Oncogene (2010) 29, 6464–6474 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Epigenetic inactivation of T-box 5, a novel tumor suppressor , is associated with colon cancer

JYu1,2,XMa1,2,3,7, KF Cheung1,2,XLi1,2, L Tian4, S Wang1,2,CWWu1,2, WKK Wu1,2,MHe5, M Wang3, SSM Ng6 and JJY Sung1,2

1Department of Medicine and Therapeutics, Institute of Digestive Disease, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, SAR China; 2Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, SAR China; 3Department of Neurology, First Hospital, Hebei Medical University, Shijiazhuang, SAR China; 4School of Public Health and Primary Care, The Chinese University of Hong Kong, Hong Kong, SAR China; 5Stanley Ho Center for Emerging Infectious Diseases, School of Public Health and Primary Care, The Chinese University of Hong Kong, Hong Kong, SAR China and 6Department of Surgery, The Chinese University of Hong Kong, Hong Kong, SAR China

T-box transcription factor 5 (TBX5) is a member of a overall survival (P ¼ 0.0007). In conclusion, we identified phylogenetically conserved family of involved in the a novel functional tumor suppressor gene TBX5 inacti- regulation of developmental processes. The function of vated by promoter methylation in colon cancer. Detection TBX5 in cancer development is largely unclear. We of methylated TBX5 may serve as a potential biomarker identified that TBX5 was preferentially methylated in for the prognosis of this malignancy. cancer using methylation-sensitive arbitrarily primed Oncogene (2010) 29, 6464–6474; doi:10.1038/onc.2010.370; PCR. We aim to clarify the epigenetic inactivation, published online 30 August 2010 biological function and clinical significance of TBX5 in colon cancer. Promoter methylation was evaluated by Keywords: T-box transcription factor 5; colon cancer; combined bisulfite restriction analysis and bisulfite tumor suppressor gene; epigenetic alteration; prognosis genomic sequencing. Cell proliferation was examined by cell viability assay and colony formation assay, apoptosis by flow cytometry and cell migration by wound-healing Introduction assay. TBX5 target genes were identified by cDNA microarray analysis. Cox regression model and log-rank Colon cancer is the fourth most common malignancy test were used to identify independent predictors of worldwide (Parkin et al., 2005). It is rapidly increasing in prognosis. TBX5 was silenced or downregulated in 88% Hong Kong and represents the second most common (7/8) colon cancer cell lines, but was expressed in normal malignancy in this area. However, the mechanism leading colon tissues. Loss of was associated with to colon cancer development remains elusive. Hyper- promoter methylation. The biological function of TBX5 in methylation of CpG islands in the promoter region of human colon cancer cells was examined. Re-expression of genes associated with gene silencing becomes crucial to the TBX5 in silenced colon cancer cell lines suppressed colony development of colon carcinogenesis (Grady and Car- formation (Po0.001), proliferation (Po0.001), migration ethers, 2008). These abnormalities caused by promoter and induced apoptosis (Po0.01). Induction of apoptosis methylation repress the binding affinity of transcrip- was mediated through cross-talk of extrinsic apoptosis tion factors by interfering with transcription initiation, pathway, apoptotic BCL2-associated X and influence chromatin structure, alter gene expression Granzyme A signaling cascades. TBX5 suppressed tumor and thereby modulate cancer pathways involved in cell cell proliferation and metastasis through the upregulation proliferation, apoptosis and metastasis. Increasing num- of cyclin-dependent kinase inhibitor 2A, metastasis bers of tumor suppressor genes associated with epigenetic suppressor 1 and downregulation of synuclein gamma alterations have been identified in colon cancers (Zitt and metastasis-associated protein 1 family member 2. et al., 2007; Grady and Carethers, 2008). The identifica- TBX5 methylation was detected in 68% (71/105) of tion of new genes functionally involved in tumor primary colon tumors. Multivariate analysis showed that development and progression may help to find alternative patients with TBX5 methylation had a significantly poor approaches for diagnostic and therapeutic evaluation. Through methylation-sensitive arbitrarily primed PCR, we have identified a novel preferentially methylated Correspondence: Professor J Yu, Department of Medicine and gene, T-box transcription factor 5 (TBX5) (also known Therapeutics, Institute of Digestive Disease, Prince of Wales Hospital, as T-box 5), in human cancer. The Chinese University of Hong Kong, Shatin, NT, Hong Kong, TBX5 is a member of a phylogenetically conserved SAR China. family of genes that share a common DNA-binding E-mail: [email protected] 7Co-first author. domain, the T-box. T-box genes encode transcription Received 12 May 2010; revised and accepted 8 July 2010; published factors involved in the regulation of developmental online 30 August 2010 processes, cell–cell signaling, negative regulation of T-box transcription factor 5 in colon cancer JYuet al 6465 cardiac muscle cell proliferation, induction of apoptosis cell lines that showed silencing or downregulation of and negative regulation of cell migration (He et al., TBX5 with 5-aza-20-deoxycytidine (5-aza). This treat- 2002; Mori et al., 2006; Plageman and Yutzey, 2006; ment resulted in the restoration of TBX5 expression Maitra et al., 2009). This gene is located on human in all cell lines examined (Figure 1d), inferring that 12q24.1. The encoded protein partici- promoter methylation directly contributed to the TBX5 pates in heart development and specification of limb silencing. identity. However, genetic mutations of TBX5 have been associated with Holt–Oram syndrome, a developmental Genetic deletion and mutation of TBX5 were not detected disorder affecting the heart and upper limbs (Newbury- in colon cancer cell lines Ecob et al., 1996). As TBX5 is expressed in colon tissues Further genetic deletion and mutation analyses of TBX5 and the ectopic expression of TBX5 contribute to coding exons by DNA direct sequencing did not show induction of apoptosis and inhibition of cell prolifera- any homozygous deletion or mutation in eight colon tion in osteosarcoma (U2OS) and lung cancer (H1299) cancer cell lines and 20 primary colon cancers, suggest- cells (He et al., 2002), aberrant inactivation of this ing that genetic alteration does not contribute to the developmental regulator may be the cause of tumor silencing or downregulation of TBX5 gene in colon development. To that end, the TBX5 gene expression cancer. patterns in human colon cancer and its molecular mechanism of tumor suppressor function are worth Ectopic expression of TBX5 suppressed colon cancer investigating. The potential relationships of TBX5 cell growth expression with patient clinicopathological features The frequent silencing of TBX5 mediated by promoter and survival data are also investigated. methylation in colon cancer cells but not in normal colon tissue implicated that TBX5 may have a role in tumor growth. To test this speculation, we examined the Results growth inhibitory effect through ectopic expression of TBX5 in SW620 and CaCO2, which showed no TBX5 Identification of TBX5 as a novel gene downregulated expression. Re-expression of TBX5 in the transient or silenced in colon cancer cells transfected SW620 and CaCO2 was confirmed by RT– To identify candidate cancer genes with promoter PCR (Figures 2a1 and a2), which caused a significant methylation, we performed methylation-sensitive arbi- decrease in cell viability in both SW620 (P 0.05) and trarily primed PCR. A preferentially methylated CpG o CaCO2 (Po0.05) (Figure 2a3). The suppressive effect island of TBX5 promoter was identified that epigeneti- on cancer cell proliferation was further confirmed by cally silences TBX5 expression in human cancer. We colony formation assay. A significant reduction of then examined the mRNA expression of TBX5 in eight colony numbers was observed in cells stably transfected colon cancer cell lines. Reverse transcript–PCR (RT– with pcDNA3.1-TBX5, compared with empty vector in PCR) showed that TBX5 transcript was silenced or monolayer culture (down to 54–65% of vector controls, reduced in seven (88%) cell lines except LS180 Po0.001) (Figure 2b). Thus, TBX5 exhibits growth (Figure 1b). In contrast, TBX5 expression was readily inhibitory ability in tumor cells and functions as a detected in normal colon tissues (Figure 1b), suggesting potential tumor suppressor. an aberrant gene silencing of TBX5 in colon cancers.

