CYLD downregulation is correlated with tumor development in patients with hepatocellular carcinoma

  • Authors:
    • Hiroki Kinoshita
    • Hirohisa Okabe
    • Toru Beppu
    • Akira Chikamoto
    • Hiromitsu Hayashi
    • Katsunori Imai
    • Kosuke Mima
    • Shigeki Nakagawa
    • Naomi Yokoyama
    • Takatoshi Ishiko
    • Satoru Shinriki
    • Hirofumi Jono
    • Yukio Ando
    • Hideo Baba
  • View Affiliations

  • Published online on: January 14, 2013     https://doi.org/10.3892/mco.2013.68
  • Pages: 309-314
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Abstract

The cylindromatosis (CYLD) gene is involved in tumor progression by acting as a negative regulator of nuclear factor‑κB (NF‑κΒ). However, the clinical significance of CYLD in patients with hepatocellular carcinoma (HCC) remains unclear. To demonstrate the clinical significance of CYLD expression, we analyzed CYLD gene expression in 124 paired HCC and non‑tumor tissues using quantitative reverse transcription‑polymerase chain reaction (qRT-PCR). CYLD gene expression was detected in the patients and the cut‑off value was determined by the median value of tumor‑to‑non‑tumor (T/N) ratio. qRT-PCR analysis showed that a low CYLD expression was associated with a high serum α‑fetoprotein (AFP) value. Patients in the low CYLD expression group exhibited poorer overall survival compared to those in the high expression group (P=0.0406). Protein expression of CYLD was also investigated in 70 patients with HCC using immunohistochemistry. The findings showed that CYLD protein expression in tumor tissue was associated with CYLD gene expression (P=0.031). The findings of the present study suggest that CYLD is clinically associated with tumor development in HCC patients.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common gastrointestinal malignancies and constitutes the leading cause of cancer-related mortality in East Asia and South Africa (1). Currently, the first-line treatment for HCC is liver transplantation or surgical resection (2). However, the overall survival rate after curative therapy is not satisfactory due to the highly chemoresistant nature of this tumor and the frequent intrahepatic recurrence. Identification of the genes responsible for the onset and progression of HCC as well as comprehension of the clinical significance of these genes are critical for the development of successful therapies.

The cylindromatosis (CYLD) gene was originally identified as a tumor suppressor, the mutation of which predisposes patients to the development of tumors of hair follicles (cylindromas) (3). It has been reported that CYLD acts as a negative regulator of the nuclear factor-κB (NF-κB) signaling pathway by deubiquitinating NF-κB essential modulator (NEMO), IκB kinase (IKK)-γ, and IKK upstream regulators, including the tumor necrosis factor (TNF), receptor-associated factor 2 (TRAF2), TRAF6, TRAF7 and receptor-interacting protein 1 (RIP1) (410). CYLD also regulates transforming growth factor-β (TGF-β) signaling via the deubiquitination of Akt in lung fibrosis (11).

Recent studies have demonstrated that CYLD deficiency may promote the development of several types of cancer in addition to skin tumors caused by mutations and loss of the heterozygosity (LOH) of CYLD. LOH of chromosome 16q, which includes the CYLD gene, has been detected in a large proportion of multiple myeloma cases and has been associated with poor overall survival (1214). Comparative genomic hybridization (CGH) assays have also suggested potential genetic abnormalities of CYLD (reduction in copy number) in HCC, uterine carcinoma and renal cancer (1517). Moreover, suppressed CYLD gene expression may contribute to tumor development in colon cancer, hepatocellular carcinoma and melanoma (18,19).

The aim of this study was to investigate the clinical importance of the CYLD gene by analyzing 124 consecutive patients with HCC who were treated with hepatic resection. Distribution of the CYLD protein expression was also examined using immunohistochemistry.

Materials and methods

Clinical tissue samples

Between 2005 and 2010, 124 patients (100 men and 24 women) with HCC were registered at the Department of Gastroenterological Surgery, of the Kumamoto University Hospital (Kumamoto, Japan). Specimens of primary HCC and adjacent normal liver tissues were obtained from the patients after written informed consent was obtained. This study was approved by the Human Ethics Review Committee of the Graduate School of Medical Sciences, Kumamoto University (Kumamoto, Japan).

