The upregulation of VGF enhances the progression of oral squamous carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a prevalent neoplasm worldwide, necessitating a deeper understanding of its pathogenesis. VGF nerve growth factor inducible (VGF), a neuropeptide, plays critical roles in nerve and endocrine cell regulation. METHODS: In this study, the TCGA datasets were initially screened, identifying the upregulation of VGF in various malignancies. We focused on OSCC cell lines, identifying the suppressor mRNA miR-432-5p as a negative regulator of VGF. Additionally, we examined the prognostic value of VGF expression in OSCC tumors and its impact on cellular functions. RESULTS: VGF expression was found to be an independent prognostic predictor in OSCC tumors. Cells expressing VGF exhibited increased oncogenicity, influencing the proliferation and migration of oral mucosal fibroblast. Transcriptome analysis revealed associations between VGF and various pathological processes, including malignancies, exosome release, fibrosis, cell cycle disruption, and tumor immune suppression. Moreover, IL23R expression, a favorable OSCC prognostic factor, was inversely correlated with VGF expression. Exogenous IL23R expression was found to suppress VGF-associated mobility phenotypes. CONCLUSIONS: This study highlights the multifaceted role of VGF in OSCC pathogenesis and introduces the miR-432-5p-VGF-IL23R regulatory axis as a critical mediator. The combined expression of VGF and IL23R emerges as a potent predictor of survival in oral carcinoma cases, suggesting potential implications for future therapeutic strategies.

Biodentine but not MTA induce DSPP expression of dental pulp cells with different severity of LPS-induced inflammation.

OBJECTIVES: To explore the inflammatory and differentiation response in inflamed dental pulp cells (DPCs) induced by lipopolysaccharide (LPS) under different conditions with Biodentine and mineral trioxide aggregate (MTA) treatment. MATERIALS AND METHODS: DPCs were treated with 0.001-1 µg/mL LPS for different periods to induce inflammation. Normal and inflamed DPCs were further treated with 0.14 mg/mL Biodentine or 0.13 mg/mL MTA for different periods. mRNA expression level of IL-6, IL-8 and ALP were analysed by qPCR. DSPP protein expression was detected by western blot. The data were analysed by the Mann-Whitney test, unpaired t test or two-way ANOVA. RESULTS: After treatment for different times and with different concentrations of LPS, different severity of pulp inflammation was revealed by the expressions of IL-6 and IL-8. Higher concentrations of LPS induced higher IL-6 and IL-8 expressions, and these expressions first increased and then decreased (p < 0.0001). At 96 and 192 h, Biodentine significantly suppressed IL-6 expression in both normal and inflamed DPCs (p < 0.05). At 48 and 96 h, Biodentine suppressed ALP expression in both normal and inflamed DPCs (p < 0.05). At 48 and 96 h, Biodentine induced DSPP expressions in both normal and inflamed DPCs (p < 0.05). CONCLUSION: Biodentine enhanced more DSPP differentiation of both normal and inflamed DPCs under different treatment durations than MTA. CLINICAL RELEVANCE: The prognosis of vital pulp therapy may depend on the severity of pulp inflammation which is difficult to be determined in clinical settings. Therefore, Biodentine may enhance odontogenic differentiation in different severity of pulp inflammation imply its clinical indications.

Identification of Somatic Mutations in Plasma Cell-Free DNA from Patients with Metastatic Oral Squamous Cell Carcinoma.

The accurate diagnosis and treatment of oral squamous cell carcinoma (OSCC) requires an understanding of its genomic alterations. Liquid biopsies, especially cell-free DNA (cfDNA) analysis, are a minimally invasive technique used for genomic profiling. We conducted comprehensive whole-exome sequencing (WES) of 50 paired OSCC cell-free plasma with whole blood samples using multiple mutation calling pipelines and filtering criteria. Integrative Genomics Viewer (IGV) was used to validate somatic mutations. Mutation burden and mutant genes were correlated to clinico-pathological parameters. The plasma mutation burden of cfDNA was significantly associated with clinical staging and distant metastasis status. The genes TTN, PLEC, SYNE1, and USH2A were most frequently mutated in OSCC, and known driver genes, including KMT2D, LRP1B, TRRAP, and FLNA, were also significantly and frequently mutated. Additionally, the novel mutated genes CCDC168, HMCN2, STARD9, and CRAMP1 were significantly and frequently present in patients with OSCC. The mutated genes most frequently found in patients with metastatic OSCC were RORC, SLC49A3, and NUMBL. Further analysis revealed that branched-chain amino acid (BCAA) catabolism, extracellular matrix-receptor interaction, and the hypoxia-related pathway were associated with OSCC prognosis. Choline metabolism in cancer, O-glycan biosynthesis, and protein processing in the endoplasmic reticulum pathway were associated with distant metastatic status. About 20% of tumors carried at least one aberrant event in BCAA catabolism signaling that could possibly be targeted by an approved therapeutic agent. We identified molecular-level OSCC that were correlated with etiology and prognosis while defining the landscape of major altered events of the OSCC plasma genome. These findings will be useful in the design of clinical trials for targeted therapies and the stratification of patients with OSCC according to therapeutic efficacy.