Promoter methylation of TBX5 was correlated TBX5 induced apoptosis with transcriptional silencing We examined the contribution of apoptosis to the To elucidate whether silencing of TBX5 is due to the observed growth inhibition of TBX5-transfected cells. epigenetic inactivation, TBX5 methylation status was Apoptosis was investigated using two-color fluores- examined by combined bisulfite restriction analysis cence-activated cell sorting analysis. Our results indi- (COBRA). Seven cell lines with decreased or silenced cated an increase in the numbers of both early apoptotic TBX5 expression displayed methylated promoter, in- cells (12.33±0.70 vs 9.97±1.02%, Po0.05) and late cluding DLD-1, HT-29, LOVO, SW480, SW620, apoptotic cells (16.2±1.48 vs 10.9±0.95%, Po0.01) in CaCO2 and CL14, whereas no methylation was detected TBX5-transfected SW620 cells than those in vector- in the normal colon tissues (Figure 1b). We further transfected SW620 cells (Figures 3a1 and a2). Induction validated the COBRA results by cloned bisulfite of apoptosis was further confirmed by the analysis of the genomic sequencing (BGS) (Figure 1c). The BGS results expression of apoptosis-related . As shown in were consistent with those of COBRA, in which dense Figure 3a3, re-expression of TBX5 enhanced the protein methylation was found in methylated cell lines (SW620, level of the active form of caspase-3, caspase-7 and HT-29), but not in unmethylated cell line (LS180) and nuclear enzyme poly-(ADP-ribose) polymerase in the normal colon tissues (Figure 1c). stably transfected SW620 cells.

Demethylation treatment with 5-aza-20-deoxycytidine TBX5 reduced the migration rates of SW620 cells restored TBX5 expression We tested TBX5 for another tumor suppressor activity, To further reveal whether methylation directly mediates namely the ability to inhibit cell migration by wound- TBX5 silencing, we randomly treated four methylated healing assay. As shown in Figure 3b, a decrease of the

Oncogene T-box transcription factor 5 in colon cancer JYuet al 6466

Figure 1 (a) A typical CpG island (CGI) spans the promoter region of TBX5 exon 1. Each vertical bar represents a single CpG site. The transcription start site is indicated by a curved arrow. A region for COBRA and BGS is shown. BstUI digestive sites are indicated. (b) TBX5 was frequently silenced or downregulated in colon cancer cell lines, but readily expressed in normal colon tissues by semiquantitative RT–PCR. Methylation of TBX5 was determined by COBRA. The undigested fragment (upper band) represents the unmethylated DNA (U). The digested fragments represent the methylated DNA (M). (c) Detailed BGS analysis confirmed the methylation status of the TBX5 in colon cancer cell lines and in normal colon tissues. Five to six colonies of cloned BGS–PCR products from each bisulfite-treated DNA samples were sequenced and each is shown as an individual row, representing a single allele of the promoter CpG island analyzed. One circle indicates one CpG site. Filled circle, methylated; open circle, unmethylated. (d) The mRNA expression of TBX5 was restored after treatment with demethylation agent 5-aza.

cell migration ability in wound closure was observed in tumorigenesis. When compared with empty vector- SW620 cells transfected with TBX5 as compared with transfected SW620 cells, the antitumorigenesis effect cells transfected with empty vector, as measured after 48 by TBX5 was mediated by regulating important genes in and 96 h of wound healing, indicating that the migration apoptosis, cell proliferation and metastasis (Supple- of SW620 cells was inhibited by TBX5. mentary Table 1). TBX5 increased the expression of pro-apoptotic extrinsic pathway-related genes, including tumor necrosis factor alpha (TNFa), tumor necrosis Identification of genes modulated by TBX5 factor superfamily member 10b (TNFRSF10B), To gain insights into the molecular basis underlying TNFRSF1A, TNFRSF25, apoptosis executor caspase-8 the tumor suppressive effect of TBX5, gene expression and apoptotic activator BCL2-associated X protein (BAX). profile in TBX5 stably transfected SW620 was analyzed TBX5 also exerted antiproliferative effect by increasing by cDNA microarray. This array contains 84 func- cyclin-dependent kinase inhibitor 2A (CDKN2A) expres- tionally well-characterized genes involved in human sion and dampening the oncogene synuclein gamma

