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Background Aim of the pilot study was the histologic classification of the inflamed peri-implant soft tissue around ceramic implants (CI) in comparison with titanium implants (TI). Methods Peri-implant tissue were retrieved from 15 patients (aged 34 to 88 years, seven males/eight females) with severe peri-implantitis (eight CI, seven TI). The peri-implant soft tissue samples were retrieved from the sites during scheduled removal of the implant and prepared for immunohistochemical analysis. Monoclonal antibodies (targeting CD3, CD20, CD138, and CD68) were used to identify T- and B-cells, plasma cells and macrophages. Quantitative assessment was performed by one histologically trained investigator. Linear mixed regression models were used. Results A similar numerical distribution of the cell population was found in peri-implantitis around CI compared with TI. CD3 (TI, 17% to 85% versus CI, 20% to 70% of total cell number) and CD138 (TI, 1% to 73% versus CI, 12% to 69% of total cell number) were predominantly expressed. Notably, patient-individual differences of numerical cell distribution were detected. Co-localization of B- and T-lymphocytes was observed. Conclusions Peri-implantitis around CI in comparison with TI seems to have a similar histological appearance. Differences in cellular composition of peri-implantitis lesions might also depend on the patient's specific immune status and not only on the material used.

Introduction Alterations of the epigenome may influence cancer initiation and progression. At the cellular level, histones are key regulators of chromatin accessibility and gene transcription; thus, the inhibition of histone deacetylase enzymes (HDACs) constitutes an attractive target for therapy. In this study, we investigated the effects of the HDAC inhibitor entinostat on oral squamous cell carcinoma (OSCC). Materials and methods We tested the effects of entinostat on OSCC cell lines. Cell viability and growth were analyzed using MTT assay. Cell cycle analysis, cell apoptosis, cancer stem cell (CSC) content, and the concentration of reactive oxygen species (ROS) in OSCC tumor cells were assessed using flow cytometry. The expression of histones and cell cycle regulatory proteins was examined by Western blot. Results The administration of entinostat resulted in reduced proliferation of OSCC cells, followed by cell cycle arrest at the G0/G1 phase, as well as substantial tumor apoptosis. We found an increase in ROS production and significant reductions in CSCs. We also found that entinostat caused increased acetylation histone H3 and histone H4, and changes in the expression of cell cycle-associated proteins such as p21. Conclusion This study indicates that entinostat is a potential novel therapeutic agent for OSCC by halting tumor proliferation, inducing cytotoxicity and intracellular ROS, and attacking the CSCs.

Purpose The purpose of this study was to provide sufficient information on the clinical outcome of zirconia implants, mainly observing the survival rate and marginal bone loss (MBL), with a minimum follow-up of 12 months, to verify the adoption of ceramics as a rational possibility for dental implants. Materials and methods A systematic electronic search through the PubMed (MEDLINE) and EMBASE databases was performed by two independent reviewers to identify clinical studies published between January 2005 and April 2019 containing a minimum of 10 patients per study and 12 months of follow-up after functional loading. References from the selected articles were manually reviewed for further studies. Results From the initial 1,225 articles retrieved, 19 met all the inclusion criteria. The marginal bone remodeling accounted for mean losses of 0.8 mm (95% CI: 0.60 to 1.00 mm) and 1.01 mm (95% CI: 0.72 to 1.29 mm) at 1 year and 2 years postloading, respectively. The failure rate of 6.8% was calculated for a mean follow-up period of 2.75 years, where the prevalence of early failure, late failure, and implant fracture was 3.4%, 1.7%, and 1.7%, respectively. The meta-analysis associated with the survival rate of one- and two-piece zirconia dental implants was hindered due to the lack of confidence interval or standard deviation information in most of the included articles. Conclusion Zirconia implants presented MBL values consistent with the standard in the global consensus, high survival rates, and considerable clinical results at short-term observation periods following prosthetic delivery.

Despite major progress in elucidating the pathobiology of head and neck squamous cell carcinoma (HNSCC), the high frequency of disease relapse correlates with unacceptably deficient patient survival. We previously showed that cancer stem-like cells (CSCs) drive tumorigenesis and progression of HNSCC. Although CSCs constitute only 2–5% of total tumor cells, CSCs contribute to tumor progression by virtue of their high tumorigenic potential and their resistance to chemo-, radio-, and immunotherapy. Not only are CSCs resistant to therapy, but cytotoxic agents actually enhance cancer stemness by activating transcription of pluripotency factors and by inducing expression of Bmi-1, a master regulator of stem cell self-renewal. We hypothesized therapeutic inhibition of interleukin-6 receptor (IL-6R) suppresses Bmi-1 to overcome intrinsic chemoresistance of CSCs. We observed that high Bmi-1 expression correlates with decreased (p = 0.04) recurrence-free survival time in HNSCC patients (n = 216). Blockade of IL-6R by lentiviral knockdown or pharmacologic inhibition with a humanized monoclonal antibody (Tocilizumab) is sufficient to inhibit Bmi-1 expression, secondary sphere formation, and to decrease the CSC fraction even in Cisplatin-resistant HNSCC cells. IL-6R inhibition with Tocilizumab abrogates Cisplatin-mediated increase in CSC fraction and induction of Bmi-1 in patient-derived xenograft (PDX) models of HNSCC. Notably, Tocilizumab inhibits Bmi-1 and suppresses growth of xenograft tumors generated with Cisplatin-resistant HNSCC cells. Altogether, these studies demonstrate that therapeutic blockade of IL-6R suppresses Bmi-1 function and inhibits cancer stemness. These results suggest therapeutic inhibition of IL-6R might be a viable strategy to overcome the CSC-mediated chemoresistance typically observed in HNSCC patients.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide and is characterized by a fast-paced growth. Like other solid tumors, the HNSCC growth rate results in the development of hypoxic regions identified by the expression of hypoxia-inducible factor 1α (HIF-1α). Interestingly, clinical data have shown that pharmacological induction of intratumoral hypoxia caused an unexpected rise in tumor metastasis and the accumulation of cancer stem cells (CSCs). However, little is known on the molecular circuitries involved in the presence of intratumoral hypoxia and the augmented population of CSCs. Here we explore the impact of hypoxia on the behavior of HNSCC and define that the controlling function of phosphatase and tensin homolog (PTEN) over HIF-1α expression and CSC accumulation are de-regulated during hypoxic events. Our findings indicate that hypoxic niches are poised to accumulate CSCs in a molecular process driven by the loss of PTEN activity. Furthermore, our data suggest that targeted therapies aiming at the PTEN/PI3K signaling may constitute an effective strategy to counteract the development of intratumoral hypoxia and the accumulation of CSCs.—Nascimento-Filho, C. H. V., Webber, L. P., Borgato, G. B., Goloni-Bertollo, E. M., Squarize, C. H., Castilho, R. M. Hypoxic niches are endowed with a protumorigenic mechanism that supersedes the protective function of PTEN.

Aim To explore the M1/M2 status of macrophage polarization from healthy, gingivitis, and periodontitis patient samples. Materials and methods Gingival biopsies were collected from 42 individuals (14 gingivitis, 18 periodontitis, and 10 healthy samples) receiving periodontal therapy. Histomorphology analysis was performed with haematoxylin and eosin staining. Immunofluorescence was performed using a combination of CD68 (macrophages), iNOS (M1), and CD206 (M2) in order to acquire changes in macrophage polarization at a single-cell resolution. Macrophages were quantified under microscopy using narrow wavelength filters to detect Alexa 488, Alexa 568, Alexa 633 fluorophores, and Hoechst 33342 to identify cellular DNA content. Results Gingivitis and periodontitis samples showed higher levels of macrophages compared with healthy samples. Unexpectedly, periodontitis samples displayed lower levels of macrophages dispersed in the stromal tissues compared with gingivitis samples; however, it remained higher than healthy tissues. The polarization of macrophages appears to be reduced in periodontitis and showed similar levels to those observed in healthy tissues. Conclusions Our study found that gingivitis and periodontitis differ from each other by the levels of macrophage infiltrate, but not by changes in macrophage polarization.

