This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ning, X. Articles by Fan, D. Search for Related Content PubMed PubMed Citation Articles by Ning, X. Articles by Fan, D.

---

Cancer Genes and Genomics

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

¹ State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Departments of ² Geriatrics and ³ Nephrology, Xijing Hospital, the Fourth Military Medical University, Xi'an, Shaanxi, China; and ⁴ Department of Gastroenterology, Wuhan General Hospital of Guangzhou Command, Wuhan, Hubei, China

Requests for reprints: Kaichun Wu and Daiming Fan, Institute of Digestive Diseases, Xijing Hospital, the Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China. Phone: 86-29-8477-5230; Fax: 86-29-8253-9041. E-mail: ningsun{at}fmmu.edu.cn

	Abstract

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Calcyclin-binding protein/Siah-1–interacting protein (CacyBP/SIP), a target protein of the S100 family, which includes S100A6, S100A1, S100A12, S100B, and S100P, has been identified as a component of a novel ubiquitinylation complex leading to β-catenin degradation. However, the function of CacyBP/SIP in gastric cancer has not been elucidated. In the present study, we prepared CacyBP/SIP overexpressing and knockdown cell lines of gastric cancer. Forced CacyBP/SIP expression inhibited the proliferation of gastric cancer cells, suppressed tumorigenicity in vitro, and prolonged the survival time of tumor-bearing nude mice. In addition, increased CacyBP/SIP repressed the invasive potential of gastric cancer cells. Conversely, the down-regulation of CacyBP/SIP by RNA interference showed the opposite effects. Further studies showed that depressed CacyBP/SIP increased the expression of total and nuclear β-catenin at the protein level and elevated the transcriptional activity of Tcf/LEF. Taken together, our results suggest that CacyBP/SIP may be a potential inhibitor of cell growth and invasion in the gastric cancer cell, at least in part through the effect on β-catenin protein expression and transcriptional activation of Tcf/LEF. (Mol Cancer Res 2007;5(12):1254–62)

	Introduction

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Calcyclin-binding protein (CacyBP), a 30 kDa protein, was identified on the basis of its ability to interact with S100 proteins, including S100A6, S100A1, S100A12, S100B, and S100P, in a calcium-dependent manner (1-3). CacyBP/SIP is highly expressed in the mouse and in rat brain, liver, spleen, and stomach, and weakly in rat lung and kidney (4). Recent studies have suggested that CacyBP/SIP is implicated in the differentiation of erythroid cells, neurons, and rat neonatal cardiomyocytes (5), the development of thymocytes (6), and in mouse endometrial events, such as the establishment of pregnancy (7). It has been reported that the expression and translocation of CacyBP/SIP has been observed in neuroblastoma NB-2a cells (8). A human orthologue of CacyBP, Siah-1–interacting protein (SIP), has been shown to be a component of the novel ubiquitin ligase complex responsible for ubiquitination and degradation of β-catenin (9, 10), which is known to be an oncogene participating in tumorigenesis in many different types of cancers (11, 12). Like β-catenin, many target proteins of Siah-1–mediated degradation have been reported, including DCC (13), N-CoR (14), PHD1/3 (15), and proto-oncogene c-Myb (16). This suggests that CacyBP/SIP may play a role in tumorigenesis by participating in the degradation of cancer-related proteins. However, the pathologic roles of human CacyBP/SIP remain unclear. The aim of this study was to explore the role of CacyBP/SIP in tumorigenesis of gastric cancer and the related mechanism. In the present study, we observed the effects of ectopic expression and knockdown of CacyBP/SIP on cell proliferation and invasion of gastric cancer cells in vitro and in vivo. Expression and distribution of β-catenin was further examined by Western blot analysis and immunofluorescence staining; Tcf/LEF transcriptional activation was also detected by the reporter gene assay. These results showed that overexpressed CacyBP/SIP inhibits proliferation, tumorigenicity, and invasion of gastric cancer cells, at least in part, via the down-regulation of β-catenin and the consequent transcriptional activation of Tcf/LEF.

