NDRG1 as a biomarker for metastasis, recurrence and of poor prognosis in hepatocellular carcinoma
Highlights
► Proteomics profile shows correlation of NDRG1 to metastasis and recurrence in HCC. ► Poor disease-free survival and overall survival are related with NDRG1 positive HCC. ► NDRG1 modulates genes involved in immune response, adhesion and proliferation.
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.
Section snippets
Patients and samples
HCC tissues and their corresponding normal liver tissues had been obtained earlier from patients in our hospital (The First Affiliated Hospital, Zhejiang University, China) with informed consent. The selection criteria were as follows: (a) confirmed by pathologic diagnosis, (b) without pre-operative anti-cancer treatment and distant metastases before liver transplantation, (c) curative liver transplantation, except perioperative death, (d) with a complete clinicopathological and follow-up data.
Differentially expressed proteins in HCC tissue among recurrence and without recurrence
In order to find a biomarker of HCC recurrence, HCC tissues which are from recurrence or without recurrence after liver transplantation were analyzed in triplicate by 2-DE. After gel-to-gel matching and normalization, 141 protein spots were found to display differential expression in HCC tissues among recurrence and without recurrence by one-way ANOVA. Fig. S1 shows a representative 2-DE gel image of HCC tissues with and without recurrence. Those deregulated spots were excised from the gels and
Discussion
Despite advances in surgical and nonsurgical therapies for hepatocellular cancer, metastasis and recurrence still remain the major challenges in clinical practice, and represent the most cause of death in patients with HCC [5], [10], [19]. The present practical evaluation of the clinical prognosis of liver cancer, including tumor size, number, pTNM stage, venous invasion, may be useful for predicting patient outcome roughly, but those indicators do not have a good linear relationship with HCC
Grant support
This study was financially supported by National Basic Research Program of China (2009CB522407) and Major National S&T Program (2008ZX10002-026).
Conflict of interest
The authors declare no conflict of interest related to this work.
Acknowledgements
We gratefully acknowledge the clinical data support by Jilei Fu, Chenxi Zhang, Shen Yan, Jun Yu; technical assistance by Prof. Bo-Xiong Zhong (Zhejiang University). The authors are grateful to Dr. Grace Marquez (Samford University, Birmingham, Al, USA) and Sunyi Ye for revision of this manuscript.
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