Qi et al. Journal of Experimental & Clinical Cancer Research 2014, 33:107 http://www.jeccr.com/content/33/1/107 RESEARCH ARTICLE Open Access Wnt3a expression is associated with epithelial-mesenchymal transition and promotes colon cancer progression Lisha Qi1,3,4†, Baocun Sun1,2,3,4*†, Zhiyong Liu1,3,4, Runfen Cheng1,3,4, Yixian Li2 and Xiulan Zhao2 Abstract Introduction: Epithelial–mesenchymal transition (EMT) contributes to the progression and metastasis of cancer cells and is associated with a more invasive phenotype of cancer. The Wnt/β-catenin signaling pathway is one of the major pathways involved in EMT regulation. Many studies provide evidence that β-catenin, the key regulator of the canonical Wnt signaling pathway, is important in regulating EMT in cancer. However, the roles of Wnt3a, the representative canonical Wnt ligand, in EMT and colon cancer progression have not yet been fully explored. Methods: The expression levels of Wnt3a and EMT-associated proteins (E-cadherin, vimentin, and β-catenin) were assessed by immunohistochemistry in human colon cancer tissues to evaluate the clinicopathological significance of Wnt3a, as well as the correlation between Wnt3a and EMT. We then upregulated Wnt3a expression in HCT116 colon cancer cells, established a nude mouse xenograft model, detected the expression of EMT and Wnt/β-catenin signaling-associated proteins, and observed invasion and clone-initiating abilities. Results: In 203 human colon cancer tissue samples, Wnt3a protein overexpression was related to colon cancer histological differentiation (P = 0.004), clinical stage (P = 0.008), presence of metastasis and recurrence (P = 0.036), and survival time (P = 0.007) of colon cancer patients. Wnt3a expression was notably concomitant with EMT immunohistochemical features, such as reduced expression of the epithelial marker E-cadherin (P = 0.012), increased expression of the mesenchymal marker vimentin (P = 0.002), and cytoplasmic distribution of β-catenin (P = 0.021). Results of in vitro and in vivo experiments showed that Wnt3a overexpression could alter cell morphology, regulate EMT-associated protein expression, and enhance clone-initiation and invasion. Dkk1 (antagonist of Wnt/β-catenin signaling) could also partially reverse the expression of EMT-associated proteins in Wnt3a-overexpressing cells. Conclusions: Wnt3a expression was associated with EMT and promoted colon cancer progression. The EMT-inducing effect was partially due to the stimulative effect of Wnt3a on the Wnt/β-catenin pathway. Keywords: Wnt3a, Epithelial-mesenchymal transition, Wnt/β-catenin pathway, Colon cancer Introduction [2]. Thus, knowledge on the treatment of this disease Colorectal carcinoma is the third most common cause would advance if its metastasis mechanism is deeply of cancer-related deaths worldwide [1]. Although colorec- understood. tal cancer diagnosis and treatment have significantly ad- Epithelial–mesenchymal transition (EMT) is an early vanced over the past two decades, the five-year survival phase of the malignant transformation of epithelial cells. rate remains below 50%. More than 50% of colorectal can- In EMT, cells lose their polarities and contacts with neigh- cer cases metastasize to the lymph nodes, liver, and lungs boring cells and then acquire mesenchymal-like and motile phenotypes [3]. Tumor-cell EMT is considered a crucial * Correspondence: [email protected] event in cancer metastasis. Numerous factors can report- † Equal contributors edly induce or mediate EMT, and these factors are com- 1Department of Pathology, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China monly associated with carcinoma progression. 2Department of Pathology, Tianjin Medical University, Tianjin 300070, China Full list of author information is available at the end of the article © 2014 Qi et al.; licensee BioMed Central Ltd. 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. Qi et al. Journal of Experimental & Clinical Cancer Research 2014, 33:107 Page 2 of 11 http://www.jeccr.com/content/33/1/107 The Wnt signaling pathway is pivotal in embryogen- Wnt3a promoted EMT by activating the Wnt/β-catenin esis and development and is also widely implicated in a signaling pathway. number of human diseases [4-6]. Deregulation of the Wnt signaling pathway has been associated with senes- Material and methods cence bypass [7], aberrant cell growth [8], and cancer Clinical samples [9]. Signaling through the Wnt pathway starts with Wnt Tissue samples of colon cancer were harvested from 203 ligands, which consist of more than 19 