Chun-Yi Chuang

Exome Sequencing of Oral Squamous Cell Carcinoma Reveals Molecular Subgroups and Novel Therapeutic Opportunities

Oral squamous cell carcinoma (OSCC), an epithelial malignancy affecting a variety of subsites in the oral cavity, is prevalent in Asia. The survival rate of OSCC patients has not improved over the past decades due to its heterogeneous etiology, genetic aberrations, and treatment outcomes. Improvement in therapeutic strategies and tailored treatment options is an unmet need. To unveil the mutational spectrum, whole-exome sequencing of 120 OSCC from male individuals in Taiwan was conducted. Analyzing the contributions of the five mutational signatures extracted from the dataset of somatic variations identified four groups of tumors that were significantly associated with demographic and clinical features. In addition, known (TP53, FAT1, EPHA2, CDKN2A, NOTCH1, CASP8, HRAS, RASA1, and PIK3CA) and novel (CHUK and ELAVL1) genes that were significantly and frequently mutated in OSCC were discovered. Further analyses of gene alteration status with clinical parameters revealed that the tumors of the tongue were enriched with copy-number alterations in several gene clusters containing CCND1 and MAP4K2. Through defining the catalog of targetable genomic alterations, 58% of the tumors were found to carry at least one aberrant event potentially targeted by US Food and Drug Administration (FDA)-approved agents. Strikingly, if targeting the p53-cell cycle pathway (TP53 and CCND1) by the drugs studied in phase I-III clinical trials, those possibly actionable tumors are predominantly located in the tongue, suggesting a better prediction of sensitivity to current targeted therapies. Our work revealed molecular OSCC subgroups that reflect etiological and prognostic correlation as well as defined the landscape of major altered events in the coding regions of OSCC genomes. These findings provide clues for the design of clinical trials for targeted therapies and stratification of OSCC patients with differential therapeutic efficacy.

Carbonic anhydrase IX overexpression regulates the migration and progression in oral squamous cell carcinoma.

Carbonic anhydrase IX (CAIX) is reportedly overexpressed in several types of carcinomas and is generally considered a marker of malignancy. The current study investigated the association between membrane expression of CAIX and the clinicopathological characteristics in oral squamous cell carcinoma (OSCC) patients. The study used immunohistochemistry to examine CAIX expression in 271 OSCC specimens by tissue microarray (TMA) and assessed the effect of CAIX overexpression and knockdown on migration of oral cancer cells in vitro. We found that CAIX expression was associated with more advanced clinical stages (p = 0.030) and positive lymph node metastasis (p = 0.026). Importantly, CAIX expression was correlated with a poorer patient prognosis in a univariate survival analysis (p = 0.025). Moreover, CAIX suppression by small interfering RNA (siRNA) significantly reduced cellular migration in OECM-1 oral cancer cell. In conclusion, our study showed that the expression of CAIX in OSCC samples can predict the progression of OSCC and survival of OSCC patients in Taiwan.

Selaginella tamariscina extract suppresses TPA-induced invasion and metastasis through inhibition of MMP-9 in human nasopharyngeal carcinoma HONE-1 cells

BackgroundNasopharyngeal carcinoma (NPC) is known for its high incidence of neck lymph node metastasis, which represents poor prognosis. The present study aimed to examine the anti-metastatic properties of Selaginella tamariscina extract (STE) in human nasopharyngeal carcinoma HONE-1 cells in vitro.MethodsCell viability was examined by MTT assay, whereas cell motility was measured by invasive, migration and would healing assays. Real-time PCR, and promoter assays confirmed the inhibitory effects of STE on matrix metalloproteinase-9 (MMP-9) mRNA level in HONE-1 cells.ResultsThe STE inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HONE-1 cell migration and invasion in a concentration-dependent manner. By zymographic and Western blot analyses, STE was shown to inhibit the activities and expression of MMP-9. Treatment of STE on TPA-induced HONE-1 cells inhibited MMP-9 expression and ERK1/2 phosphorylation without affecting JNK and p38 phosphorylation.ConclusionsSTE inhibits MMP-9 expression and HONE-1 cell metastasis. Its inhibitory effects may involve the Src/FAK/ERK 1/2 pathway. STE may have the potential of being an anti-metastatic agent against NPC.

