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Whether oral lichen planus (OLP) was potentially malignant remains controversial. Here, we examined associations of ZNF582 methylation (ZNF582This is a case-control study. ZNF582OLP lesions showed significantly lower ZNF582OLP is unlikely to be potentially malignant based on ZNF582

Background/purpose Cancer stem cells (CSCs) have been known to be implicated in tumorigenesis, metastasis, and drug resistance in oral squamous cell carcinomas (OSCC). In this study, we aimed to investigate whether magnolol, a polyphenolic component derived from Magnolia officinalis, exhibited the anti-CSCs properties. Methods The cytotoxicity of magnolol was tested using normal gingival epithelioid SG cells and sphere-forming OSCC-CSCs isolated from SAS, OECM1, and GNM cells. Secondary sphere-forming ability, the proportion of ALDH1 positive cells, Transwell migration, and invasion capacities were examined as well. The chemosensitive effects of magnolol were investigated using MTT, secondary sphere-forming, and invasion assays. Results Magnolol exerted a higher cytotoxicity of OSCC-CSCs and cancer stemness features, including self-renewal ability, the expression CSC marker, migration, and invasion capacities were all downregulated in magnolol-treated OSCC-CSCs. Moreover, administration of magnolol potentiated the effect of cisplatin, including a decrease in cell viability, self-renewal, and invasion activities. In addition, we observed that the secretion of IL-6 and phosphorylation of Stat3 were decreased in OSCC-CSCs treated with magnolol. Conclusion Our data suggest that magnolol is able to target CSCs and suppress the cancer stemness properties, at least in part, via IL-6/Stat3 signaling. Besides, a dietary supplement of magnolol may function as an adjunct to cisplatin treatment.

Background/Purpose Oral submucous fibrosis (OSF) a well-recognized oral premalignant disorder. Several studies have demonstrated that periostin, a matricellular protein, is involved in the development and pathogenesis of fibrosis diseases. Nevertheless, the contribution of periostin in OSF remains to be uncovered. The purpose of the study was to illustrate the functional role of periostin involved in OSF pathogenesis. Methods RNA-sequencing was employed to screen for differentially expressed genes in normal and OSF tissues. Validation of the upregulation of periostin in OSF specimens and fibrotic buccal mucosal fibroblasts (fBMFs) was conducted by qRT-PCR. The correlation of the gene expression of periostin and various fibrosis markers was analyzed. In addition, the functional role of periostin in myofibroblast features was tested using collagen gel contraction and transwell migration assays. Results We observed overexpression of periostin in OSF specimens using RNA-sequencing and confirmed its upregulation in OSF tissues and patient-derived fBMFs. Besides, there was a positive relationship between the expression of periostin and several fibrosis-associated markers, including ACTA2 (α-smooth muscle actin; α-SMA), COL1A1 (type 1 collagen α1 chain), TGFB1 (TGF-β1), and FN1 (fibronectin). Furthermore, we examined the effect of silencing periostin on the maintenance of myofibroblast characteristics and showed that knockdown of periostin suppressed the expression of α-SMA. Also, inhibition of periostin markedly downregulated the myofibroblast activities (collagen gel contraction and migration capacities). Conclusion Our results indicate the aberrant expression of periostin in OSF tissues and myofibroblasts. Moreover, the expression of periostin is positively associated with fibrosis markers, and repression of periostin may be a promising direction to alleviate the progression of OSF.

In approximately 50% of patients with oral cavity squamous cell carcinoma (OCSCC), the disease progresses after curative surgery. However, the role of salvage surgery (SS) is controversial, and life expectancy after SS is unknown.In this study, 262 patients with OCSCC with locoregional recurrence and second primary OCSCC were retrospectively enrolled and divided into a resectable (55.0%, 144/262) and unresectable (45.0%, 118/262) groups. After excluding neck recurrence only, SS had been performed 195 times in the resectable group. The corresponding preoperative clinicopathologic factors and postsurgery survival (PSS) of each SS were pooled for analysis.Median survival after disease progression was 64.2 and 10.4 months for the resectable and unresectable groups, respectively. In the resectable group, one-fifth (19.5%, 37/190) of the patients died within 1 year of SS (PSS 1 year), and one-third (32.8%, 64/195) of the patients had undergone SS two or more times. The interval from the last surgery ≤ 12 months, depth of invasion of the last surgery 1 cm, and clinical evidence of nodal disease at the preoperative evaluation were independent predictors of poor PSS. A scoring prediction model was established with 1 point for each factor. The results revealed 1-year postsurgery death rates of 10.3% in the low-risk group (score: 0-1) and 48.6% in the high-risk group (score: 2 or 3) (P 0.001).In conclusion, an effective scoring model predicting life expectancy after SS for patients with OCSCC was established.