	Results

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Analysis of CacyBP/SIP Expression in Gastric Cancer Cells
The levels of expression of CacyBP/SIP mRNA and protein in gastric cancer cell lines were examined by semiquantitative reverse transcription-PCR (RT-PCR) and Western blot analysis. As shown in Fig. 1 , both the mRNA and protein of CacyBP/SIP were expressed at higher levels in the well-differentiated gastric cancer cell line, MKN28, and was somewhat lower in the moderately differentiated gastric cancer cell line, SGC7901 (P < 0.001). The lowest expressions of CacyBP/SIP mRNA and protein were observed in the poorly differentiated gastric cancer cell lines, KATOIII, MKN45, MGC803, and AGS.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   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. β-Actin was designated as the internal control. Experiments were repeated in duplicate.

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   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. β-Actin was designated as the internal control. B. Detection of mRNA expression for CacyBP/SIP in all transfectants by semiquantitative RT-PCR. β-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.

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   FIGURE 3. Role of CacyBP/SIP in regulating gastric cancer cell proliferation. Monolayer 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.

View larger version (43K): [in this window] [in a new window] [Download PPT slide]   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 x 107 transfected cells. The volumes of tumors were monitored at the indicated times and calculated according to the formula: 0.5 x length x 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.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   FIGURE 5. Role of CacyBP/SIP in regulating the invasion of gastric cancer cells [SGC7901 cells (A) and AGS cells (B)]. The invasion activity of cells was assayed in a Matrigel-coated transwell. The cells that succeeded in invading the Matrigel were quantified 24 h after plating. Data were expressed after normalization against parental cells. Columns, mean from at least three separate experiments; bars, SE. *, P < 0.05; **, P < 0.001, versus corresponding control.

View larger version (55K): [in this window] [in a new window] [Download PPT slide]   FIGURE 6. CacyBP/SIP regulates β-catenin levels and Tcf/LEF activity. A. Total β-catenin mRNA in cells examined by semiquantitative RT-PCR. B. Total β-catenin protein level measured by Western blot analysis. C. The β-catenin expressions in the nuclear protein extract from the cells were tested using Western blot analysis. D. Subcellular distributions of β-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.

	Discussion

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

CacyBP/SIP has been reported to be differentially expressed in various normal tissues of the rat and human neuron cell lines. In the present study, we characterized CacyBP/SIP expression at the mRNA and protein levels in six gastric cancer cell lines and showed that it was expressed at different levels and that the level of expression was positively correlated with the differentiation status of the gastric cancer cells. Many studies have shown that poorly differentiated tumors have an increased growth rate and unfavorable prognosis (19, 20), indicating that 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 the malignant phenotype of gastric cancer cells, we established CacyBP/SIP-overexpressing and knockdown sub–cell lines using gene transfection. Our investigation on the behavior of these transfectants showed that a higher level of expression of CacyBP/SIP inhibited cell proliferation. On the contrary, reduction of CacyBP/SIP expression with RNA interference promoted cell growth, which was in accordance with the findings by Fukushima et al. (6), that CacyBP/SIP-deficient mouse embryonic fibroblasts had a faster growth rate and showed enhanced proliferation properties. Furthermore, the overexpression of CacyBP/SIP suppressed the tumorigenicity of gastric cancer cells in vitro and relieved tumor malignant behavior in nude mice, whereas inhibition of CacyBP/SIP yielded the opposite effects. Taken together, the data suggested that CacyBP/SIP may be a growth suppression molecule that acts directly or indirectly to control the proliferation of gastric cancer cells and induces a reversal of the malignant phenotype.

Cell migration is a prerequisite for cancer invasion and metastasis. Many members of the EF-hand calcium sensor family play an important role in regulating tumor cell invasiveness (21, 22). In some cases, the level of S100A6 expression correlates with high metastatic activity, suggesting its involvement in increased cell invasion (23). S100A4 has also been shown to regulate cell motility and invasion to promote cancer metastasis (24). In our present study, we found that the overexpression of CacyBP/SIP, a target for the calcium-binding S100 protein family, led to decreased invasive ability. Conversely, the siRNA-mediated CacyBP/SIP knockdown in SGC7901 cells was characterized by the increased invasive potential of cells. Therefore, CacyBP/SIP is proposed to be involved in the invasion and metastatic processes of gastric cancer. However, the mechanism by which CacyBP/SIP reduces cell invasiveness remains to be identified.