RNA extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Total RNA was obtained from the frozen tissue samples and cell lines using a mirVana™ miRNA Isolation kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Reverse transcription was performed with 1.0 μg of total RNA as previously described (20). qRT-PCR was performed on a LightCycler 480 II (Roche Diagnostics, Tokyo, Japan) using 2X PCR Master mix (Roche Diagnostics) and Universal ProbeLibrary (Roche Diagnostics). Primers were designed using the Roche website and the Universal ProbeLibrary according to the manufacturer’s instructions. The primers used were: CYLD, F: 5′-TCTATGG GGTAATCCGTTGG-3′ and R: 5′-CAGCCTGCACACTCAT CTTC-3′, and universal probe no. 83; and hypoxanthine phosphoribosyltransferase (HPRT), F: 5′-TGACCTTGATTTA TTTTGCATACC-3′ and R: 5′-CGA GCAAGACGTTCAGT CCT-3′, and universal probe no. 73. HPRT, 18S ribosomal RNA (rRNA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were examined as the internal controls (21). HPRT was proved to be the most suitable reference gene. For amplification, an initial denaturation at 95°C for 10 min was followed by 45 cycles for 15 sec at 95°C, annealing 15 sec at 60°C, and extension 13 sec at 72°C. The experiments were performed twice to confirm reproducibility.

Immunohistochemistry and evaluation of CYLD

Paraffin-embedded tissue sections were dewaxed with xylene and rehydrated using graded concentrations of ethanol. The samples were then stained for CYLD using our previously described technique (22). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide. The sections were incubated in 200X diluted primary rabbit anti-CYLD antibody (Sigma, Tokyo, Japan) overnight at 4°C. A subsequent reaction was performed with a biotin-free horseradish peroxidase enzyme-labeled polymer of the EnVision Plus detection system (Dako Co., Tokyo, Japan). A positive reaction was visualized with a 3,3′-diaminobenzidine (DAB) solution, followed by counterstaining with Mayer’s hematoxylin. Each immunohistochemical marker was independently evaluated by two blinded investigators. CYLD expression status in HCC cells was quantified as a percentage of the total number of stained cells detected in ≥5 random high-power fields (magnification, ×400) in each section. The positivity of staining cells with 10% was determined as the cut-off value.

Statistical analysis

Statistical analysis was performed using the JMP® 8.0 software (SAS Institute., Cary, NC, USA). Values were presented as the mean ± standard deviation (SD). Differences between groups were calculated using the Wilcoxon test. P<0.05 was considered to indicate a statistically significant difference.

Results

Expression of CYLD in clinical tissue specimens and their clinicopathological characteristics

We performed qRT-PCR analysis in the primary HCC specimens. CYLD expression was quantified by caluculating the ratio of CYLD to HPRT1 signal. CYLD expression was detected in the tumor and non-tumor tissues. CYLD expression of tumor tissue was not markedly different compared to that of non-tumor liver tissue. For the clinicopathological evaluation, patients were allocated into two groups based on the median value of tumor-to-non-tumor (T/N) ratio of CYLD expression. Patients with a T/N ratio larger than the median T/N ratio of CYLD expression were allocated to the high expression group, while the remaining patients comprised the low expression group. Clinicopathological characteristics associated with the CYLD expression status of the 124 patients are summarized in Table I. CYLD expression was only correlated with the serum α-fetoprotein (AFP) value (P=0.0093).

Table I

CYLD-mRNA expression and patient clinicopathological characteristics.

Table I

CYLD-mRNA expression and patient clinicopathological characteristics.

CYLD (T/N ratio)
Clinicopathological characteristicsNo. of patientsHighLowP-value
Agea (years)
  <666330330.7637
  ≥66613229
Gender
  Male10049510.4103
  Female241311
AFPb (U/ml)
  <15.26841270.0093
  ≥15.2562135
PIVKA-IIa (U/ml)
  <1086131300.5000
  ≥108693132
Tumor diametera (mm)
  <35.56232300.4288
  ≥35.5623032
No. of tumors
  Solitary9447470.5829
  Multiple301515
Differentiation
  Well/mod10352510.5000
  Poor211011
Vascular invasionc
  Negative6636300.1345
  Positive562432
HCV-Ab
  Negative7038320.1826
  Positive542430
HBs-Ag
  Negative8645410.2796
  Positive381721
Liver cirrhosisd
  Negative8743440.8444
  Positive371918