closely related patients who had undergone surgery for colon cancer in but distinct secreted cysteine-rich glycoproteins [10]. By Tianjin Medical University Cancer Institute and Hospital activating multiple intracellular signaling cascades, Wnts (Tianjin, China) between January 2002 and December control various cellular functions, including prolifera- 2004. None of the patients had received any chemother- tion, tissue homeostasis, stem cell maintenance, and cell apy or radiotherapy before their operation. Data of clini- fate decisions [11,12]. Several Wnts such as Wnt1, copathological parameters were obtained from patients’ Wnt3a, and Wnt7a stimulate the β-catenin-dependent clinical records and pathological reports. pathway, called canonical Wnt signaling [12]. Other Wnts such as Wnt4, Wnt5a, and Wnt11 may activate Cell culture reagents and animals the protein kinase c, calcium-calmodulin kinase, or Jun The human colon cancer cell line HCT116 was obtained NH (2)-terminal kinase pathway, called non-canonical from the Cell Resource Center at the Institute of Basic Wnt signaling [13]. Medical Sciences, Chinese Academy of Medical Sciences/ The canonical Wnt/β-catenin signaling pathway is well Peking Union Medical College (Beijing, China). Cells were established in colorectal oncogenesis, with >85% of these cultured in Iscove modified Dulbecco medium with 10% malignancies harboring mutations along the pathway FBS. Dkk1 recombinant protein was obtained from R&D that lead to constitutive activation. Many studies provide Systems. For Dkk1 administration in vitro, recombinant evidence that the Wnt/β-catenin signaling pathway plays Dkk1 (1 μg/mL) was added to culture medium. Typically, an important role in EMT regulation [14,15]. However, 50% of the medium was replaced every 24 h with fresh most investigations on the effects of the canonical Wnt conditioned medium containing Dkk1 at the original con- pathway on EMT have focused on β-catenin rather than centrations. Cells were harvested 48 h post-treatment, and on Wnt ligands despite the role of Wnt proteins as total cell lysates were collected for measurement by major initiating factors in the Wnt/β-catenin pathway. Western blot. The micro-Boyden chambers used were Blavier et al. found that Wnt1 overexpression in murine from NeuroProbe (Gaithersburg, MD, USA). Anti- mammary epithelial cells in vitro could promote EMT bodies to β-catenin, goat anti-rabbit, and goat anti- and cell proliferation [16]. Bo and Kanzawa have shown mouseIgG-FITCwerefromSantaCruzBiotechnology that increased Wnt5a provokes EMT of pancreatic can- (SantaCruz,CA,USA).AntibodiestoWnt3a,Snail, cer and gastric cancer cells [17,18]. Wu reported that Slug, and Twist were from Abcam (Cambridge, UK). Wnt3 activates the Wnt/β-catenin pathway and pro- Antibody to E-cadherin was from BD Biosciences (San motes EMT-like phenotypes in trastuzumab-resistant Jose, CA, USA). Antibody to vimentin was from Epitomics HER2-overexpressing breast cancer cells [19]. Bao et al. (Burlingame, CA, USA). Phalloidin was from Invitrogen found that Wnt3a could promote the EMT, migration, (Carlsbad, CA, USA). Alexa Fluor 488 and 546 were from and proliferation of human lens epithelial cells [20]. Molecular Probes (Eugene, OR, USA). BALB/C nude mice Meanwhile, the role of Wnt3a in colorectal cancer EMT (4–5 weeks old) were obtained from Wei Tong Li Hua has not been fully explored. Experimental Animal Company (Beijing, China). In this study, we evaluated the clinicopathological sig- nificance of Wnt3a and analyzed the correlation between Immunohistochemical staining Wnt3a expression and EMT immunohistochemical fea- Streptavidin–biotin–peroxidase staining was performed as tures in tissue specimens from 203 colon cancer pa- previously described [21]. In a typical procedure, the sec- tients. The effects of Wnt3a ectopic expression in the tions were pretreated with microwaves, blocked, and incu- colon cancer cell line HCT116 on the expression of epi- bated with a series of antibodies overnight at 4°C. Then, thelial and mesenchymal markers and EMT transcrip- they were immunostained with HRP-conjugated antibody tion factors were studied. We also investigated cell and signals were revealed using 3,3-diaminobenzidine proliferation and invasion in cell cultures with Wnt3a buffer as substrate. In place of primary antibodies for overexpression, as well as tumor growth and metastasis the negative control, PBS was used. in a colon cancer xenograft model. We further treated The expression of Wnt3a, E-cadherin, vimentin, and Wnt3a-overexpressing