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

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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

shorter survival time (50.33 F 3.51 days) compared with the the invasive capacity of gastric cancer cells, and thus, might control SGC7901-mU6 (75.33 F 8.51 days; P < 0.01). The inhibit tumor progression and metastasis. in vivo results were in good agreement with the results obtained in the in vitro clonogenic assay. To confirm that the effects of The Expression of b-Catenin in Transfectants CacyBP/SIP protein expression on xenograft growth were Because CacyBP/SIP is involved in h-catenin degradation as caused by the proliferation potential of transfected cells, but not a component of the ubiquitin ligase complex, we determined because of increased angiogenesis contributing to the xenograft the h-catenin expression in all transfectants to confirm the tumor growth, we performed immunohistochemical analyses on effect of CacyBP/SIP on h-catenin and to evaluate the possible various tumor sections to assess angiogenesis in mice. As mechanism by which CacyBP/SIP affects the malignant shown in Fig. 4E, the microvessel densities in the xenografts phenotype of gastric cancer cells. It was shown that the total were 14.3 F 4.5 (SGC7901), 16.3 F 4.9 (SGC7901-pFLAG), h-catenin protein levels increased greatly in SGC7901- 17.3 F 3.8 (SGC7901-CacyBP), 15.3 F 3.2 (SGC7901-mU6), siCacyBP cells by Western blot analysis and reduced the level and 17.7 F 1.5 (SGC7901-siCacyBP), and statistical analysis of expression in SGC7901-CacyBP, although no change showed no significant difference between the groups, suggest- occurred in the mRNA (Fig. 6A and B) in these cells. As a ing the CacyBP/SIP protein in gastric cancer cells did not transcription factor, h-catenin only performs transcriptional influence angiogenesis in the xenografts. Taken together, these functions when it is translocated into the nucleus, so we further results implied that CacyBP/SIP plays a suppressive role and investigated the levels of nuclear h-catenin protein using may partly reverse the malignant growth potential of gastric Western blot analysis and immunofluorescence staining. As cancer cells both in vitro and in vivo. shown in Fig. 6C, a greater amount of nuclear h-catenin in the siRNA transfectant was detected and lower nuclear h-catenin Impairment of Invasion Potential of Gastric Cancer Cells levels existed in SGC7901-CacyBP cells compared with control by CacyBP/SIP Expression cells. Immunofluorescence staining of nuclear h-catenin in To see if the different levels of expression of CacyBP/SIP SGC7901-siCacyBP cells was significantly stronger than in might have an effect on invasion potential, the Matrigel-coated control cells (Fig. 6D), in accordance with the results of the transwell assay was used to detect the invasive activity of the Western blot analysis. Because h-catenin is required as a transfectants. As shown in Fig. 5, compared with control cells, cofactor for the activation of the transcription factor, Tcf/LEF, the number of SGC7901-siCacyBP cells that succeeded in we determined the effects of CacyBP/SIP on Tcf/LEF activity invading the Matrigel was significantly increased, with a in the gastric cancer cell, SGC7901, using transient transfec- relative invasion index of 4.06 F 0.036 (P < 0.001). tion reporter gene assays. When the siRNA plasmid, mU6- Conversely, the relative invasion indices of SGC7901-CacyBP siCacyBP, was cotransfected, Tcf/LEF activity was increased and AGS-CacyBP cells were 0.1919 F 0.067 and 0.45 F 0.09, (Fig. 6E). Transfecting greater amounts of mU6-siCacyBP respectively, which was markedly lower than the corresponding plasmid further raised Tcl/LEF activity in a dose-dependent control cells. These data indicated that CacyBP/SIP may impair manner. In contrast, the overexpression-regulation of CacyBP/

FIGURE 2. Establishment of overexpression and knockdown of CacyBP/SIP in human gastric cancer cells by gene transfection. A. Examination of the level of expression of CacyBP/SIP protein in all transfectants by Western blot analysis. h-Actin was designated as the internal control. B. Detection of mRNA expression for CacyBP/SIP in all transfectants by semiquantitative RT-PCR. h-Actin was designated as the internal control. C. Identification of an inhibition effect of specific siRNAs for CacyBP/SIP by immunofluorescence staining using anti-CacyBP and FITC-conjugated goat anti-mouse IgG (green). The nuclei were stained by propidium iodine. D. Expression of CacyBP/SIP in AGS transfectants by Western blot analysis. The experiments were repeated in duplicate.

