Normal Tongue Tissues Research Articles

Diffuse reflectance spectroscopy for optical characterizations of orthotopic head and neck cancer models in vivo.

We demonstrated an easy-to-build, portable diffuse reflectance spectroscopy device along with a Monte Carlo inverse model to quantify tissue absorption and scattering-based parameters of orthotopic head and neck cancer models in vivo. Both tissue-mimicking phantom studies and animal studies were conducted to verify the optical spectroscopy system and Monte Carlo inverse model for the accurate extraction of tissue optical properties. For the first time, we reported the tissue absorption and scattering coefficients of mouse normal tongue tissues and tongue tumor tissues. Our in vivo animal studies showed reduced total hemoglobin concentration, lower tissue vascular oxygen saturation, and increased tissue scattering in the orthotopic tongue tumors compared to the normal tongue tissues. Our data also showed that mice tongue tumors with different sizes may have significantly different tissue absorption and scattering-based parameters. Small tongue tumors (volume was ∼60 mm3) had increased absorption coefficients, decreased reduced-scattering coefficients, and increased total hemoglobin concentrations compared to tiny tongue tumors (volume was ∼18 mm3). These results demonstrated the potential of diffuse reflectance spectroscopy to noninvasively evaluate tumor biology using orthotopic tongue cancer models for future head and neck cancer research.

Selinexor assists vorinostat in inhibiting HDAC activity via promoting the accumulation of maspin in the nucleus of oral tongue squamous cell carcinoma cells.

Oral tongue squamous cell carcinoma (OTSCC) is the most common oral cancer with a low overall survival rate, necessitating effective treatments. This study reports the anti-OTSCC effect of vorinostat and selinexor. OTSCC cell lines SCC-4 and SCC-25 were cultured to determine the effects of vorinostat and/or selinexor on cell survival, invasion, migration, and apoptosis. The transplanted tumor model of SCC-25 in nude mice was established to observe the therapeutic effects of vorinostat and/or selinexor. Western blotting was used to determine protein expressions in tumor cells. The results showed that histone deacetylase 1 (HDAC1) and exportin 1 (XPO1) were highly expressed, while nuclear maspin was expressed at a low rate in SCC-4 and SCC-25 compared to the normal tongue tissue. In vitro, both vorinostat and selinexor effectively inhibited cell viability, invasion, and migration, promoted cell apoptosis, down-regulated HDAC1, Matrix Metalloproteinase 2 (MMP2), and B cell leukemia/lymphoma 2 (Bcl-2), and up-regulated nuclear maspin and cleaved caspase 3. In vivo, both vorinostat and selinexor inhibited the growth of SCC-25-bearing tumors, down-regulated the expression of Ki67, HDAC1, MMP2, and Bcl-2, and promoted the expression of nuclear maspin and cleaved caspase 3. The combination of these two drugs exhibited synergistic effects both in vivo and in vitro. Our evidence shows that vorinostat combined with selinexor is an effective treatment for OTSCC. The mechanism may be that selinexor promotes the accumulation of maspin in the nucleus, an endogenous HDAC1 inhibitory protein to inhibit the HDAC1 activity of vorinostat and exert a synergistic anti-OTSCC effect.

What is Idiopathic Macroglossia? A case series and review of literature

<h3>Purpose</h3> Macroglossia is pathological condition which resulted in hypertrophy of tongue muscles. It is classified into congenital and acquired macroglossia. Idiopathic macroglossia (IM) is tongue hypertrophy without systemic or genetic causes. This condition has a significant impact on the quality of life, and often require tracheostomy and percutaneous endoscopic gastrostomy (PEG) to sustain living. Guidelines regarding treatment for this subtype are scant. The purpose of this project to present diagnosis and management of a series of patients with idiopathic macroglossia. <h3>Methods</h3> This was a retrospective case series of a cohort of patients with IM who were treated by Oral and Maxillofacial Surgery (OMS) service at University of Texas Health Science Center in Houston (UTHealth) and Emory University from 2019 to 2021. Inclusion criteria are (1) 18 years or older, (2) diagnosed with macroglossia, (3) managed with surgery, (4) with normal tongue tissue on histopathology results, and (5) with a negative COVID test. Patients were excluded if they are younger than 18 years old and diagnosed with macroglossia due to an underlying etiology such as congenital anomaly, systemic conditions, and intraoral inflammatory changes. Study variables were patient demographics, social history, medical comorbidities, clinical presentation, clinical dimensions, presence of tongue protrusion, difficulty feeding, difficulty in breathing, imaging characteristics/dimensions, pathological findings, management (tracheostomy, PEG, glossectomy), and length of inpatient stay. The outcome variables were normalization of tongue size, return of parenteral nutrition, and able to tolerate tracheostomy decannulation. Data were collected using a standardized collection form. Descriptive statistics were computed. <h3>Results</h3> Five patients (mean age, 45 years) with IM met inclusion criteria. All patients had history of hypertension, cerebral vascular disease, and prolong intubation. All patients presented with anterior tongue enlargement, with mean dimension of 13 × 6 cm, full or partial dentition, altered tongue sensation. They were all managed with tracheostomy to secure the airway, PEG and partial glossectomy. Average length of inpatients stay was 10 days. All IM achieved clinical resolution, 80% of the patients had their tracheostomy decannulated and PEG tube removed. <h3>Conclusion</h3> Management of macroglossia requires multidisciplinary approach. While etiology can often be identified and medical treatment can be initiated for reversible causes, most of the macroglossia cases previously reported required surgical management. Surgical reduction offers the best functional and cosmetic results and minimizes morbidity. In the case idiopathic macroglossia, management should involve tracheostomy and feeding access for the initial stabilization followed by reduction glossectomy for improvement of functional outcomes.