What mechanism might be responsible for the suppression of malignant behavior of gastric cancer cells by CacyBP/SIP? It has been reported that CacyBP/SIP binds Siah-1 and Skp1, which are components of the ubiquitin ligase that regulates β-catenin degradation (9, 25, 26). β-Catenin is normally maintained at low levels due to its constitutive proteasomal degradation (27). Once the degradation of β-catenin is repressed (e.g., inhibition of GSK3β kinase activity), β-catenin accumulation 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 β-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 β-catenin without a change in the mRNA level in gastric cancer cells, suggesting that CacyBP/SIP may be a critical component required in the β-catenin ubiquitin-degradation pathway. In addition, the expression of β-catenin in nuclei increased and the transcriptional activity of Tcf/LEF was enhanced in conjunction with reduced CacyBP/SIP. We suspect that CacyBP/SIP may suppress the malignant phenotype of gastric cancer cells in part by affecting the expression of β-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 β-catenin and transcriptional activity of Tcf/LEF.

	Materials and Methods

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Cell lines and Animals
The gastric cancer cell lines, SGC7901 and MGC803 (Academy 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 streptomycin (100 µg/mL) in a CO₂ incubator (Forma Scientific). BALB/c nude mice, 4 to 6 weeks old, were provided by the Shanghai Cancer Institute (Shanghai, China) for the in vivo tumorigenicity study. The mice were maintained in a laminar airflow cabinet under specific pathogen–free conditions, housed in cages, and provided with sterile food and water ad libitum. The experiments were performed along established, institutional animal welfare guidelines concordant with NIH species criteria.

Semiquantitative RT-PCR
To examine the mRNA level of CacyBP/SIP in gastric cancer cells, we did RT-PCR amplification on RNA extracted from gastric cancer cells using specific primers for CacyBP/SIP (accession AF_314752). Briefly, the total RNA was converted to cDNA by oligo(dT) 18 primer and M-MuLV reverse transcriptase. The specific primers for CacyBP/SIP were as follows: 5'-cgaatatggcttcagaagagcta-3' (sense) and 5'-tcaaaattccgtgtctcctttg-3' (antisense). The PCR conditions were as follows: 5 min at 94°C for a hot start, followed by 25 cycles of 45 s at 94°C, 45 s at 60°C, and 60 s at 72°C, with a final extension of 10 min at 72°C. The length of the PCR product was 687 bp. To normalize differences in the amount of cDNA added to the reaction, amplification of β-actin was done as an internal control. The PCR products were separated by 1% agarose gels and stained with ethidium bromide. Quantification of differences in mRNA levels was done using the Kodak Digital Science Electrophoresis Documentation and Analysis System 290 (Eastman Kodak Co.).

Western Blot Analysis
The cells were collected and lysed on ice in a buffer containing 50 mmol/L of Tris-Cl (pH 7.5), 150 mmol/L of NaCl, 0.2 mmol/L of EDTA, 1 mmol/L of phenylmethylsulfonyl fluoride, and 1% NP40, then quantified by the Bradford method. The lysates (100 µg) were electrophoresed in 12% SDS-PAGE and blotted on a nitrocellulose membrane. The membranes were blocked with 10% fat-free milk at room temperature for 2 h and were incubated overnight with primary antibodies at 4°C overnight. After washing thrice for 15 min in TBS supplemented with 0.1% Tween 20, the membranes were incubated with peroxidase-conjugated goat anti-mouse/rabbit IgG antibody (diluted 1:2,000; Amersham-Pharmacia Biotech) for 2 h at room temperature. The membranes were washed again in TBS supplemented with 0.1% Tween 20, and finally in substrate for enhanced chemoluminescence (Amersham) for 5 to 20 min. Exposed X-ray films were scanned for evaluation of the band densities. Primary antibodies included mouse anti-CacyBP/SIP antibody (diluted 1:2,000; prepared in our institute), mouse anti–β-catenin (diluted 1:4,000; Sigma), and mouse anti–β-actin (diluted 1:4,000; Sigma).

Generation of Sense and siRNA of the CacyBP/SIP Eukaryotic Expression Vector
Full-length cDNA encoding human CacyBP/SIP was generated from gastric cancer cells by RT-PCR. The specific primers for CacyBP/SIP were 5'-gggaattcgaatatggcttcagaagagcta-3' (sense) and 5'-gcgatatctcaaaattccgtgtctcctttg-3' (antisense). The PCR products were cloned into a pUCm-T vector by a thymine-adenine (T-A) cloning technique for further sequencing. Then, cDNA inserts were released from recombinant pUCm-T plasmids by restriction enzyme digestion and subcloned into the eukaryotic expression vector, pFLAG-CMV-3 (Sigma) to achieve the sense vector, pFLAG-CacyBP.