Cold Atmospheric Plasma Jet Irradiation Decreases the Survival and the Expression of Oncogenic miRNAs of Oral Carcinoma Cells.

Despite recent advancements, therapies against advanced oral squamous cell carcinoma (OSCC) remain ineffective, resulting in unsatisfactory therapeutic outcomes. Cold atmospheric plasma (CAP) offers a promising approach in the treatment of malignant neoplasms. Although the effects of CAP in abrogating OSCC have been explored, the exact mechanisms driving CAP-induced cancer cell death and the changes in microRNA (miRNA) expression are not fully understood. We fabricated and calibrated an argon-CAP device to explore the effects of CAP irradiation on the growth and expression of oncogenic miRNAs in OSCC. The analysis revealed that, in OSCC cell lines following CAP irradiation, there was a significant reduction in viability; a downregulation of miR-21, miR-31, miR-134, miR-146a, and miR-211 expression; and an inactivation of the v-akt murine thymoma viral oncogene homolog (AKT) and extracellular signal-regulated kinase (ERK) signals. Pretreatment with blockers of apoptosis, autophagy, and ferroptosis synergistically reduced CAP-induced cell death, indicating a combined induction of variable death pathways via CAP. Combined treatments using death inhibitors and miRNA mimics, alongside the activation of AKT and ERK following the exogenous expression, counteracted the cell mortality associated with CAP. The CAP-induced downregulation of miR-21, miR-31, miR-187, and miR-211 expression was rescued through survival signaling. Additionally, CAP irradiation notably inhibited the growth of SAS OSCC cell xenografts on nude mice. The reduced expression of oncogenic miRNAs in vivo aligned with in vitro findings. In conclusion, our study provides new lines of evidence demonstrating that CAP irradiation diminishes OSCC cell viability by abrogating survival signals and oncogenic miRNA expression.

The Concordant Disruption of B7/CD28 Immune Regulators Predicts the Prognosis of Oral Carcinomas.

Immune modulation is a critical factor in determining the survival of patients with malignancies, including those with oral squamous cell carcinoma (OSCC) and head and neck SCC (HNSCC). Immune escape or stimulation may be driven by the B7/CD28 family and other checkpoint molecules, forming ligand-receptor complexes with immune cells in the tumor microenvironment. Since the members of B7/CD28 can functionally compensate for or counteract each other, the concomitant disruption of multiple members of B7/CD28 in OSCC or HNSCC pathogenesis remains elusive. Transcriptome analysis was performed on 54 OSCC tumors and 28 paired normal oral tissue samples. Upregulation of CD80, CD86, PD-L1, PD-L2, CD276, VTCN1, and CTLA4 and downregulation of L-ICOS in OSCC relative to the control were noted. Concordance in the expression of CD80, CD86, PD-L1, PD-L2, and L-ICOS with CD28 members was observed across tumors. Lower ICOS expression indicated a worse prognosis in late-stage tumors. Moreover, tumors harboring higher PD-L1/ICOS, PD-L2/ICOS, or CD276/ICOS expression ratios had a worse prognosis. The survival of node-positive patients was further worsened in tumors exhibiting higher ratios between PD-L1, PD-L2, or CD276 and ICOS. Alterations in T cell, macrophage, myeloid dendritic cell, and mast cell populations in tumors relative to controls were found. Decreased memory B cells, CD8+ T cells, and Tregs, together with increased resting NK cells and M0 macrophages, occurred in tumors with a worse prognosis. This study confirmed frequent upregulation and eminent co-disruption of B7/CD28 members in OSCC tumors. The ratio between PD-L2 and ICOS is a promising survival predictor in node-positive HNSCC patients.