Objectives Mucoepidermoid carcinoma (MEC) is the most common type of salivary gland malignancy. Advanced or high-grade MECs are refractory to chemotherapy, often leading to tumor recurrence/metastasis and abysmal ~35% 5-year survival. Causal links have been established between Epithelial Growth Factor Receptor (EGFR) activation and poor outcome. Herein we investigated the therapeutic efficacy of EGFR inhibition against MEC using in vitro pre-clinical models. Materials and Methods Five human MEC cell lines were used in cell viability, cytotoxicity, apoptosis, cell cycle, 2D-clonogenicity, and 3D-spheroid formation assays following treatment with Erlotinib (EGFR inhibitor), SAHA (Histone Deacetylase inhibitor; HDAC) and CUDC-101 (dual EGFR-HDAC inhibitor). Effects on MEC cancer stem cells were evaluated using flow cytometry. Gene expression and pathway regulation were evaluated via qPCR and Western blot, respectively. Results MEC cells enter a quiescent, non-proliferative yet rapidly reversible drug tolerant state upon EGFR inhibition. Despite robust suppression of MEC cell proliferation, no discernable apoptosis is detected. Combination of EGFR and HDAC inhibitors exhibits synergistic effects, exerting ~5-fold more potent cell cytotoxicity compared to HDAC or EGFR monotherapy. CUDC-101, a single molecule with dual EGFR-HDAC inhibitor moieties, exerts irreversible and potent cytotoxic activity against MEC cells and blunts MEC cancer stem-cell tumorigenicity. Conclusion MEC cells are intrinsically tolerant to EGFR inhibition. Combining EGFR and HDAC inhibitors exerts synergistic and potent cytotoxic effects, suggesting that EGFR inhibitors still hold significant promise against MEC. Future studies are needed to assess the applicability and efficacy of dual EGFR-HDAC inhibitors for the clinical management of MEC.

INTRODUCTION: The maintenance of a stem cell pool is imperative to enable healing processes in the dental pulp tissue throughout life. As such, knowing mechanisms underlying stem cell self-renewal is critical to understand pulp pathophysiology and pulp regeneration. The purpose of this study was to evaluate the impact of stem cell factor (SCF) signaling through its receptor tyrosine kinase (c-Kit) on the self-renewal of human dental pulp stem cells (hDPSC). METHODS: hDPSC were stably transduced with lentiviral vectors expressing shRNA-c-Kit or vector control. The impact of the SCF/c-Kit axis on hDPSC self-renewal was evaluated using a pulpsphere assay in low attachment conditions and by evaluating the expression of polycomb complex protein Bmi-1 (master regulator of self-renewal) by western blot and flow cytometry. RESULTS: c-Kit-silenced hDPSC formed less pulpspheres when compared to hDPSC transduced with control vector (p

Several epidemiological investigations have found associations between poor oral health and different types of cancer, including colorectal, lung, pancreatic, and oral malignancies. The oral health parameters underlying these relationships include deficient oral hygiene, gingival bleeding, and bone and tooth loss. These parameters are related to periodontal diseases, which are directly and indirectly mediated by oral bacteria. Given the increased accessibility of microbial sequencing platforms, many recent studies have investigated the link between the oral microbiome and these cancers. Overall, it seems that oral dysbiotic states can contribute to tumorigenesis in the oral cavity as well as in distant body sites. Further, it appears that certain oral bacterial species can contribute to carcinogenesis, in particular, Fusobacterium nucleatum and Porphyromonas gingivalis, based on results from epidemiological as well as mechanistic studies. Yet, the strength of the findings from these investigations is hampered by the heterogeneity of the methods used to measure oral diseases, the treatment of confounding factors, the study design, the platforms employed for microbial analysis, and types of samples analyzed. Despite these limitations, there is an overall indication that the presence of oral dysbiosis that leads to oral diseases may directly and/or indirectly contribute to carcinogenesis. Proper methodological standardized approaches should be implemented in future epidemiological studies as well as in the mechanistic investigations carried out to explore these results.

Although the human body can heal, it takes time, and slow healing and chronic wounds often occur. Thus, identifying novel therapies to aid regeneration is needed. Here, we conducted a systematic review following the Preferred Reporting Items for Systematic Reviews guidelines and assessed preclinical studies on phosphatase and tensin homolog (PTEN) inhibitors and their effects on tissue repair and regeneration. In conditions associated with neurodegeneration, tissue injury and ischemia, the PTEN-regulated PI3K/AKT signaling pathway is activated. The use of PTEN inhibitors resulted in better tissue response by reducing the healing time and lesion sizes or inducing neuronal regeneration. Notably, all studies included in this systematic review indicated that pharmacological inhibition of PTEN enhanced the repair process of the eye, lung, muscle and nervous system.

Objectives The presence of titanium (Ti) particles around dental implants has been reported in the literature for decades. The prospective presence of Ti debris on soft tissues surrounding dental implants has not been systematically investigated and remains to be explored. Hence, this review aimed to evaluate the origin, presence, characteristics, and location of Ti particles in relation to dental implants. Material and methods Literature searches were conducted by two reviewers independently based on the PRISMA guidelines. The systematic review identified studies on Ti particles derived from dental implants. We evaluated several parameters, including anatomical location, and the suspected methods of Ti particles release. Results The search resulted in 141 articles, of which 26 were eligible and included in the systematic review of the literature. The investigations reported Ti and metal-like particles in the soft (i.e., epithelial cells, connective tissue, and inflammatory cells) and hard (bone crest and bone marrow) tissues around the dental implants. Shape and size of the particles varied. The current literature reported a size range from 100 nm to 54 µm identified by multiple particles identification methods. Conclusion Ti particles surrounding peri-implant tissues are a common finding. Peri-implantitis sites presented a higher number of particles compared to healthy implants. The particles were mostly around the implants and inside epithelial cells, connective tissue, macrophages, and bone. Various mechanisms were described as causes of Ti release, including friction during implant insertion, corrosion of the implant surface, friction at the implant-abutment interface, implantoplasty, and several methods used for implant surface detoxification.

Malignancies from the salivary glands are rare and represent 11% of all cancers from the oropharyngeal anatomical area. Mucoepidermoid Carcinomas (MEC) is the most common malignancy from the salivary glands. Low survival rates of high-grade Mucoepidermoid Carcinomas (MEC) are particularly associated with the presence of positive lymph nodes, extracapsular lymph node spread, and perineural invasion. Most recently, the presence of cancer stem cells (CSC), and the activation of the NFκB signaling pathway have been suggested as cues for an acquired resistance phenotype. We have previously shown that NFκB signaling is very active in MEC tumors. Herein, we explore the efficacy of NFκB inhibition in combination with class I and II HDAC inhibitor to deplete the population of CSC and to destroy MEC tumor cells. Our finding suggests that disruption of NFκB signaling along with the administration of HDAC inhibitors constitute an effective strategy to manage MEC tumors.

