility, which are important for metastasis [8]. In addition, low levels of vinculin may Results predict poor survival in squamous cell cancer [12], but Vinculin expression is downregulated in CRC cell lines the biological role of vinculin and its prognostic value and inversely correlated with CRC metastasis in CRC have not been fully investigated. To examine the significance of vinculin in CRC carcino- Vinculin activation is a key event in its coordination genesis, we measured the expression of vinculin in five with focal adhesion formation, which is important for human CRC cell lines (HCT116, Caco2, HT29, SW620 the suppression of cell mobility. This activation requires and SW480) and in HIEC, an immortalised colon epithe- various binding partners, such as talin [19], α-actinin lial cell line. Western blotting showed that vinculin ex- [20], α-catenin [21], β-catenin [22] and paxillin [8], to pression was significantly decreased in all five CRC cell unmask the binding sites of vinculin and continue its lines compared with HIEC (Figure 1A). Interestingly, localisation to focal adhesions. Because it is an import- compared with SW480, vinculin expression was signifi- ant cell-cell adhesion protein, β-catenin can bind to vin- cantly decreased in SW620, a cell line established from culin to stabilise E-cadherin (E-cad) at the cell surface the lymph node metastasis of the same patient as [22,23]. This important stabilising function requires the SW480 [32]. qRT-PCR also showed that mRNA expres- binding of vinculin to the N-terminus of β-catenin [23], sion of vinculin was relatively lost in various CRC cell which can bind a number of proteins that regulate the lines (Figure 1B). Furthermore, tissues from lymph node transition of cancer cells from an epithelial to a more metastases expressed lower levels of vinculin compared mesenchymal phenotype [24,25]. Recently, the epithelial- with primary CRC tissues and the adjacent normal tis- mesenchymal transition (EMT), a critical process in tumour sues, indicating the inverse relationship between the ex- invasion and metastasis, was found to be associated pression of vinculin and the metastatic status of CRC with the translocation of β-catenin from the membrane tissues (Figure 1C, D). Taken together, these results sug- to the nucleus [26]. Upregulated levels of β-catenin in gest that downregulation of vinculin is correlated with the nuclei of CRC cells were found to induce the activity increased CRC metastasis and that vinculin might inhibit of the transcription factor ZEB1, leading to EMT and a CRC progression. more invasive phenotype [27]. The EMT process is characterised by decreased levels of epithelial cell-cell Vinculin suppresses CRC cell invasion and metastasis adhesion molecules, such as E-cad [28], and increased in vitro and in vivo levels of mesenchymal cell-cell adhesion molecules, such To investigate whether vinculin regulates CRC cell invasion as vimentin (VIM) [29]. In addition, reduced membrane- and migration, we performed in vitro gain-of-function ana- bound β-catenin expression and increased cytoplasmic lyses by overexpressing vinculin with a lentiviral vector en- E-cad expression predict poor survival in gastric cancer coding vinculin in SW620 cells. Conversely, SW480 cells [30]. Decreased levels of β-catenin and E-cad on the cell were transfected with lentiviral vectors encoding vinculin membrane were also observed in CRC in a recent study siRNA or control siRNA. After cell transfection and anti- [31]. Based on these findings and our results that reveal biotic screening for 6 weeks, extracts from SW480 and the diminished levels of vinculin in CRC, we hypothe- SW620 cells transfected with the vinculin vector, siRNA or sised that the loss of vinculin and β-catenin at the cell control vector were submitted to western blotting and surface could be advantageous for the development of compared (Figure 2A, B). Transwell assays showed that ec- EMT and metastasis and may predict poor survival in topic expression of vinculin significantly suppressed the in- CRC patients. vasion and migration of SW620 cells (Figure 2C). In Li et al. Molecular Cancer 2014, 13:263 Page 3 of 15 http://www.molecular-cancer.com/content/13/1/263

Figure 1 Expression of vinculin in CRC cell lines and tissue samples. (A) The expression level of vinculin in five CRC cell lines and HIEC cells was measured by western blotting and quantified using Quantity One v4.6.2 software (one of three similar blots is shown). β-actin was used as a loading control. (B) The mRNA level of vinculin in five CRC cell lines and HIEC cells was measured by qRT–PCR using GAPDH as an internal control. (C) The expression of vinculin in adjacent non-cancerous colon tissues (N), primary CRC tissues (C) and lymph node metastatic tissues (M) from 6 patients was examined by western blotting. (D) The mRNA level of vinculin in paired tissues was measured using qRT-PCR. Vinculin expression was determined in tumour tissue relative to the patient’s adjacent normal tissue, and the relative expression of vinculin in the CRC cell lines was normalised to that in HIEC cells. Each sample was analysed in triplicate (*P < 0.05, **P < 0.01). contrast, the migration and invasion of SW480 cells sharply in mice injected with SW620-vinculin cells (Figure 2E). In increased when endogenous vinculin was silenced by contrast, inhibition of vinculin in SW480 cells increased siRNA (Figure 2D). These results suggest that vinculin sup- the rate of metastasis to liver and lung (Figure 2F). Taken presses CRC cell invasion and migration in vitro. together, these results indicate that vinculin has a suppres- To further validate whether vinculin could regulate the sor role in CRC metastasis. metastatic phenotype of CRC in vivo, we injected SW620- vinculin cells, which stably express vinculin, into nude mice Vinculin regulates CRC metastasis through β-catenin through the lateral tail vein. Liver and lung metastasis of To understand the underlying molecular mechanism by CRC was apparent in mice injected with SW620-vinculin- which vinculin suppresses CRC invasion and metastasis, control cells, while few metastatic tumours were detected we further investigated whether β-catenin, an important Li et al. Molecular Cancer 2014, 13:263 Page 4 of 15 http://www.molecular-cancer.com/content/13/1/263

