CHD8 is an independent prognostic indicator that regulates Wnt/β-catenin signaling and the cell cycle in gastric cancer

  • Authors:
    • Genta Sawada
    • Hiroki Ueo
    • Tae Matsumura
    • Ryutaro Uchi
    • Masahisa Ishibashi
    • Kosuke Mima
    • Junji Kurashige
    • Yusuke Takahashi
    • Sayori Akiyoshi
    • Tomoya Sudo
    • Keishi Sugimachi
    • Yuichiro Doki
    • Masaki Mori
    • Koshi Mimori
  • View Affiliations

  • Published online on: Monday, July 8, 2013
  • Pages: 1137-1142
  • DOI: 10.3892/or.2013.2597

Abstract

The chromodomain helicase DNA-binding (CHD) family comprises a class of chromatin remodeling enzymes. Previous studies suggest that CHD8 may negatively regulate various genes and signaling pathways, such as the Wnt/β‑catenin pathway. However, few studies have investigated the role of CHD8 in cancer cells. We analyzed the expression of CHD8 in cancer lesions and corresponding non-cancerous tissues to demonstrate the prognostic significance of CHD8 expression in 101 cases of gastric cancer. We also investigated the functional implications of aberrant CHD8 expression by conducting gene set enrichment analysis (GSEA). Expression of CHD8 mRNA was significantly lower in gastric cancer tissues compared to that in corresponding normal tissues (P=0.003). In multivariate analysis for overall survival, we found that CHD8 expression was an independent prognostic factor in gastric cancer. Moreover, GSEA revealed that CHD8 was significantly associated with genes involved in the Wnt/β‑catenin pathway and in the cell cycle. In addition, knockdown of CHD8 expression in the gastric cancer cell lines, MKN45 and NUGC4, promoted proliferation. In conclusion, the present study suggests that loss of CHD8 expression may be a novel indicator for biological aggressiveness in gastric cancer.

Introduction

Gastric cancer is one of the most common malignant tumors in the world. According to data from the National Cancer Institute (NCI), it is estimated that more than 24,000 patients are diagnosed with gastric cancer each year in the United States (1). Patients with advanced gastric cancer still face a poor prognosis. Identification of genes responsible for the development and progression of gastric cancer and a clear understanding of the clinical significance of these genes are both important for the diagnosis and adequate treatment of this disease.

Chromatin remodeling is a key mechanism regulating gene expression. The chromodomain helicase DNA-binding (CHD) family comprises a group of chromatin remodeling enzymes that use the energy from ATP hydrolysis to alter the structure or position of the nucleosome (25). In humans, 9 CHD family proteins have been identified to date (2,5). These proteins share common tandem chromodomains in the N-terminal region and an ATPase-helicase domain in the central region (2,5). Several studies have demonstrated that chromatin remodeling by CHD family members is involved in the pathogenesis of different types of cancers. For example, the CHD7 gene is known to be mutated in small cell lung cancer (SCLC) tissues and SCLC cell lines express the PVT1-CHD7 fusion gene (6). Moreover, CHD5 has been shown to control proliferation and apoptosis via the p19–p53 pathway, functioning as a tumor suppressor (7). In humans, CHD5 is inactivated not only by deletion, but also by hypermethylation in several types of cancer (7).

CHD8 was originally isolated as a negative regulator of the Wnt/β-catenin pathway when it was found to suppress β-catenin function (8,9). CHD8 has also been suggested to regulate the expression of various genes, including cyclin E2 and HOXA2(1014). In contrast, CHD8 can also bind to p53 and suppress its transactivation activity by recruiting histone H1 during embryogenesis (15). However, the role of CHD8 in solid malignant tumors has not yet been elucidated.

