SOX7, down-regulated in colorectal cancer, induces apoptosis and inhibits proliferation of colorectal cancer cells

Cancer Letters 277 (2009) 29–37

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

SOX7, down-regulated in colorectal cancer, induces apoptosis and inhibits proliferation of colorectal cancer cells Yu Zhang a, Shuyan Huang a, Wei Dong b, Lin Li a, Yunpeng Feng a, Lina Pan a, Zhenkun Han a, Xiuli Wang a, Guoling Ren a, Dongmei Su a, Baiqu Huang c, Jun Lu a,* a

The Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China The Abdominal Surgery, The Tumor Hospital of Harbin Medical University, Harbin 150040, China c The Key Laboratory of Molecular Epigenetics of MOE, Northeast Normal University, Changchun 130024, China b

a r t i c l e

i n f o

Article history: Received 5 October 2008 Received in revised form 29 October 2008 Accepted 11 November 2008

Keywords: SOX7 Colorectal cancer DNA methylation Apoptosis Wnt pathway

a b s t r a c t The sex-determining region Y-box 7 (Sox7) is a member of high mobility group (HMG) transcription factor family, essential for embryonic development and endoderm differentiation. Deregulation of Wnt signaling pathway is a hallmark of colorectal cancer. Our results showed that the expression level of SOX7 was frequently down-regulated in human colorectal cancer cell lines and in primary colorectal tumor tissues, and the SOX7 silencing was partially due to the aberrant DNA methylation of the gene. Restoration of SOX7 induced colorectal cancer cell apoptosis, inhibited cell proliferation and colony formation. In addition, SOX7 efficiently suppressed b-catenin-mediated transcriptional activity. Ó 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Colorectal cancer is the second leading cancer in the United States [1,2]. In China, the incidence of colorectal cancer has been experiencing a significant rise in recent years. In addition to environmental and dietary factors, changes in gene expression have been shown to associate with the occurrence of colorectal cancer. Recently, the correlation between anomalous expression of transcription factors, such as SOXs and HOXs, and carcinogenesis, has attracted extensive research attention in various cancers, including colorectal cancer [3,4]. Sox genes encode transcription factors with strong homology to the high-mobility group (HMG box), which are homologous to SRY in the HMG box [5]. There are at least 30 Sox members expressed in many different cell types and tissues, and at multiple stages during development [6]. * Corresponding author. Tel.: +86 431 85098729; fax: +86 431 85099768. E-mail address: [email protected] (J. Lu). 0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.11.014

Sox7, together with Sox17 and Sox18, belongs to Sox group F subfamily. Sox7 encodes an HMG box transcription factor and has been implicated in parietal endoderm differentiation [6]. Mouse Sox7 polypeptide can bind to DNA sequence AACAAT [7], which is related to the transcription of a number of target genes, including Gata-4 and Gata-6 [8]. Sox proteins generally require interacting with protein partners to function [9]. It has been reported that Xenopus Sox7 regulates Nkx2.5 gene transcription via the physical interaction of its C-terminal domain with b-catenin [10]. Moreover, SOX7 mRNA is undetected in some human cancer cell lines including HeLa S3 (cervical cancer), K562 (chronic myelogenous leukemia), SW480 (colorectal cancer), etc. [11], implicating that SOX7 might be a tumor suppressor gene in these cancers. However, the tumor suppressing function of SOX7 has not been fully investigated. The canonical Wnt signaling pathway operates by stabilizing b-catenin, enabling it to accumulate in the nuclear where it directs transcription of a range of genes in association with TCF/LEF factors [12]. Deregulation of Wnt sig-