Two siRNAs targeting 118 to 136 (CacyBP-siRNA1) and 301 to 319 (CacyBP-siRNA2) of the CacyBP/SIP coding sequence, respectively, were designed and aligned to the human genome database in a BLAST search to ensure that the chosen sequences were not highly homologous with those of other genes: for oligo-1; sense, 5'-tttgttgcatcttgttcttgatacaatcaagaacaagatgcaacttttt-3', and antisense, 5'-ctagaaaaagttgcatcttgttcttgattgtatcaagaacaagatgcaa-3'; and for oligo-2; sense, 5'-tttgcacctgcacattctcagtacaactgagaatgtgcaggtgcttttt-3', and antisense, 5'-ctagaaaaagcacctgcacattctcagttgtactgagaatgtgcaggtg-3'. The pairs were annealed in annealing buffer [0.1 mol/L NaCl and 10 mmol/L Tris (pH 7.4)] and cloned into the mU6pro vector to construct the siRNA vector of CacyBP/SIP (mU6-siCacyBP). The siRNA vector, mU6pro, was a generous gift from Professor Dave Turner (University of Michigan).

Cell Transfection
Cells were plated and grown to 70% to 90% confluency without antibiotics. Transfections were done with LipofectAMINE 2000 (Invitrogen AB), as directed by the manufacturer. The CacyBP/SIP sense vector (pFLAG-CacyBP) was introduced into SGC7901 and AGS cells. For stable expression of siRNAs, SGC7901 cells were cotransfected with 1 µg of siRNA-expressing vectors (mU6-siCacyBP) and 100 ng of pcDNA3.1(+) plasmid (Invitrogen). Clones were selected after 2 months of screening with G418. The pFLAG-CMV and mU6pro vectors were transfected into cells as controls. Semiquantitative RT-PCR, Western blot analysis, and immunofluorescence staining were used to examine the expression of CacyBP/SIP in these transfectants.

Immunofluorescence Staining
The transfectants were plated onto cleaned-up coverslips and fixed with 95% alcohol for 20 min at room temperature. The coverslips were washed with PBS and permeabilized for 10 min with 0.5% Triton X-100 in PBS. The cells were incubated with anti-CacyBP/SIP antibody (diluted 1:2,000) or anti–β-catenin antibody (diluted 1:2,000) after blocking with 3% bovine serum albumin for 1 h. The cells were then incubated with FITC-conjugated anti-mouse IgG (Santa Cruz Biotech). The immunofluorescence was analyzed under a MRC-1024 Laser Scanning Confocal Imaging System (Bio-Rad).

Cell Growth Assay
The proliferation of cells was evaluated by the cell-counting method and the MTT assay (32). Cells (2 x 10⁴ per well) were seeded in 12-well plates and allowed to grow for different times. The growth rate was determined by the cell number, counted on a hemacytometer in triplicate every day of the culture, up to the 7th day. For the MTT assay, cells were plated in a 96-well plate at 2.5 x 10³ cells/well. For 7 days, cells were incubated with 50 µL of 0.2% MTT for 4 h at 37°C in a 5% CO₂ incubator. Following MTT incubation, 150 µL of 100% DMSO was added to dissolve the crystals. Viable cells were counted every day by reading the absorbance at 490 nm using a 96-plate reader BP800 (Dynex Technologies).

Clonogenic Assay
To measure the proliferative ability of a single cell in vitro, plate clonogenic (33) and soft agar clonogenic assays (34) were done. For the plate clonogenic assay, 1 x 10³ cells were seeded into a 9-cm dish and cultured in RPMI 1640 for 2 weeks to allow colony formation. Colonies were fixed in 70% ethanol, stained with Giemsa solution, and counted. For the soft agar clonogenic assay, the cells were detached and plated in 0.3% agarose with a 0.5% agarose underlay (1 x 10³/well in a 24-well plate). The number of foci >100 µm was counted after 20 days.

