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Supplementary Figures S1-S4 from IFN-Induced Protein with Tetratricopeptide Repeats 2 Inhibits Migration Activity and Increases Survival of Oral Squamous Cell Carcinoma

The function of the IFN-stimulated gene family protein, IFN-induced protein with tetratricopeptide repeats 2 (IFIT2), is poorly understood. Here, we report that IFIT2 colocalizes with cytokeratin 18 in oral squamous cell carcinoma (OSCC) cells. Treatment of OSCC cells with IFN-β significantly increased the expression of IFIT2 and remarkably inhibited cell migration. To further explore the effect of IFIT2 on cell migration, IFIT2 expression was either silenced with a small interfering RNA or increased by ectopic expression. IFIT2 knockdown in OSCC cells led to a significantly higher level of migration in vitro (P < 0.05) compared with control cells; by contrast, IFIT2 overexpression led to a significantly lower level of migration in vitro (P < 0.05). Immunohistochemically, 71.4% of OSCC tissues had elevated IFIT2 protein levels compared with noncancerous matched tissues. Elevated IFIT2 protein expression was positively associated with tumor differentiation status and inversely associated with nodal stage in OSCC specimens (P < 0.05). Higher IFIT2 protein levels in tumor tissues were also associated with better patient survival (P < 0.01). Our present study shows an inverse correlation between IFIT2 expression and cell migration, suggesting that IFIT2 plays an important role in inhibiting this process and that its expression may be associated with better prognosis in patients with OSCC. (Mol Cancer Res 2008;6(9):1431–9)

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

Supplementary Figures 1-8, Tables 1-7 from miR-31 Ablates Expression of the HIF Regulatory Factor FIH to Activate the HIF Pathway in Head and Neck Carcinoma

Supplementary Materials and Methods. Table S1. Cell cultivation conditions. Table S2. siRNAs used in the present study. Table S3. TaqMan assay probes used in the present study. Table S4. Primers used in the present study. Table S5. shRNA clones used in the present study. Table S6. Paired OSCC samples for qRT-PCR and Western blot analysis. Table S7. OSCC samples in TMA for IHC and ISH analysis. Table S8. Primary antibodies or associated reagents used in the present study. Table S9. The complimentarity between miR-211 and the 3’UTR of TCF12 gene. Table S10. Jaspar prediction scores. Fig S1. Genotyping of K14-EGFP-miR-211 transgenic mouse model. Fig S2. GFP immunoreactivity and thickness in squamous epithelium. Fig S3. Immunohistochemistry of the cell proliferation and survival proteins in tongue epithelium. Fig S4. Induction of mouse esophageal tumorigenesis by 4NQO. Fig. S5. Representative histopathological sections of orthotopic xenografts and neck lymph nodes. Fig. S6. Analysis of pri-miR-211 expression in OSCC cells. Fig. S7. miR-211 staining and TCF12 immunoreactivity in mouse tongue tissues. Fig. S8. Analysis of TCF12 protein in subcellular fractions in FaDu cells. Fig. S9. Phenotypic analysis of OECM1 cell with TCF12 expression. Fig. S10. Correlation between miR-211 expression and TCF12 mRNA expression in OSCC tumors. Fig. S11. miR-211 staining and TCF12 immunoreactivity in OSCC. Fig. S12. TCF12 immunoreactivity in mouse multistep carcinogenesis. Fig. S13. Color diagram of Fig. 4A, a. Fig S14. Analysis of TCF12 associated biological processes and gene interaction networks. Fig. S15. Prediction of the potential regulation of TCF12 on the promoter activity of downstream genes using Jasper software. Fig. S16. qRT-PCR analysis to confirm the regulation of TCF12 on downstream genes across different OSCC cells. Fig. S17. Correlation between the expression of TCF12 and FAM213A in database. Fig. S18. FAM213A immunoreactivity. Fig. S19. Color diagram of Fig. 6D. Fig. S20. Kaplan-Meier survival analysis.

Oral cancers are the seventh most common cancer globally. While progresses in oral cancer treatment has been made, not all patients respond to these therapies in the same way. To overcome this difficulty, numerous studies have been devoted to identifying biomarkers, which enable early identification of patients who may benefit from a particular treatment modality or at risk for poor prognosis. Biomarkers are protein molecules, gene expression, DNA variants, or metabolites that are derived from tumors, adjacent normal tissue or bodily fluids, which can be acquired prior to treatment and during follow-up, thus extending their use to the evaluation of cancer progression and prediction of treatment outcome. In this review, we employed a basic significance level (< 0.05) as the minimal requirement for candidate biomarkers. Effect sizes of the biomarkers in terms of odds ratio, hazard ratio and area under the receiver operating characteristic curves were subsequently used to evaluate the potential of their clinical use. We identified the CCND1 from the tumor, human papillomavirus, HSP70, and IL-17 from the peripheral blood, and high density of CD45RO+ tumor-infiltrating lymphocytes as the clinically relevant biomarkers for oral cancers.

BACKGROUND A recent study suggested to develop and implement more interacted material for preprocedural education to decrease patients' anxiety about the atrial fibrillation (AF) ablation. This study compared the effectiveness of using either newly developed virtual reality (VR) materials (VR group) or paper-based materials (paper group) on giving AF preprocedural education. METHODS This study consequentially enrolled 33 AF patients preparing for ablation from November 2019 to October 2020. After enrollment, patients were randomized as either paper (n = 22) or VR (n = 11) groups. RESULTS In comparison with the baseline stage, at the posteducation stage, the degree of improvement in patients' self-assessed self-efficacy on AF ablation knowledge was higher among VR group patients than those in the paper group. At the posteducation stage, the patients' satisfaction to preprocedural education and used materials were higher among the VR group than that among the paper group. In addition to meet their needs and give accurate medical information, VR group patients reported that VR materials increased the effectiveness of education, increased their preparedness for AF catheter ablation, achieved paperless purposes, and willing to recommend VR materials to others. Operators subjectively reported that the periprocedure cooperation was increased both among paper and VR group patients after preprocedural education for the details of procedure. Better preparedness of VR group patients was supported by less periprocedure pain, anxiety, and impatience than those among paper group patients. CONCLUSION Interactive VR-based materials are superior to the paper-based materials to provide patients immerse and imagine the journey and detail knowledge of AF catheter ablation before the procedure and better prepared patients for the procedure.

Background: Visual oral examination (VOE) is a conventional oral cancer screening method. This study aimed to evaluate the value of methylation marker to assist VOE in identifying oral epithelial dysplasia and oral squamous cell carcinoma (OED/OSCC) from non-cancerous lesions in a real-world situation. Methods: 201 patients with high-risk personal habits who self-perceived oral anomaly were VOE examined, ZNF582 methylation (ZNF582m) tested, and histologically diagnosed. Results: Among them, 132 patients (65.7%) were histologically diagnosed OED/OSCC. Using VOE, 56.1% OED/OSCC patients had possible oral cancer, whereas 37.7% non-OED/OSCC patients had leukoplakia. ZNF582m-positive was detected in 90.2% OED/OSCC patients and 44.9% non-OED/OSCC patients. Various logistic regression models were postulated to evaluate the diagnostic performance of conventional VOE and new strategies using ZNF582m. ROC analysis and its corresponding C-index demonstrated that either triage or co-testing models of VOE and ZNF582m could improve diagnostic performance and discriminative abilities compared with the VOE only approach. Conclusions: In conclusion, methylation marker test shows equivalent performance to an experienced judgment by oral maxillofacial surgeons and plays a significantly supplementary role in increasing the efficacy in identifying oral malignant lesions. ZNF582m may be an especially important tool for family physicians or general dentists to properly diagnose suspicious oral lesions.

