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Wilms’ tumor 1 gene modulates Fas-related death signals and anti-apoptotic functions in hepatocellular carcinoma

  • Original Article—Liver, Pancreas, and Biliary Tract
  • Published:
Journal of Gastroenterology Aims and scope Submit manuscript

Abstract

Background

The Wilms’ tumor 1 (WT1) gene is known to be overexpressed in hepatocellular carcinoma (HCC) and to upregulate tumor growth and oncogenic potential, although the detailed mechanisms remain to be elucidated.

Methods

We identified host genes involved in WT1 gene modulation of human liver cancer cell lines in vitro, and further characterized genes related to apoptosis. Moreover, we evaluated the alteration of genes by WT1 in 40 HCC and 58 non-HCC human liver samples collected at resection.

Results

Analysis of the effect of small interfering RNAs-mediated knock-down of WT1 on apoptosis using an annexin V labeling assay, and on modulation of the activity of caspases-3, -8 and -9, indicated that WT1 has an anti-apoptotic role. We identified three apoptosis-related genes that were modulated by WT1; the cellular FLICE-inhibitory proteins (cFLIP) gene was upregulated, and Fas-associated death domain (FADD) and nuclear factor kappa B (NF-κB) were downregulated. Interestingly, knock-down of FADD or NF-κB resulted in the upregulation of WT1, and the expression of cFLIP changed in parallel with WT1 expression. We further evaluated WT1-mediated alteration of genes in HCC and non-HCC human liver samples. Both HCC and non-HCC tissues that expressed relatively high levels of WT1 showed cFLIP overexpression.

Conclusions

WT1 modulates cFLIP, FADD and NF-κB, and has an anti-apoptotic role in HCC. This mechanism of action of WT1 could be related to the tumor growth and oncogenic potential of HCC.

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Abbreviations

cFLIP:

Cellular FLICE-inhibitory proteins

FADD:

Fas-associated death domain protein

GAPDH:

Glyceraldehyde phosphate dehydrogenase

HCC:

Hepatocellular carcinoma

NF-κB:

Nuclear factor kappa-light-chain-enhancer of activated B cells

WT1:

Wilms’ tumor 1 gene

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Acknowledgments

We thank Dr. Shigeki Higashiyama (Department of Biochemistry and Molecular Genetics), and Dr. Mamoru Aoto (Department of Physiology) in Ehime University Graduate School of Medicine (Ehime, Japan) and Dr. Keiko Udaka (Department of Immunology, Kochi University School of Medicine, Kochi, Japan) for valuable advice, and also Ms. Satomi Yamanaka, Ms. Chie Takeichi and Ms. Sakiko Inoh (in our department) for valuable technical assistance. We thank Dr. Francis V. Chisari (Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA) for providing Huh7.5.1 cells, and Dr. Haruo Sugiyama (Department of Functional Diagnostic Science, Graduate School of Medicine, Osaka University, Osaka, Japan) for providing the plasmids encoding WT1 genes. This work was supported in part by a Grant-in-Aid for Scientific Research (Japan Society for the Promotion of Science, KAKENHI 24590980 to Y.H.) and the Program for Enhancing Systematic Education in Graduate School (to K.U.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by a Grant-in-Aid for Scientific Research and Development (to Y.H.) from the Japanese Ministry of Health, Labor and Welfare, Japan.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime, 791-0295, Japan

    Kazuhiro Uesugi, Yoichi Hiasa, Yoshio Tokumoto, Toshie Mashiba, Yohei Koizumi, Masashi Hirooka, Masanori Abe, Bunzo Matsuura & Morikazu Onji

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  1. Kazuhiro Uesugi
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  2. Yoichi Hiasa
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  3. Yoshio Tokumoto
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  5. Yohei Koizumi
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Corresponding author

Correspondence to Yoichi Hiasa.

Electronic supplementary material

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535_2012_708_MOESM1_ESM.tif

Supplementary Figure 1. Down- and upregulation of WT1 gene expression in liver cancer cell lines by WT1 siRNA and by a WT1 plasmid, respectively. WT1 mRNA was efficiently knocked-down in all three cell lines (Huh7, Huh7.5.1 and HLE cells) after WT1 siRNA transfection (black bars) compared to control siRNA transfection (white bars) as assessed by real-time RT-PCR (a). Expression of the WT1 protein was also downregulated by WT1 siRNA transfection as assessed by western blotting (b). WT1 mRNA levels remained upregulated for at least 3 days after transfection of the WT1-expressing plasmid (pWT1) compared to cells transfected with a control plasmid (p3.1) (c). Western blotting indicated that WT1 proteins were overexpressed in each cell line following plasmid transfection. (d). The mRNA data are means ± SEM of 4 independent experiments. *p < 0.05 versus control (TIFF 5883 kb)

535_2012_708_MOESM2_ESM.tif

Supplementary Figure 2. Upregulation of WT1 mRNA expression following transfection of plasmids encoding the four splicing variants of WT1 into liver cancer cell lines. Plasmids expressing the four splicing variant forms of WT1 were transfected into the indicated cell lines and WT1 mRNA was then analyzed by RT-PCR 2 days following transfection. Data are indicated as the copy ratio of WT1 mRNA/GAPDH mRNA, and each value represents the fold of the pWT1 (+/+) value, which is normalized as 1.0. Data are means ± SEM of three independent experiments (TIFF 1295 kb)

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Supplementary Table 3 (DOC 90 kb)

Supplementary Table 4 (DOC 82 kb)

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Uesugi, K., Hiasa, Y., Tokumoto, Y. et al. Wilms’ tumor 1 gene modulates Fas-related death signals and anti-apoptotic functions in hepatocellular carcinoma. J Gastroenterol 48, 1069–1080 (2013). https://doi.org/10.1007/s00535-012-0708-7

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  • DOI: https://doi.org/10.1007/s00535-012-0708-7

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