Early progression, defined as a disease-free interval (DFI) of less than 6 months after completion of adjuvant platinum-based chemoradiotherapy (CRT), leads to poor outcomes in locally advanced oral cavity squamous cell carcinoma (OCSCC). However, appropriate biomarkers for predicting early progression remain unknown.In this study, 346 patients with OCSCC, who underwent curative surgical resection and platinum-based adjuvant CRT at the Taipei Veterans General Hospital (202 patients, training cohort) and Chung Shan Medical University Hospital (144 patients, validation cohort) were enrolled. The clinical-pathological variables were compared using the χOne-fifth (20.5%, 71/346) of all patients experienced progression within 6 months. Each of the independent factors for the DFI in the training cohort, including pT3-4, extracapsular spread, and perineural invasion, were assigned a score of one point to establish a scoring system. The 6-month DFIs of the low-risk (score 0-1), intermediate-risk (score 2), and high-risk (score 3) groups were 97.8%, 78.7%, and 35.7% and 88.2%, 77.6%, and 42.1% in the training and validation cohorts, respectively. If the cutoff level was ≥2 or2, the sensitivity/specificity/area under the curve for the training and validation cohorts were 94.4%/56.1%/0.837, and 73.3%/56.6%/0.703, respectively.The established scoring system effectively predicted early progression after adjuvant CRT for locally advanced OCSCC.

Oral submucosal fibrosis (OSF) is a premalignant disorder of the oral cavity, and areca nut chewing is known to be a major etiological factor that could induce epithelial to mesenchymal transition (EMT) and activate buccal mucosal fibroblasts (BMFs). However, this detailed mechanism is not fully understood. In this study, we showed that the upregulation of Snail in OSF samples and fibrotic BMFs (fBMFs) may result from constant irritation by arecoline, a major alkaloid of the areca nut. The elevation of Snail triggered myofibroblast transdifferentiation and was crucial to the persistent activation of fBMFs. Meanwhile, Snail increased the expression of numerous fibrosis factors (e.g., &alpha, SMA and collagen I) as well as IL-6. Results from bioinformatics software and a luciferase-based reporter assay revealed that IL-6 was a direct target of Snail. Moreover, IL-6 in BMFs was found to further increase the expression of Snail and mediate Snail-induced myofibroblast activation. These findings suggested that there was a positive loop between Snail and IL-6 to regulate the areca nut-associated myofibroblast transdifferentiation, which implied that the blockage of Snail may serve as a favorable therapeutic strategy for OSF treatment.

Dysbiosis of oral microbiome may dictate the progression of oral squamous cell carcinoma (OSCC). Yet, the composition of oral microbiome fluctuates by saliva and distinct sites of oral cavity and is affected by risky behaviors (smoking, drinking and betel quid chewing) and individuals’ oral health condition. To characterize the disturbances in the oral microbial population mainly due to oral tumorigenicity, we profiled the bacteria within the surface of OSCC lesion and its contralateral normal tissue from discovery (n = 74) and validation (n = 42) cohorts of male patients with cancers of the buccal mucosa. Significant alterations in the bacterial diversity and relative abundance of specific oral microbiota (most profoundly, an enrichment for genus Fusobacterium and the loss of genus Streptococcus in the tumor sites) were identified. Functional prediction of oral microbiome shown that microbial genes related to the metabolism of terpenoids and polyketides were differentially enriched between the control and tumor groups, indicating a functional role of oral microbiome in formulating a tumor microenvironment via attenuated biosynthesis of secondary metabolites with anti-cancer effects. Furthermore, the vast majority of microbial signatures detected in the discovery cohort was generalized well to the independent validation cohort, and the clinical validity of these OSCC-associated microbes was observed and successfully replicated. Overall, our analyses reveal signatures (a profusion of Fusobacterium nucleatum CTI-2 and a decrease in Streptococcus pneumoniae) and functions (decreased production of tumor-suppressive metabolites) of oral microbiota related to oral cancer.

Background: Oral submucosal fibrosis is a premalignant disorder of the oral cavity, and areca nut chewing habit has been known as a major etiologic factor. Various studies have revealed that areca nut induced epithelial to mesenchymal transition (EMT) and activation of buccal mucosal fibroblasts (BMFs), but the detailed mechanism has not been fully understood. Methods: The expression of Snail was assessed in the OSF tissues and BMFs derived from OSF specimens (fBMFs). We examined the functional role of Snail in myofibroblast activation, and used the bioinformatics software to predict the interacting factor of Snail and studied their impact on myofibroblast activities. Findings: The upregulation of Snail in OSF samples and fBMFs may result from constant irritation of arecoline, a major alkaloid of areca nut. The elevation of Snail triggered myofibroblast activation and was crucial to the transdifferentiation of BMFs as well as the persistent activation of fBMFs. Meanwhile, Snail increased the expression of numerous fibrosis factors (e.g. α-SMA and collagen I) as well as IL-6. Results from bioinformatics software and luciferase-based reporter assay revealed that IL-6 was a direct target of Snail. Moreover, IL-6 in BMFs was found to further increase the expression of Snail and contribute to the Snail-induced myofibroblast activation. Interpretation: Our results suggested that there was a positive loop between Snail and IL-6 to regulate the areca nut-associated myofibroblast transdifferentiation. These findings implied that blockage of Snail may serve as a favourable therapeutic strategy for OSF treatment. Funding Statement: This work was supported by grants from the Chung Shan Medical University Hospital (grant number CSH-2018-C-013); the China Medical University (grant numbers CMU108-N-10, CMU108-MF-06); and the Ministry of Science and Technology (grant number MOST108-2314-B-040-001, MOST107-2314-B-040 -023) in Taiwan. Declaration of Interests: The authors declare that there are no conflicts of interest. Ethics Approval Statement: All procedures performed in this study involving human participants were in accordance with the tenets of the Declaration of Helsinki and reviewed by the Institutional Review Committee at Chung Shan Medical University, Taichung, Taiwan.