Figure 2 Vinculin suppresses CRC cell invasion and metastasis in vitro and in vivo. (A) Western blot showing vinculin expression in SW620 cells infected with lentiviral vector encoding vinculin and in SW480 cells infected with vinculin siRNA (B). β-actin was used as an internal control. (C) Transwell migration and invasion assays using SW620 cells stably expressing vinculin or control vector. Representative images are shown on the left, and the quantification of 10 randomly selected fields is shown on the right. (D) Transwell migration and invasion assays using SW480 cells stably infected with vinculin siRNA or control siRNA. (E) Representative H&E staining of livers and lungs isolated from mice that received injections of SW620-control-vector or SW620-vinculin-vector cells. Black arrows indicate metastatic intrahepatic or lung tumours. The incidences of liver and lung metastasis in 10 mice are presented on the right. (F) Representative H&E staining of livers and lungs isolated from mice that received injections of SW480-control-siRNA or SW480-vinculin-siRNA cells. Li et al. Molecular Cancer 2014, 13:263 Page 5 of 15 http://www.molecular-cancer.com/content/13/1/263

binding partner of vinculin and a key activator of cancer More importantly, the loss of vinculin was associated with malignant phenotypes, was involved in this process. Im- lymph node metastasis and organ metastasis (P = 0.0273, munofluorescence assays showed that β-catenin was P = 0.01078). Node or organ metastasis was also related primarily located in the plasma membrane in SW480 to the absence of membrane-bound β-catenin expression cells; however, following vinculin siRNA infection, (P = 0.0027, P = 0.0159), further supporting the relationship β-catenin showed less localisation in the nucleus and in- between decreased vinculin and the absence of membrane- stead localised in the nucleus and cytoplasm (Figure 3A). bound β-catenin in CRC tissues. Interestingly, we found that On the other hand, the expression of membrane-bound decreased vinculin expression was significantly associated β-catenin in SW620 cells significantly increased after with vascular invasion (P = 0.0371). Tumour depth was also vinculin restoration, while nuclear β-catenin was almost found to be associated with the absence of membrane- absent (Figure 3B). bound β-catenin expression (P = 0.0121). There were no sig- To further verify whether β-catenin accounted for the nificant differences in these molecules with regard to patient change in CRC invasion and metastasis induced by vincu- gender, age, tumour stage or location. lin, we transfected β-catenin siRNA into SW480 cells previously transfected with the vinculin siRNA vector. Transwell Low vinculin expression is an independent prognostic factor assays showed that the β-catenin siRNA significantly re- We evaluated the three-year survival rates using the duced vinculin-siRNA-induced CRC cell invasion and mi- Kaplan-Meier method. Our results showed that vinculin gration (Figure 3C). By contrast, restoration of β-catenin loss was confirmed to be an independent prognosticator for significantly abrogated the inhibition of invasion and migra- low survival of CRC patients (Figure 5A, P = 0.001). Be- tion that was induced by vinculin overexpression in SW620 cause our results indicated that vinculin and β-catenin are cells (Figure 3D). Collectively, these results suggest that vin- co-expressed in CRC, we set out to detect whether the im- culin may regulate CRC invasion and migration at least pact of vinculin on the prognosis of CRC patients was af- partially through β-catenin. fected by β-catenin expression. Thus, the patients were divided into four groups according to their expression pat- Vinculin positively correlates with membrane-bound terns of vinculin and β-catenin: vinculin(High, H)/β-catenin β-catenin in CRC (Membrane-bound, M), vinculin(Low, L)/β-catenin(Non- To further investigate the expression levels and possible Membrane-bound, NM), vinculin (H)/β-catenin(NM) and associations between vinculin and β-catenin in CRC, we vinculin(L)/β-catenin(M). Survival analysis showed that measured the expression of vinculin and β-catenin in pri- patients with vinculin(L)/β-catenin(NM) expression en- mary CRC tissue arrays containing 228 cases. Decreased dured the lowest overall survival (Figure 5B, P < 0.001). Fur- levels of vinculin and β-catenin on the cell membrane thermore, in patients with low and high expression of were observed in CRC tissues compared to adjacent vinculin, β-catenin(NM) patients showed a decreased sur- normal tissues, and immunohistochemistry showed low vival time compared to β-catenin(M) patients (Figure 5C, expression of vinculin in 145 of 228 CRC tissues P < 0.001; Figure 5D, P = 0.042). However, low vinculin was (63.6%), as opposed to 59 of 228 adjacent non-cancerous found to result in low survival rates when membrane- tissues (25.9%) (Figure 4A, B, Additional file 1: Table S1). bound β-catenin was absent (Figure 5E, P = 0.037), but not Moreover, diminished expression of membrane-bound when membrane-bound β-catenin was detected (Figure 5F, β-catenin was also detected in 138 of 228 CRC tissues P = 0.506). (60.5%), whereas lack of membrane-bound β-catenin was A univariate analysis according to the Cox proportional found in only 36 of 228 adjacent non-cancerous tissues hazard regression model further confirmed these results (15.8%) (Figure 4A, C, Additional file 1: Table S1). Fur- (Table 2). Low expression of vinculin in the primary thermore, membrane-bound β-catenin was correlated tumour was associated with an increased risk of death (HR: with high vinculin expression (Figure 4D, Additional file 2: 1.805; 95% confidence interval [CI]: 1.262-2.582). Similarly, Table S2). Taken together, these results indicated that a lack of membrane β-catenin was also associated with higher low level of vinculin was significantly correlated with the risk (HR: 2.420; 95% CI: 1.685-3.475). As expected, larger absence of membrane-bound β-catenin. tumour size, vascular invasion, node metastasis and organ metastasis at the time of primary surgery were also associ- Low vinculin expression and lack of membrane-bound ated with poorer survival. Taken together, these results sug- β-catenin are associated with tumour malignancy in CRC gest that low vinculin expression is an independent The correlations between vinculin or β-catenin expres- prognostic factor with poor prognosis in colon cancer. sion and various clinicopathological features of CRC are summarised in Table 1. There was a statistically signifi- Vinculin inhibits EMT in CRC cant correlation between differentiation and vinculin Because extensive evidence suggests that translocation expression (P = 0.0021) or β-catenin expression (P = 0.0163). of β-catenin from the cell membrane to the nucleus can Li et al. Molecular Cancer 2014, 13:263 Page 6 of 15 http://www.molecular-cancer.com/content/13/1/263

