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Atherosclerosis is a major cause of mortality worldwide. The initial change in atherosclerosis is intimal thickening due to muscle cell proliferation and migration. A correlation has been observed between periodontal disease and atherosclerosis. Here, we investigated the proliferation and migration of human aortic smooth muscle cells (HASMCs) using Porphyromonas gingivalis-derived LPS (Pg-LPS). To elucidate intracellular signaling, toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) of HASMCs were knocked down, and the role of these molecules in Pg-LPS-stimulated proliferation and migration was examined. The role of mitogen-activated protein kinase (MAPK) in HASMC proliferation and migration was further elucidated by MAPK inhibition. Pg-LPS stimulation increased the proliferation and migration of HASMCs and activated the TLR4/MyD88 pathway. TLR4 knockdown inhibited Pg-LPS stimulated HASMCs proliferation and migration. Pg-LPS stimulation led to the phosphorylation of P38 MAPK, JNK, and ERK, and MyD88 knockdown inhibited the phosphorylation of P38 MAPK and JNK but not ERK. P38 MAPK and SAPK/JNK inhibition did not suppress the proliferation of HASMCs upon Pg-LPS stimulation, but ERK inhibition significantly inhibited proliferation. SAPK/JNK and ERK inhibition suppressed Pg-LPS-stimulated migration of HASMCs. In conclusion, our findings suggest that Pg-LPS may promote atherosclerosis via the activation of MAPK through TLR4.

Immunotherapy with immune‐checkpoint therapy has recently been used to treat oral squamous cell carcinomas (OSCCs). However, improvements in current immunotherapy are expected because response rates are limited. Transforming growth factor‐β (TGF‐β) creates an immunosuppressive tumor microenvironment (TME) by inducing the production of regulatory T‐cells (Tregs) and cancer‐associated fibroblasts and inhibiting the function of cytotoxic T‐lymphocytes (CTLs) and natural killer cells. TGF‐β may be an important target in the development of novel cancer immunotherapies. In this study, we investigated the suppressive effect of TGF‐β on CTL function in vitro using OSCC cell lines and their specific CTLs. Moreover, TGFB1 mRNA expression and T‐cell infiltration in 25 OSCC tissues were examined by in situ hybridization and multifluorescence immunohistochemistry. We found that TGF‐β suppressed the function of antigen‐specific CTLs in the priming and effector phases in vitro. Additionally, TGF‐β inhibitor effectively restored the CTL function, and TGFB1 mRNA was primarily expressed in the tumor invasive front. Interestingly, we found a significant negative correlation between TGFB1 mRNA expression and the CD8+ T‐cell/Treg ratio and between TGFB1 mRNA expression and the Ki‐67 expression in CD8+ T‐cells, indicating that TGF‐β also suppressed the function of CTLs in situ. Our findings suggest that the regulation of TGF‐β function restores the immunosuppressive TME to active status and is important for developing new immunotherapeutic strategies, such as a combination of immune‐checkpoint inhibitors and TGF‐β inhibitors, for OSCCs. We found that, TGF‐β suppressed the function of antigen‐specific CTLs in the priming and effector phases in vitro. And, we found a significant negative correlation between TGFB1 mRNA expression and the CD8+ T‐cell/Treg ratio. Inhibition of TGF‐β restores the immunosuppressive TME to active status by a mechanism different from that of immune‐checkpoint inhibitors, suggesting that the combination of both may lead to a new therapeutic strategy for OSCCs.

Background/Objectives: Oral squamous cell carcinoma (OSCC) often presents at an advanced stage; therefore, the early detection of precursor lesions is crucial. However, the risk assessment of precursor lesions such as oral epithelial dysplasia (OED) remains challenging because of the subjectivity of histopathological grading. We aimed to identify molecular markers that enhance the diagnostic accuracy and prognostic stratification of OSCC and explore the differences in the molecular characterization of OED and OSCC using a few selected markers. Methods: A two-step diagnostic workflow was applied: (1) FISH evaluation of EGFR amplification and CDKN2A deletion to distinguish OED from OSCC and identify EGFR-dependent tumors, and (2) HRAS immunohistochemistry performed exclusively in EGFR-negative OSCCs to stratify EGFR-independent cases. Fluorescence in situ hybridization (FISH) was used to assess seven EGFR/cell cycle-related genes (CCND1, CDKN2A, EGFR, PIK3CA, PTEN, TP53, and 1p36 locus) in 117 formalin-fixed paraffin-embedded samples (66 OED and 51 OSCC) and 10 normal mucosa samples. HRAS expression was evaluated using immunohistochemistry (IHC) in 36 EGFR amplification-negative OSCCs samples. Results:EGFR amplification was frequent in OSCC, whereas CDKN2A deletion was common in OED. The EGFR-amplified/ CDKN2A-intact profile showed high specificity for OSCC and improved diagnostic performance (area under the curve = 0.77) when combined with the Ki-67 labeling index. It also predicted poor disease-free survival (hazard ratio [HR] = 5.08, p = 0.016) and overall survival (HR = 6.10, p = 0.047). Among EGFR-negative OSCCs, HRAS overexpression was associated with advanced-stage disease and a poor prognosis (HR = 6.15, p = 0.043). Conclusions:EGFR amplification was frequent in OSCC, and CDKN2A deletion was prevalent in OED, supporting their use as molecular markers for differential diagnoses. FISH for EGFR/CDKN2A and HRAS IHC can stratify OSCC by diagnosis and prognosis, enabling practical molecular subclassification, including EGFR-negative cases.