Identification and Confirmation of the miR-30 Family as a Potential Central Player in Tobacco-Related Head and Neck Squamous Cell Carcinoma.

Constituents of tobacco that can cause DNA adduct formation and oxidative stress are implicated in the development of head and neck squamous cell carcinoma (HNSCC). However, there are few studies on the mechanism(s) that underlie tobacco-associated HNSCC. Here, we used a model in which tumors were induced in rats using 4-nitroquinoline 1-oxide (4NQO), which mimicked tobacco-related HNSCC, and analyzed the expression profiles of microRNAs and mRNAs. Our results indicated that 57 miRNAs and 474 mRNA/EST transcripts exhibited differential expression profiles between tumor and normal tongue tissues. In tumor tissue, the expression levels of rno-miR-30 family members (rno-miR-30a, rno-miR-30a-3p, rno-miR-30b-5p, rno-miR-30c, rno-miR-30d, rno-miR-30e and rno-miR-30e-3p) were only 8% to 37% of those in the control group. The GO terms enrichment analysis of the differentially expressed miRNAs indicated that oxidation reduction was the most enriched process. Low expression of miR-30 family members in human HNSCC cell lines and tissues was validated by qPCR. The results revealed that the expression of miR-30b-5p and miR-30e-5p was significantly decreased in the TCGA HNSCC dataset and validation datasets, and this decrease in expression further distinguishes HNSCC associated with tobacco use from other subtypes of HNSCC. CCK8, colony formation, transwell migration and HNSCC xenograft tumor assays indicated that miR-30b-5p or miR-30e-5p inhibited proliferation, migration and invasion in vitro, and miR-30b-5p suppressed tumor growth in vivo. Moreover, we uncovered that KRAS might be the potential target gene of miR-30e-5p or miR-30b-5p. Thus, our data clearly showed that decreased expression of miR-30e-5p or miR-30b-5p may play a crucial role in cancer development, especially that of tobacco-induced HNSCC, and may be a novel candidate biomarker and target for this HNSCC subtype.