((3RS,5SR)- and ((3RS,5RS)-2-(2-methoxybenzyl)-3-(1,10-phenanthrolin-2-yl)isoxazolidin-5-yl)methanol have been synthesized, according to 1,3-dipolar cycloaddition methodology, as DNA intercalating agents and evaluated for their anticancer activity against human cervical carcinoma HeLa and head and neck squamous cells carcinoma cell lines. The synthesized compounds exhibited good cytotoxic activity with IC50 better than cisplatin, used as the main and effective treatment for HNSCC, and a 24.3–72.0-fold selectivity respect to the 184B5 non-cancerous immortalized breast epithelial cell lines. Unwinding assay, circular dichroism data, and Uv-vis melting experiments confirmed that these compounds act as DNA intercalators with a binding constant in the order of 104 M−1. Docking studies showed that both compounds can interact as intercalating agent with both poly-d(AT)2 and poly-d(GC)2, preferring an entrance by the minor groove of the poly-d(AT)2.

Head and neck cancer is globally challenging due to the resistance to therapy and aggressive behavior leading to high rates of mortality. Recent findings show that the tumor microenvironment plays a role in the maintenance and progression of many solid tumors, including head and neck cancer. The mechanisms involved in the modulation and regulation of the tumor microenvironment remain poorly understood. Increasing evidence suggests that epigenetic events can modulate the crosstalk between neoplastic and non-neoplastic cells during tumor progression. In this review, we explore the current understanding of the involvement of epigenetic events in the modulation of the tumor microenvironment and its impact on head and neck cancer behavior. We also explore the latest therapeutic strategies that use epigenetic-modulating drugs to manage tumor growth and progression.

Histone deacetylase inhibitors (HDACis) play an important role as valuable drugs targeted to cancer therapy: several HDACis are currently being tested in clinical trials. Two new potential HDACis 1a and 1d, characterized by the presence of a biphenyl-4-sulfonamide group as a connection unit between the N-{4-[(E)-(2-formylhydrazinylidene)methyl]-3-hydroxyphenyl} and the 2-hydroxy-N-(trifluoroacetyl)benzamide moiety, respectively, as two zinc-binding group (ZBG), have been designed, synthesized and tested for their biological activity. Surprisingly, compounds 1a and 12, this last exclusively obtained in place of 1d, exhibited a very low HDAC inhibitory activity. A serendipitous assay of these two compounds, conducted on three chemoresistant cell lines of head and neck squamous cell carcinoma (HNSCC), showed their antiproliferative activity at low nanomolar concentrations, better than cisplatin. In vitro, biological assays indicated that compounds 1a and 12 are able to increase acetylation of histone H3 and to interfere with the PI3K/Akt/mTOR pathway by inducing the accumulation of PTEN protein.

Objective To evaluate the influence of periodontal therapy on DNA methylation in chronic periodontitis patients as compared to healthy individuals. Materials & Methods Twenty patients were enrolled into two groups: 1) 10 diagnosed as clinically healthy; and 2) 10 diagnosed with chronic periodontitis. Clinical measures were recorded and gingival biopsies were harvested at baseline (both patient groups) and at 2 and 8 weeks post-baseline for diseased individuals. Molecular DNA methylation analysis was performed by pyrosequencing for the putative inflammation-associated genes LINE-1, COX-2 IFN-γ, and TNF-α. Random-intercept linear regression models were applied to evaluate methylation levels across groups at baseline and the methylation changes over time in the diseased and normal tissues. Results Periodontal therapy did not influence gene expression methylation of TNF-α, IFN-γ and LINE-1 levels at normal and periodontitis sites over time. However, it significantly reduced COX-2 methylation levels comparable to healthy individuals at both 2 and 8 weeks post-treatment (P < 0.05). Conclusions Periodontal therapy resets the DNA methylation status of inflammatory gene for COX-2 in periodontal disease patients. DNA methylation levels of TNF-α, IFN-γ and LINE-1 were sustained in periodontitis sites despite therapy. Future studies should consider an expanded panel of inflammatory genes over time. (ClinicalTrials. gov NCT02835898). This article is protected by copyright. All rights reserved.

Aims Epigenetics refers to changes in cell characteristics that occur independently of modifications to the DNA sequence. Oral carcinogenesis is influenced by modifications in epigenetic mechanisms, including changes in histones, which are proteins that support chromatin remodelling for the dynamic regulation of gene expression and silencing. The dysregulation of histone acetylation can lead to the uncontrolled activity of different genes, thereby triggering events associated with malignant transformation. The aim of this study was to analyse the expression of acetyl-histone H3 at lys9 (H3K9ac) in oral leucoplakia (OL) and oral squamous cell carcinoma (OSCC) in addition to its association with cell proliferation, epithelial–mesenchymal transition (EMT) and clinical-pathological findings. Methods and results Samples of normal oral mucosa (NOM), OL and OSCC were submitted to immunohistochemical analysis using anti-H3K9ac, Ki67 and vimentin. Slides were submitted to quantitative analysis regarding the percentage of positive cells. OSCC presented less expression of H3K9ac in comparison to OL (P < 0.01), whereas Ki67 and vimentin levels increased from OL to OSCC (P < 0.001 and P = 0.03, respectively). OSCC patients with poor prognosis had less H3K9ac expression (P = 0.04). The Kaplan–Meier cumulative survival curves also revealed lower survival rates in patients with less H3K9ac expression (P < 0.01). Conclusions The present findings suggest that changes in H3K9ac occur during the process of oral carcinogenesis along with an increase in cell proliferation and EMT. The results demonstrate that H3K9ac may be a useful novel prognostic marker for OSCC.

Adenoid cystic carcinoma (ACC) is an uncommon malignancy of the salivary glands that is characterized by local recurrence and distant metastasis due to its resistance to conventional therapy. Platinum-based therapies have been extensively explored as a treatment for ACC, but they show little effectiveness. Studies have shown that a specific group of tumor cells, harboring characteristics of cancer stem cells (CSCs), are involved in chemoresistance of myeloid leukemias, breast, colorectal and pancreatic carcinomas. Therapeutic strategies that target CSCs improve the survival of patients by decreasing the rates of tumor relapse, and epigenetic drugs, such as histone deacetylase inhibitors (HDACi), have shown promising results in targeting CSCs. In this study, we investigated the effect of the HDACi Suberoylanilide hydroxamic acid (Vorinostat), and cisplatin, alone or in combination, on CSCs and non-CSCs from ACC. We used CSCs as a biological marker for tumor resistance to therapy in patient-derived xenograft (PDX) samples and ACC primary cells. We found that cisplatin reduced tumor viability, but enriched the population of CSCs. Systemic administration of Vorinostat reduced the number of detectable CSCs in vivo and in vitro, and a low dose of Vorinostat decreased tumor cell viability. However, the combination of Vorinostat and cisplatin was extremely effective in depleting CSCs and reducing tumor viability in all ACC primary cells by activating cellular senescence. These observations suggest that HDACi and intercalating agents act more efficiently in combination to destroy tumor cells and their stem cells.

