Modern Pathology (2011) 24, 175–184 & 2011 USCAP, Inc. All rights reserved 0893-3952/11 $32.00 175 A novel role for TNFAIP2: its correlation with invasion and metastasis in nasopharyngeal carcinoma Lih-Chyang Chen1, Chia-Chun Chen2, Ying Liang1, Ngan-Ming Tsang3, Yu-Sun Chang1,2 and Chuen Hsueh4 1Chang Gung Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan; 2Graduate Institute of Basic Medical Sciences, Chang Gung University, Taoyuan, Taiwan; 3Department of Radiation Oncology, Chang Gung Memorial Hospital at Lin-Kou, Taoyuan, Taiwan and 4Department of Pathology, Chang Gung Memorial Hospital at Lin-Kou, Taoyuan, Taiwan Tumor necrosis factor alpha (TNFa) is an inflammatory cytokine that is present in the microenvironment of many tumors and is known to promote tumor progression. To examine how TNFa modulates the progression and metastasis of nasopharyngeal carcinoma, we used Affymetrix chips to identify TNFa-inducible genes that are dysregulated in this tumor. Elevated expression of TNFAIP2, which encodes TNFa-inducible protein 2 and not previously known to be associated with cancer, was found and confirmed by quantitative RT-PCR of TNFAIP2 expression in nasopharyngeal carcinoma and adjacent normal tissues. Immunohistochemical analysis showed that the TNFAIP2 protein was highly expressed in tumor cells. Analysis of 95 nasopharyngeal carcinoma biopsy specimens revealed that high TNFAIP2 expression was significantly correlated with high- level intratumoral microvessel density (P ¼ 0.005) and low distant metastasis-free survival (P ¼ 0.001). A multivariate analysis further confirmed that TNFAIP2 was an independent prognostic factor for nasopharyngeal carcinoma (P ¼ 0.002). In vitro, TNFa treatment of nasopharyngeal carcinoma HK1 cells was found to induce TNFAIP2 expression, and siRNA-based knockdown of TNFAIP2 dramatically reduced the migration and invasion of nasopharyngeal carcinoma HK1 cells. These results collectively suggest for the first time that TNFAIP2 is a cell migration-promoting protein and its expression predicts distant metastasis. Our data suggest that TNFAIP2 may serve as an independent prognostic indicator for nasopharyngeal carcinoma. Modern Pathology (2011) 24, 175–184; doi:10.1038/modpathol.2010.193; published online 5 November 2010 Keywords: metastasis; migration; nasopharyngeal carcinoma; TNFAIP2 Tumor necrosis factor alpha (TNFa), a major inflam- which TNFa increases tumor progression have not matory cytokine in the tumor microenvironment, yet been fully defined. is produced by both malignant and non-cancerous Nasopharyngeal carcinoma is a highly metastatic cells,1,2 and has been shown to mediate cancer- head and neck cancer that is relatively rare among related inflammation and promote tumor progres- Caucasians, but is common in southeastern China sion by enhancing angiogenesis and metastasis.3 and in Taiwan.8 In Hong Kong, the incidence of Increased TNFa levels in the tumor microenviron- nasopharyngeal carcinoma is as high as 20–30 per ment and plasma have been associated with poor 100 000 in men and 15–20 per 100 000 in women.8 prognosis in various cancers, including prostate According to data from the Cancer Registry of cancer,4 breast cancer,5 and nasopharyngeal Taiwan, nasopharyngeal carcinoma is the ninth carcinoma.6,7 However, the mechanisms through most common cancer, with an estimated incidence of B6 cases per 100 000 in 2002. Under the current treatment regimens, the survival rates among nasopharyngeal carcinoma patients are B92% at 1 Correspondence: Dr C Hsueh, MD, Department of Pathology, year and B50% at 5 years, with 20–25% of Chang Gung Memorial Hospital at Lin-Kou, 5, Fu-Hsing Street, patients eventually developing distant metastases.9,10 Kwei-Shan, Taoyuan 333, Taiwan. E-mail: [email protected] Nasopharyngeal carcinoma is generally sensitive Received 8 June 2010; revised 25 August 2010; accepted 30 to radiation therapy, but more advanced cases August 2010; published online 5 November 2010 may require a combination of radiotherapy and www.modernpathology.org TNFAIP2 promotes metastasis 176 L-C Chen et al chemotherapy.11 Thus, it is essential for us to previously.19 This study was reviewed and approved develop more effective diagnostic and therapeutic by the institutional review board and ethics committee strategies against nasopharyngeal carcinoma. of Chang Gung Memorial Hospital. Informed consent In an effort to clarify the underlying mechanisms was obtained from all patients. of malignancy in nasopharyngeal carcinoma, our All enrolled patients had been treated with group previously used Affymetrix-chip-based micro- definitive radiotherapy (cumulative dose of external array hybridization to identify various nasopharyn- beam radiotherapy 4R64.8 Gy). Among them, 