Figure 3 Vinculin regulates CRC metastasis via β-catenin. (A) Immunofluorescence analysis of β-catenin (green) in SW480 cells infected with vinculin or control siRNA. Merged images represent overlays of β-catenin (green) and nuclear staining by DAPI (red). (B) Immunofluorescence analysis of β-catenin (green) in SW620 cells infected with the vinculin or control vector. (C) Transwell migration and invasion assays for SW480 cells infected with control or vinculin siRNA along with lentiviral vector expressing β-catenin siRNA. Representative images are shown on the left, and the quantification of 10 randomly selected fields is shown on the right. (D) Transwell migration and invasion assays using SW620 cells infected with control or vinculin vectors along with lentiviral vector expressing β-catenin. Li et al. Molecular Cancer 2014, 13:263 Page 7 of 15 http://www.molecular-cancer.com/content/13/1/263

Figure 4 Vinculin positively correlates with membrane-bound β-catenin in CRC. Tissue microarrays of consecutive immunostained sections were performed using specific antibodies against vinculin and β-catenin. (A) Representative images of vinculin and β-catenin protein expression levels in normal and cancer specimens. (B) The expression level of vinculin was significantly lower in CRC tissue species than in normal colorectal tissue species (P < 0.0001, χ2 test). The same result was observed for membrane-bound β-catenin (P < 0.0001, χ2 test) (C). (D) Membrane-bound β-catenin was positively correlated with vinculin expression in CRC tissues (P < 0.0001, χ2 test). initiate the EMT process [26], we speculated that loss of positive, strong expression of E-cad was detected in vin- vinculin might impact EMT in CRC. To investigate this culin(H)/β-catenin(M) CRC tissues, but not in vinculin hypothesis, we detected the expression of the epithelial (L)/β-catenin(NM) tissues, whereas VIM expression was differentiation marker E-cad and the mesenchymal marker present in the latter group of CRC tissues, but not in VIM in CRC cells and tissues. Western blots showed that the former (Figure 6C, D). Moreover, the results showed membrane-bound β-catenin expression as well as E-cad that the absence of both vinculin and membrane-bound dramatically increased in SW620 cells infected with vincu- β-catenin were correlated with decreased E-cad and in- lin vectors, whereas the expression of nuclear β-catenin creased VIM (Figure 6 E-H, Additional file 3: Table S3), and VIM were inhibited. In contrast, silencing vinculin indicating that loss of vinculin and membrane-bound in SW480 cells increased the subcellular expression of β-catenin may benefit the process of EMT in CRC. β-catenin, suppressed the expression of E-cad and upregulated the expression of VIM (Figure 6A, B). Discussion To evaluate whether those results could be translated Metastasis is one of the most distinguished phenotypes to the clinical setting, immunohistochemistry on tissue of a malignant tumour, and it results in extremely poor arrays was further conducted. The results showed that prognosis and relatively high recurrence. The phenotypic Li et al. Molecular Cancer 2014, 13:263 Page 8 of 15 http://www.molecular-cancer.com/content/13/1/263

Table 1 Correlations of Clinico-pathological variables with vinculin and β-catenin expression Vinculin P value Membrane-bound β-catenin P value Low High Absent Present Total cases 145 83 138 90 Gender Male 76 49 0.4068 71 54 0.2048 Female 69 34 67 36 Age <60 55 37 0.3304 61 31 0.1677 >60 90 46 77 59 Differentiation Well 59 51 0.0021* 56 54 0.0163* Morderate 62 17 55 24 Poor 24 15 27 12 Depth of Tumor T1-T2 47 32 0.3866 39 40 0.0121* T3-T4 98 51 99 50 Tumor stage I - II 82 51 0.4885 76 57 0.2716 III - IV 63 32 62 33 Location Signoid 38 24 0.7572 40 22 0.5428 Colon /Rectum 107 59 98 68 Vascular invasion Negative 77 56 0.0371* 85 48 0.2203 Positive 68 27 53 42 Node Metastasis Negative 72 54 0.0273* 65 61 0.0027* Positive 73 29 73 29 Organ metastasis Negative 115 77 0.0078* 110 89 0.0159* Positive 30 6 28 8 Analysis by chi-square criterion or Fisher’s exact test. *P<0.05. changes of reduced cell adhesion and increased cell mo- adhesion strength and faster migration than control cells tility drive tumour metastasis [33]. Recently, accumulat- [34], and this result was also verified in vinculin-null car- ing studies have suggested that the loss of vinculin, a cinoma cells [16]. Recent studies found that poor vincu- protein contributing to cell-cell adhesion, is observed in lin labelling in tumours of squamous-epithelial origin the development of various cancers and may lead to appeared to be related to the metastatic potential of the metastatic changes. In this study, we investigated the tumour [12]. Overexpression of vinculin in cancer cells biological role and prognostic value of vinculin in CRC was found to suppress their tumourigenic ability and metastasis. metaplastic capacity [17]. These results, which revealed Vinculin is a 117-kDa actin-binding protein consisting the cell-adhesion reduction and motility increase associ- of 1066 amino acids and is localised on the cytoplasmic ated with vinculin loss, indicate the potential involve- face of integrin-mediated cell-extracellular matrix junc- ment of vinculin in tumour metastasis. However, few tions (focal adhesions) and cadherin-mediated cell-cell studies have been performed to determine the biological junctions. Early studies have found that fibroblasts iso- role of vinculin in CRC metastasis. Thus, we were inter- lated from vinculin-deficient mice showed decreased ested as to whether CRC metastasis, a multistep process Li et al. Molecular Cancer 2014, 13:263 Page 9 of 15 http://www.molecular-cancer.com/content/13/1/263

Figure 5 Survival curves of CRC patients according to vinculin and β-catenin expression. (A) Patients with low vinculin expression showed a significantly poor prognosis than those with high vinculin expression. (B) Subgroup analysis of CRC patients with low or high expression of vinculin accompanied with the absence or presence of membrane-bound β-catenin. (C) The correlation of membrane-bound β-catenin expression with overall survival among patients with tumours expressing low or high levels of vinculin (D). (E) The correlation of vinculin expression with overall survival among patients with tumours with or without membrane-bound β-catenin (F). that normally begins with the loss of cell-cell adhesion found that vinculin significantly suppressed CRC cell in- leading to the detachment of cancer cells and invasion vasion and metastasis both in vitro and in vivo. Some of the basement membrane, is related to the loss of vin- studies reported that vinculin-knockout cells were 3-fold culin. In this study, the expression of vinculin in several less invasive in three-dimensional collagen matrices CRC cell lines was determined using qRT–PCR and simulating extracellular matrix (ECM) because the western blotting. Vinculin expression was significantly connection between the ECM and the actomyosin cyto- decreased in all five CRC cell lines and was lower in skeleton through integrin-type cell-matrix adhesion re- SW620 cells compared with the immortal colon epithe- ceptors is facilitated by vinculin [35,36]. These studies lial cell line HIEC. Furthermore, the results obtained focused on tractions exerted by these cells to the ECM from clinical CRC tissue also confirmed that vinculin and cell-ECM adhesions. In our study, however, we ex- was downregulated in advanced stages of CRC, indicat- plored the effects of vinculin on tumor metastasis, which ing its possible involvement in both oncogenic trans- often develops through several essential steps including formation and tumour metastasis. We subsequently not only invading through surrounding ECM, but also Li et al. Molecular Cancer 2014, 13:263 Page 10 of 15 http://www.molecular-cancer.com/content/13/1/263