Dental pulp cells cocultured with macrophages aggravate the inflammatory conditions stimulated by LPS.

BACKGROUND: Pulp inflammation is complex interactions between different types of cells and cytokines. To mimic the interactions of different types of cells in inflamed dental pulp tissues, dental pulp cells (DPCs) were cocultured with different ratios of macrophages (THP-1) or LPS treatment. METHODS: DPCs were cocultured with various ratios of THP-1, then photographed cell morphology and determined cell viability by MTT assay at preset times. Total RNA was also extracted to measure the inflammation marker-IL-6 and IL-8 expressions by RT-Q-PCR. The DPCs and THP-1 were treated with 0.01 - 1µg/ml lipopolysaccharide (LPS) and extract RNA at preset times, and detected IL-6 and IL-8 expression. DPCs were cocultured with various ratios of THP-1 with 0.1 µg/mL LPS, and detected IL-6 and IL-8 expression after 24 and 48 h. The data were analyzed by unpaired t-test or Mann-Whitney test. Differences were considered statistically significant when p < 0.05. RESULTS: THP-1 and DPCs coculture models did not suppress the viability of DPCs and THP-1. Cocultured with various ratios of THP-1 could increase IL-6 and IL-8 expressions of DPCs (p = 0.0056 - p < 0.0001). The expressions of IL-6 and IL-8 were stronger in higher ratio groups (p = 0.0062 - p < 0.0001). LPS treatment also induced IL-6 and IL-8 expressions of DPCs and THP-1 (p = 0.0179 - p < 0.0001 and p = 0.0189 - p < 0.0001, separately). Under the presence of 0.1 µg/mL LPS, DPCs cocultured with THP-1 for 24 h also enhanced IL-6 and IL-8 expression (p = 0.0022). After cocultured with a higher ratio of THP-1 for 48 h, IL-6 and IL-8 expressions were even stronger in the presence of LPS (p = 0.0260). CONCLUSIONS: Coculturing dental pulp cells and macrophages under LPS treatment aggravate the inflammatory process. The responses of our models were more severe than traditional inflamed dental models and better represented what happened in the real dental pulp. Utilizing our models to explore the repair and regeneration in endodontics will be future goals.

The upregulation of oncogenic miRNAs in swabbed samples obtained from oral premalignant and malignant lesions.

OBJECTIVES: Oncogenic miRNAs upregulated in OSCC play a range of versatile roles in oral carcinogenesis. Oral potentially malignant disorders (OPMDs) are the antecedent lesions to oral squamous carcinoma (OSCC) and they require a definitive diagnosis and early intervention. This study hypothesizes the presence of aberrant oncogenic miRNA expression in swabbed oral lesions. MATERIALS AND METHODS: The expression of miR-21, miR-31, miR-134, miR-146a, and miR-211 in swabbed samples from 36 dysplastic or hyperplastic OPMDs and 10 OSCCs, relative to respective normal mucosa within the same patient, is analyzed with qRT-PCR to develop a diagnosis. RESULTS: Upregulation of all tested miRNAs in OPMD and OSCC samples comparing to controls is found to have occurred. Receiver operating characteristics curve analysis shows that miR-31 gives the best diagnostic accuracy of 0.91 when differentiating OPMD/OSCC from controls. An analysis of miR-134 and miR-211 expression allows the discrimination of the dysplastic state associated with OPMD, while the use of expression of the combined miRNAs further improves the analytical performances when identifying the dysplastic state. The concordant upregulation of miR-21, miR-31, and miR-146a is found to occur during an early stage of OSCC carcinogenesis. CONCLUSION: This study demonstrates the upregulation of multiple oncogenic miRNAs in swabbed OPMD and OSCC samples. miRNA expression in swabbed collectives enables the differentiation between normal mucosa and OPMD/OSCC, independent of their histopathological severity. CLINICAL RELEVANCE: This conventional and convenient sampling tool, when coupled with an assessment of miR-31 expression, would seem to be an adjuvant approach to the diagnosis of OPMD and OSCC.

miR-31-NUMB Cascade Modulates Monocarboxylate Transporters to Increase Oncogenicity and Lactate Production of Oral Carcinoma Cells.

