Jenn Ren Hsiao

Cathepsin L mediates resveratrol-induced autophagy and apoptotic cell death in cervical cancer cells.

Cathepsins have long been considered as housekeeping molecules. However, specific functions have also been attributed to each one of these lysosomal proteases. Squamous cell carcinoma antigen (SCCA) 1, widely expressed in various uterine cervical cells, is an endogenous cathepsin (cat) L inhibitor. In this study, we investigated whether the cat L-SCCA 1 lysosomal pathway and autophagy were involved in resveratrol (RSV)-induced cytotoxicity in cervical cancer cells. RSV induced GFP-LC3 aggregation as well as increased the presence of LC3-II and autophagosomes as was revealed by electron microscopy in cervical cancer cells. Prolonged treatment of RSV induced cytosolic translocation of cytochrome c, caspase 3 activation, and apoptotic cell death. This apoptotic effect was abrogated by trans-epoxysuccinyl- L-leucylamido - (4-guanidino)butane, an inhibitor of cat B and L, but not by pepstatin A, an inhibitor of cat D. As cervical cancer cells express little cat B, we further studied the role of cat L. RSV induced dissipation of the lysosomal membrane permeability (LMP), leakage and increased cytosolic expression and activity of cat L. Inhibition of cat L by small interference RNA (siRNA) protected cells from RSV-induced cytotoxicity. In contrast, inhibition of SCCA 1 by siRNA promoted RSV-induced cytotoxicity. Inhibition of autophagic response by wormannin (WT) or asparagine (ASP) resulted in decreased early LC3-II formation, reduced LMP, and abolishment of the increase in RSV-induced cell death. In conclusion, we have identified a new cytotoxic mechanism in which the lysosomal enzyme cat L acts as a death signal integrator in cervical cancer cells. Furthermore, SCCA 1 may play an anti-apoptotic role through anti-cat L activity.

Constitutive activation of STAT3 and STAT5 is present in the majority of nasopharyngeal carcinoma and correlates with better prognosis

Constitutively activated signal transducers and activators of transcription (STAT) factors, in particular STAT1, STAT3 and STAT5, have been demonstrated in a variety of human tumours and cancer cell lines. However, data on the expression of these STATs in nasopharyngeal carcinoma (NPC) are limited. In this study, the expression patterns of STAT1, STAT3 and STAT5 were immunohistochemically examined on the archival specimens from 61 patients with NPC. Staining results of each STATs were then correlated with the clinical parameters and prognosis of these patients. The results showed that constitutive activation of STAT3 and STAT5 was detected in the majority, 70.5 and 62.3%, respectively, of the 61 tumour specimens. Furthermore, coexpression of activated STAT3 and STAT5 was found in 54.1% of the specimens. In contrast, constitutive activated STAT1 could only be detected in 8 (13.1%) cases. Surprisingly, following radiotherapy, patients with constitutive STAT5 activation, or activation of both STAT3 and STAT5, had better disease-free survival and overall survival than those without activated STAT5. To our knowledge, this is the first report providing the overall expression patterns and prognostic significance of specific STATs in NPC.

Increase of programmed death-1-expressing intratumoral CD8 T cells predicts a poor prognosis for nasopharyngeal carcinoma

Intratumoral cytotoxic T lymphocytes are critical for controlling tumor recurrence, and programmed death-1 (PD-1) is a recognized marker of T-cell dysfunction. We analyzed this marker and its binding ligands in nasopharyngeal tumor tissue and non-cancerous nasopharyngeal control tissue to retrospectively evaluate the correlation between its expression and the post-treatment outcome of nasopharyngeal carcinoma patients. Using double immunofluorescence staining, we found that the expression of PD-1 in CD8 T cells in tumor tissue was significantly higher than in control tissue (mean: 28.4 vs 3.9%, P<0.0001). Although the expression rate of PD-1 in intratumoral CD8 cells was not associated with the other clinicopathological parameters examined, the higher expression rate in this subset of T cells significantly correlated with a poorer prognosis of overall survival, disease-free survival, and locoregional recurrence-free survival of the cancer patients (P=0.05, 0.007, and 0.004, respectively). Multivariate analysis confirmed it as an independent risk factor for death, treatment failure, and local recurrence of nasopharyngeal carcinoma. On the other hand, the expression of PD-1 in CD4 T cells and of its ligands in epithelial and stromal cells was not significantly different between tumor and control tissue, and its expression was not associated with clinical outcome of the cancer patients. We propose that PD-1 expression in CD8 cells reflects the selective suppression of cytotoxic lymphocytes in the tumor microenvironment and predicts recurrence of nasopharyngeal carcinoma after conventional therapies.

Epstein-Barr virus detection in neck metastases by in-situ hybridization in fine-needle aspiration cytologic studies: An aid for differentiating the primary site

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein‐Barr virus (EBV). The metastasis to cervical lymph nodes represents a frequent initial manifestation of NPC. The usefulness of EBV detection by polymerase chain reaction (PCR) in the diagnosis of occult NPC with cervical metastasis has been reported. Our previous study showed that EBER1 in‐situ hybridization was somewhat more sensitive and specific than PCR in detecting EBV in the evaluation of specimens from a population at high risk for NPC.