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FIGURE 3. Role of CacyBP/SIP in regulating gastric cancer cell proliferation. Mono- layer growth rates of cells were determined by the MTT assay (A) and cell counting method (B). Points, mean of at least three separate experiments; bars, SE. *, P < 0.01, versus corresponding empty vector control.

SIP resulted in reduced h-catenin–mediated activation of Tcf/ level of expression was positively correlated with the dif- LEF. Taken together, we inferred that CacyBP/SIP might ferentiation status of the gastric cancer cells. Many studies have participate in regulating the malignant behavior of gastric shown that poorly differentiated tumors have an increased cancer cells in part by modulating h-catenin expression and Tcf/ growth rate and unfavorable prognosis (19, 20), indicating that LEF activity. the level of expression of CacyBP/SIP may be associated with the growth of gastric cancer cells. To further explore the regulatory effect of CacyBP/SIP on Discussion the malignant phenotype of gastric cancer cells, we established S100 protein is the biggest subfamily in calcium-binding CacyBP/SIP-overexpressing and knockdown sub–cell lines proteins with EF-hands. It has also been shown that S100 using gene transfection. Our investigation on the behavior of proteins are associated with cell cycle progression, differentia- these transfectants showed that a higher level of expression of tion, metabolism (17), and metastatic phenotype (18). These CacyBP/SIP inhibited cell proliferation. On the contrary, proteins always function by interacting with their targets. reduction of CacyBP/SIP expression with RNA interference CacyBP/SIP is a new target that binds to S100A6, S100A1, promoted cell growth, which was in accordance with the S100A12, S100B, and S100P in a Ca2+-dependent manner (3). findings by Fukushima et al. (6), that CacyBP/SIP-deficient It has been reported that CacyBP/SIP may be involved in the mouse embryonic fibroblasts had a faster growth rate and differentiation of erythroid cells and neurons, and plays a role in showed enhanced proliferation properties. Furthermore, the thymocyte development by affecting the level of h-catenin (6). overexpression of CacyBP/SIP suppressed the tumorigenicity However, the pathologic roles of CacyBP/SIP, as a target for of gastric cancer cells in vitro and relieved tumor malignant S100 proteins that participate in the development and behavior in nude mice, whereas inhibition of CacyBP/SIP progression of numerous tumors and diseases, remain unclear. yielded the opposite effects. Taken together, the data suggested To better elucidate the function of CacyBP/SIP, we investigated that CacyBP/SIP may be a growth suppression molecule that the effect of this molecule on the behavior of gastric cancer acts directly or indirectly to control the proliferation of gastric cells. cancer cells and induces a reversal of the malignant phenotype. CacyBP/SIP has been reported to be differentially expressed Cell migration is a prerequisite for cancer invasion and in various normal tissues of the rat and human neuron cell lines. metastasis. Many members of the EF-hand calcium sensor In the present study, we characterized CacyBP/SIP expression family play an important role in regulating tumor cell inva- at the mRNA and protein levels in six gastric cancer cell lines siveness (21, 22). In some cases, the level of S100A6 expres- and showed that it was expressed at different levels and that the sion correlates with high metastatic activity, suggesting its

FIGURE 4. The effect of CacyBP/SIP on tumorigenicity in vitro and in vivo. A. Plate clonogenic assay for the SGC7901 cell line. *, P < 0.01; **, P < 0.001, versus controls. B. Soft clonogenic assay for the SGC7901 cell line. *, P < 0.05, versus controls. C. Plate and soft clonogenic assays for the AGS cell line. *, P < 0.05 versus controls. D. Tumor growth in nude mice. Mice were injected subcutaneously with 1 Â 107 transfected cells. The volumes of tumors were monitored at the indicated times and calculated according to the formula: 0.5 Â length Â width2.*,P < 0.05; **, P < 0.01, versus corresponding control. E. Detection of microvessel density by immunohistochemical staining. The tissue slides from xenografts were incubated with anti-factor VIII – related antigen (FVIIIRAg) antibody for detecting microvessel density in xenografts. Experiments were repeated in triplicate.