Background Epigenetic changes refer to any heritable modification in gene expression independent of alterations in the DNA sequence. Currently, it is well established that epigenetics represents a crucial player for tumor development. Nevertheless, the epigenetic mechanisms involved in the development and progression of salivary gland tumors (SGTs) remain poorly understood. Methods In this study, we analyzed the pattern of acetyl-histone H3 (lys9) expression in benign and malignant SGTs and further correlate our results with tumors' proliferative activity and clinical outcomes. We assembled tissue microarrays (TMAs) of 84 cases of SGTs and analyzed for acetyl-histone H3 (lys9) and Ki-67 using immunohistochemistry. The study comprised 42 benign and 42 malignant SGTs. Results All cases included in this study were positive to acetyl-H3 (lys9). We observed that malignant SGTs were hypoacetylated compared with benign (P = 0.04). Moreover, acetyl-H3 (lys9) expression was inversely correlated with Ki67 (**P = 0.02). Conclusion This study provides the first insight regarding histone modifications in SGTs. Our results suggest that epigenetic mechanism, particularly hypoacetylation of histone H3 (lys9), might play a role in the behavior of salivary gland tumors. Also, our findings suggest that interfering with the acetylation pattern of tumor histones represents a potential novel therapeutic strategy for the treatment of SGTs.

A mass spectrometry-based proteomics study carried out on 20 oral squamous cell carcinoma (OSCC) samples identified 26 differentially expressed proteins between high-risk human papillomavirus DNA (HR-HPV DNA) positive (+) and negative (-) tumors. Among these proteins, S100-A8 was upregulated in HR-HPV + cases and presented the most relevant correlation with survival rates. Objective: Validate the expression of S100-A8 in an expanded number of OSCC and correlate it with clinicopathological features and survival outcomes. Study Design: Immunofluorescence for S100-A8 was performed on a 91 OSCC tissue microarray. Additionally, The Cancer Genome Atlas data set of 338 OSCC samples was analyzed for S100-A8 mRNA levels. Results: Higher expression of S100-A8 protein and mRNA levels in HR-HPV DNA + tumors were confirmed. The up-regulated S100-A8 was correlated with T3/T4 tumors, advanced clinical stage and positive surgical margins. Furthermore, a high level of S100-A8 was associated with decreased disease-free survival, cancer specific, and overall survival. Conclusions: S100-A8 may play a role in HR-HPV DNA-related OSCC onset and progression. Acknowledgments: This work was supported by FAPESP grants: 2016/03248-1 and 2017/16347-0. We acknowledge the Mass Spectrometry Laboratory at Brazilian Biosciences National Laboratory, CNPEM, Campinas, Brazil for their support with the mass spectrometry analysis.

Photobiomodulation therapy (PBMT) is an emerging therapeutic modality designed to prevent and treat chemotherapy-driven oral mucositis (OM). However, the response of tumor cells to the effects of PBMT remains poorly understood. Our study explores the effects of PBMT in head and neck squamous cell carcinoma (HNSCC) based on cellular proliferation, migration, and survival of tumor cells and its population of cancer stem cells (CSC). We explored the behavior of two HNSCC cell lines (HN6 and HN13) under two distinct conditions, a physiological growing condition (10% FBS), and under stress growing condition (2% FBS) prior to irradiation using diode laser (InGaAlP; MM Optics, São Carlos, SP, Brazil). Diode laser (660 nm) was applied with a power of 100 mW delivering a total energy per point of 0.24 J. MTT and wound healing test (scratch assay) were performed to evaluate, respectively, proliferation and migration of tumor cells. Clonogenic and spheres formation assays were also performed to evaluate the survival and percentage of CSC upon irradiation. Overall, we observed that PBMT does not exacerbate the behavior of HNSCC. We could only observe a decrease in cellular proliferation of one cell line (HN6) when cultured under nutritional stress conditions (p .05). There were no significant differences between the control and the PBMT groups regarding cell migration, survival and the percentage of CSC. Collectively, our results suggest that in vitro administration of PBMT to HNSCC does not modify the behavior of tumor cells.

OBJECTIVES The mechanisms involved in the initiation and progression of peri-implantitis lesions are poorly understood. It was the aim to determine the content and activation status of macrophages present in human peri-implantitis lesions and compare the current findings with the macrophage polarization associated with periodontitis lesions. MATERIAL AND METHODS A total of 14 patients were studied in this investigation. Seven were soft tissue biopsies from dental implants affected by peri-implantitis that required explantation. Seven biopsies were from chronic periodontal disease. Immunofluorescence stains were performed using biomarkers to identify macrophages (CD68+ ) undergoing M1 polarization (iNOS+ ) and M2 polarization (CD206+ ), along with Hoechst 33,342 to identify DNA content. All samples were stained and photographed, and double-positive cells for CD68 and iNOS or CD68 and CD206 were quantified. RESULTS All peri-implantitis biopsies examined revealed a mixed population of macrophages undergoing M1 polarization and M2 polarization. Further analysis demonstrated the co-expression of iNOS and CD206, which indicates the presence of a heterogenic immune response on peri-implantitis lesions. Macrophage polarization in peri-implantitis lesions presents a distinct pattern than in periodontitis. We observed a significant increase in the population of M1 macrophages on peri-implantitis samples compared to periodontal disease samples. CONCLUSION Our results demonstrate that peri-implantitis has higher numbers of macrophages displaying a distinct macrophage M1 polarization signature compared to periodontitis lesions. This pattern may explain, in part, the distinct nature of peri-implantitis progression vs. periodontitis in humans.

A frequent observation in several malignancies is the development of resistance to therapy that results in frequent tumor relapse and metastasis. Much of the tumor resistance phenotype comes from its heterogeneity that halts the ability of therapeutic agents to eliminate all cancer cells effectively. Tumor heterogeneity is, in part, controlled by cancer stem cells (CSC). CSC may be considered the reservoir of cancer cells as they exhibit properties of self-renewal and plasticity and the capability of reestablishing a heterogeneous tumor cell population. The endowed resistance mechanisms of CSC are mainly attributed to several factors including cellular quiescence, accumulation of ABC transporters, disruption of apoptosis, epigenetic reprogramming, and metabolism. There is a current need to develop new therapeutic drugs capable of targeting CSC to overcome tumor resistance. Emerging in vitro and in vivo studies strongly support the potential benefits of combination therapies capable of targeting cancer stem cell-targeting agents. Clinical trials are still underway to address the pharmacokinetics, safety, and efficacy of combination treatment. This review will address the main characteristics, therapeutic implications, and perspectives of targeting CSC to improve current anticancer therapeutics.

A systematic review (SR) and meta-analysis were conducted to determine the prevalence of PI3K-AKT-mTOR signaling pathway mutations in patients with head and neck cancer (HNC). Overall, 105 studies comprising 8630 patients and 1306 mutations were selected. The estimated mutations prevalence was 13 % for PIK3CA (95 % confidence interval [CI] = 11-14; I2 = 82 %; p < 0.0001), 4% for PTEN (95 % CI = 3-5; I2 = 55 %; p < 0.0001), 3% for MTOR (95 % CI = 2-4; I2 = 5%; p = 0.40), and 2% for AKT (95 % CI = 1-2; I2 = 50 %; p = 0.0001). We further stratified the available data of the participants according to risk factors and tumor characteristics, including HPV infection, tobacco use, alcohol exposure, TNM stage, and histological tumor differentiation, and performed subgroup analysis. We identified significant associations between PI3K-AKT-mTOR pathway-associated mutations and advanced TNM stage (odds ratio [OR] = 0.20; 95 % CI = 0.09-0.44; I² = 71 %; p = 0.0001) and oropharyngeal HPV-positive tumors and PIK3CA mutations (OR = 17.48; 95 % CI = 4.20-72.76; I² = 69 %; p < 0.0002). No associations were found between alcohol and tobacco exposure, and tumor differentiation grade. This SR demonstrated that the PI3K-AKT-mTOR pathway emerges as a potential prognostic factor and could offer a molecular basis for future studies on therapeutic targeting in HNC patients.