CRTC1–MAML2 and CRTC3–MAML2 fusions have been associated with favorable clinicopathological features of mucoepidermoid carcinomas. However, the significance of the MAML2 gene split has not been fully clarified. In the present study, 95 mucoepidermoid carcinomas (paraffin‐embedded materials) were analyzed for CRTC1–MAML2 and CRTC3–MAML2 fusions by RT‐PCR and for the MAML2 gene split by FISH. Quantitative RT‐PCR for the CRTC1–MAML2 transcript was performed in selected cases. MLL gene involvement, which has been reported in some leukemia cases, was examined by FISH in fusion partner‐unknown cases. CRTC1–MAML2 and CRTC3–MAML2 fusions were detected in 37 and 6 cases, respectively. The MAML2 gene split was detected in 62 cases, which included all CRTC1/3–MAML2 fusion‐positive cases. The level of CRTC1–MAML2 transcript expression was highly variable, and its clinicopathological impact was unclear. The MLL gene split was not detected. Mucoepidermoid carcinomas negative for CRTC1/3–MAML2 and positive for the MAML2 gene split (n = 19) showed favorable clinicopathological tumor features similar to those positive for CRTC1/3–MAML2 fusions. Compared with negative cases (n = 33), mucoepidermoid carcinomas positive for the MAML2 split (n = 62) were associated with lower patient age, a mild female predilection, a smaller tumor size, less frequent nodal metastasis, a lower clinical stage, a lower histological grade, and longer overall and disease‐free survival. The MAML2 gene split emerged as an independent prognostic factor for both overall and disease‐free survival in multivariate prognostic analysis. The presence of the MAML2 gene split defines a distinct mucoepidermoid carcinoma subset that is associated clinicopathologically with favorable tumor features. (Cancer Sci 2013; 104: 85–92)

Mucoepidermoid carcinoma is the most common primary malignancy of the salivary gland. We and others showed that CRTC1–MAML2 gene fusion was associated with favorable clinicopathological tumor features. Recently, a novel gene fusion, CRTC3–MAML2 , was reported as a rare gene alteration in a case of mucoepidermoid carcinoma. However, its frequency and clinicopathological significance remains unclear. In all, 101 cases of mucoepidermoid carcinoma and 89 cases of non-mucoepidermoid carcinoma of the salivary gland were analyzed, and RNA was extracted from formalin-fixed, paraffin-embedded specimens. In the CRTC family, there have been three genes, CRTC1 , CRTC2 , and CRTC3 . We developed reverse transcription-polymerase chain reaction (RT-PCR) assays for CRTC1–MAML2 , CRTC2–MAML2 , and CRTC3–MAML2 fusions. Clinicopathological data of the patients were obtained from their clinical records. Of 101 cases of mucoepidermoid carcinoma, 34 (34%) and 6 (6%) were positive for CRTC1–MAML2 and CRTC3–MAML2 fusion transcripts. However, in the 89 cases of non-mucoepidermoid carcinoma, neither transcript was noted. In the former cases, CRTC1–MAML2 and CRTC3–MAML2 fusions were mutually exclusive. The other fusion, CRTC2–MAML2 , was not detected. We confirmed that the clinicopathological features of CRTC1–MAML2 -positive mucoepidermoid carcinomas indicated an indolent course. CRTC3–MAML2 -positive mucoepidermoid carcinomas also had clinicopathologically favorable features; all cases showed a less advanced clinical stage, negative nodal metastasis, no high-grade tumor histology, and no recurrence or tumor-related death after surgical resection of the tumor. It is interesting to note that patients with CRTC3–MAML2 -positive tumors (mean 36 years of age) were significantly younger that those with the CRTC1–MAML2 fusion (55 years) and those with fusion-negative tumors (58 years). In conclusion, CRTC3–MAML2 fusion, which is mutually exclusive with CRTC1–MAML2 fusion and specific to mucoepidermoid carcinoma, may be detected more frequently than previously expected. Mucoepidermoid carcinomas possessing CRTC3–MAML2 fusion may be associated with favorable clinicopathological features and patients may be younger than those with CRTC1–MAML2 fusion or those with no detectable gene fusion.