Epithelial-mesenchymal transition (EMT) has been implicated in fibrogenesis and carcinogenesis; however, the exact role of EMT-inducer Slug in the progression of precancerous oral submucous fibrosis (OSF) has not been investigated. In the current study, we showed that the expression of Slug was upregulated in OSF tissues and associated with various myofibroblast markers. After silence of Slug in fibrotic buccal mucosal fibroblasts (fBMFs), the elevated myofibroblast activities and fibrosis markers were all downregulated. Our data revealed that arecoline, an areca nut alkaloid, increased the expression of Slug in normal BMFs, and inhibition of Slug successfully prevented the arecoline-induced myofibroblast activation. Additionally, overexpression of Slug in BMFs stimulated the activities of myofibroblasts, indicating that upregulation of Slug by arecoline contributes to the myofibroblast transdifferentiation. Most importantly, Slug was able to bind to the E-box of type I collagen, leading to increased expression of type I collagen. Altogether, this study demonstrated the abnormal elevation of Slug in OSF and its significance in arecoline-induced fibrogenesis. Moreover, downregulation of Slug could be a potential target for OSF remedy via suppression of myofibroblast activities and type I collagen.

Background/Purpose: MicroRNA-200c (miR-200c) recently emerged as an important regulator of tumorigenesis and cancer metastasis, however, its role in regulating oral submucous fibrosis (OSF) remains unknown. In this study, we investigated the functional role of miR-200c in myofibroblastic differentiation activity and identified its potential target. Methods: qRT-PCR was applied to assess the expression of miR-200c in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs). Arecoline, a major areca nut alkaloid, was utilized to explore whether the expression of miR-200c would alter following stimulation. Collagen gel contraction, migration and invasion capabilities were examined in arecoline-stimulated BMFs as wells as in fBMFs. Luciferase reporter assay was conducted to show the relationship between miR-200c and ZEB1. Results: Our results showed that the expression of miR-200c was downregulated in OSF specimen and fBMFs. Arecoline treatment dose-dependently reduced the relative expression of miR-200c in normal BMFs. Overexpression of miR-200c impeded the arecoline-induced collagen gel contraction, migration, invasion and wound healing capacities. Moreover, ectopic expression of miR-200c in fBMFs successfully reduced the increased collagen gel contractility and invasion abilities. Our results demonstrated that ZEB1 was a direct target of miR-200c, and overexpression of miR-200c inhibited the expression of ZEB1 and α-SMA. Conclusion: These findings suggest that downregulation of miR-200c in OSF may be involved in the pathogenesis of areca nut-associated OSF through regulation of ZEB1. Keywords: Oral submucous fibrosis, Arecoline, miR-200c, ZEB1, Myofibroblasts

Background/Purpose: Emerging research findings suggest that long non-coding RNAs (lncRNAs) are key regulators to fibrosis formation. Nevertheless, the role of lncRNA GAS5-AS1 in the progression of precancerous oral submucous fibrosis (OSF) remains to be elucidated. Methods: Quantitative real-time PCR were used to examine the expression of GAS5-AS1 in OSF tissues. The activities of myofibroblasts, including collagen contractility and cell migration, as well as the marker α-smooth muscle actin (SMA) were assessed following overexpression of GAS5-AS1. Also, we analyzed the expression of Smad activity in order to gain insight into the downstream regulator. Results: The level of GAS5-AS1 was found significantly downregulated in the OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs). Ectopic expression of GAS5-AS1 significantly reduced the abilities of collagen gel contraction and migration in fBMFs or arecoline-treated BMFs. Moreover, we have shown that overexpression of GAS5-AS1 inhibited the expression of p-Smad and the marker of myofibroblasts. Conclusion: We showed the reduced expression of GAS5-AS1 in OSF tissues and demonstrated its effect on the myofibroblast activities and the level of p-Smad and α-SMA, indicating its potential contribution in OSF pathogenesis. Keywords: lncRNA GAS5-AS1, Myofibroblasts, Oral submucous fibrosis

Background The let-7 family of microRNAs has been considered as tumor suppressors in various cancers; however, the role of let-7c in oral squamous cell carcinoma has not been determined yet. Methods In this study, phenotypical behaviors and the radio/chemoresistance were examined subsequent to overexpression of let-7c. In addition, the expression of let-7c in cancer stem cells (CSCs) was evaluated and the effect of let-7c on stemness characteristics was assessed. Also, luciferase activity assays were performed to test whether interleukin (IL)-8 was a putative target of let-7c. Results Our results confirmed that the expression of let-7c in CSCs was reduced, while overexpression of let-7c attenuated the oncogenicity. Moreover, ectopic expression of let-7c in CSCs downregulated the stemness hallmarks and the radio/chemoresistance. Expression and secretion of IL-8 in oral CSCs were both reduced following overexpression of let-7c. Besides, the inhibitory effect of let-7c on various stemness phenotypes was reverted by IL-8, indicating that lower expression of let-7c may confer higher cancer stemness through a failure to downregulate IL-8. Conclusion These findings revealed the significance of let-7c in the contribution of oral cancer stemness and radio/chemoresistance. Targeting let-7c and its downstream IL-8 may be beneficial to prevent cancer recurrence and metastasis of oral squamous cell carcinoma.