// Camila S. Matsumoto 1, 5, * , Luciana O. Almeida 1, * , Douglas M. Guimaraes 1, 6, * , Manoela D. Martins 1, 7 , Petros Papagerakis 3, 4 , Silvana Papagerakis 2, 8 , Andreia M. Leopoldino 5 , Rogerio M. Castilho 1 , Cristiane H. Squarize 1, 2 1 Laboratory of Epithelial Biology, Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, MI, USA 2 Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI, USA 3 Department of Orthodontics and Pediatric Dentistry, School of Dentistry, University of Michigan, Ann Arbor, MI, USA 4 Center for Organogenesis, University of Michigan, Ann Arbor, MI, USA 5 Department of Clinical Analysis, Toxicology and Bromatology, School of Pharmacy, University of Sao Paulo, Ribeirao Preto, SP, Brazil 6 Department of Oral Pathology, School of Dentistry, University of Sao Paulo, SP, Brazil 7 Department of Oral Pathology, School of Dentistry, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil 8 Department of Otolaryngology, Medical School, University of Michigan, Ann Arbor, MI, USA * These authors have contributed equally to this work Correspondence to: Cristiane H. Squarize, email: csquariz@umich.edu Keywords: head and neck cancer, oral cancer Received: January 13, 2016 Accepted: May 19, 2016 Published: June 7, 2016 ABSTRACT Dysfunctional clock signaling is observed in a variety of pathological conditions. Many members of the clock gene family are upregulated in tumor cells. Here, we explored the consequences of a commonly disrupted signaling pathway in head and neck cancer on the regulation of circadian clock genes. PTEN is a key molecular controller of the PI3K signaling, and loss of PTEN function is often observed in a variety of cancers. Our main goal was to determine whether PTEN regulates circadian clock signaling. We found that oxidation-driven loss of PTEN function resulted in the activation of mTOR signaling and activation of the core clock protein BMAL1 (also known as ARNTL). The PTEN-induced BMAL1 upregulation was further confirmed using small interference RNA targeting PTEN, and in vivo conditional depletion of PTEN from the epidermis. We observed that PTEN-driven accumulation of BMAL1 was mTOR-mediated and that administration of Rapamycin, a specific mTOR inhibitor, resulted in in vivo rescue of normal levels of BMAL1. Accumulation of BMAL1 by deletion of PER2, a Period family gene, was also rescued upon in vivo administration of mTOR inhibitor. Notably, BMAL1 regulation requires mTOR regulatory protein Raptor and Rictor. These findings indicate that mTORC1 and mTORC2 complex plays a critical role in controlling BMAL1, establishing a connection between PI3K signaling and the regulation of circadian rhythm, ultimately resulting in deregulated BMAL1 in tumor cells with disrupted PI3K signaling.

To analyze the expression profile of DNA repair proteins (XRCC1 and APE1) and histone acetylation (H3K9) in oral and cutaneous lichen planus, in order to investigate potential biological markers that can clarify pathogenesis of these lesions.The total sample consisted of 89 lichen planus cases (66 oral and 23 cutaneous). Analysis of APE1 and XRCC1 expression was performed by immunohistochemistry in 44 oral and 20 cutaneous lichen planus, whereas the analysis of H3K9 acetylation was performed by immunofluorescence in 42 oral and 11 cutaneous lichen planus.Immunoreactivity for APE1 and XRCC1 was significantly higher in cutaneous lichen planus than in oral lichen planus (P = 0.003 and P = 0.034, respectively). There was a significant and moderate positive correlation between APE1 and XRCC1 in the oral group (Rho = 0.544; P 0.0001). In oral cases, there were no statistically significant results comparing APE1 and XRCC1 expression between reticular and erosive cases (P 0.05). Evaluation of H9K3 histone acetylation levels did not reveal significant results comparing oral to cutaneous lichen planus, neither comparing erosive to reticular (P 0.05).Changes in the expression profile of the DNA repair proteins exerted greater influence in pathogenesis of cutaneous lichen planus than oral lichen planus, in addition, H3K9 histone acetylation is an epigenetic event found in both lesions.

Background Patient-derived xenograft (PDX) involve the direct surgical transfer of fresh human tumor samples to immunodeficient mice. This systematic review aimed to identify publications of head and neck cancer PDX (HNC-PDX) models, describing the main methodological characteristics and outcomes. Methods An electronic search was undertaken in four databases, including publications having used HNC-PDX. Data were analyzed descriptively. Results 63 articles were yielded. The nude mouse was one most commonly animal model used (38.8 %), and squamous cell carcinoma accounted for the majority of HNC-PDX (80.6 %). Tumors were mostly implanted in the flank (86.3 %), and the latency period ranged from 30 to 401 days. The successful rate ranged from 17 % to 100 %. Different drugs and pathways were identified. Conclusion HNC-PDX appears to significantly recapitulate the morphology of the original HNC and represents a valuable method in translational research for the assessment of the in vivo effect of novel therapies for HNC.

Objective: This research aimed to evaluate the effects of the circadian clock genes in regulating wound healing (WH). Study design: Wounds were created in the dorsal skin in wild-type (WT) and BMAL1 knockout (Ko) male mice. The injury was measured daily, immediately following wounding (day 0), until wound closures were observed. For the morphologic analysis of the epithelial tongues, wounds collected at day 4 of each group were used. Quantitative analyses of BrdU-positive cells were performed by using immunohistochemistry assays. The stains for K10, pS6, and αSMA were also included in the analyses. Results: Statistically significant results were observed in the healing time between the WT mice and BMAL1 Ko mice. The epithelial migrating tongues were also reduced in size in the BMAL1 Ko group, in addition to a decrease in the number of myofibroblasts. In contrast, the number of proliferative cells was higher in WT mice. WT animals also showed higher pS6 expression. Conclusion: Overall, our results demonstrated that the absence of the BMAL1 gene negatively affects the healing time leading to delayed wound healing of the skin.

Adenoid cystic carcinoma from the salivary glands (ACC-SG) is characterized by slow-growing and metastatic potential. Also, they are resistant to chemotherapy, and mutations in NOTCH1 and MYB suggest a poor prognosis. Objective: Here we decided to test the efficiency of a novel histone deacetylase inhibitor (HDACi), entinostat, in patient-derived xenografts (PDX) of ACC-SG (ACCX9, ACCM1, ACCX6). Study Design: PDX tumors were divided into vehicle, entinostat, cisplatin, and entinostat/cisplatin. Then, tumor growth inhibition (TGI) was calculated for each tumor and frozen samples were processed for gene expression using NanoString and key markers using immunofluorescence. Results: All responded to combined therapy of entinostat/cisplatin and the overall TGI observed was 106%, 63%, and 38% for ACCX9, ACCM1, and ACCX6, respectively. Furthermore, we found that tumors with a better response to therapy had a low expression of Ki-67, p16 ink4, gamma-H2 AX, nuclear factor kappa B (NFkB), p300, and upregulation of caspase3, H3 K9, and acetyl-p53. Reduced expression of NOTCH1 was associated with better response to therapy whereas high levels of MYB were associated with reduced response. Conclusion: Overall, a subset of ACC-SG presenting with a NOTCH1 mutation had a better response to the new epigenetic therapy, and that ACC-SG is comprised of a heterogeneous group of tumors exhibiting distinct genetic and resistance behavior toward HDACi.