Oral squamous cell carcinoma (OSCC) is among the leading causes of cancer-associated death worldwide. miR-31 is an oncogenic miRNA in OSCC. NUMB is an adaptor protein capable of suppressing malignant transformation. Disruption of the miR-31-NUMB regulatory axis has been demonstrated in malignancies. Mitochondrial dysfunction and adaptation to glycolytic respiration are frequent events in malignancies. Monocarboxylate transporters (MCTs) function to facilitate lactate flux in highly glycolytic cells. Upregulation of MCT1 and MCT4 has been shown to be a prognostic factor of OSCC. Here, we reported that miR-31-NUMB can modulate glycolysis in OSCC. Using the CRISPR/Cas9 gene editing strategy, we identified increases in oncogenic phenotypes, MCT1 and MCT4 expression, lactate production, and glycolytic respiration in NUMB-deleted OSCC subclones. Transfection of the Numb1 or Numb4 isoform reversed the oncogenic induction elicited by NUMB deletion. This study also showed, for the first time, that NUMB4 binds MCT1 and MCT4 and that this binding increases their ubiquitination, which may decrease their abundance in cell lysates. The disruptions in oncogenicity and metabolism associated with miR-31 deletion and NUMB deletion were partially rescued by MCT1/MCT4 expression or knockdown. This study demonstrated that NUMB is a novel binding partner of MCT1 and MCT4 and that the miR-31-NUMB-MCT1/MCT4 regulatory cascade is present in oral carcinoma.

LncRNA MIR31HG Drives Oncogenicity by Inhibiting the Limb-Bud and Heart Development Gene (LBH) during Oral Carcinoma.

The miR-31 host gene (MIR31HG) encodes a long non-coding RNA (LncRNA) that harbors miR-31 in its intron 2; miR-31 promotes malignant neoplastic progression. Overexpression of MIR31HG and of miR-31 occurs during oral squamous cell carcinoma (OSCC). However, the downstream effectors modulated by MIR31HG during OSCC pathogenesis remain unclear. The present study identifies up-regulation of MIR31HG expression during the potentially premalignant disorder stage of oral carcinogenesis. The potential of MIR31HG to enhance oncogenicity and to activate Wnt and FAK was identified when there was exogenous MIR31HG expression in OSCC cells. Furthermore, OSCC cell subclones with MIR31HG deleted were established using a Crispr/Cas9 strategy. RNA sequencing data obtained from cells expressing MIR31HG, cells with MIR31HG deleted and cells with miR-31 deleted identified 17 candidate genes that seem to be modulated by MIR31HG in OSCC cells. A TCGA database algorithm pinpointed MMP1, BMP2 and Limb-Bud and Heart development (LBH) as effector genes controlled by MIR31HG during OSCC. Exogenous LBH expression decreases tumor cell invasiveness, while knockdown of LBH reverses the oncogenic suppression present in MIR31HG deletion subclones. The study provides novel insights demonstrating the contribution of the MIR31HG-LBH cascade to oral carcinogenesis.

Activation of the miR-371/372/373 miRNA Cluster Enhances Oncogenicity and Drug Resistance in Oral Carcinoma Cells.

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

Establishment of a p53 Null Murine Oral Carcinoma Cell Line and the Identification of Genetic Alterations Associated with This Carcinoma.