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naling pathway is a hallmark of many aggressive human cancers including colorectal cancer. APC and b-catenin, which are important Wnt pathway components, are always mutated in colorectal cancer [13]. Epigenetic silencing of Wnt antagonists in colorectal cancer progression has been identified, e.g., the secreted frizzled-related proteins (sFRP1) [14] and APC [15]. For Sox factors, it has been reported that SOX2, SOX10, SOX17, and SOX18 are methylated in different cancers [16–19]. Other SOX factors such as SOX9 has been reported as tumor-suppressive proteins in colorectal carcinomas [20]. Albeit these available data, the mechanisms of the regulation of human SOX7 gene are unclear, and whether SOX7 has the tumor suppressive function in colorectal cancer cells needs to be further validated. Here, we report that the expression of SOX7 was frequently down-regulated in human colorectal cancer cell lines and in primary colorectal tumor tissues. We also showed that the down-regulation of SOX7 gene could be partially attributed to the aberrant DNA methylation of the gene. Moreover, restoration of SOX7 induced colorectal cancer cell apoptosis, inhibited cell proliferation and colony formation. In addition, SOX7 antagonized Wnt signaling pathway in colorectal cancer cell line SW480 by suppressing b-cateninmediated transcriptional activity. Our study disclosed the role of SOX7 in suppressing colorectal cancer, providing a basis for further investigation of SOX7 as a tumor suppressor gene in colorectal cancer.

2. Materials and methods 2.1. Cell lines and tissue samples Nine human colorectal cancer cell lines (SW480, SW620, HCT116, HT29, DLD1, Lovo, Colo320, CW-2 and Ls174T) were cultured in appropriate medium with 10% FBS (fetal bovine serum), 100U/ml penicillin and 100lg/ ml streptomycin, and kept in a humidified atmosphere of 5% CO2. A total of 45 primary colorectal tumor tissue samples and their corresponding noncancerous colorectal tissues were obtained from The Abdominal Surgery, The Tumor Hospital of Harbin Medical University, China. Genomic DNAs were extracted using the standard Proteinase-K method. Total RNA was extracted by using the Trizol reagent (TAKARA). For demethylation studies, cells were treated daily with 50 -aza-20 -deoxycytidine (Sigma) for 72h. 2.2. RT-PCR For single-stranded cDNA synthesis, 1lg (for cell lines) or 2lg (for tissue samples) of total RNA was reverse transcribed using the RT-Systems supplied by Promega. Quantitative real-time RT-PCR was carried out on an ABI Prism 7000 Sequence Detection System (Applied Biosystems), and SYBR Green (TOYOBO) was used as a double-stranded DNA-specific fluorescent dye. The PCR primer sequences were as follows.  SOX7: 50 -ACCAACGGGTCCCACAGA-30 (sense) and 50 CCACTCAAGGCACAAGAAGG-30 (antisense);

 cyclinD1: 50 -CGCCCCACCCCTCCAG-30 (sense) and 50 CCGCCCAGACCCTCAGACT-30 (antisense) [21];  survivin: 50 -TCAAGGACCACCGCATCTCTA-30 (sense) and 50 -TGAAGCAGAAGAAACACTGGGC-30 (antisense) [22];  B2M: 50 -GTGCTCGCGCTACTCTCTC-30 (sense) and 50 GTCAACTTCAATGTCGGAT-30 (antisense) [23];  b-actin: 50 -TCGTGCGTGACATTAAGGAG-30 (sense) and 50 -ATGCCAGGGTACATGGTGGT-30 (antisense) [24].