The nuclear factor kappa B (NFkB) may promote tumor cell proliferation, tumor angiogenesis, and inhibition of apoptosis in oral squamous cell carcinoma through the activation of the pathway by tumor necrosis factor-alpha (TNFα). A previous study from our group identified an increase of TNFα using a multiplex immunoassay in samples of proliferative verrucous leukoplakia (PVL), a rare and particular disorder with high rates of malignant transformation and recurrence. Our aim was to evaluate the expression of NFkB through an immunofluorescence assay in paraffin, tissue samples from PVL (n = 12), and oral squamous cell carcinoma derived from PVL (OSCC-PVL; n = 12). At least 5 fields (×400) per sample were registered, and the average percentage of positive cells were statistically accessed. We found that NFkB is statistically increased in the group OSCC-PVL. These findings suggest that the NFkB pathway may be activated by TNFα in OSCC-PVL, and this activation may be important in the malignant transformation of PVL in patients. Apoio financeiro FAPESP (No Processo 2017/01798-7), CAPES (No Processo 88881.187412/2018-01) University of Michigan.

Despite multiple modalities used to management of head and neck squamous cell carcinoma (HNSCC), disease control remains unsatisfactory. Immunotherapy is emerging as a novel therapeutic approach. This systematic review assesses clinical data regarding immunotherapy efficacy and safety.Data from 11 clinical trials testing immunotherapy in HNSCC were assessed. We performed the meta-analysis to correlate the overall survival (OS), response rate (RR), adverse effects, HPV status, and PD-L1 expression.Immunotherapy extended OS (hazard ratio = 0.77, p 0.0001) and RR significantly (risk ratio = 1.41, p = 0.02). Patients with HPV-positive HNSCC exhibited a better RR (risk ratio = 1.29, p = 0.24) and OS (11.5 vs. 6.3 months). PD-L1 positive tumors showed a higher OS (9.9 vs. 6.5 months). Moreover, immunotherapy caused less adverse effects than standard therapy.Our results indicate the benefit of immunotherapy for improving RR and OS of HNSCC patients. The benefit is higher in patients with HPV and PD-L1 positive tumors.

The circadian rhythm regulates the physiology and behavior of living organisms in a time-dependent manner. Clock genes have distinct roles including the control over gene expression mediated by the transcriptional activators CLOCK and BMAL1, and the suppression of gene expression mediated by the transcriptional repressors PER1/2 and CRY1/2. The balance between gene expression and repression is key to the maintenance of tissue homeostasis that is disrupted in the event of an injury. In the skin, a compromised epithelial barrier triggers a cascade of events that culminate in the mobilization of epithelial cells and stem cells. Recruited epithelial cells migrate towards the wound and reestablish the protective epithelial layer of the skin. Although we have recently demonstrated the involvement of BMAL and the PI3K signaling in wound healing, the role of the circadian clock genes in tissue repair remains poorly understood. Here, we sought to understand the role of BMAL1 on skin healing in response to injury. We found that genetic depletion of BMAL1 resulted in delayed healing of the skin as compared to wild-type control mice. Furthermore, we found that loss of Bmal1 was associated with the accumulation of Reactive Oxygen Species Modulator 1 (ROMO1), a protein responsible for inducing the production of intracellular reactive oxygen species (ROS). The slow healing was associated with ROS and superoxide dismutase (SOD) production, and pharmacological inhibition of the oxidative stress signaling (ROS/SOD) led to cellular proliferation, upregulation of Sirtuin 1 (SIRT1), and rescued the skin healing phenotype of Bmal1&minus, /&minus, mice. Overall, our study points to BMAL1 as a key player in tissue regeneration and as a critical regulator of ROMO1 and oxidative stress in the skin.

Histone modification, such as histone 3 lysine 9 acetylation (H3K9ac), is a major posttranslational epigenetic mechanism related to physiologic process of repairing, replication, and gene expression. Changes in H3K9ac immunoexpression have been recently reported in some human pathologies, for example in oral squamous cell carcinoma, but it has not yet been described in ameloblastoma. Objectives: To report H3K9ac immunoexpression in ameloblastoma, correlating it with dental follicles. Study Design: Ten dental follicles and 38 ameloblastoma cases with 3-µm sections in silanized slides underwent immunohistochemical reactions for H3K9ac antibody (C5B11 – Cell Signaling) in order to determine the immunoexpression profile, mean positivity and staining intensity. All samples were digitalized into high-resolution images using the Aperio Scanscope CS Slide Scanner and evaluated using software for digital analysis. Results: The respective mean values of positivity and intensities (weak, medium and strong) were 71.79%, 17.93%, 38.60%, and 15.26% for dental follicles, whereas 67.84%, 18.69%, 34.08%, and 15.07% for ameloblastoma. Conclusions: Although other human pathologies show H3K9ac immunoexpression changes in relation with normal tissues, the ameloblastoma development and progression may be not correlated with epigenetic changes involving histone 3 acetylation.

Objectives Limited availability of validated human adenoid cystic carcinoma (ACC) cell lines has hindered the mechanistic understanding of the pathobiology of this malignancy and the development of effective therapies. The purpose of this work was to generate and characterize a human ACC cell line. Material and Methods Immediately after surgery, a tumor fragment from a minor salivary gland from the tongue of a female Caucasian was minced, dissociated, and a single cell suspension was plated in fibronectin-coated flasks. A culture medium containing bovine brain extract and rhEGF was optimized for these cells. Whole exome sequencing was used to evaluate the presence of MYB-NFIB translocation. Results The University of Michigan-Human Adenoid Cystic Carcinoma (UM-HACC)-2A cells showed continuous growth in monolayers for at least 180 in vitro passages while maintaining epithelial morphology. Short-tandem repeat (STR) profiling confirmed a 100% match to patient DNA. Whole exome sequencing revealed the presence of the MYB-NFIB fusion in UM-HACC-2A cells, which was confirmed by PCR analysis. Western blots revealed high expression of epithelial markers (e.g. E-cadherin, EGFR, pan-cytokeratin) and proteins associated with ACC (e.g. c-Myb, p63). Developmental therapeutic studies showed that UM-HACC-2A cells were resistant to cisplatin (IC50 = 44.7 µM) while more responsive to paclitaxel (IC50 = 0.0006 µM). In a pilot study, we observed that UM-HACC-2A cells survived orthotopic transplantation into the submandibular gland. Notably, one of the mice injected with UM-HACC-2A cells exhibited lung metastasis after 6 months. Conclusion UM-HACC-2A is a MYB-NFIB fusion-positive ACC cell line that is suitable for mechanistic and developmental therapeutics studies.