// Che-Yi Lin 1 , Pei-Ling Hsieh 2, * , Yi-Wen Liao 3, * , Chih-Yu Peng 3 , Ming-Yi Lu 3 , Ching-Hsuan Yang 2 , Cheng-Chia Yu 2, 3, 4 and Chia-Ming Liu 3, 4 1 Department of Oral and Maxillofacial Surgery, Chi Mei Hospital, Tainan, Taiwan 2 Institute of Oral Sciences, Chung Shan Medical University, Taichung, Taiwan 3 School of Dentistry, Chung Shan Medical University, Taichung, Taiwan 4 Department of Dentistry, Chung Shan Medical University, Taichung, Taiwan * These authors have contributed equally to this work Correspondence to: Cheng-Chia Yu, email: ccyu@csmu.edu.tw Chia-Ming Liu, email: y337@csmu.edu.tw Keywords: oral squamous cell carcinomas, miR-21, berberine Received: June 16, 2017 Accepted: August 04, 2017 Published: September 08, 2017 ABSTRACT Cancer recurrence and chemoresistance are two major obstacles to the treatment of oral squamous cell carcinomas (OSCC). And cancer stem cells (CSCs) have been found to possess tumor initiating, self-renewal and metastasis abilities, resulting in the relapse and chemoresistance of OSCC. In the present study, we investigated the anti-CSCs effect of berberine, a phenanthrene alkaloid isolated from the Berberis genus. Our results demonstrated that berberine dose dependently downregulated the oncogenicity in vitro , including ALDH1 activity, self-renewal property, and colony formation and invasion abilities as well as potentiated chemosensitivity of OSCC-CSCs. In addition, tumor growth in mice was attenuated after oral gavage treatment of berberine. We showed that the expression of miR-21 was suppressed following administration of berberine in OSCC-CSCs. And inhibition of endogenous miR-21 reduced the characteristics of CSCs, including self-renewal, migration, invasion capabilities and ALDH1 activity. Taken together, we demonstrated the anti-CSC effect of berberine in oral cancer and its potential to serve as adjuvant to traditional chemotherapy to improve treatment effect.

Background/Purpose Cancer stem cells (CSCs) are deemed as the driving force of tumorigenesis in oral squamous cell carcinomas (OSCCs). In this study, we investigated the chemotherapeutic effect of sulforaphane, a dietary component from broccoli sprouts, on targeting OSCC-CSCs. Methods The effect of sulforaphane on normal oral epithelial cells (SG) and sphere-forming OSCC-CSCs isolated from SAS and GNM cells was examined. ALDH1 activity and CD44 positivity of OSCC-CSCs with sulforaphane treatment was assessed by flow cytometry analysis. In vitro and in vivo tumorigenicity assays of OSCC-CSCs with sulforaphane treatment were presented. Results We observed that the sulforaphane dose-dependently eliminated the proliferation rate of OSCC-CSCs, whereas the inhibition on SG cells proliferation was limited. Cancer stemness properties including self-renewal, CD44 positivity, and ALDH1 activity were also decreased in OSCC-CSCs with different doses of sulforaphane treatment. Moreover, sulforaphane treatment of OSCC-CSCs decreased the migration, invasion, clonogenicity, and in vivo tumorigenicity of xenograghts. Sulforaphane treatment resulted in a dose-dependent increase in the levels of tumor suppressive miR200c. Conclusion These lines of evidence suggest that sulforaphane can suppress the cancer stemness and tumor-initiating properties in OSCC-CSCs both in vitro and in vivo.

Oral submucous fibrosis (OSF) is an oral precancerous condition associated with the habit of areca nut chewing and the TGF-&beta, pathway. Currently, there is no curative treatment to completely heal OSF, and it is imperative to alleviate patients&rsquo, symptoms and prevent it from undergoing malignant transformation. Arctigenin, a lignan extracted from Arctium lappa, has been reported to have a variety of pharmacological activities, including anti-fibrosis. In the present study, we examined the effect of arctigenin on the cell proliferation of buccal mucosal fibroblasts (BMFs) and fibrotic BMFs (fBMFs), followed by assessment of myofibroblast activities. We found that arctigenin was able to abolish the arecoline-induced collagen gel contractility, migration, invasion, and wound healing capacities of BMFs and downregulate the myofibroblast characteristics of fBMFs in a dose-dependent manner. Most importantly, the production of TGF-&beta, in fBMFs was reduced after exposure to arctigenin, along with the suppression of p-Smad2, &alpha, smooth muscle actin, and type I collagen A1. In addition, arctigenin was shown to diminish the expression of LINC00974, which has been proven to activate TGF-&beta, /Smad signaling for oral fibrogenesis. Taken together, we demonstrated that arctigenin may act as a suitable adjunct therapy for OSF.