Tumor Growth in Nude Mice
The logarithmically growing cells (1 x 10⁷) were trypsinized and resuspended in 0.1 mL of D'Hanks solution, and injected into the cervical hypodermis of athymic nude mice. The tumor growth was observed and the survival time of the mice was recorded from the day of injection. The size of the subcutaneous tumor mass was measured by calipers for 5 weeks. Tumor volume was calculated according to the formula: 0.5 x length x width². Each experimental group consisted of five mice.

Immunohistochemical Staining
The avidin-biotin complex immunoperoxidase method (34) was used to examine the microvessel density of xenograft tissues. In brief, the slides were blocked in 10% normal goat serum for 1 h. The slides were then incubated with anti-factor VIII–related antigen (FVIIIRAg) primary antibody (polyclonal rabbit anti-FVIIIRAg antibody diluted at 1:200; Beijing Biosynthesis Biotechnology Co.) at 4°C overnight and washed thrice in PBS for 5 min. The tissues were incubated in biotin-labeled goat anti-rabbit IgG (diluted 1:200) for 30 min, rinsed with PBS, and incubated with avidin-biotin-peroxidase complex for 1 h. The signal was detected using 3,3-diaminobenzidine as the chromogen. Microvessel density was quantified by examining areas of vascular hotspots, as previously described by Weidner et al. (35). Sections were scanned at a low magnification (x40 and x100) for the localization of vascular hotspots. The three most vascular areas of the tumor not containing necrosis were determined and then counted in a high-power field (x200). The values of the three sections were averaged and the results were analyzed. Branching structures were counted as a single vessel.

Invasion Assay
A transwell plate (Costar) precoated with Matrigel (Becton Dickinson) was used to perform the cell invasion assay (34). Briefly, cells were suspended in 0.2 mL of culture medium with 1% FCS and were added to the upper chamber. Ten percent of FCS in the culture medium was plated in the lower chamber as a chemoattractant. Cells in the invasion chambers were incubated in a humidified incubator for 24 h. The cells that traversed the membrane pore and spread to the lower surface of the filters were stained with 5% Giemsa solution for visualization.

Detection of Nuclear β-Catenin Protein
The cells were collected and washed with PBS, then resuspended in buffer A solution [10 mmol/L HEPES-NaOH (pH 7.9), 1 mmol/L MgCl₂, 15 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 µg/mL leupeptin] and incubated for 15 min at 0°C. Ten microliters of 10% NP40 was added and the mixture was vortexed for 10 s. The nuclei existed in the deposited pellet after centrifugation at 12,000 x g for 10 min. The deposit was resuspended in 50 µL of buffer B solution [20 mmol/L Hepes-NaOH (pH 7.9), 1.5 mmol/L MgCl₂, 0.42 mmol/L NaCl, 0.2 mmol/L EDTA, 10 mmol/L glycerol, 0.5 mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl fluoride, and 1 µg/mL leupeptin] and the mixture was incubated for 30 min at 0°C. The nuclear protein extracts were purified and existed in the supernatant after centrifugation at 12,000 x g for 4 min at 4°C. The extracted nuclear proteins were quantified by the Bradford method and processed for Western blot analysis.

Reporter Gene Assay
To assay the effect of CacyBP/SIP on the transcriptional activity of Tcf/LEF, the reporter gene assay was done as described (36). Briefly, SGC7901 cells in a 24-well plate (50,000 cells per well) were cotransfected with or without pFLAG-CMV, FLAG-CacyBP, mU6pro, mU6-siCacyBP, and the reporter plasmid, pTOPFLASH, containing wild-type Tcf/LEF binding sites using LipofectAMINE 2000; pRL-TK Renilla luciferase reporter served as a control for transfection efficiency. The luciferase activity was measured and quantitated in a luminometer using the Dual-Luciferase Reporter Assay System (Promega). Experiments were done in triplicate and repeated twice. Results are expressed as the mean of the ratio between the firefly luciferase activity and the renilla luciferase activity.

Statistical Analysis
Data were analyzed by one-way ANOVA followed by the S-N-K test, using SPSS software package. The Mann-Whitney test was used to compare survival times in the in vivo experiments. For all comparisons, probability values <5% (P < 0.05) were considered statistically significant.