33 geal carcinoma metastasis-associated biomarkers. patients had received additional chemotherapy in We analyzed the various TNFa-regulated genes that the Department of Medical Oncology at Chang Gung appeared to be upregulated in nasopharyngeal Memorial Hospital. Patients who presented with carcinoma tumor cells vs adjacent normal tissues, distant metastatic disease at diagnosis (M1 stage) and found differential expression of various TNFa and those who had undergone previous treatment at target genes MMP9,12 MMP12,13 MMP14,14 BIRC5,15 another institute were excluded from this study. For CXCL13,16 IL12B,17 c-FLIP,18 and thymidine phos- all enrolled patients, pathology reports were phorylase.19–21 We further showed that the upregula- obtained from the pathological database and medi- tion of thymidine phosphorylase in nasopharyngeal cal records, and the relevant slides were reviewed to carcinoma tumor cells resulted at least in part from confirm the nasopharyngeal carcinoma diagnosis cytoplasmic heterogeneous nuclear ribonucleopro- (reviews were performed by C Hsueh). Clinical data tein K (hnRNP K)-mediated stabilization of the on stage, treatment, and follow-up were collected thymidine phosphorylase mRNA.22 High levels of from hospital tumor registries and medical files. The thymidine phosphorylase and cytoplasmic hnRNP K primary end point was disease-free survival, which were significantly correlated with overall survival was calculated from the date of diagnosis to the date and distant metastasis.19 On the basis of this success, of death or last follow-up. Distant metastasis-free we have continued to search for other TNFa- survival and local recurrence-free survival were also regulated genes that could serve as potential bio- assessed. The time to local recurrence or distant markers for nasopharyngeal carcinoma prognosis. metastasis was calculated by using the date on TNFAIP2 is also a TNFa-regulated gene that is which the local recurrence or distant metastasis was expressed in endothelial cells,23 peripheral blood detected as the end point. Patients who died with- monocytes,24 and mature sperm.24 Here we investi- out local recurrence or distant metastasis were gated TNFAIP2, which is found to be highly omitted from our analyses of disease-free survival, expressed at both the mRNA and protein levels in local recurrence-free survival, and distant metasta- nasopharyngeal carcinoma tumor cells vs adjacent sis-free survival. normal tissues. TNFAIP2 encodes a protein whose precise function is not known, but appears to be Affymetrix Microarray Analysis involved in endothelial capillary tube formation in vitro.23 Although TNFAIP2 has been classified as RNA samples from nasopharyngeal carcinoma an angiogenic factor,23 its function and significance tissues were isolated using the TRIzol reagent in tumor development have not previously been (Invitrogen, Carlsbad, CA, USA). The biotinylated determined. Here, we show for the first time that oligonucleotide was hybridized to the Human increased expression of TNFAIP2 is significantly Genome U133 Plus 2.0 Array (Affymetrix, Santa correlated with shorter distant metastasis-free sur- Clara, CA, USA) by the National Yang-Ming Uni- vival in nasopharyngeal carcinoma patients, and versity Genomics Center (Taiwan), following the that TNFAIP2 promotes the migration and invasion manufacturer’s instructions. The microarray data of nasopharyngeal carcinoma cells in vitro. These were analyzed using the GeneSpring version GX results suggest that TNFAIP2 may serve as a 7.31 software package (Silcogenetics, Redwood, CA, potential diagnostic and independent prognostic USA). The relative expression level of each gene was marker for nasopharyngeal carcinoma. calculated as the ratio of the average hybridization intensity from nine individual nasopharyngeal carcinoma tissues vs that from a pool of the Materials and methods corresponding adjacent non-tumor tissues. Patients, Clinical Staging, Treatment, and Assessment of Clinical Outcome Immunohistochemical Staining Analysis The utilized retrospective cohort comprised 95 naso- Immunohistochemical analyses were performed pharyngeal carcinoma patients who had been ad- using an automatic immunohistochemical staining mitted to Chang Gung Memorial Hospital at Lin-Kou device according to the manufacturer’s instructions from 1990 to 2006. AJCC stage, T stage, and N stage (Bond-max Automated Immunostainer; Vision Bio- were defined according to the 2002 cancer-staging Systems, Melbourne, Australia). Tissue sections system revised by the American Joint Committee on were retrieved using Bond Epitope Retrieval Cancer. Histological typing was performed according Solution 1 (Vision BioSystems) and stained with to the WHO classification criteria,