Ann-Joy Cheng

Head and neck cancer in the betel quid chewing area: Recent advances in molecular carcinogenesis

Head and neck cancer (HNC) is one of the 10 most frequent cancers worldwide, with an estimated over 500 000 new cases being diagnosed annually. The overall 5‐year survival rate in patients with HNC is one of the lowest among common malignant neoplasms and has not significantly changed during the last two decades. Oral cavity squamous cell carcinoma (OSCC) shares part of HNC and has been reported to be increasing in the betel quid chewing area in recent years. During 2006, OSCC has become the sixth most common type of cancer in Taiwan, and it is also the fourth most common type of cancer among men. It follows that this type of cancer wreaks a high social and personal cost. Environmental carcinogens such as betel quid chewing, tobacco smoking and alcohol drinking have been identified as major risk factors for head and neck cancer. There is growing interest in understanding the relationship between genetic susceptibility and the prevalent environmental carcinogens for HNC prevention. Within this review, we discuss the molecular and cellular aspects of HNC carcinogenesis in Taiwan, an endemic betel quid chewing area. Knowledge of molecular carcinogenesis of HNC may provide critical clues for diagnosis, prognosis, individualization of therapy and molecular therapeutics. (Cancer Sci 2008; 99: 1507–1514)

Prognostic significance of EGFR and Her-2 in oral cavity cancer in betel quid prevalent area cancer prognosis

Although several studies have found overexpression of epidermal growth factor receptor (EGFR) proteins EGFR and Her-2 in head and neck cancers, the clinical relevance of the finding varies. We examined the expression and clinical association of these molecules with oral squamous cell carcinoma in an area where betel chewing is prevalent. EGFR and Her-2 proteins were measured in 59 paired (grossly normal and cancer) tissues by an enzyme immunoassy method. The cutoff value for gene overexpression was defined as the level of mean expression in normal tissue plus two s.d. A total of 59% of the patients consumed alcohol, 90% smoked tobacco, and 90% chewed betel quid. Of the patients assayed, 34 (58%) and 24 (41%) had EGFR and Her-2 overexpression, with average 3.5- and 1.5-fold elevations. EGFR overexpression has been shown to be statistically associated with T stage, N stage, overall TMN stage, primary tumour depth, lymph node extra-capsular spread, and poor survival. Her-2 overexpression, however, did not demonstrate a similar association with clinicopathological parameters or therapeutic outcome. On multivariant analysis, EGFR overexpression (P=0.041) and N stage (P=0.024) were the only independent factors for overall survival. These results indicate that the molecular targeting therapy to EGFR may be a treatment for oral cavity cancer in the betel quid-chewing prevalent area.

Oncogenic function and early detection potential of miRNA-10b in oral cancer as identified by microRNA profiling

The miRNA participates in a variety of biologic processes, and dysregulation of miRNA is associated with malignant transformation. In this study, we determined specific profile of miRNA associated with oral cancer by using miRNA array screening method. There were 23 miRNAs found with considerably differential expressions between six oral cancer cell lines and five lines of normal oral keratinocytes, in which, 10 miRNAs showed the highest significant difference after independent examination by reverse transcription quantitative PCR. Eight molecules were upregulated, miR-10b, miR-196a, miR-196b, miR-582-5p, miR-15b, miR-301, miR-148b, and miR-128a, and two molecules, miR-503 and miR-31, were downregulated. The most upregulated miR-10b was further examined, and its functions were characterized in two oral cancer cell lines. The miR-10b actively promotes cell migration (2.6- to 3.6-fold) and invasion (1.7- to 1.9-fold) but has minimal effect on cell growth or chemo-/radiosensitivity. Furthermore, miR-10b was considerably elevated in the plasma of xenografted tumor mice (20-fold). This upregulation of miR-10b in plasma was further shown in the patients with oral cancer [P < 0.0001, area under curve (AUC) = 0.932] and precancer lesions (P < 0.0001, AUC = 0.967), suggesting that miR-10b possesses a high potential to discriminate the normal subjects. In conclusion, we have identified at least 10 miRNAs significantly associated with oral cancer, including the most elevated miR-10b. The miR-10b actively participates in cancer formation by promoting cell migration and invasion. Our study using clinical samples suggests that plasma miR-10b has high potential as an early detection marker for oral cancer. Cancer Prev Res; 5(4); 665–74. ©2012 AACR.