Table 2 Univariate and multivariate analysis for overall survival (Cox proportional hazards regression model) Factors Univariate analysis Multivariate analysis1 HR 95%CI P HR 95%CI P Gender (male/female) 1.148 0.825-1.596 0.412 - - - Differentiation (well/ moderately, poorly) 1.340 0.966-1.860 0.080 - - - Vascular invasion (absent/present) 1.843 1.327-2.561 0.0002* 1.410 0.996-1.997 0.0527 Depth of Tumor (T1-T2/T3-T4) 1.185 0.850-1.453 0.317 - - - Node Metastasis (absent/present) 2.739 1.963-3.820 0.0015* 1.631 1.139-2.315 0.0062* Organ metastasis (absent/present) 4.509 2.915-6.975 <0.0001* 5.401 3.379-8.634 <0.0001* Vinculin expression (high/low) 1.805 1.262-2.582 0.0012* 2.048 1.401-2.995 0.0002* membrane-bound β-catenin expression (absent/present) 2.420 1.685-3.475 <0.0001* 2.024 1.402-2.923 0.0019* 1Multivariate analysis performed only for variables significant in the univariate analysis. *P < 0.05. loosening of cell-cell adhesions and other steps. Thus it Vinculin expression has been associated with squamous still requires further investigation to elaborate the spe- cell tumours, and vinculin loss might predict high meta- cific functions of vinculin on cell-cell adhesion and cell- plastic ability and poor prognosis [12]. A recent study also ECM adhesion through which tumor invasion and me- revealed that the expression of cytoskeletal proteins, in- tastasis takes place. cluding vinculin, talin and tensin, was downregulated and Vinculin exists in two conformations. In the inactive correlated with carcinogenesis, invasion and metastasis of conformation, the extensive interactions between the CRC, irrespective of its relative limited-case capacity [15]. head and tail domains prevent detectable binding to As an important binding partner and a specific activator most of its ligands [19]. In the active conformation, dis- of vinculin, β-catenin has been found to be associated with placement of the head-tail interactions leads to a signifi- CRC survival in several studies [31,41-43]. The decreased cant accumulation of ternary complexes [37]. Vinculin localisation of β-catenin on the cell membrane combined activation requires several binding partners, such as talin with its increased expression in the cytoplasm and nucleus [19], α-actinin [20], α-catenin [21], β-catenin [22] or may be involved in abnormal E-cad expression. This ex- paxillin [8], to fully unmask its binding sites and con- pression pattern also coincides with the poor clinical prog- tinue its localisation to focal adhesions. Several studies nosis of patients with CRC [31]. Our results indicated that have indicated the close interaction between vinculin vinculin was associated with membrane-bound β-catenin and β-catenin. Vinculin plays a critical role in maintain- in CRC. Through analysing the relationship between ing the beta-catenin-MAGI-2 interaction in epithelial vinculin or β-catenin and various clinicopathological cells [38]. Moreover, β-catenin was found to be required features, we found that decreased expression of vinculin to recruit vinculin to the cell cortex and to strengthen and β-catenin in the cell membrane is correlated with the junction’s association with the underlying cytoskel- poor differentiation, extensive tumour invasion and a eton in response to tension [39]. Furthermore, re- high incidence of metastasis. Moreover, our analysis of searchers found that the interaction between vinculin survival of the four groups ofCRCpatientsfurtherre- and β-catenin is crucial for the stabilisation of E-cad vealed that the loss of vinculin together with decreased at the cell surface [23], which is considered to be associ- membrane-bound β-catenin predicted the lowest level ated with inhibition of tumour metastasis [40]. In of survival. Most importantly, we found that lack of vin- this study, we found that vinculin modulates CRC me- culin expression was independently associated with tastasis through β-catenin. We confirmed that vinculin poor prognosis in colon cancer. and membrane-bound β-catenin are co-expressed in nor- Recently, the concept of the epithelial–mesenchymal mal tissues and that their expression is partially decreased transition (EMT), which was first reported in early embry- or lost in CRC tissues. Moreover, our results indicated that onic morphogenesis [44], has been extended to cancer vinculin restoration accumulates β-catenin on the cell progression and metastasis [45]. During EMT, non-motile, membrane and that silencing vinculin benefits the trans- polarised epithelial cells embedded via cell-cell junctions location of β-catenin. Finally, vinculin-induced cell migra- in a cell collective dissolve their cell-cell junctions tion and invasion were reversed by β-catenin. Taken and convert into individual, non-polarised, motile and in- together, these results establish a functional connection vasive mesenchymal cells [46]. Typically, cancer cells between vinculin and β-catenin and confirm that vinculin experiencing this process show decreased levels of epithe- functions as an anti-metastatic protein in CRC cells by af- lial cell-cell adhesion molecules such as E-cad [28] and fecting the subcellular location of β-catenin. increased levels of mesenchymal cell-cell adhesion molecules Li et al. Molecular Cancer 2014, 13:263 Page 11 of 15 http://www.molecular-cancer.com/content/13/1/263

Figure 6 (See legend on next page.) Li et al. Molecular Cancer 2014, 13:263 Page 12 of 15 http://www.molecular-cancer.com/content/13/1/263

