OBJECTIVE: We aimed at studying the role of the most deregulated miR-99a, identifying its downstream targets, and exploring the clinical potential of miR-99a and its target(s) in oral cancer. SUBJECTS AND METHODS: Following confirmation of miR-99a deregulation in nine oral lines and 26 pairwise clinical specimens, miR-99a-manipulated oral cancer cells were subjected to cell proliferation, migration, invasion, and in vivo murine metastasis assays. We characterized putative miR-99a target(s) using luciferase reporter assays and genetic manipulation. The inverse relation of miR-99a and its target(s) was examined in clinical specimens using real-time PCR and Western blot analysis. RESULTS: MiR-99a down-regulation was confirmed both in tested oral cancer cell lines and clinical specimens. Ectopic miR-99a expression inhibited oral cancer cell migration and invasion. Anti-miR-99a, silencing miR-99a functions, had the opposite effect. Myotubularin-related protein 3 (MTMR3) with one evolutionarily conserved seed region in the 3′-untranslated region was a novel miR-99a target. Depleting MTMR3 expression significantly reduced cell proliferation, migration, or invasion. There was an inverse expression of miR-99a and MTMR3 protein in oral cancer lines and clinical specimens. CONCLUSION: miR-99a repressed oral cancer cell migration and invasion partly through decreasing MTMR3 expression. MTMR3 may serve as a therapeutic target for oral cancer treatment. Oral Diseases (2014) 20, e65-e75

Background: Alcohol is a major risk factor for head and neck cancer (HNC). The metabolism of ethanol can disrupt the metabolism of retinoic acid because the two pathways share some key enzymes. Retinoic acid is involved in cell growth, differentiation, and apoptosis, which when dysregulated may lead to the development of cancer. A previous genome-wide association study reported an association between single nucleotide polymorphisms (SNPs) in two retinol transporter genes (TTR and RBP4) and serum retinol level (1). The current study evaluates the interaction between alcohol consumption and SNPs in TTR and RBP4 on the risk of HNC. Methods: 223 incident cases of HNC and 214 sex- and age- matched controls were recruited from the department of otolaryngology and department of stomatology. In-person interviews were conducted to collect information on the use of alcohol, cigarette, and betel quid. Three tag-SNPs of TTR and 8 tag-SNPs of RBP4 were genotyped using the Taqman-based real-time PCR method or mass spectrometry-based detection method. Unconditional logistic regression was performed to estimate the odds ratio (OR) and 95% confidence interval (CI) of HNC risk associated with each SNP and to evaluate the joint influence of SNPs and alcohol consumption on HNC risk, adjusted for age, sex, education, cigarette smoking and betel quid use. Results: No significant association between HNC risk and any of the 11 tag-SNPs was observed. However, three SNPs of RBP4 appeared to modify the relationship between alcohol consumption and HNC risk. After combining the three SNPs into a polygenic risk score, the results showed that those who drank alcohol daily had a different risk of HNC, depending on the number of variant alleles (0 to 2 variant alleles: OR=1.2, 95% CI: 0.7-2.1; 3 or more variant alleles: OR=2.9, 95% CI: 1.1-7.9; interaction P-value = 0.01) Conclusion: The association between HNC risk and alcohol consumption is modified by the polymorphisms of RBP4. Citation Format: Jeffrey S. Chang, Jenn-Ren Hsiao, Tung-Yiu Wong, Sen-Tien Tsai, Chun-Yen Ou, Hung-I Lo, Cheng-Chih Huang, Wei-Ting Lee, Ken-Chung Chen, Jehn-Shyun Huang, Yi-Hui Wang, Ya-Ling Weng, Han-Chien Yang. Interaction between retinol transporter genes and alcohol on the risk of head and neck cancer. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 15.