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Figure 2 (a) TBX5 inhibited tumor cell viability. Colon cancer cell lines (SW620 and CaCO2) were transiently transfected with pcDNA3.1 (Vector), pcDNA3.1-TBX5 (TBX5) or green fluorescent protein plasmid for 48 h. (a1) The transfection efficiency was estimated by green fluorescent protein under a uorescence microscope. (a2) Ectopic expression of TBX5 in SW620 and CaCO2 cell lines was evidenced by RT–PCR. (a3) Ectopic expression of TBX5 in SW620 and CaCO2 significantly suppressed cell viability. (b) TBX5 suppressed tumor cell clonogenicity. (b1) The effect of ectopic TBX5 expression on cancer cell growth was further investigated by colony formation assay. Cells were transfected with pcDNA3.1-TBX5 or control vector, and selected with G418 for 10–14 days, and stained with Gentian Violet. (b2) Ectopic expression of TBX5 in SW620 and CaCO2 was determined by RT–PCR. (b3) Quantitative analysis of colony formation. Values are expressed as the mean±s.d. from three independent experiments. *Po0.05; **Po0.001.

(SNCG) expression. Moreover, TBX5 greatly induced the outcome was analyzed in 80 colon cancer patients expression of antimetastasis gene, metastasis suppressor 1 with known survival data (median survival time is (MTSS1), and downregulated the expression of the 54.4 months, range from 2.3 to 65.6 months). There was pro-metastatic gene, metastasis associated protein 1 family no correlation between TBX5 methylation and clinico- member 2 (MTA2), both of which contributed to the pathological features of colon cancer patients, such as antimetastatic effect of TBX5. age, gender, tumor staging, lymph node metastasis, distant metastasis and overall tumor, node, metastasis (TNM) staging (Greene et al., 2002) (Table 1). Frequent TBX5 methylation in primary colon cancers The characteristics of colon cancer patients that We next examined the TBX5 methylation status in 105 related to the survival status are shown in Table 2. As primary colon cancers, 20 adjacent non-tumor tissues and expected, tumor staging (P ¼ 0.009), lymph node me- 15 normal colon tissues. Frequent methylation was tastasis (P ¼ 0.001), distant metastasis (P ¼ 0.004) and detected in primary colon cancers (71/105, 68%), but less overall TNM staging (P ¼ 0.0004) are significant prog- in paired non-tumor tissues (6/20, 30%) (Figure 4a1) nostic factors. Moreover, TBX5 methylation was found (P ¼ 0.005). Further detailed BGS methylation analysis to be significantly associated with death (P ¼ 0.0003). In showed densely methylated promoter alleles in tumor, univariate Cox regression analysis (Table 3), TBX5 which are rare in paired non-tumor tissue (Figure 4a2). methylation was associated with a significantly increased Fifteen normal colon tissues showed no methylation, risk of cancer-related death (relative risk (RR), 6.21; indicating that TBX5 methylation was tumor specific. 95% confidence interval, 2.39–16.2; P ¼ 0.0002). After the adjustment for potential confounding factors (TNM TBX5 methylation was associated with poor survival of staging), multivariate Cox regression analysis showed colon cancer patients that TBX5 methylation was a predictor of poorer The association of TBX5 methylation status and survival of colon cancer patients (RR, 5.42; 95% clinicopathological characteristics including clinical confidence interval, 2.06–14.4; P ¼ 0.0007) (Table 4).

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Figure 3 (a) TBX5 induced apoptosis of SW620 cells. Apoptosis was measured by flow cytometry analysis of Annexin V-FITC double-labeled SW620 cells transfected with pcDNA3.1-TBX5 (TBX5) or empty vector (Vector). (a1) Flow cytometry profile represents Annexin V-FITC staining in x axis and propidium iodide in y axis. Dual staining of cells with Annexin V-FITC and propidium iodide enabled categorization of cells into four regions. Region Q1 shows the necrotic cells, Q2 shows the late apoptotic cells, Q3 shows the live cells and Q4 shows the early apoptotic cells. (a2) The experiment was repeated three times and data represent the average of the early apoptotic and late apoptotic cells. *Po0.05; **Po0.001. (a3) Protein expressions of apoptotic-related genes were determined by western blot. b-Actin was used as internal control. (b) TBX5 reduces the migration rates of SW620 cells. Representative image of cell migration of SW620 stable cell lines in scratch wound-healing assay. pcDNA3.1-TBX5 or empty vector- transfected SW620 cells grew up to 90% confluence and were wounded. Photographs were taken at 0, 48 and 96 h, respectively, after the wound was made.