	Acknowledgements

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
We greatly thank Prof. Cun-Yu Wang (University of Michigan, Ann Arbor, MI) for the pTOPFLASH plasmid and Taidong Qiao, Baojun Chen, Zheng Chen, and Dan Chen for excellent experimental assistance.

	Notes

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Grant support: National Nature Science Foundation of China (no. 30572113) and Science and Technology Innovation Foundation of Xijing Hospital (no. XJCX05M21).

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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: X. Ning and S. Sun contributed equally to this work.

Received 12/20/06; revised 7/13/07; accepted 7/31/07.

	References

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 

1. Filipek A, Wojda U. p30, a novel protein target of mouse calcyclin (S100A6). Biochem J 1996;320:585–7.[Medline]
2. Filipek A, Kuznicki J. Molecular cloning and expression of a mouse brain cDNA encoding a novel protein target of calcyclin. J Neurochem 1998;70:1793–8.[Medline]
3. Filipek A, Jastrzebska B, Nowotny M, Kuznicki J. CacyBP/SIP, a calcyclin and Siah-1-interacting protein, binds EF-hand proteins of the S100 family. J Biol Chem 2002;277:28848–52.[Abstract/Free Full Text]
4. Jastrzebska B, Filipek A, Nowicka D, Kaczmarek L, Kuznicki J. Calcyclin (S100A6) binding protein (CacyBP) is highly expressed in brain neurons. J Histochem Cytochem 2000;48:1195–202.[Abstract/Free Full Text]
5. Au KW, Kou CY, Woo AY, et al. Calcyclin binding protein promotes DNA synthesis and differentiation in rat neonatal cardiomyocytes. J Cell Biochem 2006;98:555–66.[Medline]
6. Fukushima T, Zapata JM, Singha NC, et al. Critical function for SIP, a ubiquitin E3 ligase component of the β-catenin degradation pathway, for thymocyte development and G1 checkpoint. Immunity 2006;24:29–39.[CrossRef][Medline]
7. Yang YJ, Liu WM, Zhou JX, et al. Expression and hormonal regulation of calcyclin-binding protein (CacyBP) in the mouse uterus during early pregnancy. Life Sci 2006;78:753–60.[CrossRef][Medline]
8. Filipek A, Jastrzebska B, Nowotny M, et al. Ca2+-dependent translocation of the calcyclin-binding protein in neurons and neuroblastoma NB-2a cells. J Biol Chem 2002;277:21103–9.[Abstract/Free Full Text]
9. Matsuzawa SI, Reed JC. Siah-1, SIP, and Ebi collaborate in a novel pathway for β-catenin degradation linked to p53 responses. Mol Cell 2001;7:915–26.[CrossRef][Medline]
10. Filipek A. S100A6 and CacyBP/SIP—two proteins discovered in Ehrlich ascites tumor cells that are potentially involved in the degradation of β-catenin. Chemotherapy 2006;52:32–4.[CrossRef][Medline]
11. Fuchs SY, Ougolkov AV, Spiegelman VS, Minamoto T. Oncogenic β-catenin signaling networks in colorectal cancer. Cell Cycle 2005;4:1522–39.[Medline]
12. Peifer M, Polakis P. Wnt signaling in oncogenesis and embryogenesis—a look outside the nucleus. Science 2000;287:1606–9.[Abstract/Free Full Text]
13. Hu G., Zhang S, Vidal M. Mammalian homologs of seven in absentia regulate DCC via the ubiquitin-proteasome pathway. Genes Dev 1997;11:2701–14.[Abstract/Free Full Text]
14. Zhou BB, Elledge SJ. The DNA damage response: putting checkpoints in perspective. Nature 2000;408:433–9.[CrossRef][Medline]
15. Nakayama K, Frew IJ, Hagensen M. Siah2 regulates stability of prolyl-hydroxylases, controls HIF-1 abundance, and modulates physiological responses to hypoxia. Cell 2004;117:941–52.[CrossRef][Medline]
16. Tanikawa J, Ichikawa-Iwata E, Kanei-Ishii C, Nakai A. p53 suppresses the c-Myb-induced activation of heat shock transcription factor 3. J Biol Chem 2000;275:15578–15585.[Abstract/Free Full Text]
17. Zimmer DB, Cornwall EH, Landar A, Song W. The S100 protein family: History, function, and expression. Brain Res Bull 1995;37:417–29.[CrossRef][Medline]
