Identification of RKIP as an Invasion Suppressor Protein in Nasopharyngeal Carcinoma by Proteomic Analysis Yan Chen,# Guo-Liang Ouyang,# Hong Yi, Mao-Yu Li, Peng-Fei Zhang, Cui Li, Jian-Ling Li, Ying-Fu Liu, Zhu-Chu Chen, and Zhi-Qiang Xiao* Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, China Received August 7, 2008
To identify novel proteins associated with the pathogenesis of nasopharyngeal carcinoma (NPC), a proteomic approach was used to screen for differential proteins between NPC and adjacent noncancerous nasopharyngeal epithelial tissue (ANNET). As a result, 21 differential proteins were identified by two-dimensional electrophoresis and mass spectrometer. Raf kinase inhibitor protein (RKIP), one of the downregulated proteins in NPC compared to ANNET, was investigated for its role in the metastasis of NPC. Western blot analysis and immunohistochemistry were used to detect RKIP expression in 5-8F and 6-10B NPC cell lines with the different metastatic potentials, and in NNET, primary NPC and NPC metastasis. Furthermore, high metastatic 5-8F with low RKIP expression and nonmetastatic 6-10B with high RKIP expression were stably transfected with plasmids that expressed sense and antisense RKIP cDNA, respectively, or with empty vector. The effects of RKIP expression on in vitro cell invasion, and the activity of Raf-1/MEK/ERK signaling pathway were analyzed in the transfected cells. The results showed that RKIP was significantly downregulated in 5-8F compared with 6-10B, in NPC compared with NNET, and not detectable in NPC metastasis. Overexpressed RKIP in 5-8F could decrease its in vitro cell invasion, whereas downregulated RKIP in 6-10B could increase its in vitro cell invasion. RKIP negatively regulated Raf-1/MEK/ERK signaling pathway in NPC cells, and activation of this signaling pathway by RKIP downregulation increased in vitro invasion of NPC cells. Taken together, our results suggest that RKIP may be a NPC metastasis suppressor, and decreased RKIP expression is associated with the increased invasive capability of NPC cells possibly through the activation of Raf-1/MEK/ERK pathway. Keywords: Nasopharyngeal carcinoma • Proteomics • RKIP • Metastasis
Introduction Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China and Southeast Asia.1 Early metastasis is one of distinctive characteristics of NPC and the main cause of death for NPC patients.2 In this cancer, 70-80% of new cases present with lymph node metastases in the neck,3 and about 4.2% of those present with distant metastasis to the bone, lung, liver, and central nervous system.4 However, the molecular mechanism of NPC pathogenesis is not yet well-defined. To develop better diagnosis and treatment approaches, it is important to understand the molecular basis of the development and progression of NPC. High-throughput technologies such as microarrays and proteomics offer the potential ability to find alterations previously unidentified in NPC. Analyses for gene expression profiles of NPC have been reported using a cDNA array, and the genes with aberrant expressions which possibly contribute to me* To whom correspondence should be addressed. Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China. Tel: (86)7314327239. Fax: (86)731-4327321. E-mail:
[email protected]. # These authors contributed equally to this work.
5254 Journal of Proteome Research 2008, 7, 5254–5262 Published on Web 10/28/2008
tastasis of NPC have been found.5,6 Proteomics has introduced a new approach to cancer research which aims at identifying differential expression proteins associated with carcinogenesis, providing new opportunities to reveal the molecular mechanism underlying this disease.7 To identify novel proteins associated with the pathogenesis of NPC, we performed comparative proteomics on NPC and adjacent noncancerous nasopharyngeal epithelial tissue (ANNET). A potential metastasis suppressor protein was identified from this approach, Raf kinase inhibitor protein (RKIP), which was downregulated in NPC compared with ANNET. RKIP, a member of the phosphatidylethanolamine-binding protein family, is an evolutionarily conserved and widely expressed protein.8 RKIP has been identified as an endogenous inhibitor of the Raf-1/MEK/ERK signaling pathway,9 and a potential metastasis suppressor.10,11 RKIP directly interacts with Raf-1 and MEK, and disrupts the interaction between Raf-1 and MEK1, thereby preventing the activation of MEK by Raf-1 and downstream signal transduction.9 The Raf-1/MEK/ERK signaling pathway is activated in about 30% of all cancers and has been implicated in invasiveness and metastasis in many in vitro models.12 Studies have shown that loss of RKIP expression 10.1021/pr800602c CCC: $40.75
2008 American Chemical Society
research articles
Proteomics and Metastasis of NPC promotes metastasis possibly through the activation of this signaling pathway in certain cancers.13,14 Loss of function of metastasis suppressors is a key step in the cancer metastasis and progression. Studies in cell culture and animal models have demonstrated that RKIP suppresses the metastasis of prostate cancer,15 ovarian cancer16 and melanoma cells.17 RKIP downregulation or loss correlates with the metastasis of multiple human malignant tumors such as prostate,15 ovarian cancer,16 melanoma,17 and breast,18 colorectal19 and hepatocellular carcinoma.20 The accumulating evidence have also suggested that the downregulation or loss of RKIP is indicative of poor prognosis in the cancer patients.21-24 However, the function of RKIP in NPC has not been reported. Therefore, we were interested in the investigation of the role of RKIP in the metastasis of NPC, a cancer notorious for its high potential to metastasize. In this study, based on the results of the comparative proteomic analysis of NPC tissue and ANNET, we examined RKIP expression in NPC cell lines 5-8F and 6-10B with different metastatic potentials and NPC clinical specimens, and analyzed the effects of RKIP expression modulation on in vitro cell invasion and Raf-1/MEK/ERK signaling pathway in NPC. To our knowledge, this is the first report on the association of RKIP loss with NPC metastasis through activation of Raf-1/MEK/ERK signaling pathway.
Table 1. The Clinicopathological Parameters of the NPC Tissue Specimensa classification
number
Gender Male Female Age g50 1000 cells were counted for each section. The intensity of staining was graded on the following scale: 0, no staining; 1+, mild staining; 2+, moderate staining; 3+, intense staining. The area of staining was evaluated as follows: 0, no staining of cells in any microscopic fields; 1+, 60% stained positive. The minimum score when summed (extension + intensity) was therefore 0 and the maximum, 6. A combined staining score (extension + intensity) of e2 was considered to be negative staining; a score between 3 and 4 was considered to be moderate staining; that between 5 and 6 was considered to be strong staining. Statistical Analysis. The statistical software package SPSS 13.0 was used in this study. Single comparisons were performed using Student’s t test or Mann-Whitney’s U test, and multiple comparisons were performed using one-way analysis of variance (ANOVA). All statistical tests were two-sided. Differences were considered statistically significant for P-values