e18040 Background: Salvage surgery (SS) is one of the curative options for oral cavity squamous cell carcinoma (OCSCC) patients with locoregional recurrence (LRR) or secondary primary, but the role of SS should be reevaluated between life expectancy, morbidity, and quality of life. Selecting suitable OCSCC patients receiving SS is important. Methods: From 2010 to 2018, newly diagnosed OCSCC patients who progressed to LRR or secondary primary were recorded. Clinical outcomes, prognostic factors, and predictors were analyzed for the patients receiving SS. Cox regression analyses were performed for PSS, defined from the date of SS to the date of death or last follow-up. Survival was estimated using the Kaplan–Meier method and log-rank tests. Results: A total of 263 newly diagnosed OCSCC patients progressing to LRR or secondary primary were recorded. Half (55.1%, 145/263) of them received SS, and one-third (29.7%, 43/145) of the SS group received twice and more times SS. Median survivals after disease progression were 65.6 and 10.6 months for patients with or without SS, respectively (P < 0.001). A total of 214 SS events were enrolled for analysis. Nearly twenty percentage (20.1%, 39/194) of SS events would progress to death within 1 year after surgery (PSS < 1 year). PSSs of the first, secondary, third, and fourth or more times SS were 64.2, 47.6, 40.9, and 18.9 months, respectively (P = 0.217). Surgical features of the last surgery (perineural invasion and depth of invasion), the interval between the last and current surgery, and clinical N staging of the current surgery were the four independent factors for PSS. To predict the patients with PSS < 1 year, a scoring system was established that each of the independent factors was scored one point. The area under the curve of the scoring system was 0.755, and sensitivity, specificity, positive predictive value, and negative predictive value were 66.7%, 76.0%, 39.0%, and 90.8%, respectively (Table). Conclusions: A scoring system with a high negative predictive value was established to predict PSS < 1 year.[Table: see text]

// Po-Yu Yang 1, 2, 4, * , Pei-Ling Hsieh 3, * , Tong Hong Wang 5, 6, 7, * , Cheng-Chia Yu 1, 2, 3, 4, * , Ming-Yi Lu 1, 2, 3, 4 , Yi-Wen Liao 1 , Tzu-Hsin Lee 1, 2 , Chih-Yu Peng 1, 2, 3, 4 1 School of Dentistry, Chung Shan Medical University, Taichung, Taiwan 2 Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan 3 Institute of Oral Sciences, Chung Shan Medical University, Taichung, Taiwan 4 Oral Medicine Center, Chung Shan Medical University, Taichung, Taiwan 5 Tissue Bank, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan 6 Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Tao-Yuan, Taiwan 7 Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Tao-Yuan, Taiwan * These authors contributed equally to this work Correspondence to: Chih-Yu Peng, email: cyp@csmu.edu.tw Keywords: oral squamous cell carcinomas, andrographolide, miR-218, cancer stemness Received: October 31, 2016 Accepted: November 23, 2016 Published: December 01, 2016 ABSTRACT Current evidence suggests that oral cancer stem cells (OCSCs) possess high tumorigenic and metastatic properties as well as chemo- and radioresistance. In this study, we demonstrated that andrographolide, the main bioactive component in the medicinal plant Andrographis, significantly reduced oncogenicity and restored radio-sensitivity of ALDH1 + CD44 + OCSCs. Mechanistic studies showed that andrographolide treatment increased the expression of microRNA-218 (miR-218), leading to the downregulation of Bmi1. We showed that knockdown of miR-218 in ALDH1 − CD44 − non-OCSCs enhanced cancer stemness, while silencing of Bmi1 significantly counteracted it. Furthermore, we found tumor growth was reduced in mice bearing xenograft tumors after andrographolide treatment via activation of miR-218/Bmi1 axis. Together, these data demonstrated that the inhibition of tumor aggressiveness in OCSCs by andrographolide was mediated through the upregulation of miR-218, thereby reducing Bmi1 expression. These findings suggest that andrographolide may be a valuable natural compound for anti-CSCs treatment of OSCC.

// Tung Yuan Wang 1, * , Chih-Yu Peng 1, 2, * , Shiuan-Shinn Lee 4 , Ming-Yung Chou 1, 2, 3 , Cheng-Chia Yu 1, 2, 3 , Yu-Chao Chang 1, 2 1 School of Dentistry, Chung Shan Medical University, Taichung, Taiwan 2 Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan 3 Institute of Oral Sciences, Chung Shan Medical University, Taichung, Taiwan 4 School of Public Health, Chung Shan Medical University, Taichung, Taiwan * These authors contributed equally to this work Correspondence to: Cheng-Chia Yu, email: ccyu@csmu.edu.tw Yu-Chao Chang, email: cyc@csmu.edu.tw Keywords: arecoline, cancer stemness, oral squamous cell carcinomas Received: November 09, 2015 Accepted: August 13, 2016 Published: August 20, 2016 ABSTRACT Oral squamous cell carcinoma (OSCC), one of the most deadliest malignancies in the world, is caused primarily by areca nut chewing in Southeast Asia. The mechanisms by which areca nut participates in OSCC tumorigenesis are not well understood. In this study, we investigated the effects of low dose long-term arecoline (10 μg/mL, 90-days), a major areca nut alkaloid, on enhancement cancer stemness of human oral epithelial (OE) cells. OE cells with chronic arecoline exposure resulted in increased ALDH1 population, CD44 positivity, stemness-related transcription factors (Oct4, Nanog, and Sox2), epithelial-mesenchymal transdifferentiation (EMT) traits, chemoresistance, migration/invasiveness/anchorage independent growth and in vivo tumor growth as compared to their untreated controls. Mechanistically, ectopic miR-145 over-expression in chronic arecoline-exposed OE (AOE) cells inhibited the cancer stemness and xenografic. In AOE cells, luciferase reporter assays further revealed that miR-145 directly targets the 3′ UTR regions of Oct4 and Sox2 and overexpression of Sox2/Oct4 effectively reversed miR-145-regulated cancer stemness-associated phenomenas. Additionally, clinical results further revealed that Sox2 and Oct4 expression was inversely correlated with miR-145 in the tissues of areca quid chewing-associated OSCC patients. This study hence attempts to provide novel insight into areca nut-induced oral carcinogenesis and new intervention for the treatment of OSCC patients, especially in areca nut users.