In the present study, we analyzed CHD8 mRNA expression using clinical samples from 101 patients diagnosed with primary gastric cancer. We then examined the relationship between CHD8 mRNA expression and clinicopathological factors and determined the clinical significance of aberrant CHD8 expression. Moreover, we investigated the functional role of CHD8 in gastric cancer by analyzing expression array data in silico and confirmed the biological significance of CHD8 in gastric cancer cells in vitro.

Materials and methods

Clinical samples and cell lines

A total of 101 gastric cancer patients were enrolled in this study. All patients underwent surgery without preoperative treatments such as chemotherapy and radiotherapy. Tumor and adjacent normal tissues were obtained. Total RNA was extracted using the QIAamp DNA Micro Kit (Qiagen) following the manufacturer’s protocol. Patients were closely observed each month after surgery and the mean postoperative follow-up period was 2.8 years. Histopathological evaluations were assessed according to the Japanese Classification of Gastric Cancer, 3rd English edition.

MKN-45 cell lines were provided by the American Type Culture Collection and were maintained in RPMI-1640 containing 10% FBS with 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were cultured in a humidified 5% CO2 incubator at 37°C.

Real-time quantitative reverse transcription (RT)-PCR

Real-time quantitative RT-PCR was performed using a LightCycler® System and a LightCycler® 480 Probes Master kit (both from Roche Applied Science, Indianapolis, IN, USA) following the manufacturer’s protocol with the following specific CHD8 primers: forward, 5′-AGTGGTGTCTACGTT TGGTGTG-3′ and reverse, 5′-GATGGGCTCAATGAACAG GT-3′. CHD8 levels were normalized to GAPDH (primers: forward, 5′-GTCAACGGATTTGGTCTGTATT-3′ and reverse, 5′-AGTCTTCTGGGTGGCAGTGAT-3′).

siRNA transfections and proliferation assays

For siRNA knockdown studies, double-stranded RNA duplexes targeting human CHD8 (5′-AGGAGCGUCCAGUAGAUGAACACG C-3′/5′-GCGUGUUCAUCUACUGGACGCUCCU-3′; 5′-UU CAAAUGCUUAAACUUUGGGAUUG-3′/5′-CAAUCCCAA AGUUUAAGCAUUUGAA-3′) were purchased from Invitrogen (Carlsbad, CA, USA) (Stealth RNAi). Negative control siRNA (NC) was also purchased from Invitrogen. MKN-45 and NUGC4 cells were transfected with siRNA at a concentration of 20 μmol/l using Lipofectamine reagent (RNAiMax) in glucose-free Opti-MEM (both from Invitrogen). For proliferation assays, siRNA-, NC- or mock-transfected MKN-45 cells were seeded at 8.0×103 cells/well in 96-well flat-bottomed microtiter plates in a final volume of 100 μl of culture medium per well. Cells were incubated in a humidified atmosphere (37°C and 5% CO2) for 24, 48, 72 or 96 h after initiation of transfection. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Roche Diagnostics Corp.) was then used to measure cell growth inhibition according to standard protocols. Briefly, after incubation, 10 μl of MTT labeling reagent (final concentration of 0.5 mg/ml) was added to each well, and the plate was incubated for an additional 4 h in a humidified atmosphere. Solubilization solution (100 μl) was added to each well, and the plate was incubated overnight in a humidified atmosphere. After confirming that the purple formazan crystals were completely solubilized, the absorbance of each well was measured by a Model 550 Series Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 570 nm corrected to 655 nm. Each sample was run with 6 replicates.

Gene set enrichment analysis (GSEA) of gastric cancer expression

To investigate CHD8 function in gastric cancer, we obtained gastric cancer expression profiles from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (accession code GSE22377) and analyzed these profiles using GSEA.

Expression profiles were normalized with the ‘affy’ R/BioConductor package (http://www.bioconductor.org/packages/release/bioc/html/affy.html). We applied a continuous-type CLS file with the CHD8 profile to phenotype labels in GSEA. The metric for ranking genes was set as ‘Pearson’ and all other parameters were set to their default values.