(See figure on previous page.) Figure 6 Vinculin regulates CRC metastasis through β-catenin. (A) Western blot analysis of vinculin, membrane-bound β-catenin and nuclear β-catenin in SW620 cells infected with vinculin or control vector (left) and SW480 cells transfected with vinculin or control siRNA (right). (B) Western blot analysis of E-cadherin and vimentin in SW620 cells infected with vinculin or control vector (left) and of SW480 cells transfected with vinculin or control siRNA (right). (C) CRC tissue with high expression of vinculin, membrane-bound β-catenin, positive staining of E-cadherin and negative staining of vimentin. (D) CRC tissue with low vinculin expression, the absence of membrane-bound β-catenin, negative staining of E-cadherin and positive staining of vimentin. (E) E-cadherin expression was positively correlated with vinculin expression as well as (F) with membrane-bound β-catenin in CRC tissues (P < 0.0001, χ2 test; P < 0.0001, χ2 test). (G) Vimentin expression was negatively correlated with vinculin expression as well as (H) with membrane-bound β-catenin in CRC tissues (P = 0.0274, χ2 test; P = 0.0153, χ2 test). such as VIM [29]. Several studies indicated that vinculin vinculin expression is independently associated with poor strengthens the mechanical links between adhesion prognosis in CRC. Hence, we believe that vinculin could complexes (containing E-cad, β-catenin and α-catenin) be a potential new target in the development of therapies and the actin cytoskeleton. It has been reported that for CRC. vinculin plays a role in the establishment or regulation of the E-cad-based cell adhesion complex in breast cancer Materials and methods cells by directly interacting with β-catenin [22]. Recent Cell culture and tissue collection studies also suggest that vinculin is part of a protein com- HIEC, HCT116, Caco2 and HT29 cells were cultured in plex on the cytoplasmic face of E-cad, which includes RPMI-1640 medium (Thermo Scientific HyClone, Beijing, β-catenin and its binding partners MAGI-2 and α-catenin China). SW480 and SW620 cells were maintained in [38]. Researchers further confirmed that this complex is DMEM (HyClone). Paired samples of primary CRC, adja- required to stabilise a famous tumour suppressor PTEN cent normal tissues and lymph node metastatic tissues [39]. Taken together, these results suggest that vinculin, were obtained from patients who had undergone CRC through interactions with β-catenin on the cell membrane, surgery at Xijing Hospital, Xi’an, China. All samples were may act as an anti-metastatic factor in carcinogenesis. clinically and pathologically shown to be correctly labelled. Based on these findings and our results that suggest a This study was approved by the Hospital’s Protection of positive correlation between vinculin and β-catenin, we Human Subjects Committee, and informed consent was hypothesised that the loss of vinculin and β-catenin at the obtained from every patient. cell surface may be an advantage for EMT and metastasis in CRC. To test this hypothesis, two markers, i.e., E-cad Patients and VIM, which represent epithelial and mesenchymal dif- This study included 228 patients with CRC who under- ferentiation, respectively, were examined in CRC cells and went surgical therapy at Tianjin Medical University tissues, respectively. We found that restoration of vinculin Cancer Institute and Hospital between 2004 and 2008. induced the upregulation of E-cad and the downregulation Paired samples of primary CRC and adjacent normal tis- of VIM in CRC cells, while silencing vinculin in CRC cells sues were obtained from these patients with written in- decreased E-cad expression and increased VIM expres- formed consent for research purposes. The use of human sion. Using IHC on CRC microarrays, we further identi- tissues in this study was approved by the institutional re- fied positive E-cad staining in cancer tissues stained with view board of the Fourth Military Medical University and high vinculin expression and membrane-bound β-catenin, was performed in accordance with the international guide- whereas VIM staining was negative. Conversely, the tis- lines for the use of human tissues. Clinical data (including sues with low expression of vinculin and the absence of gender, age, grade, stage, tumour depth, differentiation, membrane-bound β-catenin showed low levels of E-cad lymph node and organ metastasis status) were obtained and extremely high levels of VIM. These phenomena fur- from each patient’s medical records. The average age of ther suggest that vinculin has multiple roles in EMT and the group was 61.4 years (range: 18–85 years). A 3-year CRC metastasis. follow-up was performed on the patients for survival analysis from the date of surgery until the date of death or last Conclusion follow-up, which ranged from 1 month to 36 months. All We have demonstrated that vinculin is significantly down- patients received postoperative chemotherapy using a regulated in highly metastatic cells and tissues. Vinculin fluorouracil-based regimen without neoadjuvant chemo- overexpression can inhibit CRC cell migration, invasion therapy, radiation therapy or immunotherapy. The cases and metastasis both in vitro and in vivo. Furthermore, lost to follow-up or those who died of a cause other than vinculin functions as an anti-metastatic protein, and in CRC were treated as censored data for the analysis of sur- CRC cells, it functions at least partially by regulating the vival rates. Ethical approval to perform this study was ob- subcellular location of β-catenin. Furthermore, the loss of tained from the local Human Research Ethics Committee. Li et al. Molecular Cancer 2014, 13:263 Page 13 of 15 http://www.molecular-cancer.com/content/13/1/263

Western blot analysis USA) with an 8.0-μm pore membrane. For invasion assays, Total proteins were prepared from fresh frozen tissue or 2×105 cells were placed into chambers coated with cultured cell samples by complete cell lysis (Roche, 150 μg of Matrigel (BD Biosciences, Maryland, USA). The Mannheim, Germany) with protease and phosphatase in- chambers were then inserted into the wells of a 24-well hibitors. Cytoplasmic proteins and nuclear proteins were plate and incubated for 48 h in DMEM medium with 10% isolated using the Nuclear and Cytoplasmic Protein FBS before examination. The cells remaining on the upper Extraction Kit (Beyotime, Jiangsu, China). Denatured surface of the membranes were removed, while the cells proteins (20–50 μg) were separated on SDS-PAGE gels adhering to the lower surface were fixed, stained in a dye and transferred to nitrocellulose membranes. The solution containing 0.05% crystal violet and counted under following primary antibodies were used: vinculin (Millipore, a microscope (Olympus Corp., Tokyo, Japan) to calculate Darmstadt, Germany), β-actin (Sigma-Aldrich Inc., St. Louis, their relative numbers, as described before [48]. The re- MO, US), β-catenin (BD Bioscience, San Jose, CA, US), sults of three independent experiments were averaged. Histone(SantaCruzBiotechnology,SantaCruz,US), E-cad (Cell Signaling Technology, Boston, MA, US) In vivo metastasis assays and VIM (Santa Cruz). The bands were scanned using the For the in vivo metastasis assays, 2 × 106 SW620 cells in- ChemiDocXRS+ Imaging System (Bio-Rad) and