Head and neck squamous cell carcinoma (HNSCC), including oral squamous cell carcinoma (OSCC), ranks sixth in cancer incidence worldwide. To generate OSCC cells lines from human or murine tumors, greatly facilitates investigations into OSCC. This study describes the establishing of a mouse palatal carcinoma cell line (designated MPC-1) from a spontaneous tumor present in a heterozygous p53 gene loss C57BL/6 mouse. A MPC-1-GFP cell subclone was then generated by lentivirus infection resulting in stable expression of green fluorescent protein. Assays indicated that MPC-1 was a p53 null polygonal cell that was positive for keratinocyte markers; it also expressed vimentin and showed a loss of E-cadherin expression. Despite that MPC-1 having strong proliferation and colony formation capabilities, the potential for anchorage independent growth and tumorigenesis was almost absent. Like other murine MOC-L and MTCQ cell line series we have previously established, MPC-1 also expresses a range of stemness markers, various oncogenic proteins, and a number of immune checkpoint proteins at high levels. However, the synergistic effects of the CDK4/6 inhibitor palbociclib on other therapeutic drugs were not observed with MPC-1. Whole exon sequencing revealed that there were high rates of non-synonymous mutations in MPC-1 affecting various genes, including Akap9, Arap2, Cdh11, Hjurp, Mroh2a, Muc4, Muc6, Sp110, and Sp140, which are similar to that the mutations present in a panel of chemical carcinogenesis-related murine tongue carcinoma cell lines. Analysis has highlighted the dis-regulation of Akap9, Cdh11, Muc4, Sp110, and Sp140 in human HNSCC as indicated by the TCGA and GEO OSCC databases. Sp140 expression has also been associated with patient survival. This study describes the establishment and characterization of the MPC-1 cell line and this new cell model should help to advance genetic research into oral cancer.

Overexpression of Platelet-Derived Growth Factor and Its Receptor Are Correlated with Oral Tumorigenesis and Poor Prognosis in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is a cancerous disease with poor prognosis. According to the statistics, the 5-year survival rate has not improved significantly over the past 20 years. The platelet-derived growth factor (PDGF) and its signaling pathway is a key regulator of angiogenesis and tumorigenesis. High level of PDGF and its receptor (PDGFR) have been reported in several types of malignancies. In this study, we investigated the relationship of the molecular expression levels of PDGF and PDGFR with clinicopathological parameters in OSCC. To this end, we measured the mRNA and protein levels of PDGF and PDGFR by real-time quantitative PCR (qRT-PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA), respectively. We found positive correlations of the mRNA levels of PDGFA, PDGFB, and PDGFRB with lymph node metastasis and poor overall survival (OS). High expression of PDGF, PDGFRA, and PDGFRB were remarkably associated with lymph node metastasis and poor OS, as determined by immunohistochemistry. Preoperative serum levels of PDGF-AA and PDGF-BB had a positive correlation with preoperative platelet count. Elevated serum levels of PDGF-AA. PDGF-BB, and platelet count correlated with lymph node metastasis and an unfavorable outcome. In multivariate Cox regression analysis, PDGFA mRNA, PDGFB mRNA, PDGFRB mRNA, PDGF immunoexpression, PDGFRB immunoexpression, serum PDGF-AA, serum PDGF-BB, and platelet count emerged as significant independent prognostic factors for OS. In vitro, we found that elevated PDGF promotes colony formation, migration, and invasiveness of SAS and OECM-1 cancer cell lines. Our results suggest that the expression level of serum PDGF has the potential to become a useful diagnostic marker for the prognosis of OSCC. In addition, PDGFR should be considered as a potential therapeutic target for OSCC. Furthermore, research should be undertaken to elucidate the role of PDGF and PDGFR regarding the behavior of tumor cells in OSCC.

Eicosanoids and HB-EGF/EGFR in cancer.

Eicosanoids are bioactive lipids that play crucial roles in various pathophysiological conditions, including inflammation and cancer. They include both the COX-derived prostaglandins and the LOX-derived leukotrienes. Furthermore, the epidermal growth factor receptor (EGFR) pathways family of receptor tyrosine kinases also are known to play a central role in the tumorigenesis. Various antitumor modalities have been approved cancer treatments that target therapeutically the COX-2 and EGFR pathways; these include selective COX-2 inhibitors and EGFR monoclonal antibodies. Research has shown that the COX-2 and epidermal growth factor receptor pathways actively interact with each other in order to orchestrate carcinogenesis. This has been used to justify a targeted combinatorial approach aimed at these two pathways. Although combined therapies have been found to have a greater antitumor effect than the administration of single agent, this does not exempt them from the possible fatal cardiac effects that are associated with COX-2 inhibition. In this review, we delineate the contribution of HB-EGF, an important EGFR ligand, to the cardiac dysfunction related to decreased shedding of HB-EGF after COX-2/PGE2 inhibition. A better understanding of the molecular mechanisms underlying these cardiac side effects will make possible more effective regimens that use the dual-targeting approach.

Establishing of mouse oral carcinoma cell lines derived from transgenic mice and their use as syngeneic tumorigenesis models.