Statistical analysis

The significance of differences between 2 groups was estimated with the Student’s t-test and Chi-square test. Overall survival curves were plotted according to the Kaplan-Meier method, with the log-rank test applied for comparison. Variables with a P-value of <0.05 by univariate analysis were used in subsequent multivariate analysis on the basis of the Cox proportional hazards model. All differences were considered statistically significant at the level of P<0.05. Statistical analyses were conducted using JMP 5 Software (SAS Institute).

Results

CHD8 expression in gastric cancer tissues

We examined CHD8 mRNA expression in tumor tissues and the corresponding normal mucosa collected from 101 gastric cancer patients by quantitative real-time PCR. Notably, CHD8 was significantly downregulated in tumor tissues when compared to the level in the corresponding normal mucosa (median CHD8/GAPDH ratio, 1.58 vs. 2.26, respectively; P=0.003) (Fig. 1).

Association between CHD8 mRNA expression and clinicopathological factors

As shown in Table I, we divided the patient population into a high (n=60) and a low CHD8 expression group (n=41), with a cutoff value of 1.29 for the CHD8/GAPDH ratio in the cancerous tissues. The low CHD8 expression group was significantly associated with an increased depth of tumor invasion (P=0.036) and lymph node metastasis (P=0.028). Relative to the high CHD8 expression group, the low CHD8 expression group showed an increased tendency to be associated with peritoneal dissemination (Table I). No significant differences were observed regarding histological type, lymphatic invasion, or venous invasion.

Table I

Association between CHD8 mRNA expression and clinicopathologic factors.

Table I

Association between CHD8 mRNA expression and clinicopathologic factors.

FactorsLow expression (n=41)High expression (n=60)P-value
Age (mean ± SD)63.9±11.367.2±10.6
Gender0.013
 Male3131
 Female1029
Histological grade0.44
 Well and mod1831
 Por and sig2329
Depth of invasion0.036
 T1–21230
 T3–42930
Lymph node metastasis0.028
 Negative1129
 Positive3031
Lymphatic invasion0.23
 Negative2834
 Positive1326
Venous invasion0.71
 Negative810
 Positive3350
Peritoneal dissemination0.051
 Negative3255
 Positive95

[i] CHD8, chromodomain helicase DNA-binding 8; well, well-differentiated adenocarcinoma; mod, moderately differentiated adenocarcinoma; por, poorly differentiated adenocarcinoma; sig, signet ring cell adenocarcinoma.

Association between CHD8 expression and prognosis

With regard to overall survival, patients with high CHD8 expression had a significantly better prognosis than those with low CHD8 expression (P=0.044) (Fig. 2). Univariate analysis revealed that the level of CHD8 expression, depth of tumor invasion and presence of lymph node metastasis, lymphatic invasion, or venous invasion were significantly correlated with prognosis in gastric cancer patients (Table II). These factors identified by univariate analysis were then applied to multivariate analysis, and CHD8 expression was found to be an independent prognostic indicator for overall survival in patients with gastric cancer (P=0.048; Table II).

Table II

Univariate and multivariate analyses for overall survival using the Cox proportional hazards regression model.

Table II

Univariate and multivariate analyses for overall survival using the Cox proportional hazards regression model.

Univariate analysisMultivariate analysis


FactorRR95% CIP-valueRR95% CIP-value
Age (>65 years)0.830.58–1.200.336---
Gender0.970.67–1.430.89---
Histology grade (well and mod/por and sig)1.130.79–1.660.48---
Tumor stage (T1/T2–4)2.391.49–4.410.000011.080.57–2.230.81
Lymph node metastasis (negative/positive)3.461.89–8.6302.171.04–5.740.037
Lymphatic invasion (negative/positive)2.391.42–4.900.00041.740.94–3.780.076
Venous invasion (negative/positive)2.111.43–3.040.00041.81.13–2.840.012
CHD8 mRNA expression (low/high)1.441.00–2.100.0471.551.00–2.450.048

[i] RR, relative risk; CI, confidence interval; well, well-differentiated adenocarcinoma; mod, moderately differentiated adenocarcinoma; por, poorly differentiated adenocarcinoma; sig, signet ring cell adenocarcinoma; CHD8, chromodomain helicase DNA-binding 8.