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 1994;269:18992–9.[Abstract/Free Full Text]
19. Nishida T, Tanaka S, Haruma K, et al. Histologic grade and cellular proliferation at the deepest invasive portion correlate with the high malignancy of submucosal invasive gastric carcinoma. Oncology 1995;52:340–6.[Medline]
20. Yamamoto H, Soh JW, Shirin H, et al. Comparative effects of overexpression of p27Kip1 and p21Cip1/Waf1 on growth and differentiation in human colon carcinoma cells. Oncogene 1999;18:103–15.[CrossRef][Medline]
21. Jenkinson SR, Barraclough R, West C R, et al. S100A4 regulates cell motility and invasion in an in vitro model for breast cancer metastasis. Br J Cancer 2004;90:253–62.[CrossRef][Medline]
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 patients. Cancer Res 2006;66:1199–207.[Abstract/Free Full Text]
23. Ohuchida K, Mizumoto K, Ishikawa N, et al. The role of S100A6 in pancreatic cancer development and its clinical implication as a diagnostic marker and therapeutic target. Clin Cancer Res 2005;11:7785–93.[Abstract/Free Full Text]
24. Li ZH, Bresnick AR. The S100A4 metastasis factor regulates cellular motility via a direct interaction with myosin-IIA. Cancer Res 2006;66:5173–80.[Abstract/Free Full Text]
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.[Medline]
26. Matsuzawa S, Li C, Ni CZ, et al. Structural analysis of Siah1 and its interactions with Siah-interacting protein (SIP). J Biol Chem 2003;278:1837–40.[Abstract/Free Full Text]
27. Hart M, Concordet JP, Lassot I, et al. The F-box protein β-TrCP associates with phosphorylated β-catenin and regulates its activity in the cell. Curr Biol 1999;9:207–10.[CrossRef][Medline]
28. Chesire DR, Isaacs WB. β-Catenin signaling in prostate cancer: an early perspective. Endocr Relat Cancer 2003;10:537–56.[Abstract]
29. Sangkhathat S, Kusafuka T, Miao J, et al. In vitro RNA interference against β-catenin inhibits the proliferation of pediatric hepatic tumors. Int J Oncol 2006;28:715–22.[Medline]
30. Lowy AM, Clements WM, Bishop J, et al. β-Catenin/Wnt signaling regulates expression of the membrane type 3 matrix metalloproteinase in gastric cancer. Cancer Res 2006;6:4734–41.
31. Mezhybovska M, Wikstrom K, Ohd JF, et al. The inflammatory mediator leukotriene D4 induces β-catenin signaling and its association with antiapoptotic Bcl-2 in intestinal epithelial cells. J Biol Chem 2006;281:6776–84.[Abstract/Free Full Text]
32. Yanglin P, Lina Z, Zhiguo L, et al. KCNE2, a down-regulated gene Identified by in silico analysis, suppressed proliferation of gastric cancer cells. Cancer Lett 2007;246:129–38.[CrossRef][Medline]
33. Chow M, Yao A, Rubin H. Cellular epigenetics: Topochronology of progressive "spontaneous" transformation of cells under growth constraint. Proc Natl Acad Sci U S A 1994;91:599–603.[Abstract/Free Full Text]
34. Liu N, Bi F, Pan Y, et al. Reversal of the malignant phenotype of gastric cancer cells by inhibition of RhoA expression and activity. Clin Cancer Res 2004;10:6239–47.[Abstract/Free Full Text]
35. Weidner N, Carroll PR, Flax J, Blumenfeld W, Folkman J. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol 1993;143:401–9.[Abstract]
36. Korinek V, Barker N, Morin PJ, et al. Constitutive transcriptional activation by a b-catenin-Tcf complex in APC–/– colon carcinoma. Science 1997;275:1784–7.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. C.J. Steiniger, J. A. Coppinger, J. A. Kruger, J. Yates III, and K. D. Janda Quantitative Mass Spectrometry Identifies Drug Targets in Cancer Stem Cell-Containing Side Population Stem Cells, December 1, 2008; 26(12): 3037 - 3046. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ning, X. Articles by Fan, D. Search for Related Content PubMed PubMed Citation Articles by Ning, X. Articles by Fan, D.