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Oral cancer is a subtype of head and neck cancer, the eighth common cancer type in the world. MicroRNAs (miRNAs) are small non-coding RNAs that mediate gene expression at the post-transcriptional level by degrading target messenger RNA or repressing their translation. The deregulation of certain microRNAs has been associated with the progression and metastasis of various cancer types. A total of 133 miRs were dysregulated in oral cancer cells when compared with normal oral keratinocytes by using microRNA microarray hybridization. Among them, miR-22, often silenced in breast cancer, colon cancer, and multiple myeloma, is downregulated and least known in oral carcinogenesis. In this study, we confirmed the down-regulation of miR-22 in the majority of oral cell lines and 70 % of the tested clinical specimens, as measured by real-time PCR. Both gain-of-function and loss-of-function experiments showed that increased miR-22 expression significantly reduced oral cancer cell migration and invasion, whereas decreased miR-22 expression dramatically enhanced cell migration and invasion. Snail, a transcription factor that promotes cell invasion and tumor metastasis, was predicted to be a target of miR-22. The prediction was validated by the inverse correlation of miR-22 and snail protein, and the luciferase reporter assay bearing 3’- translated region derived from snail. Silencing snail expression by using shRNA recapitulated the anti-metastatic function of miR-22, whereas restored snail expression attenuated the function of miR-22 in oral cancer cells. Together, these results suggest that miR-22 may act as a tumor suppressor through decreasing snail expression in oral cancer and thus be used as a therapeutic target to block oral cancer metastasis. Citation Format: Yi-Zih Kuo, Yun-Chu Huang, Yuh-Ling Chen, Hung-I Lo, Sen-Tien Tsai, Chi-Wu Chiang, Li-Wha Wu. Identification of dysregulated miRs and its target genes in head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4195. doi:10.1158/1538-7445.AM2013-4195

Background: Head and neck cancer (HNC), including cancer of the oral cavity, pharynx, and larynx, is one of the leading cancers in the world. The known risk factors, including alcohol, betel quid, and cigarette, account for the majority of the HNC cases. However, the biological mechanisms regarding the carcinogenic effects of these agents in the development of HNC are not completely understood. Retinoic acid is involved in cell growth, differentiation, and apoptosis, which when dysregulated may lead to the development of cancer. Studies have shown that alcohol, betel quid, and cigarette can affect either the metabolism or the function of retinoic acid pathway. The current analysis evaluated the association between serum retinol and HNC risk and assessed whether this association can be modified by alcohol, betel quid, or cigarette. Methods: 93 incident cases of HNC and 79 sex- and age- frequency matched controls were recruited from the department of otolaryngology and department of stomatology. Information on the use of alcohol, betel quid, and cigarette was collected by in-person interviews. Serum retinol levels were measured using high performance liquid chromatography. Unconditional logistic regression was performed to estimate the odds ratio (OR) and 95% confidence interval (CI) of HNC risk associated with serum retinol levels. Additional analyses were performed stratified by alcohol, betel quid, and cigarette. Results: Compared to controls, HNC cases had a lower level of serum retinol (Median for HNC cases: 811 ug/l vs. median for controls: 901 ug/l, Wilcoxon rank-sum P = 0.04). For every 100 ug/l increase, the risk of HNC was reduced by 8% (OR = 0.92, 95% CI: 0.83-1.02). The OR was 0.5 (95% CI: 0.24-1.06) comparing the highest tertile of serum retinol level to the lowest tertile. A lower HNC risk associated with higher serum retinol levels was observed among never alcohol drinkers (For every 100 ug/l increment, OR = 0.80, 95% CI: 0.66-0.96) but not among regular alcohol drinkers (For every 100 ug/l increment, OR = 0.98, 95% CI: 0.86-1.13). Conclusion: Higher levels of serum retinol are associated with a reduced risk of HNC. Alcohol consumption obliterates the inverse association between serum retinol and HNC risk, possibly by disrupting the metabolism of retinoic acid pathway. Citation Format: Jeffrey S. Chang, Jenn-Ren Hsiao, Chun-Yen Ou, Tung-Yiu Wong, Sen-Tien Tsai, Hung-I Lo, Cheng-Chih Huang, Wei-Ting Lee, Ken-Chung Chen, Jehn-Shyun Huang, Yi-Hui Wang, Ya-Ling Weng, Han-Chien Yang. Serum retinol and risk of head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 105. doi:10.1158/1538-7445.AM2013-105