Purpose: Undertaking a standard-setting exercise is a common method for setting pass/fail cut scores for high-stakes examinations. The recently introduced equal Z standard-setting method (EZ method) has been found to be a valid and effective alternative for the commonly used Angoff and Hofstee methods and their variants. The current study aims to estimate the minimum number of panelists required for obtaining acceptable and reliable cut scores using the EZ method.Methods: The primary data were extracted from 31 panelists who used the EZ method for setting cut scores for a 12-station of medical school’s final objective structured clinical examination (OSCE) in Taiwan. For this study, a new data set composed of 1,000 random samples of different panel sizes, ranging from 5 to 25 panelists, was established and analyzed. Analysis of variance was performed to measure the differences in the cut scores set by the sampled groups, across all sizes within each station.Results: On average, a panel of 10 experts or more yielded cut scores with confidence more than or equal to 90% and 15 experts yielded cut scores with confidence more than or equal to 95%. No significant differences in cut scores associated with panel size were identified for panels of 5 or more experts.Conclusion: The EZ method was found to be valid and feasible. Less than an hour was required for 12 panelists to assess 12 OSCE stations. Calculating the cut scores required only basic statistical skills.

Oral squamous cell carcinoma (OSCC) is a type of malignancy characterized by high relapse and recurrence rates in the late stage despite optimal surgical intervention and postoperative chemoradiotherapy. Because the management of relapse following definitive treatment is challenging, accurate risk stratification is of clinical significance to improve treatment outcomes. Circular RNAs (circRNAs) are noncoding RNAs featured with cell-type specificity and high stability, owing to their circular structure, making these molecules excellent biomarkers for a variety of diseases.The levels of hsa_circ_0000190 and 0001649 in plasma samples from 30 healthy controls and 66 OSCC patients were determined by droplet digital polymerase chain reaction. The same primer sets were used with PCR to examine the expression of these two circRNAs in cancerous and adjacent normal tissues. A receiver operating characteristics curve was generated to evaluate the diagnostic value. The Kaplan-Meier method with a log-rank test was used for survival analysis.We identified two circRNAs as potential biomarkers for OSCC, showing that the plasma level of hsa_circ_0000190 was significantly decreased in the late stage and marginally correlated with the development of second primary OSCC. We also found that the decreased plasma hsa_circ_0001649 was correlated with the recurrence and poor prognosis of patients. Additionally, we found that high plasma hsa_circ_0000190, but not hsa_circ_0001649, possibly predicted a better response of patients to induction chemotherapy.Our study demonstrated the potential of biomarkers in plasma to inform not just the tumor but the entire oral cavity, thereby offering a prediction for early recurrence and second primary OSCC. The plasma circRNAs remain valuable for OSCC, albeit the easy accessibility to the oral cavity.

Objective/design/settingThis study aims to develop preprocedural communication-specific framework that emphasises the use of audiovisual materials and compares its acceptability by trainees with a regular module.TraineesBetween October 2018 and July 2021, 96 medical clerks were enrolled and randomly divided into regular and intervention groups. Another 48 trainees whose did not join the framework-based training but complete self-assessments were enrolled as the control group.InterventionsIn the intervention training module, the key steps of preprocedural communication-specific skills were structuralised into a framework using the acronym of OSCAR.Primary and secondary outcome measuresThis study compared the acceptability of trainees for two modules by measuring the degree of increase in the end-of-rotation and follow up (4 weeks later) competency from baseline by trainees’ self-assessments and physician assessments after serial trainings.ResultsIn comparison with regular group trainees, greater degree of improvements (framework-1 statement: 111%±13% vs 27%±5%, pA-step: using audiovisual materials’ of the OSCAR was significantly improved (229%±13%, pConclusionsThe OSCAR framework-based intervention module is well accepted by medical clerks and motivates them to integrate the acquired skills in clinical practice, which leads to trainees’ primary care patients being satisfied with their preprocedural communication.

Although they receive regular training, the survey we administered to our junior physicians and lab technicians revealed that they lack familiarity and confidence regarding the proper segregation/disposal of clinical wastes. In this pilot prospective study, carried out between September 2017 and September 2019, we developed a virtual reality (VR) simulation and integrated it into the steps of Gangen’s training flow. Next, we analysed objective performance, recorded by the App in the VR system as well as the subjective self-assessments of 96 junior physicians and lab technicians after two practices of VR games. Before receiving training, lab technicians' self-assessed familiarity and confidence regarding proper clinical wastes segregation/disposal were higher than among physicians. Compared with the first VR game, we noted greater improvement in accuracy as well as a higher percentage of shortened time to complete 10 actions of segregation/disposal of VR clinical wastes, among junior physicians/male trainees, that of junior lab technicians/female trainees in the second VR game. Most of the trainees (81%–88%) reported that they found the VR game-based training to be useful, in contrast to poster-mass media-based training. Our initial positive results indicated the possibility of applying this VR game-based simulation system to regular institution-wide staff education in future.

Lineage commitment and tumorigenesis, traits distinguishing stem cells, have not been well characterized and compared in mesenchymal stem cells derived from human dental pulp (DP-MSCs) and bone marrow (BM-MSCs). Here, we report DP-MSCs exhibit increased osteogenic potential, possess decreased adipogenic potential, form dentin pulp-like complexes, and are resistant to oncogenic transformation when compared to BM-MSCs. Genome-wide RNA-seq and differential expression analysis reveal differences in adipocyte and osteoblast differentiation pathways, bone marrow neoplasm pathway, and PTEN/PI3K/AKT pathway. Higher PTEN expression in DP-MSCs than in BM-MSCs is responsible for the lineage commitment and tumorigenesis differences in both cells. Additionally, the PTEN promoter in BM-MSCs exhibits higher DNA methylation levels and repressive mark H3K9Me2 enrichment when compared to DP-MSCs, which is mediated by increased DNMT3B and G9a expression, respectively. The study demonstrates how several epigenetic factors broadly affect lineage commitment and tumorigenesis, which should be considered when developing therapeutic uses of stem cells., Mesenchymal stem cells derived from dental pulp (DP-MSCs) differ in oncogenesic and developmental potential compared to those from bone marrow. Here, the authors show that decreased DNA methylation and histone H3K9Me2 enrichment along with higher PTEN activation in DP-MSCs promotes osteogenesis and reduces oncogenesis.