Peripheral Contribution of BDNF/TrkB signaling in Mediating Oral Cancer Pain

Background: Oral cancer pain is debilitating and understanding mechanisms for it is critical to develop treatment strategies. Brain-derived neurotrophic factor (BDNF) signaling is elevated in oral tumor biopsies and has been reported to promote tumor progression. However, whether BDNF signaling in oral tumors contributes to cancer-induced pain is not known. Therefore, the current study evaluates a novel peripheral role of BDNF/TrkB signaling in oral cancer pain. Using an orthotopic mouse tongue cancer pain model, we determine BDNF levels and expression tumor-tongue and normal tongue tissues using ELISA and immunohistochemistry. Additionally, we evaluated the effect of local administration of BDNF neutralizing Ab as well as TrkB receptor antagonist (ANA12) in reversing pain-like behaviors in vivo. Further, we identified the sensory fiber type affected by local ANA12 treatment in tumor-bearing tongues using single-fiber tongue-nerve electrophysiology and characterized TrkB isoform expression in sensory neuronal subtypes innervating mouse tongue. Our data demonstrated that BDNF levels were up-regulated in superfusates and lysates of tumor tongues over normal tongues and that BDNF was expressed by the cancer cells within the tumor. Neutralization of BDNF or inhibition of TrkB activity (ANA12) within the tumor-bearing tongue reversed tongue-tumor induced pain-like behaviors. Single-fiber recordings of tongue-nerve preparations revealed that ANA12 reversed tumor-induced mechanical sensitivity of A-slow high threshold mechanoreceptors. Single-cell RTPCR of lingual neurons demonstrated expression of full-form TrkB and truncated TrkB in distinct neuronal subtypes. Our data suggests that BDNF is released from oral cancer cells at the site of tumor growth and activates TrkB-expressing lingual sensory fibers, thereby contributing to oral cancer pain. This is a novel finding and the first demonstration of a peripheral role for BDNF signaling in oral cancer pain. Targeting BDNF signaling peripherally may prove to be an effective treatment for cancer-induced pain as well as tumorigenesis. Background: Oral cancer pain is debilitating and understanding mechanisms for it is critical to develop treatment strategies. Brain-derived neurotrophic factor (BDNF) signaling is elevated in oral tumor biopsies and has been reported to promote tumor progression. However, whether BDNF signaling in oral tumors contributes to cancer-induced pain is not known. Therefore, the current study evaluates a novel peripheral role of BDNF/TrkB signaling in oral cancer pain. Using an orthotopic mouse tongue cancer pain model, we determine BDNF levels and expression tumor-tongue and normal tongue tissues using ELISA and immunohistochemistry. Additionally, we evaluated the effect of local administration of BDNF neutralizing Ab as well as TrkB receptor antagonist (ANA12) in reversing pain-like behaviors in vivo. Further, we identified the sensory fiber type affected by local ANA12 treatment in tumor-bearing tongues using single-fiber tongue-nerve electrophysiology and characterized TrkB isoform expression in sensory neuronal subtypes innervating mouse tongue. Our data demonstrated that BDNF levels were up-regulated in superfusates and lysates of tumor tongues over normal tongues and that BDNF was expressed by the cancer cells within the tumor. Neutralization of BDNF or inhibition of TrkB activity (ANA12) within the tumor-bearing tongue reversed tongue-tumor induced pain-like behaviors. Single-fiber recordings of tongue-nerve preparations revealed that ANA12 reversed tumor-induced mechanical sensitivity of A-slow high threshold mechanoreceptors. Single-cell RTPCR of lingual neurons demonstrated expression of full-form TrkB and truncated TrkB in distinct neuronal subtypes. Our data suggests that BDNF is released from oral cancer cells at the site of tumor growth and activates TrkB-expressing lingual sensory fibers, thereby contributing to oral cancer pain. This is a novel finding and the first demonstration of a peripheral role for BDNF signaling in oral cancer pain. Targeting BDNF signaling peripherally may prove to be an effective treatment for cancer-induced pain as well as tumorigenesis.

Profiles of elemental concentrations in human: contribution of X-ray fluorescence to discrimination between healthy and diseased tissues and prediction of alterations in tongue carcinoma

Introduction It has been shown that the concentrations of some elements, for example K, Ca, Cu, Fe, and Zn, may differ significantly between the healthy area and the tumour area in the same human tissue [1]. Most studies conducted so far are focussed on specific elements which are a priori known to be involved in physiological or pathological processes, and thus risk neglecting the potential role of the excluded elements in those processes [2]. The role of elements considered in isolation has been questioned because it ignores the important interactions amongst the various elements [3]. However, even when concentrations of various elements are obtained in the same study, comparisons between healthy and diseased tissues, or correlations between the various elements, both intrinsically multivariate, are often implemented with univariate methods, which may result in observed effects or the inability to detect such effects [4]. The methodologies in this study, which complement multielement determinations by X-ray fluorescence spectroscopy (XRF) and X-ray diffraction (XRD) in several types of biological samples, with multivariate data analysis methodologies, provide an important contribute to fill existing gaps in current knowledge of the role elements in such metabolic pathways. Materials and methods Samples consisted of five matched pairs (10 samples) of normal and tumour human tongue tissue. In the developing work, the XRF and XRD techniques are applied in the determination of the concentration profile of several elements of interest, in samples of healthy tissue and tongue carcinoma, with the objective of developing a classification system based on the profile of elemental concentrations which allows to discriminate between healthy tissue and carcinoma, and thus clarify the role of these elements in the aetiology of the disease. Results Potential differences in Ca, Fe and S were observed. Intrasampling tests determined that samples were inhomogeneous which may affect the ability to discriminate between normal and tumour tissues. Discussion and conclusions It is highlighted that the limited number of samples prevents any conclusive findings for now nevertheless results provide areas of focus for upcoming study.

Preclinical Assessment of the Effectiveness of Magnetic Resonance Molecular Imaging of Extradomain-B Fibronectin for Detection and Characterization of Oral Cancer.