quantified fected with vinculin vector lentivirus and SW480 cells using Quantity One v4.6.2 software (Bio-Rad). infected with vinculin siRNA lentivirus were suspended in 200 μl PBS and injected into the tail vein of nude Real-time quantitative RT–PCR (qRT-PCR) mice (10 in each group, female nu/nu). After 4 weeks, Total RNA extraction, quality control and one-step the mice were sacrificed, and tumour tissues derived qRT-PCR were performed as previously reported [47]. from various organs were dissected and examined histo- The data were normalised using glyceraldehyde-3- logically. The nude mice were provided by the Experi- phosphate dehydrogenase (GAPDH) as a reference gene. mental Animal Center of the Fourth Military Medical The PCR primers for vinculin and GAPDH were as fol- University. All animal studies complied with the Fourth lows: vinculin-forward 5′- CCAAAACATGTCTCCTAT Military Medical University animal use guidelines and by ATCCTGG-3′, vinculin-reverse 5′- GAAGTGTCCTTC the protocols approved by the Fourth Military Medical AGACAGGG -3′; and GAPDH-forward 5′-ATGTC GT University Animal Care Committee. GGAGTCTACTGGC-3′, GAPDH-reverse 5′-TGACCT TGCCCACAGCCTTG-3′. Reverse Transcription PCR Immunofluorescence was performed using the PrimeScript RT reagent Kit Indirect immunofluorescence staining for β-catenin in (TaKaRa, Dalian, China) following the manufacturer′s stable SW480 and SW620 cells was performed as previ- instruction. Quantitative Real-time PCR was performed ously described [49]. using the SYBR Premix Ex Taq II (TaKaRa) and measured using a LightCycler 480 system (Roche, Basel, Switzerland). Tissue microarrays Expression of GAPDH was used as an internal control. Colorectal cancer tissues or adjacent non-cancerous tis- △△ 2- CT referred to the fold of the RNA expression of one sues were made into tissue microarrays using a Tissue sample compared to the calibration sample. Microarrayer (Beecher Instruments, Silver Spring, USA ™) according to the manufacturer’s instructions. Briefly, core Lentivirus infection and oligonucleotide transfection tissue biopsies (2 mm in diameter) were taken from repre- The overexpression and siRNA constructs of vinculin and sentative areas of individual, paraffin-embedded tissues. β-catenin were purchased from GeneChem (Shanghai, The staining results of the different areas in these tissue China). The constructs containing the vinculin and β- array blocks showed excellent agreement. Two to three catenin CDS or siRNA sequence and 100 bases of the cores from each case were chosen for analysis. We defined upstream and downstream regions flanking these se- an adequate case as a tumour that occupied 10% of the quences were inserted into the pGCSIL-GFP vector. core area. Target cells (1 × 105) were subcultured in 24-well plates and infected with 2 × 107 lentivirus-transducing units in Immunohistochemistry (IHC) the presence of 5 μg/ml polybrene. Empty lentiviral Immunohistochemistry (IHC) was performed on formalin- vectorwasusedasthenegativecontrol. fixed, paraffin-embedded primary CRC and adjacent normal tissues as described previously [50]. Briefly, the slides Migration and invasion assays were subjected to antigen retrieval in Target Retrieval Solu- For the migration assays, infected cells were harvested and tion, pH 9 (DAKO) with PT Link (DAKO). Tissues were in- resuspended in serum-free DMEM medium, and 2 × 105 cubated in a mouse monoclonal antibody against vinculin cells were placed into Boyden chambers (Corning, MA, (Millipore, dilution 1:50), β-catenin (BD, dilution 1:100), Li et al. Molecular Cancer 2014, 13:263 Page 14 of 15 http://www.molecular-cancer.com/content/13/1/263

E-cad (Cell Signal Technology, dilution 1:100) or VIM Zinc-finger E-box binding homeobox 1; PCR: Polymerase chain reaction; (Santa Cruz, dilution 1:100). Negative controls were incu- GAPDH: Glyceraldehyde- 3-phosphate dehydrogenase; siRNA: short interfering RNA. bated with mouse or rabbit IgGs (DAKO). The percentage of positive cells was divided into four grades (percentage Competing interests cores) [51]: <1% (0), 1–25% (1), 26–50% (2), 51–75% (3) The authors declare that they have no competing interests. and >75% (4). The intensity of staining was divided into Authors’ contributions four grades (intensity scores): negative (0), weak (1), moder- TL, HG, YS and KW participated in the study design, performing the ate (2) and strong (3). The histological score (H-score) was experiments, data analysis and drafting of the manuscript. HG and TL determined using the following formula: overall scores = performed the in vivo experiments and immunohistochemistry. YS, SH and BX assisted with the in vitro experiments and provided clinical tissues. DF, YS, percentage score × intensity score. For the vinculin and YN and QZ gave suggestions on study design and discussed and interpreted EMT markers (E-cad and VIM), less than 10% positive the data. YK carried out the clinical sample analyses. All authors read and staining was deemed negative [51]. For membrane-bound approved the final manuscript. β -catenin, tumours were considered positive if >50% of the Acknowledgements cells exhibited membrane-boundexpressionoftheprotein We acknowledge Dr. Yong Guo and Dr. Yong Gu from Xijing Hospital for and negative if the expression was below 50%. However, in their help with pathological analyses. We acknowledge Dr. Yi Zhou from β Tianjin Medical University Cancer Institute and Hospital and Dr. Dake Chu reality, staining for membrane-bound -catenin was very from Xijing Hospital for providing the tissue microarray and clinical data. This homogenous, with a majority of tumours being either work was supported by grants from the National 973 Project of China (No. strongly positive for membrane-bound β-catenin, with 2010CB529302) and the National 863 Project of China (No. 2012AA02A504). nearly 100% of the cells expressing β-catenin at the mem- Grants support brane, or completely negative, with <5% of cells exhibiting This work was supported by the National Key and Basic Research immune-reactivity for β-catenin at the cell membrane [49]. Development Program of China (No. 2010CB529302), the National 863 Project of China (No. 2012AA02A504), and National Natural Science Foundation of China (no. 81270445, 81370484). Statistical methods Continuous data are presented as the means ± s.e.m., Author details ’ 1Department of Gastroenterology & State Key Laboratory of Cancer Biology, and two groups were compared using Student s unpaired Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, China. t-test. The correlation coefficient between the expression 2Department of Gastroenterology, Xi’an Central Hospital, College of of vinculin and β-catenin was estimated using the Medicine, Xi’an Jiaotong University, Xi’an, Shanxi, China.