The present study aimed to evaluate the effects of virtual reality (VR) simulations combined with bedside assignments on nurses’ self-efficacy in providing pre-treatment educational services. Between March 2019 and November 2020, we conducted a study of VR educational materials that were developed to cover information about the treatment of oral cancers. The effects of the VR simulation, the thinking-path tracking map method, and bedside assignments on the nurses’ treatment decision-related knowledge were evaluated in a ward for oral cancer patients at Taipei Veterans General Hospital, Taipei, Taiwan. The blended training model significantly increased nurses’ familiarity (P

Dry eye disease (DED), also called the keratoconjunctivitis sicca, is one of the most common diseases in the ophthalmology clinics. While DED is not a life-threatening disease, life quality may be substantially affected by the discomfort and the complications of poor vision. As such, a large number of studies have made contributions to the investigation of the DED pathogenesis and novel treatments. DED is a multifactorial disease featured with various phenotypic consequences; therefore, animal models are valuable tools suitable for the related studies. Accordingly, selection of the animal model to recapitulate the clinical presentation of interest is important for appropriately addressing the research objective. To this end, we systemically reviewed different murine and rabbit models of DED, which are categorized into the quantitative (aqueous-deficient) type and the qualitative (evaporative) type, based on the schemes to establish. The clinical manifestations of dry eye on animal models can be induced by mechanical or surgical approaches, iatrogenic immune response, topical eye drops, blockage of neural pathway, or others. Although these models have shown promising results, each has its own limitation and cannot fully reproduce the pathophysiological mechanisms that occur in patients. Nonetheless, the animal models remain the best approximation of human DED and represent the valuable tool for the DED studies.

Oral squamous cell carcinoma (OSCC) is among the leading causes of cancer-associated deaths worldwide. Family members in miR-371/372/373 miRNA cluster, which is localized at human chromosome 19q13.4, are co-expressed in both human stem cells and malignancies. The individual miRNA in this cluster are also involved in modulating the pathogenesis of malignancies as either oncogenes or suppressors. The 19q13 region is frequently gained in head and neck cancers. High expression of miR-372 and miR-373 are survival predictors for OSCC. However, the role of the miR-371/372/373 cluster in oral carcinogenesis remains to be fully investigated. We use the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system to establish OSCC cell subclones that had the miR-371/372/373 cluster deleted. In addition, further subclones were established that had the promoter of this cluster deleted. Concordant silencing in SAS cells of miR-371/372/373 decreased oncogenic potential, increased cisplatin sensitivity, activated p53, and upregulated the expression of Bad and DKK1. We also employed the CRISPR/dCas9 synergistic activation mediator system, which allowed robust transcriptional activation of the whole miR-371/372/373 cistron. Upregulation of endogenous miR-371/372/372 expression increased both oncogenicity and drug resistance. These were accompanied by a slight activation of AKT, &beta, catenin, and Src. This study identifies the oncogenic role of the miR-371/372/373 cluster in OSCC. Using CRISPR based strategy can be a powerful paradigm that will provide mechanistic insights into miRNA cluster functionality, which will also likely help the development of targeting options for malignancies.

BACKGROUND Atrial fibrillation (AF) is the most common cardiac arrhythmia, and increasing numbers of patients receive AF ablation nowadays. Therefore, in the cardiology division, residents need to be familiar with the AF ablation procedure-related knowledge for primary care. This study evaluated the baseline residents and patients' self-efficacy to the AF ablation procedure-related knowledge, developed the specific virtual reality (VR) informative aids, and evaluated the effects of VR training and resident-led VR aids-based patient education on their self-efficacy, in the aspects of familiarity, confidence and anxiety, and satisfaction. METHODS From 2019, April to 2020, April, this 1-year prospective prestudy and poststudy was undergone in the cardiology division. Between 2019, April and 2019, December, the experienced physicians were invited to develop VR informative aids for AF ablation procedure-related knowledge. Between January 2020 and April 2020, newly developed VR informative aids were implemented in the educational program of training rotated residents for giving patient education. RESULT A total of 20 residents and 32 patients were enrolled. The baseline self-reported self-efficacy and knowledge scores were relatively low among rotated residents and their patients. In addition to the high level of satisfaction, self-efficacy and knowledge scores of residents and their patients were increased after VR aids-based training and resident-led patient education. Higher degree of improvement in self-efficacy was noted among patients with depression/anxiety history than those without history. The follow-up assessments among residents showed that the positive effects of VR aids were sustained until 2 weeks later. CONCLUSION Overall, residents and patients reported that the VR aids increase the effectiveness of patient education, achieve the purpose of a paperless environment, and motivate them to recommend it to others. The implementation of resident-to-patient education has a positive impact on trainees and patients understanding about AF ablation procedures-related knowledge.

Oral squamous cell carcinoma (OSCC) is among the leading causes of cancer-associated deaths worldwide. Family members in miR-371/372/373 miRNA cluster, which is localized at human chromosome 19q13.4, are co-expressed in both human stem cells and malignancies. The individual miRNA in this cluster are also involved in modulating the pathogenesis of malignancies as either oncogenes or suppressors. The 19q13 region is frequently gained in head and neck cancers. High expression of miR-372 and miR-373 are survival predictors for OSCC. However, the role of the miR-371/372/373 cluster in oral carcinogenesis remains to be fully investigated. We use the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system to establish OSCC cell subclones that had the miR-371/372/373 cluster deleted. In addition, further subclones were established that had the promoter of this cluster deleted. Concordant silencing in SAS cells of miR-371/372/373 decreased oncogenic potential, increased cisplatin sensitivity, activated p53, and upregulated the expression of Bad and DKK1. We also employed the CRISPR/dCas9 synergistic activation mediator system, which allowed robust transcriptional activation of the whole miR-371/372/373 cistron. Upregulation of endogenous miR-371/372/372 expression increased both oncogenicity and drug resistance. These were accompanied by a slight activation of AKT, β-catenin, and Src. This study identifies the oncogenic role of the miR-371/372/373 cluster in OSCC. Using CRISPR based strategy can be a powerful paradigm that will provide mechanistic insights into miRNA cluster functionality, which will also likely help the development of targeting options for malignancies.

As of April 15, 2020, the US Food and Drug Administration has granted emergency use authorization to a first saliva test for diagnosis of severe acute respiratory syndrome coronavirus 2 infection, the device developed by RUCDR Infinite Biologics laboratory, Rutgers University. A key feature that distinguishes the saliva-based test from nasopharyngeal or oropharyngeal (throat) swabs is that this kit allows self-collection and can spare healthcare professionals to be at risk during collecting nasopharyngeal or oropharyngeal samples, thereby preserving personal protective equipment for use in patient care rather than sampling and testing. Consequently, broader testing than the current methods of nasal or throat swabs will significantly increase the number of people screening, leading to more effective control of the spread of COVID-19. Nonetheless, a comparison of saliva-based assay with current swab test is needed to understand what and how we can benefit from this newly developed assay. Therefore, in this mini-review article, we aimed to summarize the current and emerging tools, focusing on diagnostic power of different clinical sampling and specimens.

s Background This study aims to assess the feasibility, reliability and validity of the panel-based Equal Z-score (EZ) method applied to objective structural clinical examination (OSCE) of Chinese medical students and undertaking a comparison with the statistical techniques-based Borderline Regression Method (BRM). Methods Data received from two cohorts of 6th and 7th year medical students in Taiwan who set the mock OSCE as a formative assessment. Traditionally this medical school uses BRM to set the pass/fail cut-score. For the current study, 31 OSCE panellists volunteered to participate in the EZ method in parallel to the BRM. Results In the conduct of this study, each panel completed this task for an OSCE exam comprising 12 stations within less than 60 min. Moreover, none of the 31 panellists, whose are busy clinicians, had indicated that the task was too difficult or too time-consuming. Although EZ method yielded higher cut-scores than the BRM it was found reliable. Intraclass correlation (ICC) measuring absolute agreement, across the three groups of panellists was .893 and .937 for the first and second rounds respectively, demonstrating high level of agreement across groups with the EZ method and the alignment between the BRM and the EZ method was visually observed. The paired t-test results identified smaller differences between the cut-scores within methods than across methods. Conclusions Overall this study suggests that the EZ method is a feasible, reliable and valid standard setting method. The EZ method requires relatively little resources (takes about an hour to assess a 12 station OSCE); the calculation of the cut-score is simple and requires basic statistical skills; it is highly reliable even when only 10 panellists participate in the process; and its validity is supported by comparison to BRM. This study suggests that the EZ method is a feasible, reliable and valid standard setting method.