GSEA for analyzing CHD8 function in gastric cancer

We used 2,996 gene sets, obtained from GSEA, Qiagen and Sabioscience websites (GSEA: http://www.broadinstitute.org/gsea/index.jsp; Qiagen: https://www.qiagen.com/geneglobe/pathways.aspx; Sabioscience: http://www.sabiosciences.com/pathwaycentral.php). The results of GSEA showed that 5 gene sets were significantly enriched, with a false discovery rate (FDR) of <10% (Table III). In gastric cancer tissues with low CHD8 expression, genes in the Wnt/β-catenin pathway signature were the most highly enriched gene set (P=0.000, FDR=0.000) (Fig. 3). Similarly, genes within the cell cycle signature were also significantly enriched (P=0.041, FDR=0.069) (Fig. 3). Thus, CHD8 expression was inversely correlated with the Wnt/β-catenin gene signature and the cell cycle gene signature (Fig. 4).

Table III

Result of GSEA for CHD8 expression in gastric cancer.

Table III

Result of GSEA for CHD8 expression in gastric cancer.

Gene setESNominal P-valueFDR q-value
WNT_BETACATENIN_PATHWAY−0.520.0000.0006
GUTIERREZ_MULTIPLE_MYELOMA_DN−0.710.0000.050
ALONSO_METASTASIS_UP−0.550.0000.069
CYCLIN_AND_CELL_CYCLE_REGULATION−0.350.0410.069
CHIANG_LIVER_CANCER_SUBCLASS_UNANNOTATED_DN−0.720.0000.074

[i] GSEA, gene set enrichment analysis; CHD8, chromodomain helicase DNA-binding 8; ES, enrichment score; FDR, false discovery rate.

In contrast, p53-mediated apoptosis, which has been shown to be associated with CHD8 expression in embryonic cells (15), was not significantly associated with CHD8 in gastric cancer (Figs. 3 and 4).

In vitro assessment of CHD8 knockdown

To investigate the role of CHD8 in gastric cancer progression, CHD8 expression was suppressed by transient siRNA transfection into MKN-45 and NUGC4 cells. The reduction in CHD8 expression was confirmed by quantitative read-time RT-PCR. As revealed by MTT assays, siRNA-mediated knockdown of CHD8 promoted the proliferation of MKN-45 and NUGC4 cells (MKN-45: P=0.028; NUGC4: P=0.016 vs. control cells) (Fig. 5).

Discussion

Our present study revealed that CHD8 mRNA expression was frequently downregulated in gastric cancer tissues compared with that in the adjacent normal mucosa. In addition, we found that CHD8 played a key role as a tumor suppressor in gastric cancer, i.e. high expression of CHD8 was associated with a better prognosis. By in vitro analysis, we confirmed that knockdown of CHD8 mRNA in gastric cancer cell lines resulted in more aggressive proliferation. Moreover, GSEA based on GEO expression array data showed that CHD8 expression was significantly associated with downregulation of genes involved in the Wnt/β-catenin pathway and the cell cycle. Thus, loss of CHD8 expression was expected to enhance activation of the Wnt/β-catenin pathway and promote cell cycle progression in gastric cancer. Taken together, these data suggest that CHD8 exerts a suppressive effect on cell proliferation through negative regulation of the Wnt/β-catenin pathway and cell cycle progression in gastric cancer.

