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Loss of Vinculin and Membrane-Bound Β-Catenin

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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
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