RAP1-RAC1 Signaling Has an Important Role in Adhesion and Migration in HNSCC.

Cell-cell adhesion is a key mechanism to control tissue integrity and migration. In head and neck squamous cell carcinoma (HNSCC), cell migration facilitates distant metastases and is correlated with poor prognosis. RAP1, a ras-like protein, has an important role in the progression of HNSCC. RAC1 is an integrin-linked, ras-like protein that promotes cell migration. Here we show that loss of cell-cell adhesion is correlated with inactivation of RAP1 confirmed by 2 different biochemical approaches. RAP1 activation is required for cell-matrix adhesion confirmed by adhesion to fibronectin-coated plates with cells that have biochemically activated RAP1. This effect is reversed when RAP1 is inactivated. In addition, RAP1GTP-mediated adhesion is only facilitated through α5ß1 integrin complex and is not a function of either α5 or ß1 integrin alone. Moreover, the inside-out signaling of RAP1 activation is coordinated with RAC1 activation. These findings show that RAP1 has a prominent role in cell-matrix adhesion via extracellular matrix molecule fibronectin-induced α5ß1 integrin and supports a critical role for the RAP1/RAC1 signaling axis in HNSCC cell migration.

Women Recipients of IADR Distinguished Scientist Awards.

The International Association for Dental Research (IADR) Distinguished Scientist Awards are prestigious recognitions of outstanding scientific accomplishments in various areas of dental, oral, and craniofacial research, which correspond to several of the IADR Scientific Groups and Networks. These 17 awards were established over a period of 60 y. The objective of this report is to highlight women recipients of IADR Distinguished Scientist Awards. Additionally, we report the distribution of awards to women scientists over time and compare the number of women nominees, awardees, and gender distribution of the membership. Information about the awards was obtained from the IADR member database and press releases. Information collected included name of the award, year received, and the awardee's name, institution, and position held at the time of the award. For the last 14 y, the time span for which reliable information was available, the gender distribution of the membership of the IADR was also retrieved. Overall, only 13% of the awardees have been women; even in the last 20 y, <20% have been women. In the last 14 y, the number of women awardees paralleled the number of nominees for each award. However, the proportion of women nominees was significantly lower than the female membership each year (P < 0.001). With the exception of 1 y, the percentage of women awardees trailed the women membership of the IADR. In the past 4 y, women represented 12% to 18% of the awardees, whereas they composed 41% to 46% of the IADR's membership. Given the benefits of prestigious recognitions on recruitment and retention of faculty and on attracting new research trainees into a discipline, it is important that policies be implemented to increase the proportion of women nominees for awards to appropriately recognize the efforts of remarkable women scientists.

Oral Cancer: Integration of Studies for Diagnostic and Therapeutic Precision.

Head and neck cancers are among the 10 most common cancers in the world and include cancers of the oral cavity, hypopharynx, larynx, nasopharynx, and oropharynx. At least 90% of head and neck cancers are squamous cell carcinomas (SCCs). This summary discusses the integration of clinical and mechanistic studies in achieving diagnostic and therapeutic precision in the context of oral cancer. Specifically, based on recent mechanistic studies, a subsequent study reevaluated current diagnostic criteria of perineural invasion in patients with oral cavity SCC showing that overall survival could be associated with nerve-tumor distance; validation of the findings of this study from a small group of patients could lead to a personalized approach to treatment selection in patients with oral cavity SCC. Moreover, delineation of key pathways in SCC revealed novel treatment targets that can be exploited to develop personalized treatment strategies to achieve long-term remission.

Periodontal Treatment in Cancer Patients: An Interdisciplinary Approach.

PURPOSE OF REVIEW: Dental care is an essential component in the comprehensive treatment for the cancer patient. As such, a review of the literature was completed to determine the relationships between periodontal and dental care in the cancer patient and provide strategic suggestions. RECENT FINDINGS: Periodontal treatment must be personalized depending on the patient's current oral health status, systemic status, and progress in treatment. Oral mucositis, periodontal status, and osteonecrosis of the jaw (ONJ) remain periodontal concerns in the cancer patient. Contributing factors of ONJ include root amputation (OR= 6.64), extraction of a single tooth (OR=3.7), severe tooth mobility (OR = 3.60), and unclosed wound (OR = 2.51). SUMMARY: Preventive maintenance, oral hygiene instruction, use of fluoride and chlorhexidine are all important therapeutic strategies. If extractions are required in patients who have received bone modifying drug infusions, flap management and primary wound closure is needed to reduce the risk of complications.