lation results in its translocation into the nucleus and interaction with Tcf/LEF family transcription factors to activate the expression of target genes important for cell proliferation, such as c-myc and cyclin D1 (28). The h-catenin–dependent oncogenic signaling network is involved in the multistep process of tumorigenesis, including cell proliferation (29), invasion (30), and inhibition of apoptosis (31). The results in our study showed that the inhibition of CacyBP/SIP protein increased the expression of h-catenin without a change in the mRNA level in gastric cancer cells, suggesting that CacyBP/SIP may be a critical component required in the h-catenin ubiquitin-degradation pathway. In addition, the expression of h-catenin in nuclei increased and the transcriptional activity of Tcf/LEF was enhanced in conjunction with reduced CacyBP/SIP. We sus- pect that CacyBP/SIP may suppress the malignant phenotype of gastric cancer cells in part by affecting the expression of h-catenin and the activity of Tcf/LEF. In conclusion, we studied the biological role of CacyBP/SIP in gastric cancer and observed that overexpression of CacyBP/ SIP could inhibit the proliferation and invasive potential of SGC7901 cells, suggesting a tumor suppressor function for CacyBP/SIP by affecting the expression of h-catenin and transcriptional activity of Tcf/LEF.

Materials and Methods Cell lines and Animals The gastric cancer cell lines, SGC7901 and MGC803 (Aca- demy of Military Medical Sciences), AGS and KATO III (American Type Culture Collection), and MKN45 and MKN28 (Riken) were perpetuated in our institute. These cell lines were cultivated in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated FCS, penicillin (100 units/mL), and A FIGURE 5. Role of CacyBP/SIP in regulating the invasion of gastric streptomycin (100 g/mL) in a CO2 incubator (Forma Scientific). cancer cells [SGC7901 cells (A) and AGS cells (B)]. The invasion activity BALB/c nude mice, 4 to 6 weeks old, were provided by the of cells was assayed in a Matrigel-coated transwell. The cells that Shanghai Cancer Institute (Shanghai, China) for the in vivo succeeded in invading the Matrigel were quantified 24 h after plating. Data were expressed after normalization against parental cells. Columns, mean tumorigenicity study. The mice were maintained in a laminar from at least three separate experiments; bars, SE. *, P < 0.05; **, P < airflow cabinet under specific pathogen–free conditions, housed 0.001, versus corresponding control. in cages, and provided with sterile food and water ad libitum. The experiments were performed along established, institutional involvement in increased cell invasion (23). S100A4 has also animal welfare guidelines concordant with NIH species criteria. been shown to regulate cell motility and invasion to promote cancer metastasis (24). In our present study, we found that the Semiquantitative RT-PCR overexpression of CacyBP/SIP, a target for the calcium-binding To examine the mRNA level of CacyBP/SIP in gastric S100 protein family, led to decreased invasive ability. cancer cells, we did RT-PCR amplification on RNA extracted Conversely, the siRNA-mediated CacyBP/SIP knockdown in from gastric cancer cells using specific primers for CacyBP/SIP SGC7901 cells was characterized by the increased invasive (accession AF_314752). Briefly, the total RNA was converted potential of cells. Therefore, CacyBP/SIP is proposed to be to cDNA by oligo(dT) 18 primer and M-MuLV reverse involved in the invasion and metastatic processes of gastric transcriptase. The specific primers for CacyBP/SIP were as cancer. However, the mechanism by which CacyBP/SIP follows: 5¶-cgaatatggcttcagaagagcta-3¶ (sense) and 5¶-tcaaaattcc- reduces cell invasiveness remains to be identified. gtgtctcctttg-3¶ (antisense). The PCR conditions were as follows: What mechanism might be responsible for the suppression 5 min at 94jC for a hot start, followed by 25 cycles of 45 s of malignant behavior of gastric cancer cells by CacyBP/SIP? It at 