Kuo-Wei Chang

Exploiting salivary miR-31 as a clinical biomarker of oral squamous cell carcinoma

Oral carcinoma is an important malignancy throughout the world. MicroRNAs (miRNAs) are endogenously expressed, non‐coding RNAs that regulate post‐transcriptional levels of targeted mRNAs. MiRNA‐31(miR‐31) is significantly upregulated in oral carcinoma tissues and plays oncogenic roles in oral carcinogenesis.

miR-134 induces oncogenicity and metastasis in head and neck carcinoma through targeting WWOX gene.

Head and neck squamous cell carcinoma (HNSCC) is a prevalent disease worldwide, and the survival of HNSCC has not improved significantly over the last few decades. MicroRNAs (miRNAs) have an important regulatory role during carcinogenesis. Our study investigated the pathogenic implications of miR‐134 in head and neck carcinogenesis. The clinicopathologic implications of miR‐134 in HNSCC were investigated using expression assays and the functional role of miR‐134 in HNSCC pathogenesis was determined using ectopic expression, knockdown and reporter assay experiments. Xenographic tumorigenesis and orthotopic nodal metastasis were assayed in mouse models. In situ hybridization and immunohistochemistry were used to detect the expression of miR‐134 and the WWOX gene in human HNSCC. The results indicated that miR‐134 was upregulated in HNSCC tissues relative to control mucosa. High expression of miR‐134 was associated with nodal metastasis and mortality of patients. Decreased plasma miR‐134 levels after tumor ablation indicated a better prognosis for patients. Multivariate analysis showed that high miR‐134 expression in HNSCC was an independent predictor of poor survival. Ectopic miR‐134 expression significantly enhanced in vitro oncogenic phenotypes and the orthotopic growth and metastasis of HNSCC cells. miR‐134 targeted WW domain‐containing oxidoreductase (WWOX) gene and cell invasion enhanced by miR‐134 expression was abrogated by ectopic WWOX expression in HNSCC cells. miR‐134 expression was reversely associated with the WWOX expression in HNSCC tissues. Evidences from our study substantiated that miR‐134 expression contributes to head and neck carcinogenesis by targeting the WWOX.

Characterization of two new cell lines derived from oral cavity human squamous cell carcinomas--OC1 and OC2.

Two continuous cell lines, OC1 and OC2, have been established. Each one was derived from a surgical specimen of buccal mucosa squamous carcinoma from a Chinese man. Both cell lines grew as adherent monolayers and had an epithelioid morphology. The cell lines have now been grown in vitro for more than 200 passages over a 2-year period. Both cell lines were tumorigenic in nude mice. Histologic examination of the mice tumors showed a positive identification with those from the original human squamous cell carcinoma. Electron microscopic examination convincingly demonstrated their epithelial origin. Chromosomal numbers of the OC1 cell ranged from 58 to 78 and of the OC2 cells ranged from 62 to 82. Analysis of cytokeratin showed that all cell lines were positive. The carcinoembryonic antigen (CEA) tested negative in these two cell lines. The OC2 cell line showed the presence of HPV16, but the OC1 cell line did not. These cell lines should be useful for investigation of anticancer treatment and for their possible relationship to human papilloma virus (HPV).

Microrna aberrances in head and neck cancer: pathogenetic and clinical significance

Purpose of reviewMicroRNAs (miRNAs) play crucial roles in modulating the neoplastic process of cancers including head and neck squamous cell carcinoma (HNSCC). miRNAs modulate pathogenesis by inhibiting target genes. Understanding how aberrant miRNAs are involved in HNSCC pathogenesis should help to validate potential clinical applications that target these entities. Recent findingsmiR-21, miR-31, miR-504 and miR-10b are important oncogenic miRNAs that are involved in HNSCC and target tumour suppressor genes. The tumour suppressor roles of the let-7 family, the miR-99 family, miR-107, miR-133a, miR-137, miR-138 and miR-375 with respect to their targeting of oncogenes are unequivocal and have been confirmed by many studies. In addition, miR-21, let-7, miR-107, miR-138 and miR-200c seem to play complicated roles in regulating stemness or the epithelial-mesenchymal transition of tumour cells. The clinical implications of these tumour-associated miRNAs are generally in agreement with their functional roles. SummaryA number of pathways that become disregulated by aberrant miRNAs have been identified specifically for HNSCC. Analysis of these networks and their therapeutic interception might facilitate the prediction of disease status and help with the design of therapeutic trials.