Background/purpose Oral submucous fibrosis (OSF), a chronic progressive scarring disease, has been considered as a precancerous condition of oral mucosa. In this study, we investigated the functional role of Twist, an epithelial-mesenchymal transition (EMT) transcriptional factor, in myofibroblastic differentiation activity of OSF. Methods Arecoline, a major areca nut alkaloid, was used to explore whether expression of Twist could be changed dose-dependently in human primary buccal mucosal fibroblasts (BMFs). Collagen gel contraction and migration capability in arecoline-stimulated BMFs and primary oral submucous fibrosis-derived fibroblasts (OSFs) with Twist knockdown was presented. Results We observed that the treatment of arecoline dose-dependently increased Twist expression transcript and protein levels in BMFs. The myofibroblast activity including collagen gel contraction and migration capability also induced by arecoline, while knockdown of Twist reversed these phenomena. Importantly, inhibition of Twist led to the suppression collagen contraction and wound healing capability of primary cultivated OSFs. Clinically, Twist transcript and protein expression was higher in areca quid chewing-associated OSF tissues than in normal oral mucosa tissues. Conclusion This evidence suggests that upregulation of Twist might be involved in the pathogenesis of areca quid-associated OSF through dysregulation of myofibroblast activity.

The feature of oral squamous cell carcinomas (OSCC) is commonly metastasizing to locoreginal lymph nodes, and the involvement of lymph nodes metastasis represents the one of important prognostic factors of poor clinical outcome. MicroRNAs (miRNAs) have been shown to be key players of cancer-related hallmarks including cancer stemness, EMT (epithelial-mesenchymal transition), and metastaisis. Herein we showed that OSCC-derived ALDH1+ cancer stem cells (OSCC-CSCs) express lower level of miR-204, and miR-204 over-expression suppresses cancer stemness and in vivo tumor-growth of OSCC-CSCs. miR-204 binds on their 3'UTR-regions of Slug and Sox4 and suppressing their expression in OSCC-CSCs. On the contrary, down-regulation of miR-204 significantly increased cancer stemness and the lymph nodes incidence of orthotopic animal models. Furthermore, co-knockdown with sh-Slug and sh-Sox4 synergistically rescued miR-204-supressing cancer stemness and EMT properties. Clinical results further revealed that a miR-204lowSlughighSox4high signature predicted the worse survival prognosis of OSCC patients by Kaplan-Meier survival analyses. Up-regulated miR-204-targeting Slug and Sox4 by epigallocatechin-3-gallate (EGCG) treatment significantly inhibited the proliferation rate, self-renewal capacity, and the percentage of ALDH1+ and CD44+ cells in OSCC-CSCs Oral-feeding of EGCG effectively alleviated tumor-progression in OSCC-CSCs-xenotransplanted immunocompromised mice through miR-204 activation. In conclusion, miR-204-mediated suppression of cancer stemness and EMT properties could be partially augmented by the anti-CSCs effect of EGCG.

e18536 Background: At least 10% of patients with oral cavity squamous cell carcinoma (OCSCC) would progress to distant metastases (DM). Median overall survival (OS) after distant metastases is only one year and rare biomarkers are available to predict distant metastatic events. Methods: A total of 595 OCSCC patients who underwent curative surgical resection at Chung Shan Medical University Hospital were enrolled from Jan 2010 to Dec 2016. The clinical–pathological variables were compared using the χ2 test. Cox proportional-hazards analyses were performed for distant metastatic free survival (DMFS), defined from the date of initial diagnosis to the date of distant metastasis. Two scoring systems for predicting distant metastases were established for patients with pathologic regional lymph node –positive (pN+) or –negative (pN–), separately. Results: Nearly 10% (8.9%, 53/595) of patients with OCSCC would progress to DM during follow-up [20.6% (28/136) and 5.4% (25/459) for patients with pN+ and pN–, respectively]. Most of the distant metastatic lesions developed within 3 years after initial diagnosis (overall, 84.9%, 45/53; patients with pN+, 92.9%, 26/28; patients with pN–, 76.0%, 19/25). For patients with pN+, rapidly locoregional recurrence (≦6 months), lymphovascular invasion, and lymphocyte-monocyte ratio( 65), and close surgical margin(≦5mm) were independent for those with pN–. If each of the independent factors was scored one point to predict DM occurring within 3 years after the initial diagnosis, two scoring systems were established for the corresponding patients with pN+ or pN–, separately. The area under the curves of these two scoring systems for the corresponding patients with pN+ and pN– were 0.737 and 0.825, respectively (Table). Conclusions: The established scoring systems effectively predict distant metastases for OCSCC patients. [Table: see text]