94jC, 45 s at 60jC, and 60 s at 72jC, with a final extension has been reported that CacyBP/SIP binds Siah-1 and Skp1, of 10 min at 72jC. The length of the PCR product was 687 bp. which are components of the ubiquitin ligase that regulates To normalize differences in the amount of cDNA added to the h-catenin degradation (9, 25, 26). h-Catenin is normally main- reaction, amplification of h-actin was done as an internal tained at low levels due to its constitutive proteasomal degra- control. The PCR products were separated by 1% agarose gels dation (27). Once the degradation of h-catenin is repressed and stained with ethidium bromide. Quantification of differ- (e.g., inhibition of GSK3h kinase activity), h-catenin accumu- ences in mRNA levels was done using the Kodak Digital

Science Electrophoresis Documentation and Analysis System anti-CacyBP/SIP antibody (diluted 1:2,000; prepared in our 290 (Eastman Kodak Co.). institute), mouse anti–h-catenin (diluted 1:4,000; Sigma), and mouse anti–h-actin (diluted 1:4,000; Sigma). Western Blot Analysis The cells were collected and lysed on ice in a buffer Generation of Sense and siRNA of the CacyBP/SIP containing 50 mmol/L of Tris-Cl (pH 7.5), 150 mmol/L of Eukaryotic Expression Vector NaCl, 0.2 mmol/L of EDTA, 1 mmol/L of phenylmethylsul- Full-length cDNA encoding human CacyBP/SIP was gene- fonyl fluoride, and 1% NP40, then quantified by the Bradford rated from gastric cancer cells by RT-PCR. The specific primers method. The lysates (100 Ag) were electrophoresed in 12% for CacyBP/SIP were 5¶-gggaattcgaatatggcttcagaagagcta-3¶ SDS-PAGE and blotted on a nitrocellulose membrane. The (sense) and 5¶-gcgatatctcaaaattccgtgtctcctttg-3¶ (antisense). The membranes were blocked with 10% fat-free milk at room PCR products were cloned into a pUCm-T vector by a thymine- temperature for 2 h and were incubated overnight with primary adenine (T-A) cloning technique for further sequencing. Then, antibodies at 4jC overnight. After washing thrice for 15 min in cDNA inserts were released from recombinant pUCm-T TBS supplemented with 0.1% Tween 20, the membranes were plasmids by restriction enzyme digestion and subcloned into incubated with peroxidase-conjugated goat anti-mouse/rabbit the eukaryotic expression vector, pFLAG-CMV-3 (Sigma) to IgG antibody (diluted 1:2,000; Amersham-Pharmacia Biotech) achieve the sense vector, pFLAG-CacyBP. for 2 h at room temperature. The membranes were washed Two siRNAs targeting 118 to 136 (CacyBP-siRNA1) and again in TBS supplemented with 0.1% Tween 20, and finally 301 to 319 (CacyBP-siRNA2) of the CacyBP/SIP coding in substrate for enhanced chemoluminescence (Amersham) sequence, respectively, were designed and aligned to the human for 5 to 20 min. Exposed X-ray films were scanned for evalua- genome database in a BLAST search to ensure that the chosen tion of the band densities. Primary antibodies included mouse sequences were not highly homologous with those of other

FIGURE 6. CacyBP/SIP regulates h-catenin levels and Tcf/LEF activity. A. Total h-catenin mRNA in cells examined by semiquantitative RT-PCR. B. Total h-catenin protein level measured by Western blot analysis. C. The h-catenin expressions in the nuclear protein extract from the cells were tested using Western blot analysis. D. Subcellular distributions of h-catenin in transfectants were determined using immunofluorescence staining. E. Effect of CacyBP/ SIP on Tcf/LEF reporter activity. SGC7901 cells were transiently transfected with a reporter gene plasmid that contains a Tcf/LEF-responsive element cloned upstream of a luciferase reporter gene together with PRL-TK plasmid as a transfection-efficiency control and the indicated plasmids (pFLAG-CMV, pFLAG- CacyBP, mU6pro, and mU6-siCacyBP). Luciferase activity was measured in cell lysates 24 h later. *, P < 0.01, cotransfected pFLAG-CacyBP plasmid compared to pFLAG-CMV plasmid; #, P < 0.01, cotransfected mU6-siCacyBP plasmid compared to mU6pro plasmid. These experiments were repeated in triplicate.