ENO1, a potential prognostic head and neck cancer marker, promotes transformation partly via chemokine CCL20 induction

The success of using glycolytic inhibitors for cancer treatment depends on studying the individual role of frequently deregulated glycolytic genes in cancer. This report aims to study the prognostic implication, and determine the cellular role and action mechanism of glycolytic ENO1 overexpression in head and neck cancer. The relationship of ENO1 mRNA expression in 44-pair clinical specimens with patient clinicopathologic characteristics was analysed by semi-quantitative RT-PCR, Kaplan-Meier survival curve and Cox model analyses. Following ectopic ENO1 expression or knockdown, we studied the proliferative, migratory, invasive, colony-forming and tumourigenic abilities of ENO1-genetically altered cells. DNA microarray analysis was used to identify downstream targets responsible for the ENO1 action in the cells. The expression of ENO1 mRNA was increased in 68% of tumour (T) specimens when compared to their normal (N) counterparts, and positively associated with clinical progression (p<0.05). High ENO1 expression (T/N2) was frequently observed in the patients with large primary tumours, late clinical stages or advanced neck metastasis. Moreover, high ENO1 patients had significantly poorer clinical outcomes than low expressers (T/N<2). Ectopic ENO1 expression stimulated cell transformation, invasion and tongue tumour formation. ENO1 knockdown abrogated the stimulation. Suppression of ENO1-induced proinflammatory CCL20 chemokine expression significantly attenuated its stimulatory effects on cell transformation and invasion. A concordant expression of ENO1 and CCL20 was validated both in ENO1-expressing cells and in clinical specimens. Together, we demonstrate a prognostic role of ENO1 overexpression in head and neck cancer and ENO1-mediated promotion of cell transformation and invasion partly via induced CCL20 expression.

Reciprocal regulation of MicroRNA-99a and insulin-like growth factor I receptor signaling in oral squamous cell carcinoma cells

BackgroundMicroRNAs (miRNAs), small noncoding RNA molecules can function as oncogenes or tumor suppressors in tumorigenesis. Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers worldwide with a 5-year survival rate of approximately 50%.MethodsThe expression of microRNA-99a (miR-99a) in OSCC tissues and cell lines was investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The functions of miR-99a in migration/invasion and lung colonization were determined by transwell and tail vein injection assays, respectively. Specific targets of miR-99a were determined by software prediction, correlation with target protein expression, and luciferase reporter assay. The signaling pathways involved in regulation of miR-99a were investigated using the kinase inhibitors.ResultsWe observed reduced levels of miR-99a, identified as one of the most downregulated miRNA in OSCC and all tested OSCC cell lines compared to normal oral keratinocytes. Ectopic miR-99a expression in OSCC cells markedly reduced migration and invasion in vitro as well as lung colonization in vivo. When evaluating the specific targets of miR-99a, we found that ectopic miR-99a expression downregulates insulin-like growth factor 1 receptor (IGF1R) protein and that the expression of miR-99a correlates negatively with IGF1R protein in OSCC cells. Insertion of the 3′UTR of IGF1R mRNA into the 3′UTR of a reporter gene markedly reduced luciferase activity in OSCC cells expressing miR-99a, suggesting that miR-99a reduces luciferase activity by targeting the 3′UTR of IGF1R mRNA. When evaluating the mechanisms of miR-99a downregulation, we observed the upregulation of miR-99a expression in serum-starved conditions and its suppression in response to insulin-like growth factor (IGF1) stimulation. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) kinase inhibited IGF1-induced suppression of miR-99a, suggesting the negative regulation of miR-99a expression by IGF1R signaling.ConclusionOverall, results indicate that miR-99a functions as a tumor metastasis suppressor in OSCC cells and mutually regulates IGF1R expression in a reciprocal regulation.