As shown in the Kaplan–Meier survival curves, colon lines, whereas there was no methylation in the normal cancer patients with TBX5 methylation had significantly colon DNA. The silencing of TBX5 can be reversed by shorter survival than others (Po0.001, log-rank test) pharmacological demethylation, but no genetic deletion/ (Figure 4b). mutational inactivation of TBX5 was found in all tumor cell lines examined, inferring methylation is the pre- dominant mechanism for the downregulation of TBX5 Discussion gene. These results suggested that TBX5 could be a potential tumor suppressor and its downregulation In this study, we used methylation-sensitive arbitrarily could have some role in the development of colon primed PCR to identify genes that were differentially cancer. methylated in human cancer, in the hope that several We characterized the putative tumor suppressor of them would prove to be novel candidate tumor function of TBX5 in human colon cancer cell lines. suppressors. We showed for the first time that TBX5 is We cloned a full-length human TBX5 cDNA into widely distributed in normal colon epithelium, while pcDNA3.1 vector, and transduced it into silenced colon absent or very lowly expressed in colon cancer cells cancer cells. Restoration of TBX5 in the colorectal because of high methylation of the promoter region. cancer CaCO2 and SW620 cells significantly inhibited Bisulfite sequencing of the TBX5 promoter region colony formation and cell proliferation and induced showed it to be densely methylated in colon cancer cell apoptosis. Furthermore, ectopic expression of TBX5 in

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Figure 4 (a) TBX5 was frequently methylated in primary colon cancers. (a1) Representative gel images of TBX5 methylation in primary colon cancers and paired adjacent non-tumor tissues by COBRA. M, methylated; U, unmethylated. (a2) Detailed BGS analysis confirmed dense methylation in tumor tissues, but less in paired normal tissues. Filled circle, methylated; open circle, unmethylated. (b) Kaplan–Meier survival curves show that colon cancer patients with TBX5 methylation had poorer survival than those without TBX5 methylation. This difference is statistically significant based on log-rank test (Po0.001).

Table 1 Clinicopathological features of TBX5 methylation in colon colon cancer cells decreased migration ability. These cancer results indicate for the first time that TBX5 functions as a tumor suppressor in colon cancer. TBX5 belongs to Variable Methylated % Non-methylated % P-value (n ¼ 52) (n ¼ 28) the member of the T-box superfamily. Members of the T-box gene family have important and diverse roles Age 68.6±12.0 66.0±11.6 0.347 in development and disease (Papaioannou and Silver, (mean±s.d.) 1998; Finotto, 2008; Nemer, 2008). Mutations in T-box Gender genes are the cause of several congenital diseases and are Male 31 63.3 18 36.7 0.811 implicated in cancer (Cai et al., 2005). Our findings have Female 21 67.7 10 32.3 highlighted the importance of TBX5 as a potential tumor suppressor in colon cancer. Tumor staging We showed the molecular basis that TBX5 exerts the T1 1 33.3 2 66.7 T2 1 50.0 1 50.0 0.057 tumor suppressor property in colon cancer through T3 42 62.7 25 37.3 induction of apoptosis, control of cell proliferation and T4 8 8.0 0 0.0 inhibition of cell migration using a cDNA microarray and western blot. We observed that induction of TBX5- Lymph node Negative 22 62.9 13 37.1 0.815 mediated apoptosis occurs by the modulation of Positive 30 66.7 15 33.3 extrinsic apoptosis pathway and upregulation of pro- apoptotic BAX and Granzyme A. TBX5 induced the Metastasis expression of key genes mediating extrinsic apoptosis No 36 60.0 24 40.0 0.175 pathway, including extracellular death ligand TNFa, Yes 16 80.0 4 20.0 death receptors TNFRSF10B, TNFRSF1A, TNFRSF25 TNM stage and downstream apoptosis executors caspase-8, caspase- I 1 33.3 2 66.7 7, caspase-3 and nuclear enzyme poly-(ADP-ribose) II 18 64.3 10 35.7 0.252 polymerase. The extrinsic apoptosis pathway is initiated III 17 58.6 12 41.4 IV 16 80.0 4 20.0 by the binding of extracellular death ligands such as TNFa to transmembrane death receptors. Engagement Abbreviations: s.d., standard deviation; TBX5, T-box transcription of death receptors with their cognate ligands pro- factor 5; TNM, tumor, node, metastasis. vokes the recruitment of adaptor proteins such as

Oncogene T-box transcription factor 5 in colon cancer JYuet al 6470 Table 2 Distribution of patient characteristics by survival status Table 3 Univariate Cox regression analysis of potential poor prognostic factors for colon cancer patients Variable Alive (n ¼ 43) % Dead (n ¼ 37) % P-value Variable RR (95% CI) P-value

Age (mean±s.d.) 66.2±13.0 69.4±10.3 0.236 Age 1.02 (0.99–1.05) 0.211

Gender Gender M 24 49.0 25 51.0 0.359 Male 1.37 (0.69–2.74) 0.367 F 19 61.3 12 38.7 Female 1.00

Tumor staging Tumor staging T1 3 100 0 0 T1–T3 0.23 (0.10–0.55) 0.0009 T2 2 100 0 0 0.009 T4 1.00 T3 37 55.2 30 44.8 T4 1 12.5 7 87.5 Lymph node Negative 0.32 (0.15–0.69) 0.003 Lymph node Positive 1.00 Negative 26 74.3 9 25.7 0.001 Positive 17 37.8 28 62.2 Metastasis No 0.41 (0.21–0.79) 0.008 Metastasis Yes 1.00 No 38 63.3 22 26.7 0.004 Yes 5 25.0 15 75.0 TNM stage I–III 0.41 (0.21–0.79) 0.008 TNM stage IV 1.00 I 3 100 0 0 II 22 78.6 6 21.4 0.0004 TBX5 methylated III 13 44.8 16 55.2 Yes 6.21 (2.39–16.2) 0.0002 IV 5 25.0 15 75.0 No 1.00

TBX5 methylation Abbreviations: CI, confidence interval; RR, relative risk; TBX5, T-box Yes 20 38.5 32 61.5 0.0003 transcription factor 5; TNM, tumor, node, metastasis. No 23 82.1 5 17.9