Differential gene expression signature between primary and metastatic head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a world‐wide malignancy. This study aimed to identify differential gene expression associated with the progression of disease from primary to metastatic HNSCC. Microdissection retrieved pure epithelial cells from paired primary tumours and cervical lymph node metastasis. cDNA microarray analysis and algorithm grouping identified differential mRNA expression of 301 genes. Quantitative reverse transcription‐polymerase chain reaction analysis clarified the up‐regulation of CCL19, CR2, EGR2, FUCA1, RGS1, and SELL, as well as the down‐regulation of IGFBP6 and KLK8 in nodal metastasis compared to primary tumours. Immunohistochemistry confirmed the up‐regulation of SELL and down‐regulation of IGFBP6 in nodal metastasis relative to primary tumours. Interestingly, primary tumours exhibiting higher FUCA1 and SELL expression were associated with significantly worse patient survival. In OECM‐1 HNSCC cells, inhibition of proliferation, migration, and anchorage‐independent growth was noted following knockdown of SELL expression. In SAS HNSCC cells, expression of exogenous SELL resulted in increased invasion, anchorage‐independent growth, and xenographic tumourigenesis in nude mice. Knockdown of FUCA1 and treatment with IGFBP6 inhibited the migration of OECM‐1 cells. Knockdown of RGS1 inhibited the anchorage‐independent growth of SAS cells. Our results provide a useful gene signature profile describing the factors underlying the metastasis of HNSCC to cervical lymph nodes, which may be beneficial for the treatment of HNSCC metastasis. Copyright

The Evolving Transcriptome of Head and Neck Squamous Cell Carcinoma: A Systematic Review

Background Numerous studies were performed to illuminate mechanisms of tumorigenesis and metastases from gene expression profiles of Head and Neck Squamous Cell Carcinoma (HNSCC). The objective of this review is to conduct a network-based meta-analysis to identify the underlying biological signatures of the HNSCC transcriptome. Methods and Findings We included 63 HNSCC transcriptomic studies into three specific categories of comparisons: Pre, premalignant lesions v.s. normal; TvN, primary tumors v.s. normal; and Meta, metastatic or invasive v.s. primary tumors. Reported genes extracted from the literature were systematically analyzed. Participation of differential gene activities across three progressive stages deciphered the evolving nature of HNSCC. In total, 1442 genes were verified, i.e. reported at least twice, with ECM1, EMP1, CXCL10 and POSTN shown to be highly reported across all three stages. Knowledge-based networks of the HNSCC transcriptome were constructed, demonstrating integrin signaling and antigen presentation pathways as highly enriched. Notably, functional estimates derived from topological characteristics of integrin signaling networks identified such important genes as ITGA3 and ITGA5, which were supported by findings of invasiveness in vitro [1]. Moreover, we computed genome-wide probabilities of reporting differential gene activities for the Pre, TvN, and Meta stages, respectively. Results highlighted chromosomal regions of 6p21, 19p13 and 19q13, where genomic alterations were shown to be correlated with the nodal status of HNSCC [2]. Conclusions By means of a systems-biology approach via network-based meta-analyses, we provided a deeper insight into the evolving nature of the HNSCC transcriptome. Enriched canonical signaling pathways, hot-spots of transcriptional profiles across the genome, as well as topologically significant genes derived from network analyses were highlighted for each of the three progressive stages, Pre, TvN, and Meta, respectively.

Association between the rs2910164 polymorphism in pre-mir-146a and oral carcinoma progression

MicroRNAs are short non-coding RNAs that regulate gene expression by RNA interference. Oral squamous cell carcinoma (OSCC) is a prevalent malignancy worldwide. miR-146a has been reported to regulate Toll-like receptors and cytokine signaling, which are both crucial for inflammation and oncogenesis. This study identifies that areca nut extract, TNFα and TGFβ up-regulates miR-146a in OSCC cells. The increased expression of miR-146a enhanced the oncogenicity of OSCC cells. In addition, a G to C polymorphism (rs2910164), which is located in the pre-miR-146a and has been associated with functional alterations in miR-146a, was significantly more prevalent among OSCC patients having more advanced nodal involvement. Our analysis also suggested a higher miR-146a expression in OSCC tissues of patients carrying C polymorphism. The present study concluded a higher prevalence of the pre-mir-146a C-variant was associated with OSCC progression in patients with this disease.