MiRNAs have been recognized as crucial components in carcinogenesis, but whether miR-1246 affects the cancer stemness and drug resistance in oral squamous cell carcinoma (OSCC) has not been fully understood and its downstream targets still need to be unraveled. In the present work, we employed miRNAs RT-PCR analysis to evaluate the expression of miR-1246 in tumor tissues and oral cancer stem cells (OCSC). Stemness phenotypes, including self-renewal, migration, invasion, colony formation capacities, and in vivo oncogenicity of oral cancer cells following transfected with miR-1246 inhibitors or mimics were examined. Our results suggested that the expression level of miR-1246 was significantly upregulated in the tumor tissues and OCSC. Kaplan-Meier survival analysis of OSCC patients with high levels of miR-1246 had the worst survival rate compared to their low-expression counterparts. Inhibition of miR-1246 in OCSC significantly reduced the stemness hallmarks, while overexpression of miR-1246 enhanced these characteristics. Moreover, we showed that downregulation of miR-1246 decreased chemoresistance. In addition, we verified that miR-1246-inhibited CCNG2 contributed to the cancer stemness of OSCC. These results demonstrated the significance of miR-1246 in the regulation of OSCC stemness. Targeting miR-1246-CCNG2 axis may be beneficial to suppress cancer relapse and metastasis in OSCC patients.

Cancer recurrence and chemoresistance are two major obstacles to the treatment of oral squamous cell carcinomas (OSCC). And cancer stem cells (CSCs) have been found to possess tumor initiating, self-renewal and metastasis abilities, resulting in the relapse and chemoresistance of OSCC. In the present study, we investigated the anti-CSCs effect of berberine, a phenanthrene alkaloid isolated from the Berberis genus. Our results demonstrated that berberine dose dependently downregulated the oncogenicity in vitro, including ALDH1 activity, self-renewal property, and colony formation and invasion abilities as well as potentiated chemosensitivity of OSCC-CSCs. In addition, tumor growth in mice was attenuated after oral gavage treatment of berberine. We showed that the expression of miR-21 was suppressed following administration of berberine in OSCC-CSCs. And inhibition of endogenous miR-21 reduced the characteristics of CSCs, including self-renewal, migration, invasion capabilities and ALDH1 activity. Taken together, we demonstrated the anti-CSC effect of berberine in oral cancer and its potential to serve as adjuvant to traditional chemotherapy to improve treatment effect.

// Yu-Chao Chang 1,2,4 , Chia-Ing Jan 5,6,* , Chih-Yu Peng 1,2,4,* , Yu-Chi Lai 3 , Fang-Wei Hu 1,2,3,4 and Cheng-Chia Yu 1,2,3,4 1 School of Dentistry, Chung Shan Medical University, Taichung, Taiwan 2 Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan 3 Institute of Oral Sciences, Chung Shan Medical University, Taichung, Taiwan 4 Oral Medicine Research Center, Chung Shan Medical University, Taichung, Taiwan 5 Department of Pathology, China Medical University Hospital, Taichung, Taiwan 6 Department of Pathology, China Medical University Beigang Hospital, Yunlin, Taiwan * These authors contributed equally to this work Correspondence to: Cheng-Chia Yu, email: // Fang-Wei Hu, email: // Keywords : head and neck squamous cell carcinomas, tumor initiating cells, silibinin, microRNA-494 Received : February 26, 2015 Accepted : May 30, 2015 Published : June 08, 2015 Abstract Tumor initiating cells (TICs) possessing cancer stemness were shown to be enriched after therapy, resulting in the relapse and metastasis of head and neck squamous cell carcinomas (HNC). An effective therapeutic approach suppressing the HNC-TICs would be a potential method to improve the treatments for HNC. We observed that the treatment of silibinin (SB) dose dependently down-regulated the ALDH1 activity, CD133 positivity, stemness signatures expression, self-renewal property, and chemoresistance in ALDH1+CD44+ HNC-TICs. Using miRNA-microarray and mechanistic studies, SB increased the expression of microRNA-494 (miR-494) and both Bmi1 and ADAM10 were identified as the novel targets of miR-494. Moreover, overexpression of miR-494 results in a reduction in cancer stemness. However, knockdown of miR-494 in CD44 − ALDH1 - non-HNC-TICs enhanced cancer stemness and oncogenicity, while co-knockdown of Bmi1 and ADAM10 effectively reversed these phenomena. Mice model showed that SB treatment by oral gavage to xenograft tumors reduced tumor growth and prolonged the survival time of tumor-bearing mice by activation of miR-494-inhibiting Bmi1/ADAM10 expression. Survival analysis indicated that a miR494 high Bmi1 low ADAM10 low phenotype predicted a favourable clinical outcome. We conclude that the inhibition of tumor aggressiveness in HNC-TICs by SB was mediated by up-regulation miR-494, suggesting that SB would be a valuable anti-cancer drug for treatment of HNC.

Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the oral cavity and the fourth leading malignancy and cause of cancer-related death in the male population of Taiwan. Most cases are detected at advanced stages, resulting in poor prognosis. Therefore, improved detection of early oral health disorders is indispensable. The involvement of oral bacteria in inflammation and their association with OSCC progression provide a feasible target for diagnosis. Due to the nature of oral neoplasms, the diagnosis of epithelial precursor lesions is relatively easy compared with that of other types of cancer. However, the transition from an epithelial precursor lesion to cancer is slow and requires further and continuous follow-up. In this study, we investigated microbiota differences between normal individuals, epithelial precursor lesion patients, and cancer patients with different lifestyle habits, such as betel chewing and smoking, using next-generation sequencing. Overall, the oral microbiome compositions of five genera, Bacillus, Enterococcus, Parvimonas, Peptostreptococcus, and Slackia, revealed significant differences between epithelial precursor lesion and cancer patients and correlated with their classification into two clusters. These composition changes might have the potential to constitute a biomarker to help in monitoring the oral carcinogenesis transition from epithelial precursor lesion to cancer.

e17536 Background: Early progression, usually defined disease-free interval (DFI) less than six months after completing adjuvant platinum-based chemoradiotherapy (CRT), has very poor outcome for oral cavity squamous cell carcinoma (OCSCC). But there are no biomarkers to predict such early progression. Methods: Locally advanced OCSCC patients, after complete surgical resection and followed-up platinum-based adjuvant CRT, were retrospectively enrolled from Chung Shan Medical University Hospital (CSMUH, training cohort) and Taipei Veterans General Hospital (TPE-VGH, validating cohort) in Taiwan. Clinicopathologic variables of patients with DFI < or ≥ 6 months were compared by using the χ2 test. The Cox proportional hazards model was applied to identify independent factors for DFI. Survival was estimated using the Kaplan-Meier method and log-rank test. Results: A total of 350 high-risk OCSCC patients were enrolled, including 146 patients in training cohort and 204 in validating cohort. In multivariate Cox regression, pN > 0, extracapular spread, and depth of invasion ( ≥ 1cm) were independent factors for DFI in training cohort. If each factor scored one point, the scoring system could effectively predict early progression that sensitivity/specificity/area under curve (AUC) of training and validating cohort were 57.7%/91.2%/0.771 and 58.1%/83.9%/0.730, respectively (the cutoff level ≤ 2 or > 2). DFI between lower- (score 0–2) and high- (score 3) risk groups were also significantly different in both training (median DFI, 59.6 vs. 4.5 months, P < 0.001) and validating cohorts (NA vs. 9.3 months, P < 0.001). Conclusions: The established score system was effective to predict early progression after adjuvant CRT for locally advanced OCSCC.

Methyl antcinate A (MAA) is an ergostane-type triterpenoid extracted from the fruiting bodies of Antrodia camphorate that has been reported to be a cytotoxic agent towards some types of cancer cells, such as oral cancer and liver cancer. Cancer stem cells (CSCs) are a particular population within cancer cells which are responsible for tumor initiation, drug resistance and metastasis and targeting CSCs is an emerging area in cancer therapy. In this study, we examine the effect of MAA on cancer stem-like cells in the MCF7 human breast cancer cell line. Although MAA displayed very low cytotoxic effect towards MCF7 under normal culture conditions, it did show good inhibitory effects on the self-renewal capability which was examined by mammosphere culture including primary and secondary sphere. MAA also inhibited cell migration ability of MCF7 sphere cells. By western blot analysis, MAA was shown to suppress the expression of heat shock protein 27 and increase the expression of IkBα and p53. In conclusion, our data demonstrate that MAA has anti-CSC activity and is worthy of future development of potent anticancer agents.

Cerebro-costo-mandibular syndrome (CCMS) consists of severe micrognathia, glossoptosis, posterior rib-gap defects and developmental delay. It may cause upper airway obstruction and flail chest, resulting in neonatal hypoxia, and possibly death. Early airway management or surgical intervention to maintain a patent airway is critical to avoid hypoxia in CCMS patients. We report a newborn with CCMS who was successfully weaned from a laryngeal mask after undergoing a tongue–lip adhesion operation at 164 days of age.

Background: Green tea polyphenols are considered beneficial to human health, especially as cancer chemopreventive agents in recent years. Epigallocatechin- 3-gallate (EGCG), the most abundant polyphenol in green tea, has been proven to suppress colonic tumorigenesis in animal and epidemiological studies, whereas its role in the oral carcinogenesis remains to be elucidated. Methods: Cytotoxicity, invasion, and migration assays were used to investigate the effects of human oral cancer cell line OC2 cells exposed to EGCG. To look at the precise involvement of EGCG in cancer metastasis, gelatin zymography and casein zymography were performed to evaluate the impacts of EGCG on matrix metalloproteinase (MMP)-2, MMP-9, and urokinase plasminogen activator (uPA) secretion in OC2 cells. Results: EGCG exhibited a dose-dependent inhibitory effect on the invasion and migration of OC2 cells in the absence of cytotoxicity (P

This work presents a miniaturized tracking and focusing optical pickup head implemented using MEMS technology. The device was fabricated by poly-Si trench-refilled technology and used a UV-cured polymer droplet or micro ball lens as the objective lens. A bidirectional vertical comb-drive actuator and V-beam thermal actuator drove the objective lens for the out-of-plane (focusing) and in-plane (tracking) motion, respectively. In applications, various tracking and focusing devices have been successfully fabricated and characterized. The upward and downward displacement of the focusing optical pickup head is 2.9 and 1.7 µm, respectively; the in-plane displacement of the tracking optical pickup head is ± 46.7 µm. The resonant frequencies of the focusing and tacking systems are 2.3 and 8.5 kHz respectively. In addition, the UV-cured polymer lens and solid micro ball lens were both successfully integrated with a MEMS device to create an objective lens.