Abbreviations: s.d., standard deviation; TBX5, T-box transcription factor 5; TNM, tumor, node, metastasis. Table 4 Multivariate Cox regression analysis of potential poor prognostic factors for colon cancer patients Variable RR (95% CI) P-value Fas-associated death domain protein (Schu¨tze et al., 2008), which in turn recruits and aggregates several Age 1.02 (0.99–1.05) 0.181 molecules of caspase-8, thereby promoting its autopro- Gender cessing and activation. Activation of caspase-8 processes Male 1.27 (0.59–2.72) 0.538 other effector caspase members, including caspase-3 Female 1.00 and caspase-7 to initiate a caspase cascade. These effectors further initiate the proteolytic cleavage of the TNM stage I–III 0.48 (0.23–0.99) 0.048 nuclear enzyme poly-(ADP-ribose) polymerase, which IV 1.00 caused loss of DNA repair, cellular disassembly and finally undergo apoptosis (Figure 5). Apoptosis induced TBX5 methylated by extrinsic pathways has been considered to be an Yes 5.45 (2.06–14.4) 0.0007 important antitumor mechanism (Johnstone et al., 2008; No 1.00 Balkwill, 2009; Holoch and Griffith, 2009). Another Abbreviations: CI, confidence interval; RR, relative risk; TBX5, T-box important pro-apoptotic activator induced by TBX5 is transcription factor 5; TNM, tumor, node, metastasis. BAX. It was reported that BAX promotes mitochon- drial membrane permeability and cytochrome c release, thereby triggering the formation of the apoptosome kinases to cyclin D, thus preventing the inactivation of and cell death (Taylor et al., 2008). Moreover, ectopic retinoblastoma family members and leading to the expression of TBX5 leads to the upregulation of inhibition of cell proliferation (Kim and Sharpless, Granzyme A (a tryptase) that can induce caspase-inde- 2006). Moreover, the suppression of SNCG by TBX5 pendent cell death (Chowdhury and Lieberman, 2008; contributed to reduced cell proliferation and metastasis. Cullen et al., 2010). Collectively, these results suggested Aberrant overexpression of SNCG had been observed in that TBX5-mediated growth inhibition occurs by modu- multiple human cancers, especially in metastatic tumors. lation of apoptosis by various apoptosis-regulating Overexpression of SNCG, a potential oncogene, stimu- pathways (Figure 5). lates cancer cell proliferation and metastasis by mod- The antiproliferative effect caused by TBX5 in colon ulating mitogen-activated protein kinase pathways cancer cells is at least associated with upregulation of (Pan et al., 2002; Gupta et al., 2003a, b; Yanagawa CDKN2A, a critical cell cycle inhibitor. It was indicated et al., 2004; Liu et al., 2007). SNCG is considered to be that CDKN2A abrogates the binding of CDK4/6 an important prognostic marker for cancer progression

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Figure 5 Schematic diagram for the molecular basis of TBX5 as a tumor suppressor gene in colon cancer. Ectopic expression of TBX5 suppressed colon cancer cell growth was associated with several biological effects: (1) TBX5 induced tumor cell apoptosis through cross-talk of extrinsic apoptosis pathway, apoptotic BAX and Granzyme A signaling cascades. TBX5 induced the expression of key genes mediating extrinsic apoptosis pathway, including extracellular death ligand TNFa, death receptors TNFRSF10B, TNFRSF1A, TNFRSF25 and downstream apoptosis executors caspase-8, caspase-7, caspase-3 and nuclear enzyme poly-(ADP-ribose) polymerase (PARP); (2) increasing the expression of CDKN2A and inhibiting oncogene SBCG, which in turn suppressed cell proliferation; and (3) upregulating antimetastatic MTSS1 and downregulating pro-metastatic MTA2. and metastasis in human cancers including colon cancer an epigenetic biomarker for colon cancer patients. (Liu et al., 2005; Hu et al., 2009). Inhibition of cancer Colorectal cancer varies greatly in clinical outcome, cell migration by TBX5 may result from upregulating depending on the growth status and aggressiveness of MTSS1 and downregulating MTA2. Similar findings individual tumors. Currently, TNM staging is the were reported by others that antimetastatic MTSS1 and most important clinical predictor of patient outcome. pro-metastatic MTA2 were correlated with the meta- However, many patients succumb to disease recurrence. static potential of certain types of cancers (Hicks et al., Thus, additional prognostic biomarkers are required to 2006; Parr and Jiang, 2009; Toh and Nicolson, 2009). provide better risk assessment. Recognizing the bio- Thus, through modulating these important genes, TBX5 logical functions of TBX5, the inactivation of this gene exerted antitumorigenesis effect by promoting apoptosis by promoter methylation would favor tumor progres- and suppressing cell proliferation and metastasis. In this sion and a worse outcome. In this regard, we examined regard, upregulation of CDKN2A, MTSS1 and down- the influence of TBX5 methylation on outcome of regulation of SNCG and MTA2 by TBX5 may offer the colon cancer patients. Our results indicated that TBX5 explanation for the growth and metastasis inhibition in methylation was associated with poorer survival inde- colon cancer cells (Figure 5). pendent of patient characteristics. Our data support an To ascertain the clinical application of TBX5 in colon adverse effect of loss of TBX5 expression regulated by tumorigenesis in vivo, we examined the promoter promoter methylation on survival of colon cancer methylation of TBX5 by COBRA in 105 primary colon patients, providing an additional evidence for the role cancers, 20 adjacent non-cancer tissues and 15 normal of TBX5 as a novel candidate tumor suppressor gene in controls. We found that the TBX5 gene promoter was the development of colon cancer. highly methylated in the majority (68%) of colon cancer In conclusion, we have identified a novel functional tissues compared with adjacent non-tumor tissues tumor suppressor gene TBX5 inactivated by promoter (30%), but not methylated in normal controls. Like methylation in colon cancer lines and found that other cancers, many candidate tumor suppressor genes promoter methylation of TBX5 is frequent and specific are reported to be regulated by epigenetic mechanisms in primary colorectal cancers. TBX5 induces tumor cell (Barton et al., 2008). This study identified that TBX5 apoptosis through cross-talk of extrinsic apoptosis methylation is a common event in colon cancer. It is pathway, apoptotic BAX and Granzyme A signaling thus worthy further exploring the possible applica- cascades. TBX5 also suppresses tumor cell proliferation tion of TBX5 tumor-specific promoter methylation as and metastasis through upregulation of CDKN2A,