a Cut-off value defined as the median value;

b cut-off value defined as the maximum normal value;

c pathological vascular invasion;

d clasified as F4 stage in the Inuyama classification (25). CYLD, cylindromatosis; T/N, tumor to non-tumor; AFP, α-fetoprotein; PIVKA-II, the protein induced by vitamin K absence or antagonist-II; well/mod, well/moderately-differentiated hepatocellular carcinoma; poor, poorly-differentiated hepatocellular carcinoma; HCV-Ab, hepatitis C virus; HBs-Ag, hepatitis B virus.

Correlation between CYLD expression and prognosis

The correlation between each clinicopathological characteristic and prognosis was analyzed by univariate analyses (Table II). The data indicated that poor prognosis in HCC patients correlated with tumor a diameter of >35.5 mm (P<0.0001), multiple tumors (P=0.0048), positive vascular invasion (P=0.0021), the protein induced by vitamin K absence or antagonist (PIVKA)-II >108 (P=0.0278), and low CYLD expression (P=0.0406) (Fig. 1A). In the multivariate analysis, CYLD expression was not an independent factor for predicting poor prognosis (data not shown). Although CYLD expression was not significantly correlated with disease-free survival (P=0.1021) (Fig. 1B), the low CYLD expression group had more patients with early recurrence within 2 years (30/37 patients) compared to the high CYLD expression group (17/31 patients; P=0.016).

Table II

Univariate analysis of clinicopathological characteristics for overall survival of patients.

Table II

Univariate analysis of clinicopathological characteristics for overall survival of patients.

Clinicopathological characteristicsNo. of patientsMedian survival (months)P-value
Agea (years)
  <666336.00.4168
  ≥666121.6
Gender
  Male10646.70.5799
  Female2441.3
AFPb
  <15.26838.70.5008
  ≥15.25641.1
PIVKA-IIa
  <1086142.20.0278
  ≥1086338.2
Tumor diametera (mm)
  <35.56245.2<0.0001
  ≥35.56233.1
No. of tumors
  Solitary9441.20.0048
  Multiple3036.6
Differentiation
  Well/mod10341.70.129
  Poor2137.3
Vascular invasionc
  Negative6642.70.0021
  Positive5638.1
HCV-Ab
  Negative7242.00.8255
  Positive5844.7
HBs-Ag
  Negative9144.80.3037
  Positive3942.0
Liver cirrhosisd
  Negative8742.70.7831
  Positive3743.9
CYLD (T/N ratio)
  Low6241.10.0406
  High6237.0

a Cut-off value defined as the median value;

b cut-off value defined as the maximum normal value;

c pathological vascular invasion;

d classified as F4 stage in the Inuyama classification (25). AFP, α-fetoprotein; PIVKA-II, the protein induced by vitamin K absence or antagonist-II; well/mod, well/moderately-differentiated hepatocellular carcinoma; poor, poorly-differentiated hepatocellular carcinoma; HCV-Ab, hepatitis C virus; HBs-Ag, hepatitis B virus; CYLD, cylindromatosis; T/N, tumor to non-tumor.

Expression of CYLD protein

Among 70 HCC cases, 53 (75.7%) were positive for CYLD expression. CYLD expression was heterogeneously distributed in the tumor tissue and downregulated in tumor cells. In Fig. 2A, a representative case of HCC shows that a number of tumor cells (T1) with a high CYLD expression are well-differentiated and that they demonstrate a trabecular pattern. Conversely, other tumor cells (T2) with low CYLD expression lost their cell polarity and demonstrated dense chromatin in the nucleus. Another case of HCC comprising tumor cells with dense chromatin and a small nucleus that lost CYLD expression, despite being surrounded by CYLD-expressing tumor cells with more cytoplasm and only faint chromatin in the nucleus (Fig. 2B). However, CYLD protein expression was not associated with tumor-related factors, such as tumor size, tumor diameter, vascular invasion, tumor differentiation and prognosis (data not shown). To confirm the correlation of CYLD-mRNA expression with protein expression, CYLD-mRNA expression normalized by HPRT-mRNA expression in tumor tissue was compared between the high and low-CYLD protein expression groups. This finding showed that the high-CYLD protein expression group demonstrated a markedly higher CYLD-mRNA expression compared to the low-CYLD protein expression group (P=0.036) (Fig. 2C).