Oral squamous cell carcinoma (OSCC) has not seen a substantial improvement in patient survival despite therapeutic advances, making accurate detection and characterization of the disease a clinical priority. Here, we aim to demonstrate the effectiveness of magnetic resonance imaging (MRI) with the targeted MRI contrast agent MT218 specific to extradomain-B fibronectin (EDB-FN) in the tumor microenvironment for detection and characterization of aggressive OSCC tumors. EDB-FN expression was evaluated in human normal tongue and OSCC specimens with immunohistochemistry. Invasiveness of human CAL27, HSC3, and SCC4 OSCC cells was analyzed with spheroid formation and transwell assays. EDB-FN expression in the cells was analyzed with semiquantitative real-time PCR, western blotting, and a peptide binding study with confocal microscopy. Contrast-enhanced MRI with MT218 was performed on subcutaneous OSCC mouse models at a dose of 0.04mmol/kg, using gadoteridol (0.1mmol/kg) as a control. Strong EDB-FN expression was observed in human untreated primary and metastatic OSCC, reduced expression in treated OSCC, and little expression in normal tongue tissue. SCC4 and HSC3 cell lines demonstrated high invasive potential with high and moderate-EDB-FN expression, respectively, while CAL27 showed little invasive potential and low-EDB-FN expression. In T1-weighted MRI, MT218 produced differential contrast enhancement in the subcutaneous tumor models in correlation with EDB-FN expression in the cancer cells. Enhancement in the high-EDB-FN tumors was greater with MT218 at 0.04mmol/kg than gadoteridol at 0.1mmol/kg. The results suggest EDB-FN has strong potential as an imageable biomarker for aggressive OSCC. MRMI results demonstrate the effectiveness of MT218 and the potential for differential diagnostic imaging of oral cancer for improving the management of the disease.

LncRNA-MALAT1 is a promising biomarker for prognostic evaluation of tongue squamous cell carcinoma.

MALAT1 is recognized as an oncogenic lncRNA in various malignancies. Here, the authors aim to explore the association of MALAT1 expression and prognostic implication in tongue squamous cell carcinoma (SCC). The tongue tissues of 128 tongue SCC cases satisfying strict follow-up criteria and 28 normal cases were subjected to qRT-PCR assay for monitoring MALAT1 expression. Chi-square test was applied to explore the correlation between MALAT1 expression and clinicopathological features of tongue SCC. Kaplan-Meier analysis was used to calculate survival rates. Cox proportional hazard analysis was adopted to analyze the relationship between prognostic factors and patient survival. The expression of MALAT1 was upregulated in tongue SCC, compared to normal tongue tissues. The expression level of MALAT1 was correlated to differentiation and stage of tongue SCC, and high MALAT1 expression was associated with low disease-free survival and overall survival rates. Moreover, advanced tongue SCC patients with high MALAT1 level had lower disease-free survival and decreased overall survival rate than patients with low MALAT1 level. These results revealed that MALAT1 overexpression can be considered as a significant prognostic factor to independently predict the disease-free survival and overall survival rate of tongue SCC. The expression level of MALAT1 is closely related with progression of tongue SCC. Furthermore, MALAT1 can serve as an independent biomarker for prognostic evaluation of tongue SCC.

Keratin 36, a specific marker of tongue filiform papillae, is downregulated in squamous cell carcinoma of the mobile tongue.

Human keratin 36 (K36) is a member of the hair keratin family and is a marker of hair cortex differentiation. The human KRT36 gene is located on the long arm of chromosome 17 and belongs to the cluster of structurally unrelated acidic hair keratins. Recently, it has been reported that KRT36 mRNA is specifically expressed in normal tongue epithelium and downregulated in squamous cell carcinomas of the mobile tongue. Furthermore, KRT36 levels have been reported to be downregulated in clinically normal mobile tongue tissue that is adjacent to tumours, suggesting it could be a marker of pre-neoplastic changes. However, the exact role and the potential role of K36 in tongue tumour formation remains unclear. The aim of the present study was to investigate expression of K36 in a series of squamous cell carcinomas of the mobile tongue, normal mobile tongue and a small panel of other human tissues (normal tissue from the appendix, cervix, hair, lip, mamilla, nail, oesophagus, skin, thymus and vagina) and selected cancer tissue (cervical cancer, melanoma and basal cell carcinoma). Affinity purified polyclonal antibodies against K36 were generated and used for immunohistochemical analysis. The results revealed that in the normal tongue, K36 was detected specifically in the filiform papillae of the dorsal surface of the tongue. Additionally, none of the tongue cancer tissue samples were K36-positive. Immunostaining also revealed that K36 was expressed in nail beds, Hassal's corpuscles in the thymus and the hair cortex. However, K36 was not expressed in the squamous epithelia of the skin, cervix and oesophagus, and the squamous cells of cervical carcinomas, basal cell carcinoma or melanoma. The present data indicated that K36 may be inactivated in tumours of the tongue. However, whether this is part of the tumoural process or if it is an effect of the tumour itself remains to be elucidated.