Oncogene T-box transcription factor 5 in colon cancer JYuet al 6472 MTSS1 and downregulation of SNCG and MTA2, start site of TBX5 transcript variant 1. The primer sequences inferring them to be potential targets for the antitumor are as follows: forward, 50-TATTGTAGTTTGGTTGAGAG strategy in colorectal cancer. TBX5 methylation may AAAGGA-30 and reverse, 50-CTAAATCTAAACTTACCC serve as a potential epigenetic biomarker to predict CCAACTTC-30. This region contained 19 CpG dinucleotides outcome for colorectal cancer patients. and three BstUI restriction sites (Figure 1a). PCR products were digested with BstUI (New England Biolabs, Ipswich, MA, USA) at 60 1C overnight (New England Biolabs). BstUI 0 0 Materials and methods cleaved the sequence 5 -CGCG-3 , which was retained in the bisulfite-treated methylated DNA, but not the unmethylated DNA. The DNA digests were separated in 10% non- Tumor cell lines denaturing polyacrylamide gels and stained with ethidium Eight colon cancer cell lines (DLD-1, HT-29, LOVO, SW480, bromide. SW620, CaCO2, LS180 and CL14) were obtained from the American Type Culture Collection (Manassas, VA, USA). They were cultured in RPMI 1640 medium (Gibco BRL, Cloned bisulfite genomic sequencing Rockville, MD, USA) supplemented with 10–20% fetal bovine Cloned bisulfite sequencing was performed to identify the serum (Gibco BRL) and incubated in 5% CO2 at 37 1C. methylation status of 19 CG dinucleotide sites (Figure 1a). The above PCR products for COBRA from three colon cell lines, one colon tumor and its adjacent non-cancer tissue Primary tumor and normal tissue samples and two normal colon tissues were cloned into pCR2.1-TOPO A total of 105 colorectal cancers were obtained at the time vector (Invitrogen, Carlsbad, CA, USA). Ten colonies of operation. The median age of patients was 69 years (range, were chosen randomly for plasmid DNA extraction with 36–91 years). Tumor was staged according to the TNM staging Qiaprep Spin Mini Kit (Qiagen, Valencia, CA, USA) and were system. Twenty of their corresponding adjacent non-cancerous sequenced. tissues, which were at least 5 cm away from the tumor edge, were obtained from colon cancer patients at the time of surgery. In addition, 15 age- and gender-matched normal Demethylation with 5-aza colon mucosae from healthy subjects were collected as normal Cells were seeded at a density of 1 Â 106 cells per ml. After 24 h, control. The specimens were snap-frozen in liquid nitrogen and cells were treated with 2 mM of the DNA demethylating agent stored at À80 1C for molecular analyses. All subjects were 5-aza (Sigma-Aldrich, St Louis, MO, USA) for 5 days. 5-Aza given informed consent for obtaining the study materials. The was replenished every day. Controlled cells were treated with study protocol was approved by the Clinical Research Ethics an equivalent concentration of vehicle (dimethyl sulfoxide). Committee of the Chinese University of Hong Kong. Cells were then harvested for DNA and RNA extractions.

RNA extraction, semiquantitative RT–PCR and real-time PCR DNA mutation analysis analyses Genetic deletion and mutation analyses of TBX5 coding exons Total RNA was extracted from cell pellets and tissues using were performed by DNA direct sequencing. PCR products TRIzol Reagent (Molecular Research Center Inc., Cincinnati, were purified and sequenced according to the recommendation OH, USA). cDNA was synthesized from 2 mg total RNA using of the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Transcriptor Reverse Transcriptase (Roche Applied Sciences, Biosystems). Sequence homologies were analyzed using Indianapolis, IN, USA). For semiquantitative RT–PCR, a BLAST program of the National Centre for Biological 151-bp fragment of the TBX5 gene was amplified using Information (NCBI). AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA) as reported previously (Yu et al., Construction of TBX5 expression vector 2009a), with b-actin as internal control. The primer sequences Mammalian expression vector pcDNA3.1-TBX5 encoding the of TBX5 are as follows: forward, 50-CTCAAGCTCACCAAC full-length open-reading frame of human TBX5 gene was AACCA-30 and reverse, 50-CAGGAAAGACGTGAGTGC constructed. Briefly, RNA from human tissue (Ambion, AG-30. Real-time PCR was performed using SYBR Green Austin, TX, USA) was transcribed into cDNA. Sequence master mixture on HT7900 system according to the manu- corresponding to the open-reading frame clone of TBX5 was facturer’s instructions (Applied Biosystems). amplified and verified by DNA sequencing. PCR amplified inserts were subcloned into the pcDNA3.1 TOPO TA DNA extraction and sodium bisulfite modification expression vector (Invitrogen). Plasmids used for transfection Genomic DNA was extracted from colon cancer cell lines and were isolated using EndoFree Plasmid Maxi Kit (Qiagen). colon tissues using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and treated with sodium bisulte using a Western blot analysis Zymo DNA Modication Kit (Zymo Research, Hornby, Total protein was extracted and protein concentration was Canada). Sodium bisulfite leads to deamination of unmethy- measured by the DC protein assay method of Bradford (Bio- lated cytosines to uracils without modifying methylated sites. Rad, Hercules, CA, USA). Thirty micrograms of protein from This allows their differentiation by restriction digestion or each sample were used for western blotting. Bands were sequencing. quantified by scanning densitometry.