2.3. Plasmid constructs and transfection SOX7 gene (GenBank accession NM_031439) was cloned by reverse transcription-PCR (RT-PCR) from cDNA derived from normal human colorectal tissue using the following primers: 50 -CCCAAGCTTAGGCGAAGCGAGGCGACC30 (sense) and 50 -CGCGGATCCCGGCTCCTCTGCCACTCAAG30 (antisense). To generate SOX7 expression constructs, the entire encoding region was subcloned in frame into the pEGFP-N1 vector via HindIII and BamHI sites. The constructs were verified by DNA sequencing. pCS+b-cateninFLAG expression vector was a gift from Dr. L. Kosel (Charité-Universitätsmedizin, Berlin). TOPFLASH and FOPFLASH reporter vectors were the gifts from Dr. Bert Vogelstein (The Howard Hughes Medical Institute & Sidney Kimmel Comprehensive Cancer Center, United States). Short interfering RNA (siRNA) targeting SOX7 sequence (ACGCCGAGCTGTCGGATGG) was synthesized. Oligonucleotide that represents the siRNA was cloned into the pSliencer4.1-CMV neo vector (Ambion) between BamHI and HindIII sites following the manufacturer’s instructions. Plasmids were transfected using LipofectamineTM2000 (Invitrogen) follow the manufacture’s instructions. 2.4. Methylation analysis Bisulphite treatment of genomic DNA was carried out by using the CpGenomeTM DNA Modification Kit (CHEMICON) according to the manufacture’s instructions. Methylationspecific PCR (MSP) primers specific for the unmethylated (MSP-U) and methylated (MSP-M) promoter sequences were as follows, MSP-U: 50 -GGTTTTGGATGTTGAGTTG TTG-30 (sense) and 50 -CTTAACCCAAACCATAAAAACATT-30 (antisense); MSP-M: 50 -GTTTTGGACGTCGAGTTGTC-30 (sense) and 50 -AACCCAAACCATAAAAACGTT-30 (antisense). For bisulfite sequencing, primer sequences were synthesized as follows. BS-A: 50 -TTAATTAGGTGGTTGAGAATTAGAA-30 (sense) and 50 -TAACCATAAACCCCTCAAAACA-30 (antisense); BS-B: 50 -TTTTGGAGAGTTATTGGAGGA-30 0 (sense) and 5 -CCTTAACCCAAACCATAAAAA-30 (antisense). PCR products were gel purified and cloned into the pMD18-T vector (TaKaRa) following manufacture’s instructions. Integrated PCR fragments were verified by EcoRI/HindIII digestion, and sequenced with the M13 forward primer. 2.5. Western blot assay Western blotting was performed as described previously [24]. The primary antibodies used were mouse anti-SOX7 (1:1000, R&D system), rabbit anti-survivin (1:1000, R&D system), rabbit anti-cyclinD1 (1:500, Santa

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Cruz Biotechnology), mouse anti-b-actin (1:10000, Sigma), and mouse anti-GFP (1:1000, Abcam). 2.6. Hoechest 33342 staining Apoptosis of cells was assessed by observation of morphological changes of nuclei using Hoechest 33342 (Sigma) staining. In brief, cells were grown in 6-well plates and stained with Hoechest 33342 and examined by fluorescence microscopy. The numbers of apoptotic nuclei in 5 randomly selected fields were counted, and apoptotic characteristics of the cells were examined. 2.7. Caspase3/7 activity analysis SW480 cells were plated at 2103 cells/well on 96-well plates. At 24, 48 and 72h after transfection, and the ApoONE Caspase-3/7 Reagent (Promega) was added to culture medium following manufacture’s instructions. Cells were incubated for 1h at room temperature prior to record the fluorescence (485Ex/527Em). 2.8. MTT assay Cell proliferation was assessed by the MTT [3-(4,5dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. SW480 cells were plated at 1  103 cells/well on 96-well plates. At 24, 48, 72 and 96 h after transfection, 20 ll of MTT (5 mg/ml) was added to each well; the samples were incubated for 4 h at 37 °C and then subcultured to the medium with 100 ll dimethyl sulfoxide (DMSO). The absorbance of each well was determined at 492 nm. Survival percentage (%) was calculated relative to the control. 2.9. Colony formation assay SW480 cells were plated in 10-cm tissue culture plates 24 h before transfection. pEGFP-N1 control vector or SOX7GFP expression vector was transfected. 24 h later, the transfected cells were diluted, re-plated, and selected in 10-cm plates containing 500 mg/L G418 for 12d. Colonies were stained with crystal violet (Sigma-Aldrich). 2.10. Luciferase reporter assay Reporter gene assays were done as previously described [24]. Briefly, 5  104 cells were seeded in 24-well tissue culture plates 24 h before transfection. The TOPFLASH or FOPFLASH reporter vector was transfected at 100 ng/well and the Renilla luciferase control plasmid pREP7-RLuc was cotransfected at 50 ng/well as an internal control reporter. For reporter assays in HEK 293T cells, b-catenin was used to activate the reporter gene. Increasing amounts of SOX7-GFP expression vector were transfected into cells. Thirty hours post transfection, cells were washed and lysed in passive lysis buffer (Promega) and the transfection efficiency was normalized to the paired Renilla luciferase activity by using the Dual Luciferase Reporter Assay System (Promega) according to the manufacture’s instructions.