Rubus idaeus extract suppresses migration and invasion of human oral cancer by inhibiting MMP‐2 through modulation of the Erk1/2 signaling pathway

Raspberries (Rubus idaeus L.) have been extensively studies worldwide because of their beneficial effects on health. Recently reports indicate that crude extracts of Rubus idaeus (RIE) have antioxidant and anticancer ability. The aim of this study was to evaluate the mechanism of its antimetastatic ability in oral cancer cells. In this study, SCC‐9 and SAS oral cancer cells were subjected to a treatment with RIE and then analyzed the effect of RIE on migration and invasion. The addition of RIE inhibited the migration and invasion ability of oral cancer cells. Real time PCR, western blot and zymography analysis demonstrated that mRNA, protein expression and enzyme activity of matrix metalloproteinases‐2 (MMP‐2) were down‐regulated by RIE. Moreover, the phosphorylation of Focal adhesion kinase (FAK), src, and extracellular signal‐regulated kinase (ERK) were inhibited after RIE treatment. In conclusion, these results demonstrated that RIE exerted an inhibitory effect of migration and invasion in oral cancer cells and alter metastasis by suppression of MMP‐2 expression through FAK/Scr/ERK signaling pathway.

Hispolon suppresses migration and invasion of human nasopharyngeal carcinoma cells by inhibiting the urokinase‐plasminogen activator through modulation of the Akt signaling pathway

Hispolon has been reported to possess antioxidant, antiinflammatory, and antitumor activities. However, the effect of hispolon on the metastasis of nasopharyngeal carcinoma (NPC) remains unclear. In this study, we investigated how the antimetastatic activity and relevant signaling pathways of hispolon affected three NPC cell lines. The results revealed that hispolon significantly reduced the migration and invasion of three NPC cells in a dose‐dependent manner from 0 to 50 µM. Hispolon also significantly inhibited the activity and expression of urokinase‐plasminogen activator (uPA) as well as the phosphorylation of Akt. Moreover, blocking the Akt pathway also enhanced the antimetastatic ability of hispolon in the NPC cells. In conclusion, hispolon inhibited uPA expression and NPC cell metastasis by downregulating Akt signal pathways; therefore, hispolon exerts beneficial effects in chemoprevention.

Combined effect of genetic polymorphisms of AURKA and environmental factors on oral cancer development in Taiwan.

Background Oral squamous cell carcinoma (OSCC) is the sixth and fourth most common cause of cancer death in men worldwide and in Taiwan, respectively. AURKA, which encodes a centrosome-related serine/threonine kinase, is frequently amplified and overexpressed in many human cancers, particularly advanced OSCC. We conducted a hospital-based case-control study to estimate AURKA single-nucleotide polymorphisms (SNPs) and environmental risk factors to determine OSCC susceptibility and clinicopathological characteristics. Methodology/Principal findings We enrolled a total of 876 OSCC patients and 1200 controls. Four SNPs of AURKA, namely rs1047972, rs2273535, rs2064863, and rs6024836, were analyzed using real-time polymerase chain reaction (PCR). Among the 1420 smokers, the AURKA polymorphism carriers with the betel nut chewing habit had a higher risk of oral cancer than AURKA wild-type (WT) carriers without the betel nut chewing habit. Patients with the AURKA rs2064863 gene had a 1.365-fold higher risk of stage III or IV OSCC (95% confidence interval [CI] 1.029–1.811) than those with the rs2064863 WT gene. Furthermore, carriers of the AURKA rs1047972/rs2273535/rs2064863 C-A-T haplotype had a 1.736-fold (95% CI 1.110–2.715) higher risk of OSCC than controls (C-T-T, the most common haplotype). Among patients with the betel quid chewing habit, carriers of other haplotypes (C-T-T, C-A-G, T-A-T, T-A-G, T-T-T, and C-T-G) had a 12.857-fold (95% CI 10.731–15.404) increased risk, and carriers of the C-A-T haplotype had the highest risk (AOR: 31.120; 95% CI 13.864–69.850) of OSCC, compared with those without the betel quid chewing who harbored other haplotypes. Conclusions In conclusion, betel nut chewing combined with the AURKA C-A-T haplotypes lead to a high risk of OSCC. These findings reveal a novel genetic-environmental predisposition for oral tumorigenesis.