BACKGROUND: The survival of OSCC patient needs to be further improved. miR-211 is oncogenic in OSCC and its upregulation is associated with tumor progression and poor patient survival. K14-EGFP-miR-211 transgenic mice also exhibit augmented potential for OSCC induction. METHODS: Four murine OSCC cell lines, designated MOC-L1 to MOC-L4, are established from tongue tumors induced by 4-nitroquinoline 1-oxide using the K14-EGFP-miR-211 transgenic mouse model. The genetic disruption, in vitro oncogenicity, and the eligibilities of tumorigenesis and metastasis of the cell lines are analyzed. RESULTS: All cell lines show green fluorescence and express a range of epithelial markers. The MOC-L1, MOC-L2 and MOC-L3 cells carry missense mutations in the DNA binding domain of the p53 gene. MOC-L1 exhibits a high level of epithelial-mesenchymal transition and has the aggressive characteristics associated with this. MOC-L1 and MOC-L2 are clonogenic in vitro as well as being tumorigenic when implanted into the dermis or tongue of syngeneic recipients. Nonetheless, only MOC-L1 exhibits immense potential for local regional and distal metastasis. Since the expression of miR-196b in MOC-L1 xenografts is drastically decreased on cisplatin treatment, it would seem that targeting of miR-196b might facilitate tumor abrogation. CONCLUSIONS: As cell lines established in this study originated from the C57BL/6 mouse, the strain most suitable for transgenic engineering, exploring the interplay of these OSCC cells with other genetically modified cells in immune-competent mice would provide important insights into OSCC pathogenesis.

Targeting Cellular Metabolism Modulates Head and Neck Oncogenesis.

Considering the great energy and biomass demand for cell survival, cancer cells exhibit unique metabolic signatures compared to normal cells. Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent neoplasms worldwide. Recent findings have shown that environmental challenges, as well as intrinsic metabolic manipulations, could modulate HNSCC experimentally and serve as clinic prognostic indicators, suggesting that a better understanding of dynamic metabolic changes during HNSCC development could be of great benefit for developing adjuvant anti-cancer schemes other than conventional therapies. However, the following questions are still poorly understood: (i) how does metabolic reprogramming occur during HNSCC development? (ii) how does the tumorous milieu contribute to HNSCC tumourigenesis? and (iii) at the molecular level, how do various metabolic cues interact with each other to control the oncogenicity and therapeutic sensitivity of HNSCC? In this review article, the regulatory roles of different metabolic pathways in HNSCC and its microenvironment in controlling the malignancy are therefore discussed in the hope of providing a systemic overview regarding what we knew and how cancer metabolism could be translated for the development of anti-cancer therapeutic reagents.

FAT1 somatic mutations in head and neck carcinoma are associated with tumor progression and survival.

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

miR-134 targets PDCD7 to reduce E-cadherin expression and enhance oral cancer progression.

Oral squamous cell carcinoma (OSCC) is a common malignancy worldwide. This study clarified the oncogenic role of miR-134 in OSCC. Reporter assays, using both wild-type and mutant constructs, confirmed that Programmed Cell Death 7 (PDCD7) gene was a potential target of miR-134. The OSCC cells exogenously expressed miR-134 exhibited reduced PDCD7 expression. As expected, exogenous miRZip-134 expression increased PDCD7 expression in the OSCC cells; additionally, PDCD7 expression suppressed the oncogenicity of the OSCC cells. By contrast, PDCD7 knockout through gene editing increased in vitro oncogenicity and neck nodal metastasis in mice, and reduced E-cadherin (E-cad) expression. PDCD7 transactivated E-cad expression via the GC-box in the promoter. Moreover, miR-134-associated cellular transformation and E-cad downregulation was attenuated by PDCD7. Downregulation of both PDCD7 and E-cad and high levels miR-134 expression was observed in OSCC tumor tissues. Activation of the miR-134-PDCD7-E-cad pathogenesis cascade occurred early during the human and murine oral carcinogenesis process. In conclusion, the oncogenic effect of miR-134 in oral carcinoma is mediated by reducing PDCD7 and E-cad expression.

Portland cement induces human periodontal ligament cells to differentiate by upregulating miR-146a.