NIT2 overexpression predicts poor prognosis in tongue squamous cell carcinoma patients

There is disputable on the role of nitrilase-like 2 (NIT2) in cancer. Its expression and its relationship with clinicopathological features in tongue squamous cell carcinoma (TSCC) are not yet clear. The purpose of this study is to investigate the expression of NIT2 in TSCC and its correlation with clinicopathological characteristics in TSCC patients. Through proteomic identification, we found that the protein NIT2 was related to the development of TSCC. q-PCR, western blot and immunohistochemistry techniques were applied to detect the expression of NIT2 in TSCC. The relationship between the expression of NIT2 and clinicopathological features was analyzed by Chi square tests. The results showed the expression of NIT2 in TSCC was significantly higher than that in normal tongue tissues (p < 0.05). Univariate and multivariate analysis showed that the positive expression of NIT2 and N classification were associated with decreased disease-free survival rate (DFS) and overall survival (OS) (p < 0.05). The results suggested that NIT2 is overexpressed in TSCC and NIT2 may be a potential therapeutic target for TSCC.

MTA3-SOX2 Module Regulates Cancer Stemness and Contributes to Clinical Outcomes of Tongue Carcinoma.

Cancer cell plasticity plays critical roles in both tumorigenesis and tumor progression. Metastasis-associated protein 3 (MTA3), a component of the nucleosome remodeling and histone deacetylase (NuRD) complex and multi-effect coregulator, can serve as a tumor suppressor in many cancer types. However, the role of MTA3 in tongue squamous cell cancer (TSCC) remains unclear although it is the most prevalent head and neck cancer and often with poor prognosis. By analyzing both published datasets and clinical specimens, we found that the level of MTA3 was lower in TSCC compared to normal tongue tissues. Data from gene set enrichment analysis (GSEA) also indicated that MTA3 was inversely correlated with cancer stemness. In addition, the levels of MTA3 in both samples from TSCC patients and TSCC cell lines were negatively correlated with SOX2, a key regulator of the plasticity of cancer stem cells (CSCs). We also found that SOX2 played an indispensable role in MTA3-mediated CSC repression. Using the mouse model mimicking human TSCC we demonstrated that the levels of MTA3 and SOX2 decreased and increased, respectively, during the process of tumorigenesis and progression. Finally, we showed that the patients in the MTA3low/SOX2high group had the worst prognosis suggesting that MTA3low/SOX2high can serve as an independent prognostic factor for TSCC patients. Altogether, our data suggest that MTA3 is capable of repressing TSCC CSC properties and tumor growth through downregulating SOX2 and MTA3low/SOX2high might be a potential prognostic factor for TSCC patients.

Xenotropic and polytropic retrovirus receptor 1 (XPR1) promotes progression of tongue squamous cell carcinoma (TSCC) via activation of NF-\u03baB signaling

BackgroundXenotropic and polytropic retrovirus receptor 1 (XPR1), a previously identified cellular receptor for several murine leukemia viruses, plays a role in many pathophysiological processes. However, the role of XPR1 in human cancers has not yet been characterized.MethodsReal-time PCR and western blotting assay were used to measure the expression of XPR1 in tongue squamous cell carcinoma (TSCC) tissues. Expression of XPR1 and p65 in clinical specimens was analyzed using immunohistochemical assay. The function of XPR1 on progression of TSCC was explored using in vitro and in vivo experiments. The molecular mechanism by which XPR1 helps to cancer progression was investigated by luciferase reporter activity, ELISA, PKA activity assay, immunofluorescence, western blotting and qPCR assay.ResultsHerein, we find that XPR1 is markedly upregulated in TSCC tissues compared to normal tongue tissues. High expression of XPR1 significantly correlates with the malignant features and poor patient survival in TSCC. Ectopic expression of XPR1 increases, while silencing of XPR1 reduces the proliferation, invasion and anti-apoptosis capacities of TSCC cells. Importantly, silencing of XPR1 effectively inhibits the tumorigenecity of TSCC cells. Moreover, we identified that XPR1 increased the concentration of intracellular cAMP and activated PKA. Thus, XPR1 promoted phosphorylation and activation of NF-κB signaling, which is required for XPR1-mediated oncogenic roles and significantly correlates with XPR1 expression in clinical specimens.ConclusionsThese findings uncover a critical role of XPR1 in TSCC progression via activation of NF-κB, and suggest that XPR1 might be a potential prognostic marker or therapeutic target.

A Feedback Loop between MicroRNA 155 (miR-155), Programmed Cell Death 4, and Activation Protein 1 Modulates the Expression of miR-155 and Tumorigenesis in Tongue Cancer.