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.

Neck treatment of patients with early stage oral tongue cancer : comparison between observation, supraomohyoid dissection, and extended dissection

The role of elective and therapeutic selective neck dissection in patients with early stage cancer of the oral tongue remains controversial. The purpose was to investigate the role of neck treatment in the management of this condition.

miRNA-491-5p and GIT1 Serve as Modulators and Biomarkers for Oral Squamous Cell Carcinoma Invasion and Metastasis

MicroRNAs offer tools to identify and treat invasive cancers. Using highly invasive isogenic oral squamous cell carcinoma (OSCC) cells, established using in vitro and in vivo selection protocols from poorly invasive parental cell populations, we used microarray expression analysis to identify a relative and specific decrease in miR-491-5p in invasive cells. Lower expression of miR-491-5p correlated with poor overall survival of patients with OSCCs. miR-491-5p overexpression in invasive OSCC cells suppressed their migratory behavior in vitro and lung metastatic behavior in vivo. We defined the G-protein-coupled receptor kinase-interacting protein 1 (GIT1)-as a direct target gene for miR-491-5p control. GIT1 overexpression was sufficient to rescue miR-491-5p-mediated inhibition of migration/invasion and lung metastasis. Conversely, GIT1 silencing phenocopied the ability of miR-491-5p to inhibit migration/invasion and metastasis of OSCC cells. Mechanistic investigations indicated that miR-491-5p overexpression or GIT1 attenuation reduced focal adhesions, with a concurrent decrease in steady-state levels of paxillin, phospho-paxillin, phospho-FAK, EGF/EGFR-mediated extracellular signal-regulated kinase (ERK1/2) activation, and MMP2/9 levels and activities. In clinical specimens of OSCCs, GIT1 levels were elevated relative to paired normal tissues and were correlated with lymph node metastasis, with expression levels of miR-491-5p and GIT1 correlated inversely in OSCCs, where they informed tumor grade. Together, our findings identify a functional axis for OSCC invasion that suggests miR-491-5p and GIT1 as biomarkers for prognosis in this cancer.

A Novel Molecular Signature Identified by Systems Genetics Approach Predicts Prognosis in Oral Squamous Cell Carcinoma

Molecular methods for predicting prognosis in patients with oral cavity squamous cell carcinoma (OSCC) are urgently needed, considering its high recurrence rate and tendency for metastasis. The present study investigated the genetic basis of variations in gene expression associated with poor prognosis in OSCC using Affymetrix SNP 6.0 and Affymetrix GeneChip Human Gene 1.0 ST arrays. We identified recurrent DNA amplifications scattered from 8q22.2 to 8q24.3 in 112 OSCC specimens. These amplicons demonstrated significant associations with increased incidence of extracapsular spread, development of second primary malignancies, and poor survival. Fluorescence in situ hybridization, in a validation panel consisting of 295 cases, confirmed these associations. Assessment of the effects of copy number variations (CNVs) on genome-wide variations in gene expression identified a total of 85 CNV-associated transcripts enriched in the MYC-centered regulatory network. Twenty-four transcripts associated with increased risk of second primary malignancies, tumor relapse, and poor survival. Besides MYC itself, a novel dysregulated MYC module plays a key role in OSCC carcinogenesis. This study identified a candidate molecular signature associated with poor prognosis in OSCC patients, which may ultimately facilitate patient-tailored selection of therapeutic strategies.