Spearman correlation method. The chi-squared value Received: 23 July 2014 Accepted: 27 November 2014 was used to confirm the correlation between EMT Published: 11 December 2014 markers and vinculin or β-catenin. The association between clinicopathological variables and vinculin/β-catenin References 2 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D: Global cancer expression were examined by χ tests. The categorical data statistics. CA Cancer J Clin 2011, 61:69–90. were analysed by a Fisher’s exact test. Overall survival 2. Center MM, Jemal A, Smith RA, Ward E: Worldwide variations in colorectal – (OS) curves were analysed using the Kaplan–Meier cancer. CA Cancer J Clin 2009, 59:366 378. 3. Mitry E, Bouvier AM, Esteve J, Faivre J: Benefit of operative mortality method, and differences were examined using log-rank reduction on colorectal cancer survival. Br J Surg 2002, 89:1557–1562. tests. Cox’s proportional hazard regression test was used 4. Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. – to estimate univariate and multivariate hazard ratios for Cell 2011, 144:646 674. 5. Cavallaro U, Christofori G: Cell adhesion and signalling by cadherins and prognosis. P values were two sided, and those <0.05 were Ig-CAMs in cancer. Nat Rev Cancer 2004, 4:118–132. considered statistically significant. All analyses were per- 6. Ziegler WH, Liddington RC, Critchley DR: The structure and regulation of – formed with the SPSS software (version 14.0). vinculin. Trends Cell Biol 2006, 16:453 460. 7. Humphries JD, Wang P, Streuli C, Geiger B, Humphries MJ, Ballestrem C: Vinculin controls focal adhesion formation by direct interactions with Additional files talin and actin. J Cell Biol 2007, 179:1043–1057. 8. Subauste MC, Pertz O, Adamson ED, Turner CE, Junger S, Hahn KM: Vinculin modulation of paxillin-FAK interactions regulates ERK to β Additional file 1: Table S1. Expression of vinculin and -catenin in control survival and motility. J Cell Biol 2004, 165:371–381. CRC and adjacent tissues(n = 228). Analysis by chi-square criterion test or 9. Wen KK, Rubenstein PA, DeMali KA: Vinculin nucleates actin ’ Fisher s exact test. polymerization and modifies actin filament structure. J Biol Chem 2009, Additional file 2: Table S2. Correlation of vinculin and β-catenin 284:30463–30473. expression in CRC and adjacent tissues(n = 228). Analysis by chi-square 10. Carisey A, Tsang R, Greiner AM, Nijenhuis N, Heath N, Nazgiewicz A, Kemkemer R, criterion test or Fisher’s exact test. Derby B, Spatz J, Ballestrem C: Vinculin regulates the recruitment and release Additional file 3: Table S3. Correlation of vinculin and β-catenin with of core focal adhesion proteins in a force-dependent manner. Curr Biol 2013, – EMT indicators in CRC tissues (n = 228). Analysis by chi-square criterion or 23:271 281. Fisher’s exact test. 11. Kawahara E, Tokuda R, Nakanishi I: Migratory phenotypes of HSC-3 squamous carcinoma cell line induced by EGF and PMA: relevance to migration of loosening of adhesion and vinculin-associated focal contacts with Abbreviations prominent filopodia. Cell Biol Int 1999, 23:163–174. CRC: Colorectal cancer; EMT: Epithelial-mesenchymal transition; E-cad: Epithelia 12. Lifschitz-Mercer B, Czernobilsky B, Feldberg E, Geiger B: Expression of the -cadherin; VIM: Vimentin; ERK: Extracellular signal-regulated kinase; ZEB1: adherens junction protein vinculin in human basal and squamous cell Li et al. Molecular Cancer 2014, 13:263 Page 15 of 15 http://www.molecular-cancer.com/content/13/1/263

tumors: relationship to invasiveness and metastatic potential. 35. Mierke CT, Kollmannsberger P, Paranhos Zitterbart D, Smith J, Fabry B, Hum Pathol 1997, 28:1230–1236. Goldmann WH: Mechano-coupling and regulation of contractility by the 13. Meyer T, Brinck U: Immunohistochemical detection of vinculin in human vinculin tail domain. Biophys J 2008, 94:661–670. rhabdomyosarcomas. Gen Diagn Pathol 1997, 142:191–198. 36. Mierke CT, Kollmannsberger P, Zitterbart DP, Diez G, Koch TM, Marg S, 14. Somiari RI, Sullivan A, Russell S, Somiari S, Hu H, Jordan R, George A, Ziegler WH, Goldmann WH, Fabry B: Vinculin facilitates cell invasion into Katenhusen R, Buchowiecka A, Arciero C, Brzeski H, Hooke J, Shriver C: three-dimensional collagen matrices. J Biol Chem 2010, 285:13121–13130. High-throughput proteomic analysis of human infiltrating ductal 37. Chen H, Choudhury DM, Craig SW: Coincidence of actin filaments and carcinoma of the breast. Proteomics 2003, 3:1863–1873. talin is required to activate vinculin. J Biol Chem 2006, 281:40389–40398. 15. Yang HJ, Chen JZ, Zhang WL, Ding YQ: Focal adhesion plaque associated 38. Subauste MC, Nalbant P, Adamson ED, Hahn KM: Vinculin controls PTEN cytoskeletons are involved in the invasion and metastasis of human protein level by maintaining the interaction of the adherens junction colorectal carcinoma. Cancer Invest 2010, 28:127–134. protein beta-catenin with the scaffolding protein MAGI-2. J Biol Chem 16. Coll JL, Ben-Ze’Ev A, Ezzell RM, Rodriguez FJ, Baribault H, Oshima RG, Adamson ED: 2005, 280:5676–5681. Targeted disruption of vinculin genes in F9 and embryonic stem cells changes 39. Ray S, Foote HP, Lechler T: beta-Catenin protects the epidermis from cell morphology, adhesion, and locomotion. Proc Natl Acad Sci U S A 1995, mechanical stresses. J Cell Biol 2013, 202:45–52. 92:9161–9165. 40. Solanas G, Porta-de-la-Riva M, Agusti C, Casagolda D, Sanchez-Aguilera F, 17. Rodriguez FJ, Geiger B, Salomon D, Sabanay I, Zoller M, Ben-Ze’Ev A: Larriba MJ, Pons F, Peiro S, Escriva M, Munoz A, Dunach M, De Herreros AG, Suppression of tumorigenicity in transformed cells after transfection Baulida J: E-cadherin controls beta-catenin and NF-kappaB transcriptional with vinculin cDNA. JCellBiol1992, 119:427–438. activity in mesenchymal gene expression. J Cell Sci 2008, 121:2224–2234. 