Precision Therapy of Head and Neck Squamous Cell Carcinoma.

Precision medicine is an approach to disease prevention and treatment that takes into account genetic variability and environmental and lifestyle influences that are unique to each patient. It facilitates stratification of patient populations that vary in their susceptibility to disease and response to therapy. Shared databases and the implementation of new technology systems designed to advance the integration of this information will enable health care providers to more accurately predict and customize prevention and treatment strategies for patients. Although precision medicine has had a limited impact in most areas of medicine, it has been shown to be an increasingly successful approach to cancer therapy. Despite early promising results targeting aberrant signaling pathways or inhibitors designed to block tumor-driven processes such as angiogenesis, limited success emphasizes the need to discover new biomarkers and treatment targets that are more reliable in predicting response to therapy and result in better health outcomes. Recent successes in the use of immunity-inducing antibodies have stimulated increased interest in the use of precision immunotherapy of head and neck squamous cell carcinoma. Using next-generation sequencing, the precise profiling of tumor-infiltrating lymphocytes has great promise to identify hypoimmunogenic cancer that would benefit from a rationally designed combinatorial approach. Continued interrogation of tumors will reveal new actionable targets with increasing therapeutic efficacy and fulfill the promise of precision therapy of head and neck cancer.

Perineural Invasion in Head and Neck Cancer.

Perineural invasion (PNI) is a mechanism of tumor dissemination that can provide a challenge to tumor eradication and that is correlated with poor survival. Squamous cell carcinoma, the most common type of head and neck cancer, has a high prevalence of PNI. This review provides an overview of clinical studies on the outcomes and factors associated with PNI in head and neck cancer and on findings on cancer-nerve crosstalk.

Biomarkers of epithelial-mesenchymal transition in squamous cell carcinoma.

An understanding of the process by which tumor cells destroy the basement membrane of the surface epithelium, invade, and metastasize is essential to the development of novel treatment of head and neck squamous cell carcinoma (HNSCC). In recent years, there has been increased interest in the role of epithelial-mesenchymal transition (EMT) in invasion. EMT is a process that describes the development of motile, mesenchymal-like cells from non-motile parent epithelial cells. There are 3 known types of EMT that mediate development, wound healing, and carcinogenesis. This review summarizes studies of known EMT biomarkers in the context of HNSCC progression. The biomarkers discussed come from a wide range of proteins, including cell-surface proteins (E-cadherin, N-cadherin, and Integrins), cytoskeletal proteins (α-Smooth Muscle Actin, Vimentin, and ß-catenin), extracellular matrix proteins (Collagens, Fibronectin, and Laminin), and transcription factors (SNAIL1, SNAIL2, TWIST, and LEF-1). Overall, the findings of these studies suggest that EMT mediates HNSCC progression. The mechanistic role of the EMT markers that have been associated with HNSCC should be more clearly defined if new anti-HNSCC therapies to block EMT progression are to be developed.

A novel approach to biomarker discovery in head and neck cancer using an autoantibody signature.

Despite the dismal prognosis for patients with squamous cell carcinoma of the head and neck (SCCHN), there have been no novel treatments in over 40 years. Identification of novel tumor antigens in SCCHN will facilitate the identification of potential novel treatment targets. Tumor antigens are proteins selectively expressed by tumor cells and recognized by the host immune system. Phage-displayed tumor antigens were enriched by biopanning with normal and then SCCHN-specific serum. Ninety-six phage clones were sequenced for identification, and 21 clones were validated using Luminex. One of these proteins, L23, a novel tumor antigen in SCCHN, was validated as an oncogene. L23 is upregulated in SCCHN compared with normal keratinocytes. Knockdown of L23 inhibited proliferation, invasion and cell survival. Overexpression of L23 had the reverse effect. Overexpression of L23 in non malignant cells led to transformation. Injection of SCCHN cells with knockdown of L23 in mice, induced tumors that were significantly smaller than control tumors. In conclusion, the immunomic screen yielded a panel of antigens specific to SCCHN; one of these proteins, L23, is a novel oncogene in SCCHN.

Control of cytokine mRNA expression by RNA-binding proteins and microRNAs.