miR-146a Enhances the Oncogenicity of Oral Carcinoma by Concomitant Targeting of the IRAK1, TRAF6 and NUMB Genes

MicroRNAs are short non-coding RNAs that regulate gene expression and are crucial to tumorigenesis. Oral squamous cell carcinoma (OSCC) is a prevalent malignancy worldwide. Up-regulation of miR-146 has been identified in OSCC tissues. However, the roles of miR-146 in carcinogenesis are controversial as it is suppressive in many other malignancies. The present study investigated the pathogenic implications of miR-146a in oral carcinogenesis. Microdissected OSCC exhibits higher levels of miR-146a expression than matched adjacent mucosal cells. The plasma miR-146a levels of patients are significantly higher than those of control subjects; these levels decrease drastically after tumor resection. miR-146a levels in tumors and in patients’ plasma can be used to classify OSCC and non-disease status (sensitivity: >0.72). Exogenous miR-146a expression is significantly increased in vitro oncogenic phenotypes as well as during xenograft tumorigenesis and OSCC metastasis. The plasma miR-146a levels of these mice parallel the xenograft tumor burdens of the mice. A miR-146a blocker abrogates the growth of xenograft tumors. miR-146a oncogenic activity is associated with down-regulation of IRAK1, TRAF6 and NUMB expression. Furthermore, miR-146a directly targets the 3′UTR of NUMB and a region within the NUMB coding sequence when suppressing NUMB expression. Exogenous NUMB expression attenuates OSCC oncogenicity. Double knockdown of IRAK1 and TRAF6, and of TRAF6 and NUMB, enhance the oncogenic phenotypes of OSCC cells. Oncogenic enhancement modulated by miR-146a expression is attenuated by exogenous IRAK1 or NUMB expression. This study shows that miR-146a expression contributes to oral carcinogenesis by targeting the IRAK1, TRAF6 and NUMB genes.

Passenger strand miRNA miR-31∗ regulates the phenotypes of oral cancer cells by targeting RhoA

OBJECTIVESnMicroRNAs (miRNAs) are endogenous small non-coding RNAs that negatively regular target gene expression by RNA interference. The processing of the pre-miRNA hairpin generates a miRNA duplex, which consists of a miRNA (guide strand) and a miRNA(*) (passenger strand). miR-31 is an oncogenic miRNA and is up-regulated in oral squamous cell carcinoma (OSCC). miR-31(*) shows a high level of conservation across species and, based on this, this study hypothesized that miR-31(*) is a functional miRNA.nnnMATERIALS AND METHODSnThe expression of miR-31 and miR-31* in OSCC tissues and oral cells were analyzed. Functional studies were performed on OSCC cells.nnnRESULTSnmiR-31(*) is up-regulated in OSCC tissues, but its expression is less abundant than miR-31. miR-31(*) decreases the proliferation and migration of both SAS and Fadu cells. Furthermore, miR-31(*) targets the 3UTR of RhoA and is able to down-regulate RhoA expression. Knockdown of RhoA expression is known to decrease the proliferation and migration of OSCC cells. However, up-regulation of both miR-31 and miR-31(*) by delivery of pre-mir-31 does still enhance OSCC oncogenicity.nnnCONCLUSIONnmiR-31(*) is a functional miRNA involving in regulating RhoA, and the activity of miR-31(*)s activity seems to counteract the functions of miR-31 during OSCC tumorigenesis.

miR-211 promotes the progression of head and neck carcinomas by targeting TGFβRII

miR-211 up-regulation and transforming growth factor-β type II receptor (TGFβRII) down-regulation are associated with poor prognosis of head and neck squamous cell carcinoma (HNSCC). miR-211 directly targets TGFβRII with the miR-211-TGFβRII-c-Myc axis promoting HNSCC progression. An inverse correlation of miR-211 and TGFβRII expression was found in metastatic HNSCC samples. After 4-nitroquinoline 1-oxide induction, more severe epithelial tumorigenesis was detected on K14-miR-211 transgenic mouse dorsal tongues. Human metastatic lesions and mouse tongue tumors showed increased nuclear c-Myc expression. A novel role for miR-211 in the regulation of TGFβRII and c-Myc during tumorigenesis being revealed should help to develop anti-HNSCC therapies.