genes: for oligo-1; sense, 5¶-tttgttgcatcttgttcttgatacaatcaagaacaa- stained with Giemsa solution, and counted. For the soft agar gatgcaacttttt-3¶, and antisense, 5¶-ctagaaaaagttgcatcttgttcttgattg- clonogenic assay, the cells were detached and plated in 0.3% tatcaagaacaagatgcaa-3¶; and for oligo-2; sense, 5¶-tttgcacctgca- agarose with a 0.5% agarose underlay (1 Â 103/wellina cattctcagtacaactgagaatgtgcaggtgcttttt-3¶, and antisense, 5¶-ctaga- 24-well plate). The number of foci >100 Am was counted after aaaagcacctgcacattctcagttgtactgagaatgtgcaggtg-3¶.Thepairs 20 days. were annealed in annealing buffer [0.1 mol/L NaCl and 10 mmol/L Tris (pH 7.4)] and cloned into the mU6pro vector Tumor Growth in Nude Mice to construct the siRNA vector of CacyBP/SIP (mU6-siCacyBP). The logarithmically growing cells (1 Â 107) were trypsi- The siRNA vector, mU6pro, was a generous gift from Professor nized and resuspended in 0.1 mL of D’Hanks solution, and Dave Turner (University of Michigan). injected into the cervical hypodermis of athymic nude mice. The tumor growth was observed and the survival time of the Cell Transfection mice was recorded from the day of injection. The size of Cells were plated and grown to 70% to 90% confluency the subcutaneous tumor mass was measured by calipers for without antibiotics. Transfections were done with Lipofect- 5 weeks. Tumor volume was calculated according to the 2 AMINE 2000 (Invitrogen AB), as directed by the manufacturer. formula: 0.5 Â length Â width . Each experimental group The CacyBP/SIP sense vector (pFLAG-CacyBP) was intro- consisted of five mice. duced into SGC7901 and AGS cells. For stable expression of siRNAs, SGC7901 cells were cotransfected with 1 Agof Immunohistochemical Staining siRNA-expressing vectors (mU6-siCacyBP) and 100 ng of The avidin-biotin complex immunoperoxidase method (34) pcDNA3.1(+) plasmid (Invitrogen). Clones were selected after was used to examine the microvessel density of xenograft 2 months of screening with G418. The pFLAG-CMV and tissues. In brief, the slides were blocked in 10% normal goat mU6pro vectors were transfected into cells as controls. serum for 1 h. The slides were then incubated with anti-factor Semiquantitative RT-PCR, Western blot analysis, and immu- VIII–related antigen (FVIIIRAg) primary antibody (polyclonal nofluorescence staining were used to examine the expression rabbit anti-FVIIIRAg antibody diluted at 1:200; Beijing of CacyBP/SIP in these transfectants. Biosynthesis Biotechnology Co.) at 4jC overnight and washed thrice in PBS for 5 min. The tissues were incubated in biotin- Immunofluorescence Staining labeled goat anti-rabbit IgG (diluted 1:200) for 30 min, rinsed The transfectants were plated onto cleaned-up coverslips and with PBS, and incubated with avidin-biotin-peroxidase com- fixed with 95% alcohol for 20 min at room temperature. The plex for 1 h. The signal was detected using 3,3-diaminobenzi- coverslips were washed with PBS and permeabilized for 10 min dine as the chromogen. Microvessel density was quantified by with 0.5% Triton X-100 in PBS. The cells were incubated with examining areas of vascular hotspots, as previously described anti-CacyBP/SIP antibody (diluted 1:2,000) or anti–h-catenin by Weidner et al. (35). Sections were scanned at a low Â Â antibody (diluted 1:2,000) after blocking with 3% bovine serum magnification ( 40 and 100) for the localization of vascular albumin for 1 h. The cells were then incubated with FITC- hotspots. The three most vascular areas of the tumor not conjugated anti-mouse IgG (Santa