Background Several mTOR pathway proteins are involved in the regulation of cellular anabolism, growth, proliferation, and survival. Activated proteins in the mTOR pathway are deregulated in multiple types of cancers and could influence prognosis. However, it is unclear whether deregulation of mTOR pathway proteins serves a prognostic role in patients with head and neck cancer (HNC). Furthermore, proteins in the mTOR pathway may be important targets for anticancer therapy. The aim of this study was to summarize existing cohort studies to determine whether immunoexpression of mTOR pathway proteins are important prognostic factors for survival in patients with HNC. Materials and methods A systematic review was performed using the Cochrane, Lilacs, PubMed, ScienceDirect, Scopus, and Web of Science databases (up to 23 January 2015). A meta-analysis was conducted to measure the frequency of protein expression in head and neck cancer patient samples and the prognostic value of mTOR pathway proteins for overall survival (OS) and disease-free survival (DFS). Results Twelve studies were included in our final analysis. The meta-analysis revealed that the frequency of overall expression of mTOR pathway proteins was 74.42% (CI: 63.3 to 84.0, P

Periodontitis is a chronic infectious disease driven by dysbiosis, an imbalance between commensal bacteria and the host organism. Periodontitis is a leading cause of tooth loss in adults and occurs in about 50% of the US population. In addition to the clinical challenges associated with treating periodontitis, the progression and chronic nature of this disease seriously affect human health. Emerging evidence suggests that periodontitis is associated with mechanisms beyond bacteria-induced protein and tissue degradation. Here, we hypothesize that bacteria are able to induce epigenetic modifications in oral epithelial cells mediated by histone modifications. In this study, we found that dysbiosis in vivo led to epigenetic modifications, including acetylation of histones and downregulation of DNA methyltransferase 1. In addition, in vitro exposure of oral epithelial cells to lipopolysaccharides resulted in histone modifications, activation of transcriptional coactivators, such as p300/CBP, and accumulation of nuclear factor–κB (NF-κB). Given that oral epithelial cells are the first line of defense for the periodontium against bacteria, we also evaluated whether activation of pathogen recognition receptors induced histone modifications. We found that activation of the Toll-like receptors 1, 2, and 4 and the nucleotide-binding oligomerization domain protein 1 induced histone acetylation in oral epithelial cells. Our findings corroborate the emerging concept that epigenetic modifications play a role in the development of periodontitis.

A small sub-population of cells characterized by increased tumorigenic potential, ability to self-renew and to differentiate into cells that make up the tumor bulk, has been characterized in some (but not all) tumor types. These unique cells, namedcancer stem cells, are considered drivers of tumor progression in these tumors. The purpose of this work is to understand if cancer stem cells play a functional role in the tumorigenesis of salivary gland mucoepidermoid carcinomas. Here, we investigated the expression of putative cancer stem cell markers (ALDH, CD10, CD24, CD44) in primary human mucoepidermoid carcinomas by immunofluorescence, in vitro salisphere assays, and in vivo tumorigenicity assays in immunodeficient mice. Human mucoepidermoid carcinoma cells (UM-HMC-1, UM-HMC-3A, UM-HMC-3B) sorted for high levels of ALDH activity and CD44 expression (ALDHhighCD44high) consistently formed primary and secondary salispheres in vitro, and showed enhanced tumorigenic potential in vivo (defined as time to tumor palpability, tumor growth after palpability), when compared to ALDHlowCD44low cells. Cells sorted for CD10/CD24, and CD10/CD44 showed varying trends of salisphere formation, but consistently low in vivo tumorigenic potential. And finally, cells sorted for CD44/CD24 showed inconsistent results in salisphere formation and tumorigenic potential assays when different cell lines were evaluated. Collectively, these data demonstrate that salivary gland mucoepidermoid carcinomas contain a small population of cancer stem cells with enhanced tumorigenic potential and that are characterized by high ALDH activity and CD44 expression. These results suggest that patients with mucoepidermoid carcinoma might benefit from therapies that ablate these highly tumorigenic cells.

The human papillomavirus (HPV) is an etiologic agent associated with the development of head and neck squamous carcinoma (HNSCC)—in particular, oropharyngeal squamous cell carcinoma. The HPV-positive HNSCC is characterized by genetic alterations, clinical progression, and therapeutic response, which are distinct from HPV-negative head and neck cancers, suggesting that virus-associated tumors constitute a unique entity among head and neck cancers. Malignant stem cells, or cancer stem cells, are a subpopulation of tumor cells that self-renew, initiate new tumors upon transplantation, and are resistant to therapy, and their discovery has revealed novel effects of oncovirus infection in cancer. In this review, we provide a virus-centric view and novel insights into HPV-positive head and neck pathogenesis. We discuss the influence of cancer stem cells, HPV oncoproteins, altered molecular pathways, and mutations in cancer initiation and cancer progression. We compiled a catalogue of the mutations associated with HPV-positive HNSCC, which may be a useful resource for genomic-based studies aiming to develop personalized therapies. We also explain recent changes in mass vaccination campaigns against HPV and the potential long-term impact of vaccinations on the prevention and treatment of HPV-positive head and neck cancers.

Objective The aim of this study was to analyze the expression pattern of proteins in the HGF/c-MET/PI3K signaling pathway in salivary gland tumors (SGTs) and to correlate the findings with the proliferative index and clinical parameters. Study Design We assembled tissue microarrays (TMAs) of 108 cases of SGTs, including 69 cases of pleomorphic adenoma (PA), 24 cases of adenoid cystic carcinoma (AdCC), and 15 cases of mucoepidermoid carcinoma (MEC). An immunohistochemical analysis of hepatocyte growth factor (HGF), MET phosphorylation (p-MET), protein kinase B (AKT) phosphorylation (p-AKT), and Ki-67 proteins was performed. Results Benign and malignant SGTs presented similar scores of HGF-positive cells (P = .36), whereas, malignant SGTs exhibited higher levels of p-MET (P = .001) and p-AKT (P = .001) than benign SGTs. No correlation of HGF, p-MET, or p-AKT expression was observed with clinical parameters. PA had a lower proliferative index than either AdCC (P = .001) or MEC (P = .001). Conclusions The salivary gland carcinomas exhibited increased activation of the HGF pathway, as evidenced by the phosphorylation of the MET receptor, and increased activation of the PI3K pathway, as indicated by p-AKT. These data suggest that the HGF/c-MET/PI3K signaling pathway is active in SGTs, especially in malignant neoplasms.

The immune response to oral bacteria and the subsequent activation of inflammatory signaling is not only dependent on genetic factors. The importance of so-called epigenetic mechanisms presents additional regulatory pathways of genes involved in maintaining chronic inflammation, including gingivitis and periodontitis. The term epigenetics relates to changes in gene expression that are not encoded in the DNA sequence itself and include chemical alterations of DNA and its associated proteins. These changes lead to remodeling of the chromatin and subsequent activation or inactivation of a gene. Epigenetic mechanisms have been found to contribute to disease, including cancer and autoimmune or inflammatory diseases. In this state-of-the art review, the authors provide the latest findings on the involvement of epigenetic modifications in the development of periodontal disease and present emerging therapeutic strategies aimed at epigenetic targets (epidrugs) associated with the disruption of tissue homeostasis and the development of periodontitis.

Purpose To evaluate the effect of titanium (Ti) particles on oral epithelial cell homeostasis and the potential of dental implants to release Ti debris upon insertion. Materials and methods Dental implants with varying surface treatments were employed to determine the feasibility of particle release during implant placement as well as the impact of free Ti debris on oral epithelial cells. Ti particles derived from implant surfaces were isolated and cultured in direct contact with normal oral epithelial cells for 48 hours. Further, cells were fixed and processed for immunofluorescence assay to detect the activation of the DNA damage response (DDR) using CHK2 and BRCA1 molecular markers. Positive cells demonstrating DNA damage were quantified and statistically analyzed. Results Ti particles derived from implants containing phosphate-enriched titanium oxide (PETO), fluoride-modified (FM), and grit-blasted (GB) surface treatments were able to activate CHK2 and trigger the recruitment of BRCA1 in oral epithelial cells. Also, implants with GB surfaces were able to release Ti particles upon implant placement. Conclusion The results indicate that Ti debris may be detached from the implant surface upon placement. Also, free Ti particles can trigger DDR signaling in oral epithelial cells. These findings suggest that Ti particles/debris released into a surgical wound may contribute to the disruption of epithelial homeostasis, and potentially compromise the oral epithelial barrier.