From the GSEA study, we found significant associations between CHD8 expression and Wnt/β-catenin signaling and the cell cycle; this result is consistent with several studies that have described CHD8 as a negative regulator of the Wnt/β-catenin pathway. For example, Thompson et al showed that CHD8 is an ATP-dependent chromatin remodeling factor that regulates β-catenin target genes in colon cancer cells (HCT116) (8). Additionally, Nishiyama et al(16) suggested that CHD8 negatively regulates β-catenin function by recruiting histone H1 to the promoters of Wnt target genes. Together with our current data, these studies suggest that CHD8 is an essential mediator of Wnt/β-catenin signaling in several different cellular contexts.

Studies have also suggested that CHD8 controls the expression of cyclin E2 (CCNE2) and thymidylate synthetase (TYMS), two genes expressed during G1/S transition of the cell cycle (11). Indeed, in the present study, both genes were upregulated in gastric cancer tissues with low CHD8 expression (data not shown). Moreover, a previous study also found that p53-dependent apoptosis was suppressed by histone H1, which is recruited by CHD8 during embryogenesis (15); however, to date, no studies have investigated the involvement of CHD8 in p53 activation and signaling in malignancies. Our study demonstrated that CHD8 expression was not associated with the p53 pathway in gastric cancer.

The Wnt/β-catenin signaling pathway is an important functional pathway in development, specification of cell fate and adult stem cell proliferation (1720). Abnormal Wnt signaling has been demonstrated in a variety of human cancers. For example, Ooi et al(21) demonstrated that 3 oncogenic pathways (proliferation/stem cell, NF-κB and Wnt/β-catenin) were deregulated in the majority of gastric cancers and that increased activation of the Wnt/β-catenin pathway was associated with poor patient survival in gastric cancer. The present study is the first report to show that CHD8 interacts with the Wnt/β-catenin pathway in gastric cancer, with low CHD8 mRNA expression contributing to a poor prognosis.

In conclusion, our data demonstrate that CHD8 functions as a tumor suppressor by regulating Wnt/β-catenin signaling and the cell cycle. Moreover, loss of CHD8 expression, commonly observed in gastric cancer, may represent a novel indicator for the biological aggressiveness of gastric cancer.

Acknowledgements

We would like to thank T. Shimooka and M. Kasagi for their technical assistance. This study was funded in part by the Funding Program for Next Generation World-Leading Researchers (LS094).

References

1 

Jemal A, Tiwari RC, Murray T, et al: Cancer statistics, 2004. CA Cancer J Clin. 54:8–29. 2004.

2 

Marfella CG and Imbalzano AN: The Chd family of chromatin remodelers. Mutat Res. 618:30–40. 2007.

3 

Lusser A and Kadonaga JT: Chromatin remodeling by ATP-dependent molecular machines. Bioessays. 25:1192–1200. 2003.

4 

Tsukiyama T: The in vivo functions of ATP-dependent chromatin-remodelling factors. Nat Rev Mol Cell Biol. 3:422–429. 2002.

5 

Hall JA and Georgel PT: CHD proteins: a diverse family with Strong Ties. Biochem Cell Biol. 85:463–476. 2007.

6 

Pleasance ED, Stephens PJ, O’Meara S, et al: A small-cell lung cancer genome with complex signatures of tobacco exposure. Nature. 463:184–190. 2010.

7 

Bagchi A, Papazoglu C, Wu Y, et al: CHD5 is a tumor suppressor at human 1p36. Cell. 128:459–475. 2007.

8 

Thompson BA, Tremblay V, Lin G and Bochar DA: CHD8 is an ATP-dependent chromatin remodeling factor that regulates β-catenin target genes. Mol Cell Biol. 28:3894–3904. 2008.

9 

Sakamoto I, Kishida S, Fukui A, et al: A novel β-catenin-binding protein inhibits β-catenin-dependent Tcf activation and axis formation. J Biol Chem. 275:32871–32878. 2000.

10 

Yates JA, Menon T, Thompson BA and Bochar DA: Regulation of HOXA2 gene expression by the ATP-dependent chromatin remodeling enzyme CHD8. FEBS Lett. 584:689–693. 2010.