Cytokines are critical mediators of inflammation and host defenses. Regulation of cytokines can occur at various stages of gene expression, including transcription, mRNA export, and post- transcriptional and translational levels. Among these modes of regulation, post-transcriptional regulation has been shown to play a vital role in controlling the expression of cytokines by modulating mRNA stability. The stability of cytokine mRNAs, including TNFα, IL-6, and IL-8, has been reported to be altered by the presence of AU-rich elements (AREs) located in the 3'-untranslated regions (3'UTRs) of the mRNAs. Numerous RNA-binding proteins and microRNAs bind to these 3'UTRs to regulate the stability and/or translation of the mRNAs. Thus, this paper describes the cooperative function between RNA-binding proteins and miRNAs and how they regulate AU-rich elements containing cytokine mRNA stability/degradation and translation. These mRNA control mechanisms can potentially influence inflammation as it relates to oral biology, including periodontal diseases and oral pharyngeal cancer progression.

Bisphosphonates inhibit expression of p63 by oral keratinocytes.

Osteonecrosis of the jaw (ONJ), a side-effect of bisphosphonate therapy, is characterized by exposed bone that fails to heal within eight weeks. Healing time of oral epithelial wounds is decreased in the presence of amino-bisphosphonates; however, the mechanism remains unknown. We examined human tissue from individuals with ONJ and non-bisphosphonate-treated control individuals to identify changes in oral epithelium and connective tissue. Oral and intravenous bisphosphonate-treated ONJ sites had reduced numbers of basal epithelial progenitor cells, as demonstrated by a 13.8±1.1% and 31.9±5.8% reduction of p63 expression, respectively. No significant differences in proliferation rates, vessel density, or macrophage number were noted. In vitro treatment of clonal and primary oral keratinocytes with zoledronic acid (ZA) inhibited p63, and expression was rescued by the addition of mevalonate pathway intermediates. In addition, both ZA treatment and p63 shRNA knock-down impaired formation of 3D Ex Vivo Produced Oral Mucosa Equivalents (EVPOME) and closure of an in vitro scratch assay. Analysis of our data suggests that bisphosphonate treatment may delay oral epithelial healing by interfering with p63-positive progenitor cells in the basal layer of the oral epithelium in a mevalonate-pathway-dependent manner. This delay in healing may increase the likelihood of osteonecrosis developing in already-compromised bone.

The tumor suppressor gene rap1GAP is silenced by miR-101-mediated EZH2 overexpression in invasive squamous cell carcinoma.

Rap1GAP is a critical tumor suppressor gene that is downregulated in multiple aggressive cancers, such as head and neck squamous cell carcinoma, melanoma and pancreatic cancer. However, the mechanistic basis of rap1GAP downregulation in cancers is poorly understood. By employing an integrative approach, we demonstrate polycomb-mediated repression of rap1GAP that involves Enhancer of Zeste Homolog 2 (EZH2), a histone methyltransferase in head and neck cancers. We further demonstrate that the loss of miR-101 expression correlates with EZH2 upregulation, and the concomitant downregulation of rap1GAP in head and neck cancers. EZH2 represses rap1GAP by facilitating the trimethylation of histone 3 at lysine 27, a mark of gene repression, and also hypermethylation of rap1GAP promoter. These results provide a conceptual framework involving a microRNA-oncogene-tumor suppressor axis to understand head and neck cancer progression.

An orthotopic floor-of-mouth model for locoregional growth and spread of human squamous cell carcinoma.

The molecular investigation of head and neck cancer targets requires the utilization and optimization of established animal models to characterize the effects of gene transcription and protein expression on invasion and metastasis. Floor-of-the-mouth murine models have been developed to study tumor growth, invasion, and metastasis of murine squamous cell carcinoma (SCC) cells in immunocompetent mice and invasion and metastasis of human SCC cells in nude mice. However, there are tumor cell lines that do not produce tumors in mice, using standard techniques, thus reducing the utility of the model to study specific genetic or treatment conditions. Furthermore, these techniques require large tumor volumes raising the possibility of airway compromise. In this report, we detail significant modifications to the orthotopic floor-of-mouth murine model for human SCC to facilitate predictable growth of a large panel of University of Michigan SCC cell lines. Furthermore, we describe the use of bioluminescence and micro-computed tomography to monitor tumor growth and bony invasion.

Rap1, a small GTP-binding protein is upregulated during arrest of proliferation in human keratinocytes.