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2.11. Statistical analysis Student test was used to calculate the statistical significance of the experimental results. The significance level was set as *P < 0.05 and **P<0.01. Error bars denote the standard deviations (SDs). 3. Results 3.1. SOX7 was down-regulated in colorectal cancer cell lines and in primary colorectal tumor tissues We first assessed the expression of SOX7 gene in 9 human colorectal cancer cell lines by using quantitative RT-PCR and western bloting. Compared with the normal colorectal tissue, all the 9 colorectal cell lines exhibited low levels of SOX7 mRNA and protein (Fig. 1A). We next examined SOX7 expression in primary colorectal tumor samples. Total RNA of colorectal tumor and corresponding adjacent non-cancerous tissues were obtained from 20 different patients. The b-2-microgobulin (B2M), which exhibits minor changes during colorectal cancer progression [23] was used as an internal reference to normalize gene expression data. The results demonstrated that the SOX7 mRNA expression was frequently reduced in colorectal tumor tissues compared with their adjacent noncancerous tissues (16 out of 20 cases) (Fig. 1B). These data implicated that down-regulation of SOX7 gene may be a characteristic marker for colorectal cancer. 3.2. Epigenetic silencing of SOX7 gene in colorectal cancer cell lines and colorectal tumor tissues Next, we intended to explore the mechanisms underlying the downregulation of SOX7 expression in colorectal cancer. The fact that there is a dense CpG island near the transcription start site of SOX7 gene (Fig. 2A) makes it likely that the SOX7 gene may be regulated through DNA methylation. To clarify the possible roles of this epigenetic mechanism in SOX7 silencing in colorectal cell lines, we treated two colorectal cancer cell lines (SW480 and HCT116) with 5-aza-dC, a methyltransferase inhibitor. We found that treatment of cells with 5-aza-dC up-regulated both SOX7 mRNA (Fig. 2B) and protein (Fig. 2C) levels in SW480 cells. Similar results were observed in HCT116 cell line (data not shown). To further analyze the methylation status of the CpG sites at SOX7 promoter, we designed primers for MSP assays (Fig. 2A). Hypermethylation signals of SOX7 promoter were detected in the 9 colorectal cancer cell lines by MSP assays (Fig. 2D). Bisulphate sequencing results of SW480 and HCT116 were consistent with MSP analysis, indicating that these colorectal cancer cell lines exhibited the hypermethylation signals with the BS-B primer set (Fig. 2D, lower panel). To investigate the correlation between SOX7 expression and methylation status in primary colorectal tumor tissues, we examined SOX7 methylation status in colorectal tissues from patient No.31 (Case 31). The results revealed that hypermethylation signals of SOX7 were detected in Case 31T (tumor tissue) but not in Case 31N (adjacent noncancerous tissue) (Fig. 2E). These data confirmed the correlation between the SOX7 hypermethylation and its silencing in primary colorectal cancer. 3.3. Restoration of SOX7 expression induced apoptosis and inhibited cell proliferation in colorectal cancer cell line SW480 To address the functional significance of SOX7 down-regulation in colorectal cancer, we tested the effect of SOX7 on cell apoptosis, proliferation and colony formation in SW480. As evidenced in Fig. 3A, overexpression of SOX7 increased the percentage of apoptosis in SW480 cells as revealed by Hoechest staining. Fluorescence microscopy revealed that the cell nuclei in the control cells transfected with pEGFP-N1 were round in shape and homogeneously stained (Fig. 3A, lower panel, left). Meanwhile, cells transfected with SOX7-GFP revealed typical morphologic features of apoptosis, such as cell nuclei shrinkage, chromatin condensation and fragmentated nuclei; some of which possessed apoptotic bodies (Fig. 3A, lower panel, right). Next, we studied the influence of SOX7 on apoptotic effector caspases. We showed that the Caspase 3/7 activities were up-regulated upon SOX7 overexpression (Fig. 3B). To test the ability of SOX7 in inhibiting cell proliferation, MTT assays were

