NDRG1 as a biomarker for metastasis, recurrence and of poor prognosis in hepatocellular carcinoma

Abstract

N-myc downstream-regulated gene 1 (NDRG1) has been reported to be a multifunctional protein associated with carcinogenesis, however, the cellular function of NDRG1 remains elusive in human cancers. Here, our proteomics profile analysis of HCC tissues with different metastatic capabilities revealed that NDRG1 was correlated with metastasis and recurrence in HCC patients after liver transplantation (LT). Immunohistochemical staining of 143 HCC patients after LT showed that NDRG1-positive expression had poor prognosis, either for shorter disease-free survival or overall survival (P < 0.001), compared with NDRG1-negative expression. Multivariate analysis confirmed NDRG1 as an independent prognostic value (P < 0.001). In addition, in vitro experiments HCC cells with small interfering RNA against NDRG1 significantly suppressed its proliferation, colony formation, invasion and migration ability. Microarray analysis revealed that NDRG1 modulated the expression of genes associated with transmembrane transporter activity, chemoattractant activity, immune response, cell adhesion and cell proliferation process. Taken together, these results suggested that NDRG1 was an important molecule in controlling HCC metastasis and thus suggested as a novel biomarker for predicting HCC recurrence after LT.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, accounting for the third cause of cancer-related death due to poor prognosis. There were 626,000 new cases estimated around the world annually, with about half in China alone [1]. The overwhelming majority of HCC is caused by exposure to aflatoxins, infection with hepatitis B and C viruses. It has been shown that small size of HCC can be cured by surgical resection or liver transplantation. Unfortunately, the disease is often diagnosed at an advanced stage when conventional and effective treatment options become unavailable [2]. Localized disease can be treated by means of surgical resection, liver transplantation, radiofrequency ablation and chemoembolization [2], [3], [4], [5]. However, HCC represents a tumor type of high invasion and aggression. Recurrence of HCC following treatments remains to be one of the most prevalent causes leading to poor long-term survival. Therefore, studying the molecular pathogenesis of cancer recurrence can guide our evaluation of the treatment and prognosis of patients.

Hepatocarcinogenesis is a complex process with mutations in numerous protooncogenes and tumor suppressor genes, such as p53, Rb, PTEN, GSTP1, Smad2/4, IGF-2, β-catenin, c-myc, and cyclin D1 [6]. In addition, our center and some other groups have found that aberrant expression of microRNAs were also involved in the process of HCC development [7]. The major signaling pathways, such as Wnt/β-catenin-signaling pathway, MAPK pathway, PI3K/Akt/mTOR pathway, activated in HCC also play pivotal roles in the carcinogenesis. In spit that many molecules in the pathogenesis of liver cancer have been in-depth studied, they are still insufficient for recognizing those patients who are at high risk of recurrence [8]. Currently, we still use vascular invasion, the number and size of nodules, and high serum alpha-fetoprotein levels as indicators of recurrence, which are not fully reliable yet [9]. Therefore, molecular “signatures” of recurrence will be essentially important for properly control of HCC.

Identification of new biomarkers of recurrence for early detection, prediction of prognosis, and the response to treatment will be greatly useful to guide the treatment procedure of HCC. Proteomics analysis is currently considered to be a powerful tool for the discovery of new cancer biomarkers and prognostic targets. We undertook this systemic biological approach to identify protein “signatures” that significantly discriminate HCC with recurrence from that without recurrence. A total of 89 differently expressed proteins were identified. N-myc downstream-regulated gene 1 protein (NDRG1), a hypoxia-associated protein, also altered by DNA damage, oncogenes, and tumor-suppressor genes, was chosen to verify its biological and clinicopathologic significance [10], [11], [12]. We have performed immunohistochemistry analysis for clinical HCC materials. Furthermore, the function of NDRG1 in the HCC cells was studied by RNA interference assay. The results revealed that NDRG1 had over-expression in the most of HCC tissues, and NDRG1 protein level in HCC was significantly associated with HCC recurrence. NDRG1 could play a significant role in cancer cell growth and invasion, and be a potential target for the development of therapeutic agents, as well as a novel biomarker for recurrence.