Rap1 is a small GTP-binding protein (SMG) that exists in two 95% homologous isoforms, rap1A and rap1B. The functions of the rap1 proteins are not well understood. In this report we examined expression and function of rap1 in primary (HOKs) and immortalized (IHOKs) human oral keratinocytes under different growth conditions. In HOKs, rap1 increased with passage number, suggesting a role in differentiation and arrest of proliferation. Similarly, when inhibition of proliferation and differentiation were induced in HOKs by 1.2 mM CaCl2, both rap1 and involucrin increased with increasing concentrations of CaCl2. However, when similar experiments were done with IHOKs, which continue to proliferate in the presence of 1.2 mM CaCl2, the increase in involucrin expression was similar to HOKs but there was no substantial increase in rap1, suggesting that increased expression of rap1 is linked to inhibition of proliferation rather than differentiation of keratinocytes. Upon transfection of immortalized keratinocytes with rapGAP, which inactivates both isoforms of endogenous active rap1, enhanced proliferation was observed. Thus, we conclude that rap1 inhibits proliferation in keratinocytes.

Beta-adrenergic-induced cytosolic redistribution of Rap1 in rat parotid acini: role in secretion.

Rap1 has recently been identified on the secretory granule membrane and plasma membrane of rat parotid acinar cells (N. J. D'Silva, D. DiJulio, C. B. Belton, K. L. Jacobson, and E. L. Watson. J. Histochem. Cytochem. 45: 965-973, 1997). In the present study, we examined the cellular redistribution of Rap1 following treatment of acini with isoproterenol (ISO), the beta-adrenergic agonist, and determined the relationship between translocation and amylase release. In the presence of ISO, Rap1 translocated to the cytosol in a concentration- and time-dependent manner; this effect was not mimicked by the muscarinic agonist, carbachol. Translocation was maximal at 1 microM ISO and paralleled amylase release immediately after ISO stimulation. Rap1 translocation and amylase release were blocked by the beta-adrenergic antagonist, propranolol, whereas okadaic acid, a downstream secretory inhibitor, significantly blocked amylase release but did not inhibit Rap1 redistribution. Results suggest that the translocation of Rap1 is causally related to secretion and that the role of Rap1 in secretion is at a site proximal to the exocytotic event.

Immunolocalization of rap1 in the rat parotid gland: detection on secretory granule membranes.

The objective of this study was to localize rap1 in the rat parotid gland. Rap1 is a small GTP-binding protein that has been linked to phagocytosis in neutrophils and various functions in platelets. In this study, we used [alpha-32P]-GTP-blot overlay analysis, immunoblot analysis, and immunohistochemistry to identify rap1 in rat parotid gland. The immunohistochemical techniques included immunoperoxidase and widefield microscopy with image deconvolution. Rap1 was identified in the secretory granule membrane (SGM), plasma membrane (PM), and cytosolic (CY) fractions, with the largest signal being in the SGM fraction. The tightly bound vs loosely adherent nature of SGM-associated rap1 was determined using sodium carbonate, and its orientation on whole granules was assessed by trypsin digestion. Rap1 was found to be a tightly bound protein rather than a loosely adherent contaminant protein of the SGM. Its orientation on the cytosolic face of the secretory granule (SG) is of significance in postulating a function for rap1 because exocytosis involves the fusion of the cytoplasmic face of the SG with the cytoplasmic face of the PM, with subsequent release of granule contents (CO). Therefore, the localization and high concentration of rap1 on the SGM and its cytosolic orientation suggest that it may play a role in the regulation of secretion.

Globulomaxillary cyst revisited.

Classically, the globulomaxillary cyst was considered to be an inclusion or developmental cyst that arises from entrapped nonodontogenic epithelium in the globulomaxillary suture. Subsequently Christ disputed the existence and histogenesis of this lesion stating that the evidence indicated that facial processes per se did not exist. The development of the anterior maxilla was attributed to the merging of growth centers rather than fusion of facial processes, and hence ectodermal entrapment was ruled out. Recent embryologic studies, however, have demonstrated that Christ's view of facial development was incorrect. Fusion of facial processes does occur, and epithelium is entrapped in areas that later will lie between the maxillary lateral incisors and canines. This review argues that embryologically and histopathologically the globulomaxillary cyst should again be considered as an identifiable clinicopathologic entity.