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Fig. 1. Down-regulation of SOX7 in colorectal cancer cells and tissues. (A) SOX7 mRNA and protein levels in 9 colorectal cancer cell lines were significantly lower than in the normal colorectal tissue, as judged by quantitative PCR and Western blotting. b-actin was used as the internal reference. (B) Comparison of SOX7 mRNA expression in paired clinical normal human colon and tumor samples as revealed by quantitative PCR. B2M was used as the internal reference. performed and the results revealed that ectopic expression of SOX7 inhibited SW480 cell growth (Fig. 3C). Colony formation assays also confirmed that restoration of SOX7 expression markedly decreased the number of SW480 cell colonies (Fig. 3D, left). Furthermore, colonies were examined by fluorescence microscopy. Cells transfected with control vector pEGFP-N1 formed numerous colonies that displayed GFP signal, whereas transfection with SOX7 expression vector SOX7-GFP failed to form GFP-positive colonies (Fig. 3D, right), indicating the inhibitory effect of SOX7 expression on colony formation. To further evaluate the role of SOX7 on cell proliferation, we analyzed the effects of SOX7 siRNA on HEK 293T cell proliferation. In HEK 293T cells expressing high level of SOX7, cell proliferation was promoted (Fig. 3F) by SOX7 siRNA (Fig. 3E). These date suggested that expression of SOX7 induced apoptosis of colorectal cancer cell line SW480, and inhibited its growth and colony formation. Also, interference of SOX7 expression promoted HEK 293T cell proliferation.

tify the function of human SOX7 in inhibiting b-catenin-mediated Wnt activation, HEK 293T cells were transfected with TOPFLASH or FOPFLASH reporter construct, concomitantly with b-catenin expression vector and increasing amounts of SOX7 expression vector. Expression of b-catenin significantly induced TOPFLASH/FOPFLASH activities, which could be repressed by overexpression of SOX7 in a dose dependent manner (Fig. 4A). In SW480 cell line, which has ectopic active Wnt signaling pathway, SOX7 was able to repress TOPFLASH/FOPFLASH activities (Fig. 4B). Moreover, mRNA (Fig. 4C) and protein (Fig. 4D) expressions levels of Wnt-b-catenin-targeted survivin and cyclinD1 were also reduced upon SOX7 overexpression in SW480 cells. These data demonstrated that SOX7 inhibited canonical Wnt signaling pathway in colorectal cancer cell line SW480 cells.

3.4. SOX7 inhibited b-catenin/TCF-driven transcription

In the present study, we observed the down-regulation of SOX7 in all the human colorectal cancer cell lines tested, as well in primary colorectal tumor tissues (Fig. 1). We also discovered that silencing of SOX7 in colorectal cancer was partially due to the aberrant DNA

Previous studies have shown that several members of the Sox family can antagonize the canonical Wnt signaling pathway [3,25–27]. Moreover, mouse Sox7 was identified as an antagonist of b-catenin activation of the b-catenin/TCF regulated OT reporter in HEK 293 cells [28]. To jus-