Cruz Biotech). The immu- containing necrosis were determined and then counted in a Â nofluorescence was analyzed under a MRC-1024 Laser high-power field ( 200). The values of the three sections were Scanning Confocal Imaging System (Bio-Rad). averaged and the results were analyzed. Branching structures were counted as a single vessel. Cell Growth Assay The proliferation of cells was evaluated by the cell-counting Invasion Assay method and the MTT assay (32). Cells (2 Â 104 per well) were A transwell plate (Costar) precoated with Matrigel (Becton seeded in 12-well plates and allowed to grow for different Dickinson) was used to perform the cell invasion assay (34). times. The growth rate was determined by the cell number, Briefly, cells were suspended in 0.2 mL of culture medium with counted on a hemacytometer in triplicate every day of the 1% FCS and were added to the upper chamber. Ten percent of culture, up to the 7th day. For the MTT assay, cells were plated FCS in the culture medium was plated in the lower chamber as a in a 96-well plate at 2.5 Â 103 cells/well. For 7 days, cells were chemoattractant. Cells in the invasion chambers were incubated incubated with 50 AL of 0.2% MTT for 4 h at 37jCina5% in a humidified incubator for 24 h. The cells that traversed the CO2 incubator. Following MTT incubation, 150 AL of 100% membrane pore and spread to the lower surface of the filters were DMSO was added to dissolve the crystals. Viable cells were stained with 5% Giemsa solution for visualization. counted every day by reading the absorbance at 490 nm using a 96-plate reader BP800 (Dynex Technologies). Detection of Nuclear b-Catenin Protein The cells were collected and washed with PBS, then Clonogenic Assay resuspended in buffer A solution [10 mmol/L HEPES-NaOH To measure the proliferative ability of a single cell in vitro, (pH 7.9), 1 mmol/L MgCl2, 15 mmol/L KCl, 0.1 mmol/L plate clonogenic (33) and soft agar clonogenic assays (34) were EDTA, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl done. For the plate clonogenic assay, 1 Â 103 cells were seeded fluoride, and 1 Ag/mL leupeptin] and incubated for 15 min at into a 9-cm dish and cultured in RPMI 1640 for 2 weeks to 0jC. Ten microliters of 10% NP40 was added and the mixture allow colony formation. Colonies were fixed in 70% ethanol, was vortexed for 10 s. The nuclei existed in the deposited pellet

after centrifugation at 12,000 Â g for 10 min. The deposit was ascites tumor cells that are potentially involved in the degradation of h-catenin. Chemotherapy 2006;52:32 – 4. resuspended in 50 AL of buffer B solution [20 mmol/L Hepes- 11. Fuchs SY, Ougolkov AV, Spiegelman VS, Minamoto T. Oncogenic h-catenin NaOH (pH 7.9), 1.5 mmol/L MgCl2, 0.42 mmol/L NaCl, signaling networks in colorectal cancer. Cell Cycle 2005;4:1522 – 39. 0.2 mmol/L EDTA, 10 mmol/L glycerol, 0.5 mmol/L DTT, 12. Peifer M, Polakis P. Wnt signaling in oncogenesis and embryogenesis— 0.5 mmol/L phenylmethylsulfonyl fluoride, and 1 Ag/mL a look outside the nucleus. Science 2000;287:1606 – 9. leupeptin] and the mixture was incubated for 30 min at 0jC. 13. HuG., Zhang S, Vidal M. Mammalian homologs of seven in absentia regulate The nuclear protein extracts were purified and existed in the DCC via the ubiquitin-proteasome pathway. Genes Dev 1997;11:2701 – 14. supernatant after centrifugation at 12,000 Â g for 4 min at 4jC. 14. ZhouBB, Elledge SJ. The DNA damage response: puttingcheckpoints in perspective. Nature 2000;408:433 – 9. The extracted nuclear proteins were quantified by the Bradford 15. Nakayama K, Frew IJ, Hagensen M. Siah2 regulates stability of prolyl- method and processed for Western blot analysis. hydroxylases, controls HIF-1a abundance, and modulates physiological responses to hypoxia. Cell 2004;117:941 – 52. 