A reverse transcription comparative real-time PCR method for quantitative detection of angiogenic growth factors in head and neck cancer patients

OBJECTIVES Head and neck cancer is one of the ten most frequent cancers in the world. The angiogenic growth factors VEGF, PDGF and bFGF play a role in cancer aggressiveness. We developed a sensitive method to quantify the gene expression of these factors in the tissues of head and neck cancer patients. DESIGN AND METHODS All assays were performed using real-time RT-PCR, which yields a value (Ct) denoting the threshold cycle of PCR amplification at which product is first detected by fluorescence. The Ct is dependent on the quantity of the target molecule in the sample. To control for variation in RNA quantity and quality, we used 18S ribosome RNA as an internal control to calculate a relative Ct for the target molecules of interest, VEGF, PDGF and bFGF. A serially diluted positive control sample was analyzed by linear regression to determine the sensitivity and linearity of the assay. Paired normal and cancerous tissue samples from 115 head and neck cancer patients were assayed to ascertain the relative levels of the growth factors. RESULTS The CVs of within-run and between-run assays for VEGF, PDGF and bFGF were all less than 3%. The correlation coefficient of the RNA concentrations and Ct values were 0.9987, 0.9977, and 0.9996 respectively for VEGF, PDGF and bFGF. The assay was sensitive to as little as 10(-3) ng of RNA. All three growth factors were significantly increased in tumor tissue as compared to normal tissue. VEGF, PDGF and bFGF levels were elevated in 71.3%, 58.2% and 54.0% of cancerous tissue samples, with average levels of over-expression of 35.1, 24.6 and 13. sixfold, respectively. CONCLUSION This method provides sensitive, quantitative, high-throughput analysis for direct comparison of gene expression levels between samples, while adjusting for factors that may influence quantity determination. It should be applicable to molecules other than angiogenic growth factors, as well.

Identification of differentially expressed genes in oral squamous cell carcinoma (OSCC) : Overexpression of NPM, CDK1 and NDRG1 and underexpression of CHES1

To identify cellular genes that could potentially serve as predictive molecular markers for human oral cancer, we employed differential display analysis to compare the gene expression profiles between oral squamous cell carcinoma (OSCC) and histopathologically normal epithelium tissues. Comparative real‐time RT‐PCR was used to confirm the gene expression in 52 OSCC patients, and a 2‐fold difference was defined as over‐ or underexpression. A total of 7 genes were identified: NPM, CDK1, NDRG1, HMGCR, EF1A, NAC and CHES1. In the cancer tissues, NPM, CDK1 and NDRG1 were significantly overexpressed (an average of 7.6‐, 17.2‐ and 12.9‐fold, respectively), and CHES1 was underexpressed (15‐fold). The frequencies of the differential expression were 40, 56, 67 and 46%, respectively in NPM, CDK1, NDRG1 and CHES1. In Western blot analysis, the protein expressions of NPM, CDK1 and NDRG1 were also increased in the cancer tissues, consistent with the mRNA expression results. To further evaluate clinicopathological associations in these genes, Pearson chi‐square analysis was employed. Levels of CDK1 and NDRG1 were associated with poorly differentiated tumors (p = 0.043 and 0.023), suggesting that these genes participate in the mechanism of tumor transformation. Expressions of CDK1 and NDRG1, and CDK1 and CHES1 were mutually statistically correlated (p = 0.001 and 0.014), indicating that these genes share a very close regulatory relationship or interact synergistically in oncogenesis. In conclusion, we identified 7 genes that are differentially expressed in OSCC, and we provide the first evidence that NPM, CDK1 and NDRG1 are overexpressed and CHES1 is underexpressed in oral cancer. These results serve as a fundamental base for employing these genes in future clinical applications.

Salvage therapy in relapsed squamous cell carcinoma of the oral cavity: How and when?

Relapse of tumors in patients with oral cavity squamous cell carcinoma (OSCC) is associated with a poor prognosis. In addition, salvage therapy may be a significant source of morbidity in patients with relapsing OSCC. The objective of the current study was to determine prognostic factors that predict which patients may benefit from such treatment.