Yes-Associated Protein Expression in Head and Neck Squamous Cell Carcinoma Nodal Metastasis

et al. (2011) Yes-Associated Protein Expression in Head and Neck Squamous Cell Carcinoma Nodal Metastasis. PLoS ONE 6(11): e27529. doi:10.1371/journal.pone.0027529 Yes-Associated Protein Expression in Head and Neck Squamous Cell Carcinoma Nodal Metastasis Lin Ge 0 Matthew Smail 0 Wenxia Meng 0 Yu Shyr 0 Fei Ye 0 Kang-Hsien Fan 0 Xiaohong Li 0 Hong-Mei 0 Zhou 0 Neil A. Bhowmick 0 Torbjorn Ramqvist, Karolinska Institutet, Sweden 0 1 Department of Medicine, Cedars-Sinai Medical Center , Los Angeles , California, United States of America, 2 State Key Laboratory of Oral Diseases, Sichuan University , Chengdu, Sichuan , China , 3 Department of Biostatistics, Vanderbilt University , Nashville , Tennessee, United States of America, 4 Department of Cancer Biology, Vanderbilt University , Nashville , Tennessee, United States of America, 5 Department of Oral Medicine, West China Hospital of Stomatology, Sichuan University , Chengdu, Sichuan , China Introduction: Yes-associated protein (YAP) is considered an oncogene found amplified in multiple tumors, including head and neck squamous cell carcinoma (HNSCC). However, the role for YAP expression in HNSCC is not understood. Based on the central role of YAP in the hippo pathway, we tested if YAP was associated with the stage of HNSCC progression and metastatic potential. Methods: To determine the expression of YAP in human benign and HNSCC tissue specimens, immunohistochemical analyses were performed in whole tissue samples and tissue microarrays. The expression of YAP in tissues of microarray was first associated with clinic-pathologic factors and results verified in samples from whole tissue sections. To investigate the role of YAP and p63 in regulating HNSCC epithelial to mesenchymal transition, epithelial and mesenchymal markers were assayed in Fadu and SCC-25 cells, HNSCC cells with endogenously elevated YAP expression and siRNA-mediated expression knockdown. Results: Analysis of human HNSCC tissues suggested YAP expression was elevated in tumors compared to benign tissues and specifically localized at the tumor invasive front (p value ,0.05). But, indexed YAP expression was lower with greater tumor grade (p value = 0.02). In contrast, p63 expression was primarily elevated in high-grade tumors. Interestingly, both YAP and p63 was strongly expressed at the tumor invasive front and in metastatic HNSCC. Strikingly, we demonstrated YAP expression in the primary HNSCC tumor was associated with nodal metastasis in univariate analysis (p value = 0.02). However, the knockdown of YAP in Fadu and SCC-25 cell lines was not associated with changes in epithelial to mesenchymal transdifferentiation or p63 expression. Conclusion: Together, YAP expression, in combination with p63 can facilitate identification of HNSCC tumors from hyperplastic and benign tissues and the metastatic function of YAP in HNSCC may not be a result of epithelia to mesenchymal transdifferentiation. - Funding: This study was supported by grants from the National Natural Science Foundation of China (No. 30872873 to H-MZ), the Doctoral Fund of the Ministry of Education of China (No. 20070610067 to H-MZ), and the State Scholarship Fund of China (No. 2008624092 to LG). It was also supported by the National Cancer Institute/National Institute of Health grant (RO1 CA108646 to NAB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Head and neck squamous cell carcinoma (HNSCC) can involve oral cavity, larynx and laryngeal pharynx. Oral squamous cell carcinoma (OSCC) represents 90% of oral cancers. Alterations in the 11q22 amplicon is detected in 515% of OSCC [1]. The gene, Yes-associated protein (YAP), located in 11q22, is specifically amplified in 4/23 of OSCC [2]. YAP was initially proposed as an oncogene following the identification of its expression in mouse tumor models [3,4]. Later, multiple groups reported the up regulation of YAP in human cancers including those in the prostate, ovary, and colon [5,6]. YAP is a critical transcription factor in the Hippo signaling pathway, initially identified for sensing and regulating organ size [7]. As such, YAP disregulation was further proposed as candidate oncogene in hepatocellular carcinoma, non-small-cell lung carcinoma, esophageal squamous cell carcinoma, ovarian cancer, gastric cancer [711]. However, in breast cancer, YAP is considered a tumor suppressor, based on reduced xenografted mammary tumor size in the context of YAP knockdown [8]. Opposing functions in different anatomical tissues might be due to its role with p53 family of proteins including p73 and p63, as well as interactions with transcription enhancer factor (TEF/TEAD) [1217]. YAP is reported to cause EMT, stimulate proliferation, inhibit apoptosis, and promote tumor progression in a tissue-specific manner [7,9]. The specific role of YAP in HNSCC is examined in this study. A hallmark of HNSCC progression is elevated expression of the protein p63. P63 has two mRNA products from independent promoters, with each mRNA having three splice variants giving altered C-termini. Of the six p63 isoforms, delta-Np63 alpha is dominant in high grade HNSCC [10,11]. Recently YAP was reported to promote the degradation of delta-Np63 alpha in head and neck carcinoma cell lines [12]. Further, Np63 binds the YAP promoter and repress its expression [21]. Thus, an inverse correlation between Np63 and YAP was speculated in patient tissues. However, the inverse relationship between YAP and p63 expression was not supported in human HNSCC tissues or cell lines. Our data indicated that elevated YAP expression differentiates hyperplastic and low grade HNSCC from benign head and neck tissues, with an unexpected decrease in expression in high grade HSCC. However, there was consistent elevation of YAP expression localized at the HNSCC tumor invasive edge. Interestingly, YAP expression or its knockdown did not affect epithelial-mesenchymal transition (EMT) progression in HSCC cell lines, nor did it affect p63 expression. The role of YAP expression in differentiating benign head and neck tissue from HNSCC can support its use as a marker to complement p63 in identifying tumor margins. Ethics Statement The study was performed with the written approval of the Ethics Committee of West China College of Stomatology, Sichuan University. We acquired written consent from all patients. The use of the data from the tissues was considered exempt by the Institutional Review Board at Vanderbilt University. Patients and samples Twenty-three OSCC patients and six oral leukoplakia patients confirmed by pathologic diagnosis were` included in this study. Among them, 22 primary tumor samples of OSCC had been Recurrence or metastasis obtained from 2004 to 2008, with a 34-month median follow-up period (2573 month range). Six patients undergoing ortho (...truncated)