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Calcyclin-Binding Protein Inhibits Proliferation, Tumorigenicity, and Invasion of Gastric Cancer

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Calcyclin-Binding Protein Inhibits Proliferation, Tumorigenicity, and Invasion of Gastric Cancer Calcyclin-Binding Protein Inhibits Proliferation, Tumorigenicity, and Invasion of Gastric Cancer Xiaoxuan Ning,1,2 Shiren Sun,1,3 Liu Hong,1 Jie Liang,1 Lili Liu,1 Shuang Han,1 Zhiguo Liu,1 Yongquan Shi,1 Yuan Li,2 Weiqin Gong,2 Shanhong Zhang,2 Yu Chen,1 Xueyan Guo,1 Yi Cheng,4 Kaichun Wu,1 and Daiming Fan1 1State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Departments of 2Geriatrics and 3Nephrology, Xijing Hospital, the Fourth Military Medical University, Xi’an, Shaanxi, China; and 4Department of Gastroenterology, Wuhan General Hospital of Guangzhou Command, Wuhan, Hubei, China Abstract S100P, in a calcium-dependent manner (1-3). CacyBP/SIP is Calcyclin-binding protein/Siah-1–interacting protein highly expressed in the mouse and in rat brain, liver, spleen, (CacyBP/SIP), a target protein of the S100 family, and stomach, and weakly in rat lung and kidney (4). Recent which includes S100A6, S100A1, S100A12, S100B, studies have suggested that CacyBP/SIP is implicated in the and S100P, has been identified as a component of a differentiation of erythroid cells, neurons, and rat neonatal novel ubiquitinylation complex leading to B-catenin cardiomyocytes (5), the development of thymocytes (6), and degradation. However, the function of CacyBP/SIP in mouse endometrial events, such as the establishment of in gastric cancer has not been elucidated. In the pregnancy (7). It has been reported that the expression and present study, we prepared CacyBP/SIP overexpressing translocation of CacyBP/SIP has been observed in neuroblas- and knockdown cell lines of gastric cancer. Forced toma NB-2a cells (8). A human orthologue of CacyBP, Siah- CacyBP/SIP expression inhibited the proliferation of 1–interacting protein (SIP), has been shown to be a component gastric cancer cells, suppressed tumorigenicity in vitro, of the novel ubiquitin ligase complex responsible for ubiqui- and prolonged the survival time of tumor-bearing tination and degradation of h-catenin (9, 10), which is known to nude mice. In addition, increased CacyBP/SIP be an oncogene participating in tumorigenesis in many different h repressed the invasive potential of gastric cancer types of cancers (11, 12). Like -catenin, many target proteins cells. Conversely, the down-regulation of CacyBP/SIP of Siah-1–mediated degradation have been reported, including by RNA interference showed the opposite effects. DCC (13), N-CoR (14), PHD1/3 (15), and proto-oncogene Further studies showed that depressed CacyBP/SIP c-Myb (16). This suggests that CacyBP/SIP may play a role in increased the expression of total and nuclear B-catenin tumorigenesis by participating in the degradation of cancer- at the protein level and elevated the transcriptional related proteins. However, the pathologic roles of human activity of Tcf/LEF. Taken together, our results suggest CacyBP/SIP remain unclear. The aim of this study was to that CacyBP/SIP may be a potential inhibitor of cell explore the role of CacyBP/SIP in tumorigenesis of gastric growth and invasion in the gastric cancer cell, at least cancer and the related mechanism. In the present study, we in part through the effect on B-catenin protein observed the effects of ectopic expression and knockdown expression and transcriptional activation of Tcf/LEF. of CacyBP/SIP on cell proliferation and invasion of gastric (Mol Cancer Res 2007;5(12):1254–62) cancer cells in vitro and in vivo. Expression and distribution of h-catenin was further examined by Western blot analysis and immunofluorescence staining; Tcf/LEF transcriptional activa- Introduction tion was also detected by the reporter gene assay. These results Calcyclin-binding protein (CacyBP), a 30 kDa protein, was showed that overexpressed CacyBP/SIP inhibits proliferation, identified on the basis of its ability to interact with S100 tumorigenicity, and invasion of gastric cancer cells, at least in proteins, including S100A6, S100A1, S100A12, S100B, and part, via the down-regulation of h-catenin and the consequent transcriptional activation of Tcf/LEF. Results Received 12/20/06; revised 7/13/07; accepted 7/31/07. Grant support: National Nature Science Foundation of China (no. 30572113) Analysis of CacyBP/SIP Expression in Gastric Cancer and Science and Technology Innovation Foundation of Xijing Hospital Cells (no. XJCX05M21). The levels of expression of CacyBP/SIP mRNA and protein The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in in gastric cancer cell lines were examined by semiquantitative accordance with 18 U.S.C. Section 1734 solely to indicate