16. Tanikawa J, Ichikawa-Iwata E, Kanei-Ishii C, Nakai A. p53 suppresses the Reporter Gene Assay c-Myb-induced activation of heat shock transcription factor 3. J Biol Chem 2000; To assay the effect of CacyBP/SIP on the transcriptional 275:15578 – 15585. activity of Tcf/LEF, the reporter gene assay was done as 17. Zimmer DB, Cornwall EH, Landar A, Song W. The S100 protein family: described (36). Briefly, SGC7901 cells in a 24-well plate History, function, and expression. Brain Res Bull 1995;37:417 – 29. (50,000 cells per well) were cotransfected with or without 18. Gibbs FE, Wilkinson MC, Rudland PS, et al. Interactions in vitro of p9Ka, the rat S-100-related, metastasis-inducing, calcium-binding protein. J Biol Chem pFLAG-CMV, FLAG-CacyBP, mU6pro, mU6-siCacyBP, and 1994;269:18992 – 9. the reporter plasmid, pTOPFLASH, containing wild-type Tcf/ 19. Nishida T, Tanaka S, Haruma K, et al. Histologic grade and cellular LEF binding sites using LipofectAMINE 2000; pRL-TK Renilla proliferation at the deepest invasive portion correlate with the high malignancy of luciferase reporter served as a control for transfection efficiency. submucosal invasive gastric carcinoma. Oncology 1995;52:340 – 6. 20. Yamamoto H, Soh JW, Shirin H, et al. Comparative effects of overexpression The luciferase activity was measured and quantitated in a of p27Kip1 and p21Cip1/Waf1 on growth and differentiation in human colon luminometer using the Dual-Luciferase Reporter Assay System carcinoma cells. Oncogene 1999;18:103 – 15. (Promega). Experiments were done in triplicate and repeated 21. Jenkinson SR, Barraclough R, West C R, et al. S100A4 regulates cell motility twice. Results are expressed as the mean of the ratio between the and invasion in an in vitro model for breast cancer metastasis. Br J Cancer 2004; 90:253 – 62. firefly luciferase activity and the renilla luciferase activity. 22. Wang G, Platt-Higgins A, Carroll J, et al. Induction of metastasis by S100P in a rat mammary model and its association with poor survival of breast cancer Statistical Analysis patients. Cancer Res 2006;66:1199 – 207. 23. Ohuchida K, Mizumoto K, Ishikawa N, et al. The role of S100A6 in Data were analyzed by one-way ANOVA followed by the pancreatic cancer development and its clinical implication as a diagnostic marker S-N-K test, using SPSS software package. The Mann-Whitney and therapeutic target. Clin Cancer Res 2005;11:7785 – 93. test was used to compare survival times in the in vivo expe- 24. Li ZH, Bresnick AR. The S100A4 metastasis factor regulates cellular riments. For all comparisons, probability values <5% (P < 0.05) motility via a direct interaction with myosin-IIA. Cancer Res 2006;66:5173 – 80. were considered statistically significant. 25. Bhattacharya S, Lee YT, Michowski W, et al. The modular structure of SIP facilitates its role in stabilizing multiprotein assemblies. Biochemistry 2005;44: 9462 – 71. Acknowledgments 26. Matsuzawa S, Li C, Ni CZ, et al. Structural analysis of Siah1 and its We greatly thank Prof. Cun-Yu Wang (University of Michigan, Ann Arbor, MI) interactions with Siah-interacting protein (SIP). J Biol Chem 2003;278:1837 – 40. for the pTOPFLASH plasmid and Taidong Qiao, Baojun Chen, Zheng Chen, and 27. Hart M, Concordet JP, Lassot I, et al. The F-box protein h-TrCP associates Dan Chen for excellent experimental assistance. with phosphorylated h-catenin and regulates its activity in the cell. Curr Biol 1999;9:207 – 10. References 28. Chesire DR, Isaacs WB. h-Catenin signaling in prostate cancer: an early 1. Filipek A, Wojda U. p30, a novel protein target of mouse calcyclin (S100A6). perspective. Endocr Relat Cancer 2003;10:537 – 56. Biochem J 1996;320:585 – 7. 