MicroRNA 155 (miR-155) is an oncomir, generated as a noncoding RNA from the BIC gene whose promoter activity is mainly controlled via activation protein 1 (AP-1) and NF-κB transcription factors. We found that the expression levels of miR-155 and programmed cell death 4 (Pdcd4) exhibit inverse relationships in tongue cancer cells (SAS and AWL) and tumor tissues compared to their relationships in normal FBM cells and normal tongue tissues, respectively. In silico and in vitro studies with the 3' untranslated region (UTR) of Pdcd4 via luciferase reporter assays, quantitative PCR (qPCR), and Western blotting showed that miR-155 directly targets Pdcd4 mRNA and blocks its expression. Ectopic expression of Pdcd4 or knockdown of miR-155 in tongue cancer cells predominantly reduces AP-1-dependent transcriptional activity of the BIC promoter and decreases miR-155 expression. In this study, we demonstrate that miR-155 expression is modulated by a feedback loop between Pdcd4, AP-1, and miR-155 which results in enhanced expression of miR-155 with a consequent progression of tongue tumorigenesis. Further, miR-155 knockdown increases apoptosis, arrests the cell cycle, regresses tumor size in xenograft nude mice, and reduces cell viability and colony formation in soft-agar and clonogenic assays. Thus, the restoration of Pdcd4 levels by the use of molecular manipulation such as using a miR-155 sponge has an essential role in the therapeutic intervention of cancers, including tongue cancer.

CIZ1 Expression Is Upregulated in Hemangioma of the Tongue.

Hemangioma is a vascular neoplasm and one of the most common benign tumors. The pathogenesis of hemangioma has not been fully understood. CIZ1 (Cip1-interacting zinc finger protein 1) is a nuclear protein and the binding partner of p21. Dysregulation of CIZ1 expression has been reported in various types of cancerous tissues. In this study, we examined CIZ1 expression in hemangioma of the tongue and explored its function in vascular endothelial cells, the proliferative cell type in hemangioma. Immunohistochemistry showed that CIZ1 was highly expressed in hemangioma of the tongue while its expression is minimal in the normal tongue tissues. In vitro, knockdown of CIZ1 expression by shRNA transfection significantly reduced the proliferation and migration of human umbilical vein endothelium cells (HUVECs), suggesting a positive role of CIZ1 in endothelial cell proliferation and migration. Therefore, CIZ1 might involve in pathogenesis of hemangioma of the tongue by regulation of endothelial cell functions.

High expression of TMEM40 contributes to progressive features of tongue squamous cell carcinoma.

Transmembrane protein 40 (TMEM40) is a 23-kDa protein and its association with tongue squamous cell carcinoma (TSCC) remains unclear. This study aimed to investigate the expression and clinical significance of TMEM40 in TSCC and its roles in TSCC cells. Immunohistochemical analysis was performed to detect the expression levels of TMEM40 in 60 tongue tissue samples. Furthermore, TMEM40 was overexpressed and inhibited in two TSCC cell lines by transfection with pEZ-M98-TMEM40 plasmid or TMEM40 small interfering RNA, respectively. Cell Counting Kit-8 and colony formation assays were used to investigate the effects of TMEM40 on cell proliferation and colony formation ability, respectively. Flow cytometry was performed to determine cell apoptosis and cycle conditions of transfected cells. Wound-healing and Transwell assays were processed to explore the effects of TMEM40 on cell migration and invasion, respectively. The results indicated that TMEM40 expression levels were significantly increased in TSCC tissues compared with adjacent normal tongue tissues (P<0.01). Clinicopathological analysis revealed that TMEM40 expression was positively correlated with pathological TNM (pTNM) status (P<0.05), histological grade (P<0.001) and clinical stage (P<0.01), but not with sex or age. Results of cell proliferation, apoptosis, migration and invasion assays indicated that when TMEM40 had been successfully overexpressed or knocked down in CAL27 and SCC9 TSCC cell lines, cell growth and invasion increased in the TMEM40 overexpressing cells, while they decreased in TMEM40-knockdown cells. Furthermore, experiments revealed that TMEM40 knockdown resulted in increased levels of p53 and Bax, and decreased levels of MMP-9, which indicated that TMEM40 regulated cell apoptosis and migration via involvement of p53, Bax and MMP-9 in TSCC cells. Our findings indicated that increased expression of TMEM40 contributed to progressive features of TSCC via regulation of p53, Bax and MMP-9.