Combined bisulfite restriction analysis Cell viability assay The methylation level of the promoter region of TBX5 gene Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-5- was determined by COBRA, a semiquantitative methylation (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Hot start PCR amplification with 1.5 ml of bisulfite- assay (Promega, Madison, WI, USA). Briefly, cells (4.5 Â 103 treated DNA gives a PCR product of 298 bp, spanning per well) were seeded in 96-well plates and transient transfected promoter region À902 to À603 bp relative to the transcription with TBX5-expressing vector (pcDNA3.1-TBX5), empty vector

Oncogene T-box transcription factor 5 in colon cancer JYuet al 6473 (pcDNA3.1) or green fluorescent protein plasmid (1.6 mg per Human Cancer PathwayFinder RT2 Profiler PCR Array each). The transient transfection efficiency was estimated by (SABiosciences, Frederick, MD, USA). This array contains green fluorescent protein under a uorescence microscope. After 84 functionally well-characterized genes involved in human 48 h of transfection, 20 ml of reaction solution was added to tumorigenesis (http://www.sabiosciences.com). Gene expres- cultured cells in 100 ml culture medium and incubated at 37 1C sion with fold changes X1.5 or p1.5 was considered to be of for 1.5 h. The optical density was measured at a wavelength of biological significance. 490 nm using a VICTOR3 multilabel counter (Perkin-Elmer, Fremont, CA, USA). The cell viability assay was carried out in Statistical analysis triplicate wells for three independent experiments. Data were expressed as mean±standard deviation. Non- parametric data between two groups were computed by w2 Colony formation assay test or Fisher’s exact test. Multiple group comparisons were Cells (2  105 per well) were plated in a 12-well plate and made by one-way analysis of variance after Bonferroni’s transfected with pcDNA3.1-TBX5 or empty vector using correction or Kruskal–Wallis test where appropriate. The Lipofectamine 2000 (Invitrogen). After 48 h of transfection, difference for two different groups was determined by Mann– cells were collected and plated in a six-well plate and selected Whitney U-test. The Fisher’s exact test was used for with 0.6 mg/ml of G418 (Calbiochem, Darmstadt, Germany) comparison of patient characteristics by methylation status for 10–14 days, and then with 0.15 mg/ml of G418 for an and distributions of methylation and covariates by vital status. additional 1 week to establish stable clones. Surviving colonies Patients’ age (at entry of follow-up) by vital status was (X50 cells per colony) were counted after fixed with methanol/ compared using t-test. RRs of death associated with TBX5 acetone (1:1) and stained with 5% Gentian Violet (ICM methylation and other predictor variables were estimated from Pharma, Singapore, Singapore) (Yu et al., 2009a, b). The univariate Cox proportional hazards model first. Multivariate experiment was carried out in triplicate wells for three times. Cox models were also constructed to estimate the RR for TBX5 methylation. Overall survival in relation to methylation Annexin V apoptosis assay status was evaluated by Kaplan–Meier survival curve and Apoptosis was determined by dual staining with Annexin log-rank test. All analyses were performed using SAS for V:FITC and propidium iodide (Invitrogen). Briefly, Windows, version 9, software (SAS Institute Inc., Cary, NC, pcDNA3.1-TBX5 or empty vector-transfected cells (5  105 cells USA). P-value o0.05 was taken as statistical significance. per well) were harvested at 48 h post-transfection. Annexin V:FITC and propidium iodide were added to the cellular suspension according to the manufacturer’s instructions, and Abbreviations sample fluorescence of 10 000 cells was analyzed using FACS- Calibur System (Becton Dickinson Pharmingen, San Jose, CA, BAX, BCL2-associated X protein; BGS, bisulfite genomic USA). The relative proportion of Annexin V-positive cells was sequencing; CDKN2A, cyclin-dependent kinase inhibitor 2A; determined using the ModFitLT software (Becton Dickinson, CI, confidence interval; COBRA, combined bisulfite restriction San Diego, CA, USA) and counted as apoptotic cells. The assay analysis; MTA2, metastasis-associated 1 family member 2; was carried out in triplicate for three times. MTSS1, metastasis suppressor 1; RR, relative risks; RT–PCR, reverse transcript–PCR; TBX5, T-box transcription factor 5; Wound-healing assay SNCG, synuclein gamma; TNFa, tumor necrosis factor alpha; Cell migration was assessed using a scratch wound assay. TNFRSF, tumor necrosis factor receptor superfamily member; Briefly, pcDNA3.1-TBX5 or empty vector stably transfected 5-aza, 5-aza-20-deoxycytidine. SW620 cells were cultured in six-well plates (5  105 cells per well). When the cells grew up to 90% confluence, a single wound was made in the center of cell monolayer using a P-200 Conflict of interest pipette tip. At 0, 48 and 96 h of incubation, the wound closure areas were visualized under phase-contrast microscope with a The authors declare no conflict of interest. magnification  100, and the migrated cells were counted. The experiment was performed in triplicate wells for three times. cDNA expression array Acknowledgements Gene expression profiles of SW620 cells stably transfected with pcDNA3.1-TBX5 or pcDNA3.1 empty vector were analyzed This work was supported by research grant of Council by a commercial gene expression array system named the Competitive Earmarked Research Grant CUHK (473008).

References

Balkwill F. (2009). Tumour necrosis factor and cancer. Nat Rev Cancer Cullen SP, Brunet M, Martin SJ. (2010). Granzymes in cancer and 9: 361–371. immunity. Cell Death Differ 17: 616–623. Barton CA, Hacker NF, Clark SJ, O’Brien PM. (2008). DNA Finotto S. (2008). T-cell regulation in asthmatic diseases. Chem methylation changes in ovarian cancer: implications for early Immunol Allergy 94: 83–92. diagnosis, prognosis and treatment. Gynecol Oncol 109: 129–139. Grady WM, Carethers JM. (2008). Genomic and epigenetic instability Cai CL, Zhou W, Yang L, Bu L, Qyang Y, Zhang X et al. (2005). in colorectal cancer pathogenesis. Gastroenterology 135: 1079–1099. T-box genes coordinate regional rates of proliferation and regional Greene FL, Page DL, Fleming ID, Fritz A, Balch CM, Haller DG specification during cardiogenesis. Development 132: 2475–2487. et al. (2002). TNM Classification of Malignant Tumours. AJCC Chowdhury D, Lieberman J. (2008). Death by a thousand cuts: Cancer Staging Manual, 6th edn. Springer-Verlag: New York. granzyme pathways of programmed cell death. Annu Rev Immunol Gupta A, Godwin AK, Vanderveer L, Lu A, Liu J. (2003a). 26: 389–420. Hypomethylation of the synuclein gamma gene CpG island