BACKGROUND Oral squamous cell carcinoma (OSCC) leads to thousands of deaths every year in Taiwan. Nearly 40% of OSCC patients are diagnosed with stage IV disease, which has a poor prognosis. Multimodality treatments including surgery and adjuvant therapy have been utilized, but their treatment outcomes are generally poor. In this study, we sought to identify possible clinical impact factors that may contribute to the survival of stage IV OSCC. METHODS Data for patients with malignant neoplasms of the oral cavity registered in the Cancer Registry Database of Taipei Veterans General Hospital between 2002 and 2011 were retrieved. The study patients consisted of OSCC patients with clinical stage IV disease who had undergone a surgery and adjuvant therapy. The primary endpoints were the 5-year disease-free survival (DFS) and overall survival (OS) rates. The clinicopathological characteristics of the patients were also stratified and compared. RESULTS A total of 191 OSCC patients were included for retrospective analysis. The different subgroups of stage IV disease presented different treatment outcomes. The 5-year OS versus DFS rates of each subgroup were as follows: T4N0: 70.9% versus 52.6%; T1-3N23: 66.1% versus 49.8%; T4N1: 49.6% versus 31.6%; and T4N23: 40.9% versus 31.0% (p < 0.01). Patients with diabetes, moderate or poor cell differentiation, perineural invasion, and extracapsular spread presented lower 5-year OS rates (hazard ratio [HR] = 1.87, 1.65, 2.42, and 2.14, respectively), and patients with perineural invasion, positive cut margin, and extracapsular spread presented lower 5-year DFS rates (HR = 1.57, 1.62, and 1.71, respectively). CONCLUSION In this study, we elucidated the different survival rates of different subgroups of stage IV OSCC following the same treatment scheme. The results of the study provide clinical physicians with references by which to evaluate prognosis and determine post-operative disease monitoring timetables based upon different characteristics.

miR-372 has been shown a potent oncogenic miRNA in the pathogenesis of oral squamous cell carcinoma (OSCC). The zinc finger and BTB domain containing 7A protein (ZBTB7A) is a transcriptional regulator that is involved in a great diversity of physiological and oncogenic regulation. However, the modulation of ZBTB7A in OSCC remains unclear. Tissue analysis identifies a reverse correlation in expression between miR-372 and ZBTB7A in OSCC tumors. When OSCC cells have stable knockdown of ZBTB7A, their oncogenic potential and drug resistance is increased. By way of contrast, such an increase is attenuated by expression of ZBTB7A. Screening and validation confirms that ZBTB7A is able to modulate expression of the death receptors TRAIL-R1, TRAIL-R2, Fas and p53 phosphorylated at serine-15. In addition, ZBTB7A transactivates TRAIL-R2, which sensitizes cells to cisplatin-induced apoptosis. The ZBTB7A-TRAIL-R2 cascade is involved in both the extrinsic and intrinsic cisplatin-induced pathways of apoptosis. Database analysis indicates that the expression level of and the copy status of ZBTB7A and TRAIL-R2 are important survival predictors for head and neck cancers. Collectively, this study indicates the importance of the miR-372-ZBTB7A-TRAIL-R2 axis in mediating OSCC pathogenesis and in controlling OSCC drug resistance. Therefore, silencing miR-372 and/or upregulating ZBTB7A would seem to be promising strategies for enhancing the sensitivity of OSCC to cisplatin therapy.

BACKGROUND MicroRNAs (miRNAs) play crucial roles in head and neck squamous cell carcinoma (HNSCC). This study investigates whether miR-31, miR-96, and miR-182 are involved in targeting Numb during HNSCC. METHODS The expression of miR-31/96/182 in tumor tissues was analyzed. Reporter assay, knockdown, expression, and oncogenic analysis were carried out in cell lines. RESULTS Upregulation of miR-31/96/182 was detected in tumor tissues. In addition, advanced tumors showed higher expression levels of these miRNAs. The expression of these miRNAs was upregulated after treatment with areca ingredients (P

Periodontal disease may cause considerable destruction of alveolar bone, periodontal ligaments (PDLs) and cementum and even lead to progressive oral dysfunction. Periodontal tissue regeneration is the ultimate goal of periodontal disease treatment to reconstruct both structures and functions. However, the regenerative efficiency is low, possibly due to the lack of a proper periodontal microenvironment. In this study, we applied an injectable and thermosensitive chitosan/gelatin/glycerol phosphate hydrogel to provide a 3D environment for transplanted stem cells and to enhance stem cell delivery and engraftment. The iPSCs-BMP-6-hydrogel complex promoted osteogenesis and the differentiation of new connective tissue and PDL formation. In animal models of maxillary-molar defects, the iPSCs-BMP-6-hydrogel-treated group showed significant mineralization with increased bone volume, trabecular number and trabecular thickness. Synergistic effects of iPSCs and BMP-6 increased both bone and cementum formation. IPSCs-BMP-6-hydrogel-treated animals showed new bone synthesis (increased ALP- and TRAP-positive cells), new PDL regeneration (shown through Masson’s trichrome staining and a qualification assay), and reduced levels of inflammatory cytokines. These findings suggest that hydrogel-encapsulated iPSCs combined with BMP-6 provide a new strategy to enhance periodontal regeneration. This combination not only promoted stem cell-derived graft engraftment but also minimized the progress of inflammation, which resulted in highly possible periodontal regeneration.

Background The Accreditation Council for Graduate Medical Education (ACGME) core competencies (CC) in general medicine-based primary care are essential for junior medical trainees. In this country, a regular faculty development (FD) program aimed at training faculty in instructing (teaching and assessing) these CC had operated. However, leadership was not emphasized. In a new intervention module, the roles and associated responsibilities of clinical instructors to conduct, design, and lead CC-based education were emphasis. Aims This follow-up explanatory case study compares the effectiveness of intervention module with that of the previous regular module. Methods The regular group (n = 28) comprised clinical instructors who participated in the FD module during the 2013–2014 year while the intervention group (n = 28) was composed of 2015–2016 participants. Prior to the formal (hands-on) training, participants in the intervention group were asked to study the online materials of the regular module. These participants then received a 30-h hands-on training in conducting, designing, and leading skills. Finally, they prepared a 10-h reflective end-of-module presentation of their real-world practices. Results Following the training, a higher degree improvement in participants self-reported familiarity with CC education, self-confidence in their ability to deliver CC education and sustained involve CC education were noted among the intervention FD group, compared with the regular FD group. In the intervention group, senior academicians (associate and full professor) are more substantially involved in designing and leading CC-based courses than junior academicians (lecturers and assistant professors). Among non-teaching award winners of in the intervention FD group, the follow-up degree of sustained involvement in delivering, designing and leading CC-based courses was significantly higher than that of the regular group. Conclusions Our study demonstrated that leadership training in the intervention FD modules substantially motivated clinical instructors to become leaders in CC education.