IL‐1β Promotes Malignant Transformation and Tumor Aggressiveness in Oral Cancer

Chronic inflammation, coupled with alcohol, betel quid, and cigarette consumption, is associated with oral squamous cell carcinoma (OSCC). Interleukin‐1 beta (IL‐1β) is a critical mediator of chronic inflammation and implicated in many cancers. In this study, we showed that increased pro‐IL‐1β expression was associated with the severity of oral malignant transformation in a mouse OSCC model induced by 4‐Nitroquinolin‐1‐oxide (4‐NQO) and arecoline, two carcinogens related to tobacco and betel quid, respectively. Using microarray and quantitative PCR assay, we showed that pro‐IL‐1β was upregulated in human OSCC tumors associated with tobacco and betel quid consumption. In a human OSCC cell line TW2.6, we demonstrated nicotine‐derived nitrosamine ketone (NNK) and arecoline stimulated IL‐1β secretion in an inflammasome‐dependent manner. IL‐1β treatment significantly increased the proliferation and dysregulated the Akt signaling pathways of dysplastic oral keratinocytes (DOKs). Using cytokine antibodies and inflammation cytometric bead arrays, we found that DOK and OSCC cells secreted high levels of IL‐6, IL‐8, and growth‐regulated oncogene‐α following IL‐1β stimulation. The conditioned medium of IL‐1β‐treated OSCC cells exerted significant proangiogenic effects. Crucially, IL‐1β increased the invasiveness of OSCC cells through the epithelial‐mesenchymal transition (EMT), characterized by downregulation of E‐cadherin, upregulation of Snail, Slug, and Vimentin, and alterations in morphology. These findings provide novel insights into the mechanism underlying OSCC tumorigenesis. Our study suggested that IL‐1β can be induced by tobacco and betel quid‐related carcinogens, and participates in the early and late stages of oral carcinogenesis by increasing the proliferation of dysplasia oral cells, stimulating oncogenic cytokines, and promoting aggressiveness of OSCC. J. Cell. Physiol. 230: 875–884, 2015.

Detection of cell free Epstein-Barr virus DNA in sera from patients with nasopharyngeal carcinoma.

The detection of tumor‐derived DNA within the circulation of patients with malignant disease using polymerase chain reaction (PCR)‐based strategies has opened a new avenue for the diagnosis and monitoring of these patients. Because of the universal association of Epstein–Barr virus (EBV) with the nonsquamous type of nasopharyngeal carcinoma (NPC; World Health Organization types II and III), the detection of cell free EBV DNA in sera from patients with NPC may be a valuable tool for monitoring the progress of tumors or to provide advanced warning of tumor recurrence.

Apoptotic mechanism of paclitaxel-induced cell death in human head and neck tumor cell lines

BACKGROUND Paclitaxel (taxol) is clinically used to treat various human tumors. However, the cellular and molecular mechanism regarding apoptotic effect of paclitaxel on head and neck squamous cell carcinoma (HNSCC) remains elusive. METHODS The apoptotic effect and the mechanism of paclitaxel on FaDu hypopharyngeal cancer cell line, OEC-M1 gingival cancer cell line, and OC3 betel quid chewing-related buccal cancer cell lines were investigated by morphological observations, cell viability assay, flow cytometry assay and Western blotting methods. RESULTS Rounded-up cell number increased with membrane blebbing as the treatment of paclitaxel (50-500 nM) increased from 24 to 48 h among these cell lines. In cell viability assay, cell surviving rate significantly decreased from 87 to 27% as the dosage and duration of paclitaxel treatment increased (P < 0.05). Flow-cytometry analysis further demonstrated that 50 nM paclitaxel induced G2/M phase cell arrest among these cell lines within 8 h treatment, and then G2/M phase cell fraction significantly decreased as subG1 phase cell fraction significantly increased after 24 h treatment (P < 0.05), suggesting that cells underwent apoptosis. Furthermore, the activated caspases-8, -9, -3, -6 and poly ADP-ribose polymerase cleavage could all be significantly detected in FaDu, OEC-M1 and OC3 cells (P < 0.05). CONCLUSION Paclitaxel activated cell cycle arrest and caspase protein expressions to induce apoptosis in HNSCC cell lines.

Epstein-Barr Virus Latent Membrane Protein 2A Promotes Invasion of Nasopharyngeal Carcinoma Cells through ERK/Fra-1-Mediated Induction of Matrix Metalloproteinase 9

ABSTRACT Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is highly metastatic, and this malignant feature may be promoted by an EBV oncoprotein, latent membrane protein 2A (LMP2A). Acting as a signal regulator, LMP2A can enhance invasiveness and motility of epithelial cells. Downstream from the LMP2A-triggered signaling events, it is largely unknown what key effector proteins are induced and essentially promote cell invasion. In the present study, we found that in NPC cells, LMP2A upregulated matrix metalloproteinase 9 (MMP9), a metastasis-associated protease. LMP2A increased MMP9 expression at both the mRNA and protein levels. It also activated the MMP9 promoter, in which two AP-1 elements were required for the promoter activation. Among AP-1 transcription factors, Fra-1 was induced by LMP2A and is essential for LMP2A-triggered MMP9 expression. Induction of Fra-1 was dependent on the LMP2A-activated ERK1/2 pathway, and induction of the ERK1/2–Fra-1–MMP9 axis required PY motifs in the amino-terminal domain of LMP2A. Notably, LMP2A-promoted invasion of NPC cells was blocked when MMP9 expression, Fra-1 induction, or ERK1/2 activation was inhibited. In addition, we found an association of LMP2A with MMP9 expression in NPC tumor biopsy specimens, where Fra-1 was a major mediation factor. This study reveals an underlying mechanism of LMP2A-induced cell invasion, from signal transduction to upregulation of a critical protease. Considering that MMP9 can also be upregulated by another EBV oncoprotein, LMP1, this protease may be a pivotal effector at which the EBV-induced, invasion-promoting mechanisms converge, serving as an attractive therapeutic target for NPC treatment.