Oncogene T-box transcription factor 5 in colon cancer JYuet al 6474 promotes its aberrant expression in breast carcinoma and ovarian Newbury-Ecob RA, Leanage R, Raeburn JA, Young ID. (1996). Holt– carcinoma. Cancer Res 63: 664–673. Oram syndrome: a clinical genetic study. JMedGenet33: 300–307. Gupta A, Inaba S, Wong OK, Fang G, Liu J. (2003b). Breast cancer- Pan ZZ, Bruening W, Giasson BI, Lee VM, Godwin AK. (2002). specific gene 1 interacts with the mitotic checkpoint kinase BubR1. Gamma-synuclein promotes cancer cell survival and inhibits stress- Oncogene 22: 7593–7599. and chemotherapy drug-induced apoptosis by modulating MAPK He ML, Chen Y, Peng Y, Jin D, Du D, Wu J et al. (2002). Induction of pathways. J Biol Chem 277: 35050–35060. apoptosis and inhibition of cell growth by developmental regulator Papaioannou VE, Silver LM. (1998). The T-box gene family. Bioessays hTBX5. Biochem Biophys Res Commun 297: 185–192. 20: 9–19. Hicks DG, Yoder BJ, Short S, Tarr S, Prescott N, Crowe JP et al. Parkin DM, Bray F, Pisani P. (2005). Global cancer statistics, 2002. (2006). Loss of breast cancer metastasis suppressor 1 protein CA Cancer J Clin 55: 74–108. expression predicts reduced disease-free survival in subsets of breast Parr C, Jiang WG. (2009). Metastasis suppressor 1 (MTSS1) cancer patients. Clin Cancer Res 12: 6702–6708. demonstrates prognostic value and anti-metastatic properties in Holoch PA, Griffith TS. (2009). TNF-related apoptosis-inducing breast cancer. Eur J Cancer 45: 1673–1683. ligand (TRAIL): a new path to anti-cancer therapies. Eur J Plageman Jr TF, Yutzey KE. (2006). Microarray analysis of Tbx5- Pharmacol 625: 63–72. induced genes expressed in the developing heart. Dev Dyn 235: Hu H, Sun L, Guo C, Liu Q, Zhou Z, Peng L et al. (2009). Tumor cell– 2868–2880. microenvironment interaction models coupled with clinical valida- Schu¨tze S, Tchikov V, Schneider-Brachert W. (2008). Regulation of tion reveal CCL2 and SNCG as two predictors of colorectal cancer TNFR1 and CD95 signalling by receptor compartmentalization. hepatic metastasis. Clin Cancer Res 15: 5485–5493. Nat Rev Mol Cell Biol 9: 655–662. Johnstone RW, Frew AJ, Smyth MJ. (2008). The TRAIL apoptotic Taylor RC, Cullen SP, Martin SJ. (2008). Apoptosis: controlled pathway in cancer onset, progression and therapy. Nat Rev Cancer demolition at the cellular level. Nat Rev Mol Cell Biol 9: 231–241. 8: 782–798. Toh Y, Nicolson GL. (2009). The role of the MTA family and their Kim WY, Sharpless NE. (2006). The regulation of INK4/ARF in encoded proteins in human cancers: molecular functions and clinical cancer and aging. Cell 127: 265–275. implications. Clin Exp Metas 26: 215–227. Liu H, Liu W, Wu Y, Zhou Y, Xue R, Luo C et al. (2005). Loss of Yanagawa N, Tamura G, Honda T, Endoh M, Nishizuka S, epigenetic control of synuclein-gamma gene as a molecular indicator Motoyama T. (2004). Demethylation of the synuclein gamma gene of metastasis in a wide range of human cancers. Cancer Res 65: CpG island in primary gastric cancers and gastric cancer cell lines. 7635–7643. Clin Cancer Res 10: 2447–2451. Liu YE, Pu W, Jiang Y, Shi D, Dackour R, Shi YE. (2007). Chaperoning Yu J, Cheng YY, Tao Q, Cheung KF, Lam CN, Geng H et al. (2009a). of and induction of mammary gland proliferation by Methylation of protocadherin 10, a novel tumor suppressor, is neuronal protein synuclein gamma. Oncogene 26: 2115–2125. associated with poor prognosis in patients with gastric cancer. Maitra M, Schluterman MK, Nichols HA, Richardson JA, Lo CW, Gastroenterology 136: 640–651. Srivastava D et al. (2009). Interaction of Gata4 and Gata6 with Tbx5 is Yu J, Tao Q, Cheng YY, Lee KY, Ng SS, Cheung KF et al. (2009b). critical for normal cardiac development. Dev Biol 326: 368–377. Promoter methylation of the Wnt/beta-catenin signaling antagonist Mori AD, Zhu Y, Vahora I, Nieman B, Koshiba-Takeuchi K, Dkk-3 is associated with poor survival in gastric cancer. Cancer 115: Davidson L et al. (2006). Tbx5-dependent rheostatic control of 49–60. cardiac gene expression and morphogenesis. Dev Biol 297: 566–586. Zitt M, Zitt M, Mu¨ller HM. (2007). DNA methylation in colorectal Nemer M. (2008). Genetic insights into normal and abnormal heart cancer—impact on screening and therapy monitoring modalities? development. Cardiovasc Pathol 17: 48–54. Dis Markers 23: 51–71.

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