Yin-Ju Chen

DSG3 is overexpressed in head neck cancer and is a potential molecular target for inhibition of oncogenesis

To identify genes that could potentially serve as molecular therapeutic markers for human head and neck cancer (HNC), we employed differential display analysis to compare the gene expression profiles between HNC and histopathologically normal epithelial tissues. Using reverse transcription–polymerase chain reaction and Western blot analysis, desmoglein 3 (DSG3) was identified as being differentially expressed at both the RNA and protein levels. Of 56 patients assayed, 34 (61%) had overexpression of DSG3, which correlated statistically with T stage (P=0.009), N stage (P=0.047), overall stage (P=0.011), tumor depth (P=0.009) and extracapsular spread in lymph nodes (P=0.044), suggesting that DSG3 participates in carcinogenesis of HNC. Consistent with the clinical findings, inhibition of DSG3 by RNA interference (RNAi) significantly reduced cell growth and colony formation to 57–21% in three HNC cell lines. Use of an in vitro wound healing and Matrigel invasion assays, we found that cell migration and invasive ability were also inhibited to 30–48% in three cell lines tested. An in vivo xenograft study showed that administration of DSG3-RNAi plasmid significantly inhibited tumor growth for 2 months in BALB/C nude mice. In conclusion, DSG3 is identified overexpressed in HNC, with the degree of overexpression associated with clinicopathologic features of the tumor. Inhibition of DSG3 significantly suppresses carcinogenic potential in cellular and in vivo animal studies. These findings suggest that DSG3 is a potential molecular target in the development of adjuvant therapy for HNC.

Downregulation of Ches1 and other novel genes in oral cancer cells chronically exposed to areca nut extract

This study was undertaken to identify the genes in response to areca nut extract, a potential carcinogen of oral cancer.

Transcriptome profiling and network pathway analysis of genes associated with invasive phenotype in oral cancer

The aim of this study was to clarify relevant alterations of gene expression associated with the invasive phenotype of oral cancer. To reduce heterogeneity and to obtain data on genes specifically involved in invasive mechanism, we established a highly invasive ORC subline through in vitro Matrigel invasion method. Affymetrix microarrays were used for transcriptome profiling between parental and the highly invasive subline. Seventy-nine genes were differentially expressed at least 2-fold, including 38 up-regulated and 41 down-regulated. After analyzing the microarray data by MetaCore algorithm, a total of 12 regulatory pathways were found to be associated with invasive phenotype (p<0.001). Two functional pathways were most significant: the cell adhesion through extracellular matrix remodeling (p=4.964e-06), and MHC-class-I mediated antigen presentation (p=9.843e-05). To shed more light on the biological functions of invasiveness, two genes highly over-expressed in the invasive subline, Cyr61 and CD44 were further validated. RNAi knockdown of these two genes led to significant suppression of cell growth (32% and 31%, respectively at day 3), cell migration (45% and 96%, respectively at 24 h), and cell invasion (83% and 87%, respectively at day 3). These results suggested important roles of these genes in regulating invasive phenotype, and demonstrated the confidence of this study design in the search of invasive associated genes. The identified pathways associated with invasion mechanism may be novel targets for manipulation of the cancer behavior with consequences on treatment outcome.

GP96 is over-expressed in oral cavity cancer and is a poor prognostic indicator for patients receiving radiotherapy

BackgroundOral cavity cancers (ORC) are the most common cancers, and standard treatment is radical surgery with postoperative radiotherapy. However, locoregional failure remains a major problem, indicating radioresistance an important issue. Our previous work has shown that GP96 contributed to radioresistance in nasopharyngeal and oral cancer cell lines. In this study, we determined clinical significance of GP96 in ORC by evaluation of GP96 expression and its association with disease prognosis in patients receiving radiotherapyMethodsTotal of 79 ORC patients (77 males, median age: 48 years old) receiving radical surgery and postoperative radiotherapy between Oct 1999 and Dec 2004 were enrolled. Patients in pathological stages II, III and IV were 16.5%, 16.5% and 67%, respectively. For each patient, a pair of carcinoma tissue and grossly adjacent normal mucosa was obtained. GP96-expression was examined by western blot analysis, and the association with clinicopathological status was determined.ResultsThree-year locoregional control (LRC), distant metastasis-free survival (DMFS), disease-specific survival (DSS) and overall survival (OS) rates were 69%, 79%, 63% and 57%, respectively. We found that 55 patients (70%) displayed GP96-overexpression in the tumor tissue, which correlated with a higher pN stage (p = 0.020) and tumor depth (> 10 mm) (p = 0.045). Nodal extracapsular spreading (ECS) and GP96-overexpression predicted adverse LRC (p = 0.049 and p = 0.008). When stratified by nodal ECS, the adverse impact of GP96 remained significant in three-year LRC (p = 0.004). In multivariate analysis, GP96-overexpression was also an independent predictor of LRC, DSS and OS (p = 0.018, p = 0.011 and p = 0.012).ConclusionGP96 may play roles in radioresistance which attributes to tumor invasiveness in oral cancer patients receiving radiotherapy. GP96 may serve as a novel prognostic marker of radiotherapy. However, further independent studies are required to validate our findings in a larger series.