Compositional and functional variations of oral microbiota associated with the mutational changes in oral cancer

OBJECTIVES Both genetic and environmental factors are conceivably required to assess the prognosis of oral squamous cell carcinoma (OSCC), yet little is known regarding the relationship between oral microbiome and the mutational spectrum of OSCC. MATERIALS AND METHODS Here, we used 16S rRNA amplicon sequencing to study the composition of oral microorganisms in OSCC patients, whose cancer mutational profiles were previously defined by whole-exome sequencing, to evaluate the relationship between oral microbiome and the mutational changes in OSCC. RESULTS Analyzing the contributions of the five mutational signatures extracted from the primary tumors revealed three groups of OSCC (mutational signature cluster, MSC1-3) that were significantly associated with demographic and clinical features. Taxonomic analysis of the predominant phyla in salivary samples showed variation in the relative abundance of Firmicutes and Bacteroidetes in the three MSC groups. In addition, significant differences in bacterial species richness (alpha diversity) and slight sample-to-sample dissimilarities in bacterial community structures (beta diversity) were noted among different MSC groups. Further, predicting the functional capabilities of microbial communities by reconstruction of unobserved states showed that many pathways related to cell motility were differentially enriched among the three MSC groups. CONCLUSION Collectively, these results indicate a potential association of oral microbiome with the mutational changes in OSCC.

Association of matrix metalloproteinase-11 polymorphisms with susceptibility and clinicopathologic characteristics for oral squamous cell carcinoma.

The purpose of this study was to investigate the influence of genetic polymorphisms of the matrix metalloproteinase‐11 (MMP‐11) gene on the susceptibility of oral squamous cell carcinoma (OSCC).

A functional variant at the miRNA binding site in HMGB1 gene is associated with risk of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a common malignancy that has been causally associated with both hereditary and acquired factors. The high mobility group box 1 (HMGB1) gene plays an important role as a DNA chaperone to help maintain nuclear homeostasis. Altered expression of HMGB1 has been implicated in a wide range of pathological processes, including inflammation and cancer. The present study explores the impact of HMGB1 gene polymorphisms, combined with environmental risks regarding susceptibility to oral tumorigenesis. Four single-nucleotide polymorphisms (SNPs) of the HMGB1 gene, rs1412125, rs2249825, rs1045411, and rs1360485, were evaluated in 1,200 normal controls and 772 patients with OSCC. We found an association between the wild-type allele of rs1045411 and genotypes CT and CT/TT (AOR=0.754, 95% CI=0.582-0.978 and AOR=0.778, 95% CI=0.609-0.995, respectively). Additionally, bioinformatics analysis was used to characterize the functional relevance of these variants for the miRNA-505-5p binding site and transcriptional regulation by the HMGB1 3’-UTR and promoter regions. Moreover, in considering behavioral exposure to environmental carcinogens, the presence of the four HMGB1 SNPs, combined with/without betel quid chewing and smoking showed, profoundly synergistic effects on the risk of OSCC. In conclusion, we present a potential clinical relevance for HMGB1 variants in OSCC, as well as associations between HMGB1 polymorphisms, haplotypes and environmental risk factors. The finding may help in development of optimal therapeutic approaches for OSCC patients.

Tricetin suppresses human oral cancer cell migration by reducing matrix metalloproteinase-9 expression through the mitogen-activated protein kinase signaling pathway

Tricetin is a flavonoid derivative and a potent anti‐inflammatory and anticancer agent. However, the molecular mechanism underlying the effects of tricetin on human oral cancer cell migration remains unclear. The cell migration and invasion abilities of three oral cancer cell lines (SCC‐9, HSC‐3, and OECM‐1) were analyzed using Boyden chamber migration assays. Our results demonstrated that tricetin attenuates 12‐O‐tetradecanoylphorbol‐13‐acetate‐induced SCC‐9, HSC‐3, and OECM‐1 cell invasiveness and migration by reducing matrix metalloproteinase (MMP)‐9 enzyme activity. The reverse transcription polymerase chain reaction and luciferase reporter assay revealed that tricetin downregulates the mRNA expression and promoter activity of MMP‐9. In addition, Western blot analysis revealed that tricetin significantly reduced the levels of phosphorylated c‐Jun N‐terminal kinase (JNK) 1/2 and p38 levels but not those of extracellular signal‐regulated kinase 1/2. In conclusion, this study demonstrated that tricetin suppresses MMP‐9 enzymatic activity by downregulating the p38/JNK1/2 pathway and might be a beneficial chemopreventive agent.