Restin suppressed epithelial-mesenchymal transition and tumor metastasis in breast cancer cells through upregulating mir-200a/b expression via association with p73

Lu et al. Molecular Cancer Restin suppressed epithelial-mesenchymal transition and tumor metastasis in breast cancer cells through upregulating mir-200a/b expression via association with p73 Zhenduo Lu 1 Dechuang Jiao 1 Jianghua Qiao 1 Sen Yang 2 Min Yan 1 Shude Cui 1 Zhenzhen Liu 1 0 127 Dongming Road , Zhengzhou, Henan 450008 , People's Republic of China 1 Department of Breast surgery, Breast Cancer Center, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital 2 Department of Pathogen Biology, Basic Medical College of Zhengzhou University 3 100 Science Road , Zhengzhou 450001 , People's Republic of China Background: Restin belongs to MAGE superfamily and is known as MAGE H1. Restin was firstly cloned from HL-60 cells treated with all-trans retinoic acid (ATRA). Previous studies showed a pro-apoptotic role of Restin in several cell lines. However, little information is available on its expression patterns and functions in vivo. Our study was performed to detect if Restin plays a role in breast cancer cells in vitro and in vivo. Methods and results: Real-time PCR and western blot were conducted to detect Restin expression in multiple breast cancer cell lines and Restin level was negatively related with cell motility. Restin overexpression and knockdown stable cell lines were established by transducing lentivirus into MCF-7 and MDA-MB-231 cells. Cell morphology, wound closure assay, transwell migration and invasion assays were performed to detect if Restin inhibited EMT. Our data showed that Restin overexpressed cells exhibited classical epithelial cell morphology, and Restin overexpression resulted in activation of epithelial markers and suppression of mesenchymal markers, and inhibition of cell migration and invasion. Tumor xenograft model was used to characterize the biological functions of Restin in vivo. We found that Restin overexpression led to reduced lung metastasis. Real-time PCR, western blot, luciferase assay and ChIP assay were performed to identify the potential targets of Restin and the underlying molecular mechanisms. Among several master regulators of EMT, only ZEB1/2 levels were dramatically inhibited by Restin. Unexpectedly, Restin indirectly regulated ZEB1/2 expression at post-transcriptional level. We further identified mir-200a/b, well-characterized mediators controlling ZEB1/2 expression, were transcriptionally activated by Restin and the regulation was dependent on the p53 binding site in mir-200b/a/429 promoter. Further mechanical studies demonstrated Restin interacted with p73, one of p53 family members, which contributed to Restin-mediated activation of mir-200a/b and suppression of ZEB1/2. Conclusions: Taken together, our results suggest that Restin inhibits EMT and tumor metastasis by controlling the expression of the tumor metastasis suppressor mir-200a/b via association with p73. Our findings not only establish a mechanistic link between Restin, EMT and tumor metastasis, but also provide strong evidence supporting the notion that MAGE Group II proteins may exert a tumor suppressive effect in vivo. Restin; EMT; Tumor metastasis; mir-200a/b; p73; Breast cancer; MAGE superfamily - Background The melanoma-associated antigen (MAGE) is one of the well-characterized members of the CTA family that contains at least 60 closely related proteins [1], including MAGE-A, B, C, D, E, F, G, H, L2, Necidin, I and J. According to the protein expression patterns and functions, the MAGE family has been divided into groups I and II [2,3]. Group I proteins, including MAGEs A, B and C, are expressed in many types of tumor tissues, but are not expressed in normal tissues [1]. Moreover, their expression is closely correlated with aggressive clinical course, the acquisition of resistance to chemotherapy, the occurrence, and poor clinical outcomes [1,3,4]. Contrast to group I proteins, Group II proteins, such as Necdin and Mage-D1, are universally expressed in all normal tissues but rarely in tumor tissues [5]. They are more likely associated with cell growth inhibition, cell cycle arrest, apoptosis, or cell differentiation [6]. For example, Necdin has been shown to be down-regulated in both carcinoma cell lines and primary tumors [7-9], suggesting Necdin is a potent tumor growth suppressor. Ectopic overexpression of Necdin in a mouse tumor cell line is reported to attenuate tumorigenicity and metastasis in vivo [10]. In addition, MAGE-D1 inhibited cell proliferation, migration and invasion of multiple human cancer cells [11]. Although Group II proteins emerge as novel tumor suppressor candidates in a wide range of human cancers, their roles in cancers remain poorly defined. Restin belongs to MAGE Group II proteins and is known as MAGE H1 [12]. Restin was firstly cloned from the differentiated HL-60 cells induced by all-trans retinoic acid (ATRA) [13], an apoptosis and differentiation inducer. Bioinformatics analysis showed that Restin shared 49% homology with Necdin [14] and both of them were basic proteins. Further analysis found that Restin, Necdin and Mage-D1 had an alkaline conservative region, which is lowly expressed in tumor tissues [14]. Above data indicated that, similar to Necdin and Mage-D1, Restin belongs to Group II proteins. Bioinformatics data from GEO profiles show that Restin is rarely expressed in a variety of cancer cells, while its expression level is pretty high in normal cells. Restin was identified as one of pro-apoptotic genes that determined the response of multiple tumor cells to CD95-mediated apoptosis [15]. Fu HY et al. found that Restin overexpression in Hela cells promoted apoptosis [16]. Denis Selimovic et al. disclosed that Restin overexpression induced apoptosis of melanoma cells via interacting with p75 neurotrophin receptor (p75NTR), leading to the disruption of both NF-B and extracellular signal-regulated kinase (ERK) pathways [12]. Thus, Restin may function as a tumor suppressor, which is similar to Necdin and Mage-D1. Nevertheless, little information is available on its expression patterns and functions, particularly its roles in tumorigenesis in vitro and in vivo. The epithelial-mesenchymal transition (EMT) and the reverse process, termed the mesenchymal-epithelial transition (MET), play critical roles in embryogenesis, wound healing, tissue fibrosis, and carcinoma progression [17-20]. EMT is known to be a central mechanism responsible for invasiveness and metastasis of a variety of cancers [19-21]. During tumor development, epithelial cells undergo dynamic cytoskeletal rearrangement, and lose cell adhesion and epithelial components while acquiring mesenchymal and migratory phenotypes [20,22]. Therefore, targeting EMT may serve as an efficient strategy for the treatment of malignant and metastatic tumors. Our present study, for the first time, demonstrated that Restin remarkably suppressed breast cancer metastasis through inhibiting EMT by controlling the expression and function of the tumor me (...truncated)