BACKGROUND/PURPOSE: Bioaggregates such as Portland cement (PC) can be an economical alternative for mineral trioxide aggregate (MTA) with additional benefit of less discoloration. MTA has been known to induce differentiations of several dental cells. MicroRNAs are important regulators of biological processes, including differentiation, physiologic homeostasis, and disease progression. This study is to explore how PC enhances the differentiation of periodontal ligament (PDL) cells in microRNAs level. METHODS: PDL cells were cultured in a regular PC- or MTA-conditioned medium or an osteoinduction medium (OIM). Alizarin red staining was used to evaluate the extent of mineralization. Transfection of microRNA mimics induced exogenous miR-31 and miR-146a expression. The expression of microRNAs and differentiation markers was assayed using reverse-transcriptase polymerase chain reaction. RESULTS: PC enhanced the mineralization of PDL cells in a dose-dependent manner in the OIM. Exogenous miR-31 and miR-146a expression upregulated alkaline phosphatase (ALP), bone morphogenic protein (BMP), and dentin matrix protein 1 (DMP1) expression. However, miR-31 and miR-146a modulates cementum protein 1 (CEMP1) expression in different ways. PC also enhanced ALP and BMP but attenuated CEMP1 in the OIM. Although the OIM or PC treatment upregulated miR-21, miR-29b, and miR-146a, only miR-146a was able to be induced by PC in combination with OIM. CONCLUSION: This study demonstrated that PC enhances the differentiation of PDL cells, especially osteogenic through miR-146a upregulation. In order to control the ankylosis after regenerative endodontics with the usage of bioaggregates, further investigations to explore these differentiation mechanisms in the miRNA level may be needed.

Increased Plasma Circulating Cell-Free DNA Could Be a Potential Marker for Oral Cancer.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a disease that affects patients worldwide. DNA of dead cells is released into the blood stream and may be isolated from plasma or serum samples. This DNA is termed cell-free DNA (cfDNA). cfDNA is increased in several types of malignancies. We investigated if there was a correlation between cfDNA levels and the progression of OSCC. METHODS: Using quantitative spectrometry, we measured plasma cfDNA in 121 patients with OSCC and 50 matched controls. Mann Whitney and Wilcoxon tests were used to compare differences among various clinical variants. Receiver operating characteristic (ROC) analysis was used to obtain levels suitable for the separation of the clinical subsets. Kaplan-Meier analysis was used to assess correlation with survival. RESULTS: Plasma cfDNA was significantly elevated in patients with OSCC relative to controls. Plasma cfDNA levels correlated with larger tumor size, cervical lymph node metastasis and late stage. Higher plasma cfDNA levels were associated with a poor prognosis of OSCC, which is a new finding. CONCLUSION: Plasma cfDNA could serve as a novel and easily accessible biomarker in OSCC, providing diagnostic and prognostic value.

K14-EGFP-miR-31 transgenic mice have high susceptibility to chemical-induced squamous cell tumorigenesis that is associating with Ku80 repression.

Squamous cell carcinoma (SCC) occurring in the head and neck region and the esophagus causes tremendous cancer mortality around the world. miR-31 is among the most eminently upregulated MicroRNAs in SCC, when it occurs in the head and neck region and the esophagus. We established miR-31 transgenic mouse lines, in which miR-31 is under the control of the K14 promoter. 4-nitroquinoline 1-oxide (4NQO) is a mutagen that causes double strand breaks. The transgenic mice exhibited a higher potential for tumor induction than wild-type (Wt) mice of the tongue and esophagus after 4NQO treatment. After 4NQO treatment or irradiation, p-γH2AX expression in squamous epithelium of transgenic mice was increased more than in Wt mice. Exogenous expression of miR-31 was also found to be associated with the higher p-γH2AX expression induced by 4NQO in human oral SCC (OSCC) cell lines. The repair genes PARP1 and Ku80 were validated as new targets of miR-31 in human OSCC cell lines, and were found to be downregulated in the squamous epithelium of the tongue in transgenic mice. However, only the downregulation of Ku80 was essential for maintaining the high level of p-γH2AX induced by 4NQO in OSCC cells. Inverse expression profiles for miR-31 and Ku80 were noted in human OSCC tissue. Our study identifies the high sensitivity of K14-EGFP-miR-31 transgenic mice to chemical carcinogen-induced squamous cell tumorigenesis and shows that this seems to be associated with the downregulation of Ku80 and an impairment of repair activity in squamous cells, which are mediated by miR-31.