12 DOC1-dependent recruitment of NURD reveals antagonism with SWI/SNF during epithelial-mesenchymal transition in oral cancer cells

IntroductionThe molecular basis of the association between mutations in specific remodeler subunits and particular types of cancer is poorly understood. To understand how remodelers are targeted in a cell-type specific manner, we aim to explore the mechanisms of the Nucleosome Remodelling and Deacetylase (NURD) complex during cancer progression. Loss of the NURD subunit Deleted in Oral Cancer 1 (DOC1) is associated with human oral squamous cell carcinomas (OSCCs). Here, we show that restoration of DOC1 expression in OSCCs leads to a reversal of Epithelial-mesenchymal transition (EMT).Material and methodsWe examined DOC1 expression in normal and cancerous tongue tissue with immunohistochemistry. Besides, 4 different human OSCC cell lines were examined using immunofluorescence and immunoblotting. To study its role in these cells, we re-expressed DOC1 and utilised shRNA-mediated knockdowns (remodelers, EMT regulators). Subsequently we carried out proteomics, genomics, cell-based and biochemistry assays.Results and discussionsThe loss of DOC1 in oral cancer cells leads to a failure of NURD to bind and repress master transcriptional regulators of EMT. Re-expression of DOC1 in OSCC cells restores NURD recruitment to key target genes, a switch from open to closed chromatin, transcriptional repression, and reversal of EMT (MET). We speculate that, during the development of oral cancer, DOC1 is lost after the inactivation of p53 and INK4a. Our genome-wide binding site analysis showed that DOC1 is crucial for NURD recruitment to a subset of target loci; in particular, promoter regions harbouring CpG islands. DOC1-mediated NURD binding to the Twist1/2 and Zeb2 promoters leads to eviction of SWI/SNF involving nucleosome repositioning, epigenetic reprogramming, and shutdown of transcription. Binding of either SWI/SNF or NURD determines opposite epigenetic states, thereby committing OSCC cells to either EMT or MET.ConclusionRe-expression of DOC1 in OSCC cells restores the switch from open- to closed chromatin and reversal of EMT. We suggest that subunit-dependent gene selection is a major cause of the association between the loss of specific remodeler subunits and particular types of cancer. Our results emphasise that gene control involves a dynamic equilibrium between opposing chromatin modulating enzymes rather than a static chromatin state. Disturbances in this balance can initiate a cascade of chromatin reprogramming events that drives oncogenesis.

CD63 inhibits the cell migration and invasion ability of tongue squamous cell carcinoma.

The present study evaluated the expression and potential role of CD63 in the migration and invasion of tongue squamous cell carcinoma (TSCC) cells. Immunohistochemistry (IHC) was used to investigate the association between the expression level of CD63 protein and the histological differentiation of samples from 40 patients with TSCC and four normal tongue tissue specimens. RNA interference (RNAi) and gene transfection technology were used to alter the expression of CD63 in TCA8113 cells. The stable silencing and overexpression of CD63 in the TCA8113 cell line was used to assess the impact of the CD63 expression level on the migratory and invasive abilities of TCA8113 cells in a wound healing assay and a Transwell invasion assay. The effect of CD63 on the expression of matrix metalloproteinase (MMP)-2 and −9 were evaluated by western blot analysis. The results of IHC revealed a positive association between the CD63 expression level and the histopathological differentiation of TSCC and a negative association between the CD63 expression level and lymph node metastasis in TSCC. Western blotting revealed that the expressions of MMP-2 and MMP-9 were clearly upregulated in CD63-silenced TCA8113 cells but reduced in CD63-overexpressing TCA8113 cells, compared with the control. The wound-healing speed and the number of cells invading Matrigel-coated filters were negatively associated with the CD63 expression level. In summary, the results of the present study revealed that CD63 may be an inhibitor of TSCC malignancy and lymph node metastasis and may have applications in the prediction of prognosis and gene therapy for patients of TSCC.