this fact. reverse transcription-PCR (RT-PCR) and Western blot analysis. Note: X. Ning and S. Sun contributed equally to this work. Requests for reprints: Kaichun Wu and Daiming Fan, Institute of Digestive As shown in Fig. 1, both the mRNA and protein of CacyBP/SIP Diseases, Xijing Hospital, the Fourth Military Medical University, Xi’an 710032, were expressed at higher levels in the well-differentiated gastric Shaanxi Province, China. Phone: 86-29-8477-5230; Fax: 86-29-8253-9041. cancer cell line, MKN28, and was somewhat lower in the E-mail: [email protected] Copyright D 2007 American Association for Cancer Research. moderately differentiated gastric cancer cell line, SGC7901 doi:10.1158/1541-7786.MCR-06-0426 (P < 0.001). The lowest expressions of CacyBP/SIP mRNA and 1254 Mol Cancer Res 2007;5(12). December 2007 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. CacyBP Inhibits the Proliferation of Gastric Cancer 1255 FIGURE 1. Expression analysis of CacyBP/SIP in gastric cancer cell lines. A. Total RNA extracted from six gastric cancer cell lines was subjected to semiquantitative RT-PCR. The PCR products were separated with agarose gel electrophoresis. B. Proteins isolated from these cells were processed for Western blot analysis with anti-CacyBP antibody. h-Actin was designated as the internal control. Experiments were repeated in duplicate. protein were observed in the poorly differentiated gastric cancer cells (SGC7901-siCacyBP) proliferated at a faster rate than did cell lines, KATOIII, MKN45, MGC803, and AGS. control cells (SGC7901-mU6; P < 0.01), whereas SGC7901- CacyBP and AGS-CacyBP cells with a higher expression of The Expression of CacyBP/SIP in Sense and Short CacyBP/SIP exhibited a greater reduction in the proliferative Interfering RNA Transfectants rate than in control and parental cells (P < 0.01; Fig. 3). Our To investigate whether CacyBP/SIP affected the growth and data suggest that the growth of gastric cancer cells was nega- proliferation of gastric cancer cells, we selected the SGC7901 tively correlated with the level of expression of CacyBP/SIP. cell line, which moderately expresses CacyBP/SIP, as a model and introduced the CacyBP/SIP sense and short interfering Inhibition of Tumorigenicity of Gastric Cancer Cells RNA (siRNA) vectors in an attempt to establish the up- In vitro and In vivo by Up-Regulation of CacyBP/SIP regulation and down-regulation of CacyBP/SIP transfectants. The clonogenic assay is an effective method to evaluate the As shown in Fig. 2A and B, CacyBP/SIP was expressed at a proliferative ability and tumorigenicity of a single cell in vitro. higher level in the sense transfectant as compared with the Therefore, we applied the plate and soft agar clonogenic assays parental and empty vector cells. In contrast, specific siRNAs to investigate the role of CacyBP/SIP in the development of efficiently down-regulated CacyBP/SIP in the SGC7901 cells gastric cancer. As shown in Fig. 4A, B, and C, the forced at both the protein and mRNA levels, with an inhibitory overexpression of CacyBP/SIP dramatically reduced the rate exceeding 85% in the mU6-siCacyBP2 transfectant, as number and size of surviving colonies from the two gastric shown by immunofluorescence staining (Fig. 2C). The mU6- cancer cell lines compared with the empty vector–transfected siCacyBP2 and pFLAG-CacyBP transfectants were then used control cells, whereas SGC7901-siCacyBP cells showed a to test the functional outcomes of CacyBP/SIP. In addition, the notably increased plating efficiency compared with the controls gastric cancer cell, AGS, which endogenously expresses (parental cell and SGC7901-mU6 cell). To confirm this effect CacyBP/SIP protein at a relatively lower level was transfected in vivo, we injected transfected SGC7901 cells into the cervical with pFLAG-CacyBP plasmid to obtain the CacyBP/SIP- hypodermis of nude mice. It was found that tumor formation overexpressed AGS cells (Fig. 2D). occurred in all groups, but the tumor volumes of the mice injected with SGC7901-CacyBP cells were smaller than in the Growth Inhibition of Gastric Cancer Cells by CacyBP/SIP control groups (Fig. 4D). In addition, the survival time was Protein 89.67 F 9.50 days for the SGC7901-CacyBP–injected mice, The proliferation of the transfected cells was shown by which was markedly longer than in the mice injected with the methyl thiazolyl tetrazolium (MTT) assay and cell count SGC7901-pFLAG (68.67 F 3.05 days; P < 0.01). In contrast, method. Both results showed that siRNA-transfected SGC7901 the mice injected with SGC7901-siCacyBP had a significantly Mol Cancer Res 2007;5(12). December 2007 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. 1256 Ning et al. shorter survival time (50.33 F 3.51 days) compared with the the invasive capacity
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