Discussion

In this study, we showed that reduced CYLD-mRNA expression is associated with a poor prognosis in HCC patients, since the incidence of early recurrence (i.e., within 2 years) was higher in the low compared to the high-CYLD expression group. The pattern of recurrence was similar between the two groups. Since intrahepatic recurrence within 2 years is considered an intrahepatic metastasis from the primary tumor, this outcome suggests that CYLD is associated with metastatic potential and, thus, a poor prognosis. CYLD-mRNA expression demonstrated no correlation with tumor-related factors with the exception of serum AFP. AFP production has been strongly associated with specific molecular subtypes of HCC, such as hepatoblastoma (23), while a reduced CYLD expression may therefore be associated with a specific molecular phenotype.

A recent in vivo study demonstrated that a liver-specific conditional knockout of CYLD induced apoptosis in hepatocytes via the chronic activation of TGF-β-activated kinase 1 and c-Jun N-terminal kinase (JNK) in the periportal area. As a result, this promoted progressive fibrosis and inflammation, resulting in cancer development (24). Although CYLD expression was expected to be potentially associated with certain types of carcinogenesis from viral hepatitis or liver cirrhosis due to chronic inflammation, no correlation was observed between CYLD expression and non-tumor liver tissue. A previous in vitro study demonstrated that HCC cells transfected with the CYLD gene showed an increased NF-κB reporter activity (18). The present study supports the clinical and oncological importance of CYLD in HCC progression.

A limited number of clinical studies have investigated the protein expression and distribution of CYLD in solid types of cancer such as HCC. Notably, in this study, immunohistochemical analysis showed that CYLD expression was distributed according to tumor cell morphology within the same tumor, and tumor cells that lost their cell polarity tended to lose CYLD expression. The mechanism underlying staining pattern remains unclear, and further investigation is required to better understand the role of CYLD in dysplastic cell morphology and chromatin structure.

In conclusion, the present study suggests that CYLD is associated with tumor development in HCC patients. This is a preliminary study and, as a result, the functional aspect of CYLD in HCC patients needs to be further investigated. However, the present study is considered to be useful in investigating whether CYLD may be a future molecular target in HCC patients.

Abbreviations:

AFP

α-fetoprotein;

CYLD

the cylindromatosis gene;

HCC

hepatocellular carcinoma;

PIVKA-II

protein induced by vitamin K absence or antagonist-II;

qRT-PCR

quantitative reverse transcription-polymerase chain reaction

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Spandidos Publications style
Kinoshita H, Okabe H, Beppu T, Chikamoto A, Hayashi H, Imai K, Mima K, Nakagawa S, Yokoyama N, Ishiko T, Ishiko T, et al: CYLD downregulation is correlated with tumor development in patients with hepatocellular carcinoma. Mol Clin Oncol 1: 309-314, 2013.
APA
Kinoshita, H., Okabe, H., Beppu, T., Chikamoto, A., Hayashi, H., Imai, K. ... Baba, H. (2013). CYLD downregulation is correlated with tumor development in patients with hepatocellular carcinoma. Molecular and Clinical Oncology, 1, 309-314. https://doi.org/10.3892/mco.2013.68
MLA
Kinoshita, H., Okabe, H., Beppu, T., Chikamoto, A., Hayashi, H., Imai, K., Mima, K., Nakagawa, S., Yokoyama, N., Ishiko, T., Shinriki, S., Jono, H., Ando, Y., Baba, H."CYLD downregulation is correlated with tumor development in patients with hepatocellular carcinoma". Molecular and Clinical Oncology 1.2 (2013): 309-314.
Chicago
Kinoshita, H., Okabe, H., Beppu, T., Chikamoto, A., Hayashi, H., Imai, K., Mima, K., Nakagawa, S., Yokoyama, N., Ishiko, T., Shinriki, S., Jono, H., Ando, Y., Baba, H."CYLD downregulation is correlated with tumor development in patients with hepatocellular carcinoma". Molecular and Clinical Oncology 1, no. 2 (2013): 309-314. https://doi.org/10.3892/mco.2013.68