11 

Rodríguez-Paredes M, Ceballos-Chávez M, Esteller M, García-Domínguez M and Reyes JC: The chromatin remodeling factor CHD8 interacts with elongating RNA polymerase II and controls expression of the cyclin E2 gene. Nucleic Acids Res. 37:2449–2460. 2009.

12 

Rodenberg JM, Hoggatt AM, Chen M, Touw K, Jones R and Herring BP: Regulation of serum response factor activity and smooth muscle cell apoptosis by chromodomain helicase DNA-binding protein 8. Am J Physiol Cell Physiol. 299:C1058–C1067. 2010.

13 

Yuan CC, Zhao X, Florens L, Swanson SK, Washburn MP and Hernandez N: CHD8 associates with human Staf and contributes to efficient U6 RNA polymerase III transcription. Mol Cell Biol. 27:8729–8738. 2007.

14 

Menon T, Yates JA and Bochar DA: Regulation of androgen-responsive transcription by the chromatin remodeling factor CHD8. Mol Endocrinol. 24:1165–1174. 2010.

15 

Nishiyama M, Oshikawa K, Tsukada Y, et al: CHD8 suppresses p53-mediated apoptosis through histone H1 recruitment during early embryogenesis. Nat Cell Biol. 11:172–182. 2009.

16 

Nishiyama M, Skoultchi AI and Nakayama KI: Histone H1 recruitment by CHD8 is essential for suppression of the Wnt-β-catenin signaling pathway. Mol Cell Biol. 32:501–512. 2012.

17 

Bienz M and Clevers H: Linking colorectal cancer to Wnt signaling. Cell. 103:311–320. 2000.

18 

Moon RT, Kohn AD, De Ferrari GV and Kaykas A: WNT and β-catenin signalling: diseases and therapies. Nat Rev Genet. 5:691–701. 2004.

19 

Nelson WJ and Nusse R: Convergence of Wnt, β-catenin, and cadherin pathways. Science. 303:1483–1487. 2004.

20 

Willert K and Jones KA: Wnt signaling: is the party in the nucleus? Genes Dev. 20:1394–1404. 2006.

21 

Ooi CH, Ivanova T, Wu J, et al: Oncogenic pathway combinations predict clinical prognosis in gastric cancer. PLoS Genet. 5:e10006762009.

Journal Cover

September 2013
Volume 30 Issue 3

Print ISSN: 1021-335X
Online ISSN:1791-2431

2013 Impact Factor: 2.191
Ranked #33/202 Oncology
(total number of cites)

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
APA
Sawada, G., Ueo, H., Matsumura, T., Uchi, R., Ishibashi, M., Mima, K., Kurashige, J., Takahashi, Y., Akiyoshi, S., Sudo, T., Sugimachi, K., Doki, Y., Mori, M., & Mimori, K. (2013). CHD8 is an independent prognostic indicator that regulates Wnt/β-catenin signaling and the cell cycle in gastric cancer. Oncology Reports, 30(3), 1137-1142.
MLA
Sawada, Ueo, Matsumura, Uchi, Ishibashi, Mima, Kurashige, Takahashi, Akiyoshi, Sudo, Sugimachi, Doki, Mori, and Koshi Mimori. "CHD8 is an independent prognostic indicator that regulates Wnt/β-catenin signaling and the cell cycle in gastric cancer." Oncology Reports Oncology Reports 30.3 (2013): 1137-1142.
Chicago
Sawada, Ueo, Matsumura, Uchi, Ishibashi, Mima, Kurashige, Takahashi, Akiyoshi, Sudo, Sugimachi, Doki, Mori, and Koshi Mimori. "CHD8 is an independent prognostic indicator that regulates Wnt/β-catenin signaling and the cell cycle in gastric cancer." Oncology Reports Oncology Reports 30 no. 3 (2013): 1137-1142.