Cancer stem cells (CSC) are a subpopulation of tumor cells that have superior capacities of self-renewal, metastatic dissemination, and chemoresistance. These characteristics resemble, to some extent, the outcome of certain biological processes, including epithelial-mesenchymal transition (EMT), autophagy, and cellular stress response. Indeed, it has been shown that the stimuli that induce these processes and CSC are overlapping, and CSC and tumor cells that underwent EMT or autophagy are much alike. However, as the cross talk between CSC, EMT, autophagy, and cellular stress is further explored, these processes are also found to have an opposing role in CSC, depending on the condition and status of cells. This contextual effect is likely due to overwhelming reliance on CSC markers for their identification, and/or discrepancies in recognition of CSC as a particular cell population or cellular state. In this review, we summarize how EMT, autophagy, and cellular stress response are tied or unwound with CSC. We also discuss the current view of CSC theory evolved from the emphasis of heterogenicity and plasticity of CSC.

It is importance to train the interprofessional collaboration (IPC) and team-efficiency (TE) of medical trainees. This prospective study evaluates whether implementation of scenario/video-created workshops in integrated IPC and TE (IIT) program provides additional benefits for IPC-TE skills of nursing trainees.Mock simulation with two IIT scenarios was held as preintervention IPC-TE assessment. Basic and advanced workshops were arranged for teams of intervention groups for creation of discipline-specific scenario and video. Thirty-six nursing trainees were randomized into teams of five members (three nursing students, one standardized medical student, and one standardized trainees of other profession) in either intervention (scenario plus video and scenario) or control groups. After intervention, all groups received the formal simulation-based assessment using another two IIT scenario. In addition to instructors-based assessment of team' performance in mock and formal IIT simulation using interprofessional team collaboration scale (AITCS), self-assessment of attitudes and program-value score were completed by each trainee, using attitudes toward interprofessional health care teams scale (ATIHCTS) at all stages.Nursing trainees in intervention group gave high satisfaction score to this IIT intervention. In comparison with control group, greater increase in instructor-assessed team performance in the "partnership," "cooperation," and "shared decision making" domains of AITCS and the self-assessed "quality of care delivery" and TE domains of ATIHCTS were noted in the intervention groups. The overall improvement was greater in the scenario plus video group than those in the scenario group. Further, these improvements among nursing trainees persisted until follow-up stage at 4-weeks later.For nursing trainees, our study suggested that implementation of a scenario creation-based training resulted in additional improvement in trainee' IPC and TE behaviors and attitudes. Additionally, making video of newly created nurse-specific scenario enhances partnership and cooperation among nursing trainees and their interprofessional team members.

Background For patient safety, this study aims to evaluate the effectiveness of additional objective structured clinical examination (OSCE)-based medical simulation courses to establish the "emergency-stabilization" subcompetency of postgraduate first year (PGY-1) residents. Methods In the simulation course, trainees were randomly divided into three groups: intervention, regular, and control group as Trios-OSCE trainees, Single-OSCE trainees, or OSCE observers (feedback-givers) after attending the pre-OSCE common simulation workshop. Three PGY-1 residents rotated through the Trios OSCE long-station together, while single PGY-1 residents rotated through regular OSCE alone and the control group gave feedback after observation of their peers' OSCE performance. Using Queen's simulation assessment tool, either in Trios-OSCE or Single-OSCE, performance levels were rated as either inferior, novice, competent, advanced or superior in the "therapeutic actions" and "communication" domains. The "overall performances" of all trainees were graded by qualified assessors, experienced facilitators, and standardized senior nurse. Results The proportion of "overall performance" of trainee's, rated by an experienced facilitator as "above competent level," was significantly higher in intervention group A than in regular group B. After training, the degree of increase in self-efficacy scores was higher among the intervention group than the regular and control groups. In the follow-up stage, a trend of increasing self-efficacy scores was noted in both the interventional and regular groups. For all trainees among the three groups, high postcourse value scores confirm that the new Trios-OSCE model meets the needs of trainees and also motivates the self-directed learning and self-reflection of trainees. Conclusion Our results provide initial evidence that the new emergency-stabilization-enhanced Trios-OSCE-based medical simulation course including the additional training capacity offered by adding an observer group had positive effects on PGY-1 residents' self-efficacy and clinical transfer.

// Shu-Chun Lin 1, 3 , Shou-Yen Kao 1, 3 , Jennifer Chen-Yu Chang 1 , Ying-Chieh Liu 1 , En-Hao Yu 2 , Ssu-Hsueh Tseng 1 , Chung-Ji Liu 2, 4 , Kuo-Wei Chang 1, 3 1 Institute of Oral Biology, National Yang-Ming University, Taipei, Taiwan 2 Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan 3 Department of Stomatology, Taipei Veterans General Hospital, Taipei, Taiwan 4 Department of Dentistry, MacKay Memorial Hospital, Taipei, Taiwan Correspondence to: Kuo-Wei Chang, email: ckcw@ym.edu.tw Chung-Ji Liu, email: cjliu@ms2.mmh.org.tw Keywords: BARX2, carcinoma, invasion, metastasis, miR-187 Received: March 02, 2016 Accepted: August 09, 2016 Published: August 17, 2016 ABSTRACT Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide. Aberrations in miRNA regulation are known to play important roles in OSCC pathogenesis. miR-187 was shown to be up-regulated in head and neck malignancies in our previous screening. This study further investigated the oncogenic potential, clinical implications, and targets of miR-187 in OSCC. We observed that miR-187 increased oncogenicity, particularly migration, of OSCC cells. miR-187 expression increased the xenografic tumorigenicity and metastasis in mice. In addition, metastatic human OSCC had higher miR-187 expression than did non-metastatic tumors. Through vigorous screening, we confirmed BarH-like Homeobox 2 ( BARX2 ) gene as an miR-187 target. BARX2 expression suppressed the migration, invasion, anchorage-independent colony formation, and orthotopic tumorigenesis of OSCC cells. The migratory phenotype and neck metastasis induced by miR-187 was rescued by BARX2 expression. BARX2 expression was down-regulated in the vast majority of OSCC, and this down-regulation was particularly conspicuous in tumors with advanced nodal metastasis. In addition, plasma miR-187 was significantly higher in OSCC patients than in normal individuals. This study highlights the roles of miR-187 -BARX2 in driving the carcinogenesis of OSCC. The results suggest that miR-187 is a potential serological marker for OSCC and that targeting of miR-187 might prove effective in attenuating nodal metastasis.

miR-211 expression in human oral squamous cell carcinoma (OSCC) has been implicated in poor patient survival. To investigate the oncogenic roles of miR-211, we generated K14-EGFP-miR-211 transgenic mice tagged with GFP. Induction of oral carcinogenesis in transgenic mice using 4-nitroquinoline 1-oxide (4NQO) resulted in more extensive and severe tongue tumorigenesis compared with control animals. We found that 4NQO and arecoline upregulated miR-211 expression in OSCC cells. In silico and experimental evidence further revealed that miR-211 directly targeted transcription factor 12 (TCF12), which mediated suppressor activities in OSCC cells and was drastically downregulated in tumor tissues. We used GeneChip analysis and bioinformatic algorithms to identify transcriptional targets of TCF12 and confirmed through reporter and ChIP assays that family with sequence similarity 213, member A (FAM213A), a peroxiredoxin-like antioxidative protein, was repressed transcriptionally by TCF12. FAM213A silencing in OSCC cells diminished oncogenic activity, reduced the ALDH1-positive cell population, and increased reactive oxygen species. TCF12 and FAM213A expression was correlated inversely in head and neck carcinoma samples according to The Cancer Genome Atlas. OSCC patients bearing tumors with high FAM213A expression tended to have worse survival. Furthermore, 4NQO treatment downregulated TCF12 and upregulated FAM213A by modulating miR-211 both in vitro and in vivo. Overall, our findings develop a mouse model that recapitulates the molecular and histopathologic alterations of human OSCC pathogenesis and highlight a new miRNA-mediated oncogenic mechanism. Cancer Res; 76(16); 4872–86. ©2016 AACR.