29. Sangkhathat S, Kusafuka T, Miao J, et al. In vitro RNA interference against 2. Filipek A, Kuznicki J. Molecular cloning and expression of a mouse brain cDNA h-catenin inhibits the proliferation of pediatric hepatic tumors. Int J Oncol 2006; encoding a novel protein target of calcyclin. J Neurochem 1998;70:1793 – 8. 28:715 – 22. 3. Filipek A, Jastrzebska B, Nowotny M, Kuznicki J. CacyBP/SIP, a calcyclin 30. Lowy AM, Clements WM, Bishop J, et al. h-Catenin/Wnt signaling regulates and Siah-1-interacting protein, binds EF-hand proteins of the S100 family. J Biol expression of the membrane type 3 matrix metalloproteinase in gastric cancer. Chem 2002;277:28848 – 52. Cancer Res 2006;6:4734 – 41. 4. Jastrzebska B, Filipek A, Nowicka D, Kaczmarek L, Kuznicki J. Calcyclin 31. Mezhybovska M, Wikstrom K, Ohd JF, et al. The inflammatory mediator (S100A6) binding protein (CacyBP) is highly expressed in brain neurons. leukotriene D4 induces h-catenin signaling and its association with antiapoptotic J Histochem Cytochem 2000;48:1195 – 202. Bcl-2 in intestinal epithelial cells. J Biol Chem 2006;281:6776 – 84. 5. AuKW, KouCY, Woo AY, et al. Calcyclin binding protein promotes DNA 32. Yanglin P, Lina Z, Zhiguo L, et al. KCNE2, a down-regulated gene Identified synthesis and differentiation in rat neonatal cardiomyocytes. J Cell Biochem by in silico analysis, suppressed proliferation of gastric cancer cells. Cancer Lett 2006;98:555 – 66. 2007;246:129 – 38. 6. Fukushima T, Zapata JM, Singha NC, et al. Critical function for SIP, a 33. Chow M, Yao A, Rubin H. Cellular epigenetics: Topochronology of ubiquitin E3 ligase component of the h-catenin degradation pathway, for progressive ‘‘spontaneous’’ transformation of cells under growth constraint. Proc thymocyte development and G1 checkpoint. Immunity 2006;24:29 – 39. Natl Acad Sci U S A 1994;91:599 – 603. 7. Yang YJ, LiuWM, ZhouJX, et al. Expression and hormonal regulationof 34. LiuN, Bi F, Pan Y, et al. Reversal of the malignant phenotype of gastric calcyclin-binding protein (CacyBP) in the mouse uterus during early pregnancy. cancer cells by inhibition of RhoA expression and activity. Clin Cancer Res 2004; Life Sci 2006;78:753 – 60. 10:6239 – 47. 8. Filipek A, Jastrzebska B, Nowotny M, et al. Ca2+-dependent translocation of 35. Weidner N, Carroll PR, Flax J, Blumenfeld W, Folkman J. Tumor the calcyclin-binding protein in neurons and neuroblastoma NB-2a cells. J Biol angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Chem 2002;277:21103 – 9. Pathol 1993;143:401 – 9. 9. Matsuzawa SI, Reed JC. Siah-1, SIP, and Ebi collaborate in a novel pathway 36. Korinek V, Barker N, Morin PJ, et al. Constitutive transcriptional activation h for -catenin degradation linked to p53 responses. Mol Cell 2001;7:915 – 26. by a b-catenin-Tcf complex in APCÀ/À colon carcinoma. Science 1997;275: 10. Filipek A. S100A6 and CacyBP/SIP—two proteins discovered in Ehrlich 1784 – 7.

Mol Cancer Res 2007;5(12). December 2007 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Calcyclin-Binding Protein Inhibits Proliferation, Tumorigenicity, and Invasion of Gastric Cancer

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