Fascin is a circulating tumor marker for head and neck cancer as determined by a proteomic analysis of interstitial fluid from the tumor microenvironment.

Abstract Background: Head and neck cancer (HNC) is a prevalent cancer worldwide; however, clinically useful tumor markers for HNC have not been identified. Here, we aimed to identify secretory proteins from the tumor microenvironment as candidate circulating tumor markers. Methods: Samples derived from seven pairs of tumor interstitial fluid (TIF) and normal interstitial fluid (NIF) samples from patients with HNC were analyzed. The proteomes were determined by gel-based-mass-spectrometry proteomic methods. The most up-regulated protein, fascin was confirmed in the cancer tissues and cell culture supernatant by immunoblotting and immunohistochemistry assays. Serum fascin was determined in 40 HNC and 40 normal individuals by ELISA. Results: After proteomics analysis, 189 peptides were identified, corresponding to 75 proteins. Of the 21 proteins which were identified more than twice, five up-regulated proteins identified most frequently including fascin. The most elevated fascin was over-expressed in cancer tissues and cell culture supernatant. Serum fascin was significantly up-regulated in the cancer patients (p<0.001) and correlated with pathological lymph node metastasis (p=0.022). To assess the diagnostic efficacy, serum levels of fascin and another potential biomarker SCCA were determined. Fascin showed a high predictable value with an area under the curve (AUC) of 0.808 (95% CI 0.723–0.901) in the receiver operator curve (ROC), compared to 0.501 (95% CI 0.378–0.634) for SCCA. Conclusions: We have identified 75 potential circulating tumor markers associated with HNC, including fascin. Serum fascin could discriminate cancer patients from healthy individuals; thus, it may serve as a circulating biomarker for HNC.

Abstract 4026: Suppression of carcinogenic phenotypes by DSG3 silencing occurs via a plakoglobin-mediated signaling pathway

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC DSG3 has been reported to act as an oncogene in head and neck cancers (HNCs), but the underlying mechanism of its oncogenic activity is unclear. In this study, we aimed to validate the proposed oncogenic function of DSG3 and investigate its downstream regulatory mechanisms. When we stably transfected HNC cells with a short hairpin DSG3-RNAi (shDSG3) to suppress DSG3 expression, reductions in cell growth (70%) and colony formation (>90%) were observed, in addition to cell cycle arrest at the G0/G1 phase. Both cell migration and invasion abilities were also decreased, to 10% and 16%, respectively, after one day. Because DSG3 is associated with plakoglobin in desmosomes, we examined whether or not the phenotypic alterations that occur following DSG3 knockdown were mediated through the plakoglobin signaling pathway. Immunoprecipitation and immunofluorescent staining analyses revealed that DSG3 knockdown disrupted the interaction of DSG3 with plakoglobin and induced the translocation of plakoglobin from the cytoplasm to the nucleus. Using a luciferase reporter assay and western blot analysis, we found that DSG3 knockdown significantly reduced TCF/LEF transcriptional activity and subsequently suppressed the expression of the downstream target genes of the TCF/LEF transcription factors, including c-myc, cyclin D1, and MMP7, which are involved in the regulation of cell proliferation and invasion. An in vivo xenograft study showed that knockdown DSG3 expression significantly inhibited tumor growth for 2 months in BALB/C nude mice and also reduces c-myc, cyclin D1 and MMP7 expression. In conclusion, we found that DSG3 has an oncogenic function in HNC. The suppression of DSG3 expression can interfere with the plakoglobin-mediated signaling pathway, subsequently leading to a reduction of pathogenicity in HNC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4026.

Abstract 3067: Differential proteomic profilingidentifies HNSCCinvasion genes: GANAB is negative regulator in HNSCC invasion

Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent cancer worldwide, however, highly invasive or metastatic characteristics are the common cause of treatment failure. In order to investigate the invasive mechanism of cancer, we established highly invasive sublines of five HNC cell lines by using Matrigel and Transwell selection method. After confirmed the invasion phenotype, the differential proteomes were determined using proteomic methods. Total of 420 protein bands were identified by mass spectrometry analysis, which corresponds to 184 proteins. There were 52 proteins identified more than twice of the four independent experiments, indicating the significance of these proteins in regulation of invasion phenotype. In which, 18 (35%) functions as cytoskeleton or adhesion molecules, 10 (19%) function as molecular chaperone, 9 (17%) belongs to metabolic enzymes, 8 (15%) involve in transcriptional or translational regulation, 7 (13%) involve in cellular signalling pathway. RT-PCR analysis revealed 13 genes that were consistently differentially expressed in the HNSCC invasion sublines. One of the most significantly change genes is GANAB. The treatment of GANAB-shRNA also resulted in augment of cell growth in all HNSCC cell lines, with approximately increase to 1.34 fold at day 5 (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3067.