// Wen-Cheng Lu 1 , Chung-Ji Liu 2, 3 , Hsi-Feng Tu 2 , Yu-Tung Chung 1 , Cheng-Chieh Yang 1, 2, 4 , Shou-Yen Kao 2, 4 , Kuo-Wei Chang 1, 2, 4 , Shu-Chun Lin 1, 2, 4 1 Institute of Oral Biology, National Yang-Ming University, Taipei, Taiwan 2 Department of Dentistry, National Yang-Ming University, Taipei, Taiwan 3 Department of Dentistry, MacKay Memorial Hospital, Taipei, Taiwan 4 Department of Stomatology, Taipei Veterans General Hospital, Taipei, Taiwan Correspondence to: Kuo-Wei Chang, email: ckcw@ym.edu.tw Shu-Chun Lin, email: sclin@ym.edu.tw Keywords: ARID1A, cancer, miR-31, stem cell, suppressor Received: April 13, 2016 Accepted: July 27, 2016 Published: August 9, 2016 ABSTRACT miR-31 is oncogenic for head and neck squamous cell carcinoma (HNSCC). Proteins containing the AT-rich interacting domain (ARID) modulate the accessibility of chromatin to the transcription machinery needed for gene expression. In this study, we showed that miR-31 was able to target ARID1A in HNSCC. HNSCC tumors had an inverse miR-31 and ARID1A expression. miR-31 associated oncogenicities were rescued by ARID1A expression in HNSCC cells. Furthermore, ARID1A repressed the stemness properties and transcriptional activity of Nanog/OCT4/Sox2/EpCAM via the protein’s affinity for AT-rich sites within promoters. HNSCC patients with tumors having high level of miR-31 expression and high levels of Nanog/OCT4/Sox2/EpCAM expression, together with low level of ARID1A expression, were found to have the worst survival. This study provides novel mechanistic clues demonstrating that miR-31 inhibits ARID1A and that this enriches the oncogenicity and stemness of HNSCC.

Summary Objectives When treating advanced HNSCC, a cisplatin-based systemic regimen benefit patient survival. However, chemoresistance will greatly reduce the effectiveness of this approach. The identification of molecules that contribute to cisplatin resistance may potentially improve the survival. Both HB-EGF and COX-2 have been reported to increase cisplatin-resistance. Here, we have focused on the regulation of HB-EGF/COX-2 and their roles in cisplatin resistance. Materials and Methods IHC staining was used to measure the expression levels of HB-EGF and COX-2 on the tissue microarray from 43 tissue samples of patients with advanced HNSCC. siRNA, western blot and qRT-PCR were used to dissect the regulation between EGF, Akt, COX-2, PGE2, and cisplatin sensitivity. The correlation between HB-EGF, COX2 and HNSCC progression was analyzed by the receiver operating characteristic (ROC) curve and Kaplan–Meier disease free survival. Results Patients of advanced HNSCC patients with increased HB-EGF and COX-2 expression have higher tumor recurrent rates that was related to cisplatin resistance. The resistance was mediated via an increased expression of HB-EGF and COX-2. The activation of Akt by either EGF or areca nut extract were able to upregulate COX-2, which would increase the expression of HB-EGF in a PGE2 dependent manner. Inhibition and knockdown of COX-2 resulted in a decrease in HB-EGF. In the tissue samples from HNSCC patients, there was a significant positive correlation between the expression of COX-2 and HB-EGF. Conclusion Our results suggested that COX-2 and HB-EGF are important in development of HNSCC cisplatin resistance. These findings may help the development of new strategies for overcoming cisplatin resistance.

Purpose To investigate the efficacy and safety of fractionated boron neutron capture therapy (BNCT) for recurrent head and neck (H&N) cancer after photon radiation therapy. Methods and Materials In this prospective phase 1/2 trial, 2-fraction BNCT with intravenous L-boronophenylalanine (L-BPA, 400 mg/kg) was administered at a 28-day interval. Before each fraction, fluorine-18-labeled-BPA–positron emission tomography was conducted to determine the tumor/normal tissue ratio of an individual tumor. The prescription dose (D80) of 20 Gy-Eq per fraction was selected to cover 80% of the gross tumor volume by using a dose volume histogram, while minimizing the volume of oral mucosa receiving >10 Gy-Eq. Tumor responses and adverse effects were assessed using the Response Evaluation Criteria in Solid Tumors v1.1 and the Common Terminology Criteria for Adverse Events v3.0, respectively. Results Seventeen patients with a previous cumulative radiation dose of 63-165 Gy were enrolled. All but 2 participants received 2 fractions of BNCT. The median tumor/normal tissue ratio was 3.4 for the first fraction and 2.5 for the second, whereas the median D80 for the first and second fraction was 19.8 and 14.6 Gy-Eq, respectively. After a median follow-up period of 19.7 months (range, 5.2-52 mo), 6 participants exhibited a complete response and 6 exhibited a partial response. Regarding acute toxicity, 5 participants showed grade 3 mucositis and 1 participant showed grade 4 laryngeal edema and carotid hemorrhage. Regarding late toxicity, 2 participants exhibited grade 3 cranial neuropathy. Four of six participants (67%) receiving total D80 > 40 Gy-Eq had a complete response. Two-year overall survival was 47%. Two-year locoregional control was 28%. Conclusions Our results suggested that 2-fraction BNCT with adaptive dose prescription was effective and safe in locally recurrent H&N cancer. Modifications to our protocol may yield more satisfactory results in the future.

Up-regulation of ASB6 has been previously associated with late-stage and poor prognosis of oral squamous cell carcinoma (OSCC) patients. To explore the cellular and molecular basis of how ASB6 enhances the malignancy of OSCC, we employed the clonogenicity and migration assays, murine pulmonary metastasis model, Western blot, and immunofluorescence microscopy to characterize the phenotypes of OSCC cells with lentiviral-based stable overexpression or knockdown of ASB6. We found that ASB6 overexpression increases, whereas ASB6 knockdown decreases, the potential of tumor-sphere formation, colony formation, and expression of Oct-4 and Nanog. While knockdown of ASB6 decreases cell migration in vitro and lung metastasis in mice, the migratory potential was however not promoted by ASB6 overexpression. ASB6 knockdown down-regulates the level of vimentin, and the loss of filopodia formation became more prominent following CRISPR/Cas9-directed knockout of ASB6. Moreover, ASB6 was up-regulated when cells were grown in selective condition featured with a collateral effect of enhancing intracellular stress, and the level of endoplasmic reticulum (ER) stress was further increased by knockdown of ASB6. Thus, ASB6 may attenuate ER stress that would otherwise accumulate and subsequently impede the potential of cells to acquire or sustain the stemness properties and metastatic capacity, thereby enhancing the malignancy of OSCC by increasing the population of cancer stem or stem-like cells.

