Elsevier

Human Pathology

Volume 43, Issue 7, July 2012, Pages 1012-1019
Human Pathology

Original contribution
NEMO expression in human hepatocellular carcinoma and its association with clinical outcome

https://doi.org/10.1016/j.humpath.2011.08.009Get rights and content

Summary

The nuclear factor κ-light-chain enhancer of activated B-cells (NF-κB) signaling pathway is regarded as an important factor in inflammation and carcinogenesis. Recently, a role in hepatocarcinogenesis has been attributed to the NF-κB regulatory subunit IKKγ (NEMO) using knockout mice. However, a detailed investigation of NEMO expression in human hepatocellular carcinomas (HCCs) has not yet been reported. We selected 85 HCC patients who had undergone curative liver resection and analyzed NEMO expression of the respective tumors by immunohistochemistry, Western blotting, and real-time PCR. NEMO expression was correlated with clinicopathological parameters, and the impact on 5-year disease-free survival and 5-year overall survival was calculated using multivariate Cox proportional models. In our study, complete loss of NEMO immunoreactivity was found in 34 (40%) of 85 HCCs compared with their adjacent nonneoplastic tissue (P < .05). NEMO messenger RNA (mRNA) expression was detected in all HCC cases; however, no correlation between NEMO immunoreactivity and mRNA level was found. Five-year overall survival rates for patients with low and high NEMO expression were 22% and 50%, respectively (P = .049). However, high tumor stage, but not level of NEMO expression, was confirmed as an independent poor prognostic factor for 5-year disease-free survival (hazards ratio [HR] = 2.1, 95% confidence interval [CI] = 1.3-3.6, P = .009) and 5-year overall survival (HR = 2.5, CI = 1.4-4.4, P = .002). In conclusion, a loss of NEMO immunoreactivity occurs in a substantial proportion of human HCCs. Although low NEMO expression is correlated with a poor 5-year overall survival in patients with HCC, NEMO cannot be regarded as an independent prognostic marker for predicting the clinical outcome of patients suffering from HCC.

Introduction

Hepatocellular carcinoma (HCC) represents the fifth most common cancer worldwide and the third leading cause of cancer-related mortality [1]. Complete tumor resection is considered the criterion standard in curative treatment of HCC [2]. Despite successful primary tumor resection, HCC patient prognosis substantially differs among patients of the same clinical stage. The reason for this variation in clinical outcome remains largely unknown. Many studies have attempted to evaluate clinicopathological features or discover novel biomarkers that enable a better prediction of the clinical course of the disease [3], [4], [5], [6]. HCC frequently arises as a consequence of malignant transformation of hepatocytes in the setting of liver cirrhosis, which is the result of chronic inflammatory processes [7], [8], [9].

The transcription factor nuclear factor κB (NF-κB) is considered to be a master regulator that translates external inflammatory stimuli into the activation of hundreds of responsive genes [10]. NF-κB is localized in the cytoplasm in an inactive state, tightly bound to its inhibitor protein IκB. Upon stimulation of the corresponding receptors by various external stimuli like proinflammatory factors, an intracellular signaling cascade is triggered. This leads to the activation of the IκB kinase (IKK) complex. The IKK complex consists of 2 catalytic subunits, namely IKK1, also known as IKKα, and IKK2, which is also known as IKKβ, and a regulatory subunit NF-κB essential modifier (NEMO, also known as IKKγ) [10]. IKK complex activation results in IκB phosphorylation and ubiquitination, followed by the subsequent proteasome-dependent IκB degradation. NF-κB translocates to the nucleus, where it then induces the transcription of multiple target genes [10].

A role for NEMO in hepatocarcinogenesis has recently been suggested by data exclusively derived from animal models, where the selective ablation of NEMO in liver cells causes spontaneous liver tumor development [11]. Based on these data, a potential tumor suppressive role for NEMO was proposed. Tumor development in this mouse model was preceded by chronic liver disease resembling human nonalcoholic staetohepatitis (NASH) [11]. Considering the important role that NF-κB/NEMO exhibits in carcinogenesis, strategies for NEMO-directed pharmacological agents have been initiated [12].

Given the therapeutic potential of this regulatory protein and the controversial role of NEMO in different animal models, further research to elucidate the role of NEMO in human HCC is warranted. Until now, the expression pattern of NEMO in human HCC and its prognostic significance in HCC has not been explored. We examined NEMO expression in human HCC by immunohistochemistry, Western blotting, and real-time PCR (RT-PCR) approaches. The role of NEMO as useful immunohistochemical prognostic marker was evaluated by correlating its expression level with several clinicopathological features. Finally, we analyzed the influence of these parameters on 5-year disease-free survival and overall survival in HCC patients.

Section snippets

Patients and tissue samples

A total of 85 patients with primary HCC who underwent a curative liver resection at the Department of Surgery of the Medical University of Graz, Austria, were included in this retrospective study. Tissues used in the study were retrieved from the BioBank of the Medical University of Graz. We included consecutive patients diagnosed with HCC between January 1988 and December 2009. The median follow-up period was 8.8 years; none of the patients received neoadjuvant therapy or preoperative local

Patients' characteristics and histopathology

Clinicopathological patient characteristics including sex, age, tumor stage, tumor grade, growth pattern, presence of liver cirrhosis, and underlying liver disease are detailed in Table 1. The majority of tumors showed vascular invasion or multiple nodules within the liver. Tumor grades were G1 in 20 cases (23.5%), G2 in 50 cases (58.8%), and G3 in 15 cases (17.6%). Overall, there was more HCC in male patients (74%), in cirrhotic livers (66%), and with trabecular growth pattern (89%).

NEMO immunohistochemical staining in HCC

Setting

Discussion

In the present study, we provide the first analysis of NEMO immunohistochemical staining and mRNA expression in human HCC tissue and its association with patient clinical outcome. NEMO immunoreactivity was significantly reduced in a substantial proportion of HCC cases compared with their surrounding nonneoplastic liver tissue. In contrast to the steatohepatitic features found in NEMO knockout animals, we were not able to confirm such an association by correlating the presence of steatohepatitis

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    These authors contributed equally to this work.

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