Chromatin decondensation is a key mechanism that guarantees gene transcription and repair of the genome, regulated mainly by the acetylation of histones. Emerging evidence has pointed out to histones as a new controlling mechanism of stem cell maintenance and fate. In this chapter, we will focus on the methods used to enrich tumor cell lines for cancer stem cells, and in the methods to identify the status of the histone acetylation in cancer cells and stem cells using immunofluorescence, invasion, and adhesion assays and identification of nuclear size.

Head and neck squamous carcinoma (HNSCC) is the sixth most prevalent cancer and one of the most aggressive malignancies worldwide. Despite continuous efforts to identify molecular markers for early detection, and to develop efficient treatments, the overall survival and prognosis of HNSCC patients remain poor. Accumulated scientific evidences suggest that epigenetic alterations, including DNA methylation, histone covalent modifications, chromatin remodeling and non-coding RNAs, are frequently involved in oral carcinogenesis, tumor progression, and resistance to therapy. Epigenetic alterations occur in an unsystematic manner or as part of the aberrant transcriptional machinery, which promotes selective advantage to the tumor cells. Epigenetic modifications also contribute to cellular plasticity during tumor progression and to the formation of cancer stem cells (CSCs), a small subset of tumor cells with self-renewal ability. CSCs are involved in the development of intrinsic or acquired therapy resistance, and tumor recurrences or relapse. Therefore, the understanding and characterization of epigenetic modifications associated with head and neck carcinogenesis, and the prospective identification of epigenetic markers associated with CSCs, hold the promise for novel therapeutic strategies to fight tumors. In this review, we focus on the current knowledge on epigenetic modifications observed in HNSCC and emerging Epi-drugs capable of sensitizing HNSCC to therapy.

Summary The long-term outcome of patients with mucoepidermoid carcinoma is poor. Limited availability of cell lines and lack of xenograft models is considered a major barrier to improved mechanistic understanding of this disease and development of effective therapies. Objective To generate and characterize human mucoepidermoid carcinoma cell lines and xenograft models suitable for mechanistic and translational studies. Methods Five human mucoepidermoid carcinoma specimens were available for generation of cell lines. Cell line tumorigenic potential was assessed by transplantation and serial in vivo passaging in immunodeficient mice, and cell line authenticity verified by short tandem repeat (STR) profiling. Results A unique pair of mucoepidermoid carcinoma cell lines was established from a local recurrence (UM-HMC-3A) and from the metastatic lymph node (UM-HMC-3B) of the same patient, 4 years after surgical removal of the primary tumor. These cell lines retained epithelial-like morphology through 100 passages in vitro, contain the Crtc1–Maml2 fusion oncogene (characteristic of mucoepidermoid carcinomas), and express the prototypic target of this fusion (NR4A2). Both cell lines generated xenograft tumors when transplanted into immunodeficient mice. Notably, the xenografts exhibited histological features and Periodic Acid Schiff (PAS) staining patterns that closely resembled those found in human tumors. STR profiling confirmed the origin and authenticity of these cell lines. Conclusion These data demonstrate the generation and characterization of a pair of tumorigenic salivary mucoepidermoid carcinoma cell lines representative of recurrence and lymph node metastasis. Such models are useful for mechanistic and translational studies that might contribute to the discovery of new therapies for mucoepidermoid carcinoma.

The sequencing of the head and neck cancer has provided a blueprint of the most frequent genetic alterations in this cancer type. They include inactivating mutations in Notch, p53, and p16ink4a tumor suppressor genes, in addition to nonoverlapping activating mutations of the PIK3CA and RAS oncogenes or inactivation of the tumor suppressor gene PTEN. Notably, these genetic alterations, along with epigenetic changes, result in increased activity of phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, which is present in most head and neck squamous cell carcinomas (HNSCCs). Moreover, we show here that approximately 30% of HNSCCs exhibit reduced PTEN expression. We challenged the biologic relevance of this finding by combining the intraoral administration of a tobacco surrogate, 4-nitroquinoline 1-oxide, with a genetically defined animal model displaying reduced PTEN expression, achieved by the conditional deletion of Pten using the keratin promoter 14 CRE-lox system. This provided a specific genetic and environmentally defined animal model for HNSCC that resulted in the rapid development of oral-specific carcinomas. Under these experimental conditions, control mice did not develop HNSCC lesions. In contrast, most mice harboring Pten deficiency developed multiple SCC lesions in the lateral border and ventral part of the tongue and floor of the mouth, which are the preferred anatomic sites for human HNSCC. Overall, our study highlights the likely clinical relevance of reduced PTEN expression and/or inactivation in HNSCC progression, while the combined Pten deletion with exposure to tobacco carcinogens or their surrogates may provide a unique experimental model system to study novel molecular targeted treatments for HNSCC patients.

Nature Communications 5 Article number: 452710.1038/ncomms5527 (2014); Published July312014; Updated February242016 In Fig. 4e,f of this Article, the plotted tumour volumes are incorrect due to an error in the formula used to convert length and width measurements into volume. The formula used was ((length*width)^2)*(22/7)*(1/6), and this should have read (length*(widtĥ2))*(22/7)*(1/6). The resulting smaller tumour volumes do not change the original conclusions. The correct version of Fig. 4 appears below. Figure 4

Background: Adenoid Cystic Carcinoma (ACC) is a rare cancer of secretory glands, most typically occurring in the salivary glands. No approved standard of care exists for systemic therapy. To identify potentially useful therapies, we screened Food and Drug Administration (FDA)-approved and investigational therapies using a well characterized panel of low passage ACC PDX models. Based on these results, combination treatments were evaluated for additive or super-additive effects. In line with the clinical chemo resistance observed in ACC patients, we previously reported that ACC tumor models are insensitive to platinum therapies. Interestingly, histone deacetylase (HDAC) inhibitors have been shown to sensitize cancer cells to anti-cancer agents in vitro. Entinostat is an oral class 1 selective HDAC inhibitor in Phase 3 testing for ER+ breast cancer in combination with hormone therapy. To better understand the potential effects of entinostat in ACC, we evaluated this agent alone and in combination with cisplatin in ACC PDX tumor models. Methods: Low passage ACC models were established in immune-deficient mice from primary or metastatic patient tissue and confirmed by histologic comparative analysis. Drug sensitivity studies evaluating entinostat, cisplatin and the combination were performed in three models, including ACCx5M1, ACCx6 and ACCx9. Study endpoints included tumor volume and time from treatment initiation with tumor growth inhibition, delay and regression reported at study completion. Results: Entinostat, cisplatin and the combination were all well tolerated with minimum cycle-based weight loss. Cisplatin tested alone (3 mg/kg weekly) was similar to control in all models, while single agent entinostat (5 mg/kg daily) showed significant anti-tumor activity in the ACCx5M1 (p Conclusion: Our studies identify entinostat as an agent of potential benefit in treating ACC and demonstrate that entinostat can induce low-dose cisplatin activity in the ACCx9 model, which harbors a mutation in the NOTCH1 gene that has been linked with chemo resistance and worse prognosis. Genomic and other characterization methods are currently underway to correlate sensitivity and resistance between models. Citation Format: Amanda Mangold, Melissa Rundle, Nicole Spardy Burr, Alyssa Moriarty, Priscila Goncalves, Rogerio Castilho, Peter Ordentlich, Jeffrey Kaufman, Kyriakos Papadopoulos, Michael J. Wick. Activity of entinostat alone and in combination with cisplatin in a panel of low passage adenoid cystic carcinoma patient-derived xenograft (PDX) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2146.