4. Discussion

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Fig. 2. Methylation status of SOX7 gene in colorectal cancer cell lines and in primary colorectal tumor tissues. (A) Schematic illustration of CpG site structure of SOX7 promoter. The arrow denotes the transcription start site (+1). Vertical bars indicate the CpG sites. Arrowed bars below define the regions that were subjected to MSP or bisulphate sequencing (BS-A and BS-B). (B) 5-aza-dC up-regulated SOX7 mRNA expression in SW480 cells, as measured by RT-PCR and quantitative PCR. (C) 5-aza-dC up-regulated SOX7 protein expression in SW480 cells. Western blots of SOX7 protein level after treatment with 5-aza-dC, as indicated. (D) MSP analysis and sodium bisulphate DNA sequencing of SOX7 gene in colorectal cancer cell lines. MSP primer region is depicted in Panel A. Bands in lanes marked ‘‘M” and ‘‘U” are PCR products obtained with methylated-specific and unmethylated-specific primers, respectively. Circles in each horizontal row represent the analysis of a single clone of bisulphate-treated DNA, of 20 or 35 CpG sites (for BS-A or BS-B, respectively) contained in the regions. Open and solid circles represent unmethylated and methylated CpG sites, respectively. (E) MSP analysis and sodium bisulphate DNA sequencing of SOX7 in noncancerous colorectal tissue (Case 31N) and colorectal tumor tissue (Case 31T). Representative results of MSP of SOX7 in noncancerous colorectal (lane N) and colorectal tumor (lane T) tissue samples are shown. MSP primers used are shown in Fig. 2A. The bisulphate DNA sequencing analyses were carried out by using BS-B primers shown in Fig. 2A.

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Fig. 3. Restoration of SOX7 expression induced apoptosis and inhibited proliferation of SW480 cells. (A) Effect of SOX7 expression on apoptosis. SW480 cells were transfected with pEGFP-N1 or SOX7-GFP vector. Cells were stained with Hoechest 33342, examined by fluorescence microscopy and apoptotic cells were counted. (B) Effect of SOX7 expression on Caspase 3/7 activities. Caspase3/7 activities were detected at different time points after transfection with pEGFP-N1 or SOX7-GFP. (C) Effect of SOX7 expression on cell proliferation. MTT assay was used to estimate the proliferation at different time points after transfection. (D) Effect of SOX7 expression on colony formation. Cells were transfected and cultured with 0.5 mg/L G418 for 12d. Colonies were stained with crystal violet. (E) Western blot verification of the interfering efficiency of SOX7 siRNA in HEK 293T cells. (F) Effect of SOX7 knockdown on HEK 293T cell proliferation as measured by MTT assays.

methylation of the gene (Fig. 2). Furthermore, restoration of SOX7 expression induced apoptosis of colorectal cancer cells, and inhibited cell proliferation and colony formation (Fig. 3). We also provided evidence that SOX7 efficiently suppressed b-catenin-mediated transcriptional activity (Fig. 4).

A previous study has suggested that SOX7 mRNA was not expressed in normal colon [11]. Our experiments with 20 colorectal tumor tissues and their corresponding adjacent noncancerous tissues demonstrated that, although the expression level of SOX7 was very low in normal colorectal tissues in a few patients (e.g. Case 7, Fig. 1B), the

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Fig. 4. SOX7 inhibited the canonical Wnt-b-catenin signaling pathway in SW480 cells. (A) SOX7 inhibited b-catenin-activated transcription. HEK 293T cells were transfected with various expression vectors as indicated, and relative luciferase activity was determined after culturing for 30 h. (B) SOX7 inhibited TOPFLASH/FOPFLASH activities in SW480 cells. (C) SOX7 reduced cyclinD1 and survivin mRNA levels, as detected by using RT-PCR and quantitative PCR. bactin was used as the internal reference. (D) SOX7 reduced cyclinD1 and survivin protein levels as detected by Western blotting.

majority of patients showed higher levels of SOX7 in normal colorectal tissues compared with corresponding carcinomas tissues (Fig. 1B). The discrepancy between our data and other’s may probably be due to the individual differences among the colorectal cancer patients. It has been well established that loss of function of specific genes through gene silencing contributes to colorectal cancer initiation and progression. Wnt signaling inhibitors, such as sFRPs, DKK1, APC and SOX17, have been reported to be frequently hypermethylated in primary colorectal cancers. For instance, sFRPs and DKK1 as secreted Wnt antagonists acting at cell membrane to prevent ligandreceptor interactions, have been previously described to be hypermethylated in primary colorectal tumors [14,29].