In recent years, the incidence and mortality rates of head and neck squamous cell carcinoma (HNSCC) have increased worldwide. Therefore, understanding genomic alterations in HNSCC carcinogenesis is crucial for appropriate diagnosis and therapy. Protocadherin FAT1, which encodes 4588 amino acid residues, regulates complex mechanisms to promote oncogenesis or suppression of malignancies. Multiplex PCR-based next-generation sequencing (NGS) revealed FAT1 somatic mutations. The clinicopathologic implications of FAT1 in HNSCC were investigated using expression assays, and the functional role of FAT1 in HNSCC pathogenesis was determined using ectopic expression and knockdown experiments. Approximately 29% patients with HNSCC harbored damaging FAT1 mutations. InVEx algorithm identified FAT1 as a significant functional mutation burden. Each type of mutation (missense, nonsense and frameshift) accounted for nearly one-third of deleterious mutations. FAT1 mutations correlated with lower FAT1 expression in tumors. The knockdown of the endogenous expression of FAT1 and exogenous expression of crucial FAT1 domains unequivocally indicated that FAT1 suppressed the migration and invasion capability of HNSCC cells. Functional analysis suggested that nonsense mutations in FAT1 result in the loss of the suppression of tumor progression. FAT1 mutations and downregulation defined nodal involvement, lymphovascular permeation and tumor recurrence. In addition, FAT1 mutations and downregulation are independent predictors of poor disease-free survival in patients with HNSCC. This study is the first to perform multiplex PCR-based NGS to indicate marked non-synonymous FAT1 mutations in HNSCC, which are prognostic indicators. The gene analysis strategy proposed for detecting FAT1 mutations may be a valid method for mutation screening.

Summary Objectives This study investigated the oncogenic miRNA level in the tissue and biofluids in the Nitroquinoline 1-Oxide (4NQO)-induced mouse tongue carcinogenesis model for potential diagnostic or therapeutic application. Materials and methods The histological examination, immunohistochemistry, in situ hybridization, quantitative PCR analysis and bioinformatic algorithms were performed to unravel the signaling activation and miRNA expression in female murine samples. Results The increase of miR-21 and miR-31 staining, and EGFR activation paralleled the severity of 4NQO-induced epithelial pathogenesis in tongue epithelium. A progressive increase of miR-21 , miR-31 and miR-146a in both saliva and plasma samples was also noted. miR-31 was the earliest emerging miRNA in the saliva. The increase of plasma miR-146a , miR-184 and miR-372 was detectable early in the induction, and it was particularly eminent at the most advanced lesion state. The combined analysis of the multiple oncogenic miRNAs in the plasma signified a potent discriminative capacity between normal and pathological states. As the blockage of EGFR or AKT activation drastically reverted the miR-21 , miR-31 and miR-146a expression induced by 4NQO in human oral carcinoma cell lines, the results implicated a mechanistic linkage of the oncogenic miRNAs’ induction through EGFR/AKT activation. Conclusions In this study, we show the dysregulation of oncogenic miRNAs in murine tongue tumorigenesis, which simulates human counterparts. Increased multiple miRNAs in the biofluids may be valuable non-invasive markers in detecting oral carcinogenesis at an early stage. This animal model may also be useful for developing liquid biopsies and prevention strategies against oral carcinoma by abrogating EGFR or oncogenic miRNAs.

Background MicroRNAs (miRNAs) are short, noncoding RNAs that inhibit the expression of target genes that play roles in tumorigenesis. MiR-21, miR-31, and miR-184 are oncogenic miRNAs for head and neck squamous cell carcinoma (HNSCC). Factor-inhibiting hypoxia (FIH)-inducible factor is known to inactivate hypoxia-induced downstream effectors and is involved in HNSCC suppression. This study investigates whether miR-21, miR-31, and miR-184 target FIH in HNSCC. Methods Reporter assays, Western blot analysis, quantitative reverse transcriptase-polymerase chain reaction (PCR) analysis, and phenotypic assays were used to prove that miR-21, miR-31, and miR-184 directly target FIH. Clinicopathological implications of the gene expression were also analyzed. Results MiR-21, miR-31, and miR-184 directly bind to various sites in the 3′ untranslated region (UTR) of FIH transcript, and this binding is associated with decreased FIH protein expression in HNSCC cells. Treatment with the precursors of these miRNAs increases the proliferation and migration of HNSCC cells. Concomitant treatment with precursors repressed FIH and enhanced oncogenicity most profoundly. Upregulation of miR-21, miR-31, and miR-184 expression is found in more than 80% of HNSCC tumors and 72% of tumors have concordant upregulation of these 3 oncogenic miRNAs. The highest expression of these miRNAs is present in T4b and stage IVB tumors. Downregulation of FIH mRNA expression is noted in 69% of HNSCC tumors, and in tumors exhibiting high expression of these miRNAs, the FIH mRNA expression is consistently downregulated. Conclusion This study provides novel clues indicating that miR-21, miR-31, and miR-184 co-target FIH tumor suppressor during pathogenesis in the vast majority of HNSCC. © 2014 Wiley Periodicals, Inc. Head Neck, 2015

Cancer-initiating cells (CICs) are responsible for tumor initiation, progression, and therapeutic resistance; moreover, redox homeostasis is important in regulating cancer stemness. Previously, we have identified that cancer cells containing low intracellular reactive oxygen species levels (ROSLow cells) display enhanced features of CICs. However, the specific metabolic signatures of CICs remain unclear and are required for further characterization by systemic screenings. Herein, we first showed CICs mainly relying on glycolysis that was important for the maintenance of stemness properties. Next, we revealed that NRF2, a master regulator of antioxidants, was able to maintain low intracellular ROS levels of CICs, even though in the absence of oxidative stress. We further characterized that NRF2 activation was required for the maintenance of CICs properties. Of ROSLow cells, NRF2 activation not only directly activates the transcription of genes encoding glycolytic enzymes but also inhibited the conversion of pyruvate to acetyl-CoA by directly activating pyruvate dehydrogenase kinase 1 (PDK1) to lead to inhibition of tricarboxylic acid (TCA) cycle; therefore, to promote Warburg effect. A positive regulatory ROS-independent ER stress pathway (GRP78/p-PERK/NRF2 signaling) was identified to mediate the metabolic shift (Warburg effect) and stemness of CICs. Lastly, co-expression of p-PERK and p-NRF2 was significantly associated with the clinical outcome. Our data show that NRF2 acting as a central node in the maintenance of low ROS levels and stemness associated properties of the CICs, which is significantly associated with the clinical outcome, but independent from ROS stress. Future treatments by inhibiting NRF2 activation may exhibit great potential in targeting CICs.