High Fat Diet Increases HMGB1 Levels in 4NQO‐Induced Oral Cancer

IntroductionHMGB1 is a nuclear protein that binds to DNA and regulates gene transcription. HMGB1 can be released from the nucleus to function as a stress signal and/or a chemokine. HMGB1 overexpression has been reported in various cancers. The purpose of this study was to characterize the patterns of cytoplasmic HMGB1 expression in normal and cancerous mouse tongue tissues. Oral squamous cell carcinoma was induced by administration of 4‐nitroquinonline 1‐oxide (4NQO) in drinking water. 4NQO mimics the effects of tobacco and is a well‐established model of oral cancer in mice. In addition, we examined whether gender and high and low in saturated fat diets (HF and LF, respectively) could modify cytoplasmic levels of HMGB1. We hypothesized that higher levels of cytoplasmic HMGB1 will be present in tumor lesions and that gender and diet will affect the levels of expression.Materials and methodsMale and female C57Bl/6 mice (36 each gender), 5 weeks old, were divided into a low fat (10kcal% fat; LF) or HF (60 kcal% fat) diet. Within each dietary group, mice were randomly assigned to one of 3 water treatment groups for 17 weeks: water alone (control); propylene glycol in water (1.25%; PG‐H2O); or 4NQO in PG‐H2O (50 mg/ml; 4NQO). After 17 weeks, all mice were given water alone for 6 weeks prior to euthanasia. The presence of oral lesions, their size and location were monitored and recorded weekly. Upon sacrifice, tongues were harvested, formalin fixed, and processed for histological evaluation. Tongue tissue sections were stained with anti‐HMGB1 antibody (AB18256, Abcam Inc.) and examined microscopically. To evaluate localization and intensity of HMGB1 staining ImageJ program IHC profiler was used. The scale 0–3 was employed to measure the intensity of HMGB1 cytoplasmic staining.ResultsEpithelial cancer lesions were characterized by significantly higher levels of cytoplasmic HMGB1 than control epithelium (1.4–1.8 versus 0.8, p&lt;0.01). Expression of cytoplasmic HMGB1 was limited to cancer lesions since unaffected areas of tongue epithelium from 4NQO animals showed low levels of HMGB1 (0.74–1.0), similar to the control animals (0.8). HMGB1 levels were higher in cancer lesions from HF diet than from LF diet animals (1.8 in HF, 1.4 in LF, p=0.0003). In addition, cancer lesions in females (LF and HF diets combined) showed higher levels of HMGB1 than in males (1.7 versus 1.3, p=0.008).ConclusionCytoplasmic expression of HMGB1 is limited to oral squamous carcinoma cells. HMGB1 may contribute to the tumor progression. Higher levels of HMGB1 in tumors from HF diet animals provide more evidence that HF diet may exacerbate cancer development. HMGB1 could serve as a cancer biomarker and as a therapeutic target.Support or Funding InformationWe recognize the Midwestern University Deans of CDMI and the Basic Science Division for their support of this research. Student research was supported by the Office of Research and Sponsored Programs and the Biomedical Sciences Division.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An invitro study of the long non-coding RNA TUG1 in tongue squamous cell carcinoma.

This study sought to study the expression of the long non-coding RNA (lncRNA) taurine-upregulated gene 1 (TUG1) in tongue squamous cell carcinoma (TSCC) and reveal its possible function. qRT-PCR was used to evaluate 27 samples of fresh TSCC tissues and adjacent normal tongue tissues. siRNA technology was employed to downregulate TUG1 expression in CAL-27 and SCC-9 cell lines. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was utilized to assess cell proliferation ability; apoptosis and cell-cycle phases were analysed via flow cytometry. qRT-PCR findings indicated that the lncRNA TUG1 was upregulated in TSCC tissues compared with adjacent normal tongue tissues (P<.05). After TUG1 expression was downregulated using siRNA technology, cell proliferation was significantly inhibited (P<.05), and the number of cells in S phase was reduced (P<.05). The lncRNA TUG1 may represent a potential oncogene in TSCC.

Expression of von Hippel\u2013Lindau tumor suppressor protein (pVHL) characteristic of tongue cancer and proliferative lesions in tongue epithelium

BackgroundPatients with tongue cancer frequently show loss of heterozygosity (LOH) of the von Hippel–Lindau (VHL) tumor suppressor gene. However, expression of VHL protein (pVHL) in tongue cancer has rarely been investigated and remains largely unknown. We performed immunohistochemical staining of pVHL in tongue tissues and dysplasia, and examined the association with LOH and its clinical significance.MethodsImmunohistochemical staining of pVHL in formalin-fixed, paraffin-embedded sections of cancerous and other tissues from 19 tongue cancer patients showed positivity for LOH of VHL in four samples, negativity in four samples, and was non-informative in 11 samples. The staining pattern of pVHL was also compared with those of cytokeratin (CK) 13 and CK17.ResultsIn normal tongue tissues, pVHL staining was localized to the cytoplasm of cells in the basal layer and the area of the spinous layer adjacent to the basal layer of stratified squamous epithelium. Positive staining for pVHL was observed in the cytoplasm of cancer cells from all 19 tongue cancer patients. No differences as a result of the presence or absence of LOH were found. Notably, cytoplasm of poorly differentiated invasive cancer cells was less intensely stained than that of well and moderately differentiated invasive cancer cells. pVHL staining was also evident in epithelial dysplasia lesions with pVHL-positive cells expanding from the basal layer to the middle of the spinous layer. However, no CK13 staining was noted in regions of the epithelium, which were positive for pVHL. In contrast, regions with positive staining for CK17 closely coincided with those positive for pVHL.ConclusionsPositive staining for pVHL was observed in cancerous areas but not in normal tissues. pVHL expression was also detected in lesions of epithelial dysplasia. These findings suggest that pVHL may be a useful marker for proliferative lesions.