It has been reported that the promoter hypermethylation of APC, a key gene in colorectal cancer development functioning to degrade b-catenin or export b-catenin from the nuclear to cytoplasm, was an important mechanism for its inactivation besides mutations [15]. Also, nuclear Wnt antagonists, such as SOX17, were hypermethylated in colorectal cancer [19]. In a latest study, Guo et al. demonstrated that SOX7 gene was down regulated in 47% of the prostate adenocarcinomas, and tumor-specific inactivation of Sox7 by promoter hypermethylation was found in 48% of the primary prostate tumors tested [30]. These authors also suggested that Sox7 served as an independent checkpoint for b-cantenin function in prostate and colon epithelial cells[30]. Coincided with this recent study, our data

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revealed that SOX7 as a Wnt signaling antagonist was methylated at its promoter in colorectal cancer cell lines and in colorectal tumor tissues (Fig. 2), implicating that SOX7 down-regulation may occur at the transcriptional level in colorectal cancer. Our results have raised the potential possibility of the application of SOX7 hypermethylation as markers for colorectal cancer detection. Besides SOX7, SOX18 was reported to be methylated in lung carcinoma [17], SOX2 in gastric cancer [18] and SOX17 in colorectal cancer [19]. Our finding from this study that SOX7 promoter was frequently hypermethylated in colorectal cancers, is in accordance with the previous study showing that SOX7 mRNA was undetected in many human cancer cell lines including colorectal cancer cell line SW480 [11]. These data implicated that SOX7 may function as a tumor suppressor gene in these cancer cell lines. It has been reported that SOX family members, such as SOX4, SOX9 and SOX2, are able to induce apoptosis in bladder, prostate, colorectal and gastric carcinomas [18,20,31,32]. Similarly, we demonstrated that SOX7 induced cell apoptosis, repressed cell proliferation and reduced colony formation in colorectal cancer cells SW480 (Fig. 3). Since SOX7 could up-regulate Caspase 3/7 activities at as early as 24h, we speculated that SOX7 may act as a tumor suppressor in colorectal cancer cell line SW480 mainly through its ability to induce apoptosis. Collectively, our data suggested that SOX7 gene may play a crucial role in colorectal cancer development as a tumor suppressor. In many colorectal cancer cell lines, b-catenin is accumulated in nuclear, where it interacts with members of the TCF/LEF HMG box family to activate target genes including c-myc, cyclinD1 and survivin [33,34]. Aberrant Wnt pathway is associated with cell proliferation, differentiation, development, metastasis and apoptosis [35]. Sox factors may function either as Wnt/b-catenin antagonists or as activators. Sox2, Sox3, Sox9, Sox10 and Sox17 are strong antagonists of Wnt/b-catenin signaling pathway, whereas Sox4, Sox5, and Sox11 enhance b-catenin activity [3]. Mouse Sox7 was reported to repress Wnt/b-cateninstimulated transcription in HEK 293 cell line [28]. Here we showed that SOX7 repressed Wnt signaling pathway in human SW480 cells. The canonical Wnt target genes cyclinD1 and survivin are tightly associated with cell cycle progression, proliferation and apoptosis [36,37]. Our study demonstrated that SOX7 could repress cyclinD1 and survivin expression at both mRNA and protein levels (Fig. 4C and D). All these data have led us to believe that SOX7 may affect cell apoptosis and proliferation through antagonizing Wnt signals in colorectal cancer cells. In conclusion, we established in this study that SOX7 gene was frequently silenced in colorectal cancer, which was partially due to the DNA hypermethylation of the gene. SOX7 could induce colorectal cancer cell apoptosis and inhibit cell proliferation, and this may be achieved through antagonizing the Wnt signaling pathway by suppressing b-catenin-mediated transcriptional activity. Data presented in this report represent the attempt to disclose the role of SOX7 in suppressing colorectal cancer, which may be useful in the development of new therapeutic strategies for colorectal cancer.

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