Summary Many cell therapies currently being tested are based on mesenchymal stromal cells (MSCs). However, MSCs start to enter the senescent state upon long-term expansion. The role of retinoblastoma (RB) protein in regulating MSC properties is not well studied. Here, we show that RB levels are higher in early-passage MSCs compared with late-passage MSCs. RB knockdown induces premature senescence and reduced differentiation potentials in early-passage MSCs. RB overexpression inhibits senescence and increases differentiation potentials in late-passage MSCs. Expression of DNMT1, but not DNMT3A or DNMT3B, is also higher in early-passage MSCs than in late-passage MSCs. Furthermore, DNMT1 knockdown in early-passage MSCs induces senescence and reduces differentiation potentials, whereas DNMT1 overexpression in late-passage MSCs has the opposite effect. These results demonstrate that RB expressed in early-passage MSCs upregulates DNMT1 expression and inhibits senescence in MSCs. Therefore, genetic modification of RB could be a way to improve the efficiency of MSCs in clinical use., Graphical Abstract, Highlights • RB levels are higher in early-passage MSCs than in late-passage MSCs • RB knockdown induces a premature senescence and reduces differentiation potentials • RB overexpression reverses senescence and increases differentiation potentials • RB upregulates DNMT1 to inhibit senescence and promote differentiation potentials, In this article, Hung, Kao, and colleagues show that retinoblastoma (RB) levels are higher in early-passage mesenchymal stromal cells (MSCs) than in late-passage MSCs, which serves to regulate senescence and differentiation potentials. Expression of DNMT1 is also higher in early-passage MSCs than in late-passage MSCs. This study demonstrates that RB expressed in early-passage MSCs upregulates DNMT1 expression and inhibits senescence in MSCs.

Hypercalcaemia and leucocytosis are common in our patients with progressive oral squamous cell carcinoma (SCC). However, the precise incidence, prognostic value, and correlation with the condition of the tumour remain obscure. A total of 618 patients with oral SCC who were treated primarily between 2007 and 2012 and had serum calcium concentrations and white blood cell count (WCC) measured postoperatively were included in the study. Primary TNM stage, pathological features, and the presence of locoregional recurrence or distant metastasis after comprehensive surgical treatment were recorded. The incidence of hypercalcaemia was 9.1% and that of leucocytosis 7.2%. Hypercalcaemia correlated significantly with size of primary tumour (T status), nodal involvement (N status), TNM stage, perineural invasion, lymphovascular permeation, and recurrence or metastasis of disease. Leucocytosis, however, correlated only with T status, lymphovascular permeation, and recurrence or metastasis. In multivariate analysis of survival, recurrence, metastasis, hypercalcaemia, and leucocytosis were strong independent prognostic factors. Median survival was low if the patient had hypercalcaemia or leucocytosis (179 (range 3–73) days if the patient had distant metastasis, and 43 (range 3–102) days if the patient had locoregional recurrence). The incidence of hypercalcaemia and leucocytosis was high during the course of the disease, and both conditions have an adverse impact on survival from oral SCC. Periodic evaluation of serum calcium concentrations and WCC should be routine during the postoperative period.

Oral squamous cell carcinoma (OSCC) is a prevalent malignancy worldwide. MicroRNAs are short non-coding RNAs that regulate gene expression and are crucial for tumorigenesis. Previously, we have identified that miR-31 is frequently upregulated in OSCC and that this miR-31 increase, together with downstream effector modulation, enhances oral carcinogenesis. We have identified higher levels of miR-31 expression in oral potential malignant disorder (OPMD) tissues compared with normal oral mucosa. Exogenous miR-31 and human telomerase reverse transcriptase (hTERT) expression were introduced into cultured normal oral keratinocytes (NOKs), which led to the immortalization; these lines were designated M31OK1 and M31OK3. These immortalized lines maintained the capability to undergo squamous differentiation. In addition, migration by both cell lines was attenuated by hTERT and miR-31 knockdown. M31OK1 carries a p53 gene mutation at codon 273. A serum-tolerant subline, M31OK1-D, exhibits potent anchorage-independent growth that is attenuated by knockdown of both hTERT and miR-31. miR-31-targeted factors inhibiting HIF (FIH), which upregulated vascular endothelial growth factor (VEGF), was found crucial for oral tumorigenesis. The proliferation, migration and epithelial-mesenchymal transition of M31OK1-D are associated with downregulation of FIH and upregulation of VEGF, which require miR-31 expression. High miR-31 expression is correlated with higher VEGF expression and lower E-cadherin expression in OPMD tissue. It can be concluded that miR-31 collaborates with hTERT to immortalize NOKs and that this may contribute to early stage oral carcinogenesis. The targeting of downstream factors by miR-31 may further advance the neoplastic progression of immortalized NOKs, allowing them to become malignant.

Oral squamous cell carcinoma (OSCC) is one of the most prevalent malignancies worldwide. Despite the advances in diagnosis and treatment, the survival of OSCC remains to be improved. miR-211 is an oncogenic microRNA frequently disrupted in human OSCC and its high expression is a determinant of patient’s poor survival. Herein, we established four murine OSCC cell lines, designated MOC-L1 - MOC-L4 from the tongue tumor tissues induced by 4-nitroquinoline 1-oxide in K14-EGFP-miR-211 transgenic mice. All cell lines appear green fluorescence and express epithelial markers. The gene expression profiles among cell lines are complex and diverse, while MOC-L1 - MOC-L3 cells carry missense mutations in p53 gene. MOC-L1 exhibits tremendous epithelial-mesenchymal transition and the associated aggressive characteristics. On the contrary, MOC-L4 displays the least invasiveness. Both MOC-L1 and MOC-L2 are clonogenic in vitro and tumorigenic when implemented into dermis or tongue in syngeneic recipients. But, only MOC-L1 exhibits high potential for local regional and distal metastasis. Since the expression of miR-196b in MOC-L1 xenografts drastically decreased upon cisplatin treatment, targeting of miR-196b might facilitate tumor abrogation. As these cell lines originate from the C57BL/6 mouse, which is the strain most suitable for transgenic engineering, to approach the interplay of these OSCC cells with other type of genetically modified cells in immune-competent mice would bestow profound insight on OSCC pathogenesis. Note: This abstract was not presented at the meeting. Citation Format: Yi-Fen Chen, Chung-Ji Liu, Shou-Yen Kao, Shu-Chun Lin, Kuo-Wei Chang. Newly established mouse oral carcinoma cell lines and their syngeneic tumorigenesis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4635.

In the version of Supplementary Fig. 3c originally published with this Article, the authors mistakenly duplicated a blot from Supplementary Fig. 3b. The correct versions of these figures are shown below. In addition, two independent repeats of the experiments presented in Supplementary Figs. 3b and 3c, showing results consistent with those originally reported, have been deposited in Figshare ( 10.6084/m9.figshare.7545263 ).

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.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world, and metastasis is the major cause of cancer-related mortality. Prevention or elimination of metastasis may improve the survival of cancer patients; however, the availability of systemic HNSCC cell model with which to investigate the mechanisms of metastasis is limited. In the present study, we established a set of metastatic cell lines from HNSCC cells. In combination with their low-tumorigenic and high-tumorigenic ancestor cell lines, a cell model containing cell lines with varying malignant characteristics was established. Transcriptome analysis revealed distinct signatures among the metastatic cell lines, in comparison to the ancestor cell lines. Signaling of GTPase RhoA and mammalian embryonic stem cell pluripotency were identified in the metastatic cells. Moreover, we examined the expression of genes related to epithelial-mesenchymal transition (EMT) (Snail, Slug, Twist, vimentin and fibronectin) and stemness (Oct4, Nanog and Bmi1). The capabilities of the cells for migration, invasion, spheroid formation and pulmonary colonization in nude mice were determined. Together, we demonstrated gain of Slug expression, filopodium formation and stem-like properties in metastatic HNSCC cells, rendering this model a powerful tool for the development of diagnostic biomarkers and identification of therapeutic targets for HNSCC.