18. Gu S, Papadopoulou N: Activation of membrane androgen receptors in 41. Kinugasa T, Akagi Y, Ochi T, Tanaka N, Kawahara A, Ishibashi Y, Gotanda Y, colon cancer inhibits the prosurvival signals Akt/Bad InVitroand InVivoand Yamaguchi K, Shiratuchi I, Oka Y, Kage M, Shirouzu K: Increased claudin-1 blocks migration via Vinculin/Actin signaling. Mol Med 2011, 17:1. protein expression in hepatic metastatic lesions of colorectal cancer. 19. Bakolitsa C, Cohen DM, Bankston LA, Bobkov AA, Cadwell GW, Jennings L, Anticancer Res 2012, 32:2309–2314. Critchley DR, Craig SW, Liddington RC: Structural basis for vinculin 42. Morikawa T, Kuchiba A, Yamauchi M, Meyerhardt JA, Shima K, Nosho K, activation at sites of cell adhesion. Nature 2004, 430:583–586. Chan AT, Giovannucci E, Fuchs CS, Ogino S: Association of CTNNB1 20. Bois PR, O’Hara BP, Nietlispach D, Kirkpatrick J, Izard T: The vinculin binding (beta-catenin) alterations, body mass index, and physical activity with – sites of talin and alpha-actinin are sufficient to activate vinculin. survival in patients with colorectal cancer. JAMA 2011, 305:1685 1694. J Biol Chem 2006, 281:7228–7236. 43. Choi HN, Kim KR, Lee JH, Park HS, Jang KY, Chung MJ, Hwang SE, Yu HC, 21. Imamura Y, Itoh M, Maeno Y, Tsukita S, Nagafuchi A: Functional domains Moon WS: Serum response factor enhances liver metastasis of colorectal of alpha-catenin required for the strong state of cadherin-based cell adhesion. carcinoma via alteration of the E-cadherin/beta-catenin complex. – J Cell Biol 1999, 144:1311–1322. Oncol Rep 2009, 21:57 63. 22. Hazan RB, Kang L, Roe S, Borgen PI, Rimm DL: Vinculin is associated with 44. Shook D, Keller R: Mechanisms, mechanics and function of epithelial- the E-cadherin adhesion complex. J Biol Chem 1997, 272:32448–32453. mesenchymal transitions in early development. Mech Dev 2003, – 23. Peng X, Cuff LE, Lawton CD, DeMali KA: Vinculin regulates cell-surface 120:1351 1383. E-cadherin expression by binding to beta-catenin. J Cell Sci 2010, 45. Polyak K, Weinberg RA: Transitions between epithelial and mesenchymal 123:567–577. states: acquisition of malignant and stem cell traits. Nat Rev Cancer 2009, – 24. Lal M, Song X, Pluznick JL, Di Giovanni V, Merrick DM, Rosenblum ND, 9:265 273. Chauvet V, Gottardi CJ, Pei Y, Caplan MJ: Polycystin-1 C-terminal tail 46. Yilmaz M, Christofori G: EMT, the cytoskeleton, and cancer cell invasion. – associates with beta-catenin and inhibits canonical Wnt signaling. Cancer Metastasis Rev 2009, 28:15 33. Hum Mol Genet 2008, 17:3105–3117. 47. Li T, Lu YY, Zhao XD, Guo HQ, Liu CH, Li H, Zhou L, Han YN, Wu KC, Nie YZ, Shi YQ, Fan DM: MicroRNA-296-5p increases proliferation in gastric 25. Stemmer V, de Craene B, Berx G, Behrens J: Snail promotes Wnt target cancer through repression of Caudal-related homeobox 1. Oncogene gene expression and interacts with beta-catenin. Oncogene 2008, 2014, 33:783–793. 27:5075–5080. 48. Zhao X, Dou W, He L, Liang S, Tie J, Liu C, Li T, Lu Y, Mo P, Shi Y, Wu K, 26. Clevers H, Nusse R: Wnt/beta-catenin signaling and disease. Cell 2012, Nie Y, Fan D: MicroRNA-7 functions as an anti-metastatic microRNA in 149:1192–1205. gastric cancer by targeting insulin-like growth factor-1 receptor. 27. Sanchez-Tillo E, de Barrios O, Siles L, Cuatrecasas M, Castells A, Postigo A: Oncogene 2013, 32:1363–1372. beta-catenin/TCF4 complex induces the epithelial-to-mesenchymal 49. Salim T, Sjolander A, Sand-Dejmek J: Nuclear expression of glycogen synthase transition (EMT)-activator ZEB1 to regulate tumor invasiveness. kinase-3beta and lack of membranous beta-catenin is correlated with poor Proc Natl Acad Sci U S A 2011, 108:19204–19209. survival in colon cancer. Int J Cancer 2013, 133:807–815. 28. Dorudi S, Sheffield JP, Poulsom R, Northover JM, Hart IR: E-cadherin 50. Neumann J, Horst D, Kriegl L, Maatz S, Engel J, Jung A, Kirchner T: expression in colorectal cancer: an immunocytochemical and in situ A simple immunohistochemical algorithm predicts the risk of distant hybridization study. Am J Pathol 1993, 142:981–986. metastases in right-sided colon cancer. Histopathology 2012, 60:416–426. 29. Ngan CY, Yamamoto H, Seshimo I, Tsujino T, Man-i M, Ikeda JI, Konishi K, 51. Uchikado Y, Natsugoe S, Okumura H, Setoyama T, Matsumoto M, Ishigami S, Takemasa I, Ikeda M, Sekimoto M, Matsuura N, Monden M: Quantitative Aikou T: Slug Expression in the E-cadherin preserved tumors is related evaluation of vimentin expression in tumour stroma of colorectal cancer. to prognosis in patients with esophageal squamous cell carcinoma. Br J Cancer 2007, 96:986–992. Clin Cancer Res 2005, 11:1174–1180. 30. Ramesh S, Nash J, McCulloch PG: Reduction in membranous expression of beta-catenin and increased cytoplasmic E-cadherin expression predict doi:10.1186/1476-4598-13-263 poor survival in gastric cancer. Br J Cancer 1999, 81:1392–1397. Cite this article as: Li et al.: Loss of vinculin and membrane-bound β-catenin 31. Stanczak A, Stec R, Bodnar L, Olszewski W, Cichowicz M, Kozlowski W, promotes metastasis and predicts poor prognosis in colorectal cancer. Szczylik C, Pietrucha T, Wieczorek M, Lamparska-Przybysz M: Prognostic Molecular Cancer 2014 13:263. significance of Wnt-1, beta-catenin and E-cadherin expression in advanced colorectal carcinoma. Pathol Oncol Res 2011, 17:955–963. 32. Hewitt RE, McMarlin A, Kleiner D, Wersto R, Martin P, Tsokos M, Stamp GW, Stetler-Stevenson WG: Validation of a model of colon cancer progression. J Pathol 2000, 192:446–454. 33. Valastyan S, Weinberg RA: Tumor metastasis: molecular insights and evolving paradigms. Cell 2011, 147:275–292. 34. Xu W, Baribault H, Adamson ED: Vinculin knockout results in heart and brain defects during embryonic development. Development 1998, 125:327–337.