Recently, the possibility that oral microbiomes is associated with oral squamous cell carcinoma (OSCC) initiation and progression has attracted attention; however, this association is still unclear. Here, we comprehensively analyze the microbiome profiles of saliva samples using next-generation sequencing followed by determining the association between oral microbiome profiles and OSCC. Microbiome profiles in saliva samples from patients with OSCC, oral leukoplakia (OLK), and postoperative OSCC (Post) were analyzed. Candidate OSCC-associated bacteria were identified by comparing the bacterial diversity and relative abundance of each group based on these microbiome profiles, and their applicability as OSCC detection tools were evaluated. There were significant differences in genus abundances (Streptococcus, Aggregatibacter, and Alloprevotella) among the groups from saliva samples. In the OSCC group, compared with the OLK and Post groups, abundances of the genus Fusobacterium, phylum Fusobacteria and phylum Bacteroidetes were markedly increased and that of the genus Streptococcus and phylum Firmicutes were decreased. The results suggested a strong association of these bacteria with OSCC. Especially, phylum Fusobacterium was significantly associated with early recurrence of OSCC. Thus, oral microbiome analysis may have a potential of novel OSCC detection and prognostic tool.

The expression of programmed death ligand-1 (PD-L1) is controlled by complex mechanisms. The elucidation of the molecular mechanisms of PD-L1 expression is important for the exploration of new insights into PD-1 blockade therapy. Detailed mechanisms of the in situ expression of PD-L1 in tissues of oral squamous cell carcinomas (OSCCs) have not yet been clarified. We examined the mechanisms of PD-L1 expression focusing on the phosphorylation of downstream molecules of epidermal growth factor (EGF) and interferon gamma (IFN-γ) signaling in vitro and in vivo by immunoblotting and multi-fluorescence immunohistochemistry (MF-IHC), respectively. The in vitro experiments demonstrated that PD-L1 expression in OSCC cell lines is upregulated by EGF via the EGF receptor (EGFR)/PI3K/AKT pathway, the EGFR/STAT1 pathway, and the EGFR/MEK/ERK pathway, and by IFN-γ via the JAK2/STAT1 pathway. MF-IHC demonstrated that STAT1 and EGFR phosphorylation was frequently shown in PD-L1-positive cases and STAT1 phosphorylation was correlated with lymphocyte infiltration and EGFR phosphorylation. Moreover, the phosphorylation pattern of the related molecules in PD-L1-positive cells differed among the cases investigated. These findings indicate that PD-L1 expression mechanisms differ depending on the tissue environment and suggest that the examination of the tissue environment and molecular alterations of cancer cells affecting PD-L1 expression make it necessary for each patient to choose the appropriate combination drugs for PD-1 blockade cancer treatment.

Oral cancer may become advanced because of delay in diagnosis. In order to promote oral cancer screening, simple and highly reliable screening methods that can be implemented at general dental clinics are required. Herein we investigated differential salivary gene expression between oral squamous cell carcinoma (OSCC) patients and healthy volunteers (HV) to identify new biomarkers for OSCC detection. Candidate genes were selected by microarrays, nuclear undecaprenyl pyrophosphate synthase 1 (NUS1) and reticulocalbin 1 (RCN1) were selected for further investigation. We used real-time quantitative reverse transcription PCR (qRT-PCR) to determine NUS1 and RCN1 expression levels in saliva and tissues. qRT-PCR analysis of clinical samples revealed that OSCC patients had significantly higher expression of salivary NUS1 and RCN1 than HV. A combination of NUS1 and RCN1 accurately distinguished patients from controls, and this combination can be implemented as a screening test for OSCC.

Heated tobacco products (HTPs) appear to be less harmful to health than conventional cigarettes (CCs). However, limited analytical data are available to support this claim. This study aimed to compare the cytotoxic, genotoxic, and toxicogenomic effects of HTPs and CCs in carcinogenesis via multistep gene mutations in the oral mucosal cells.INTRODUCTIONHeated tobacco products (HTPs) appear to be less harmful to health than conventional cigarettes (CCs). However, limited analytical data are available to support this claim. This study aimed to compare the cytotoxic, genotoxic, and toxicogenomic effects of HTPs and CCs in carcinogenesis via multistep gene mutations in the oral mucosal cells.Cigarette smoke extract (CSE) was obtained from HTPs and CCs. Primary human oral keratinocytes (HOKs) were treated with 5% and 20% CSE from HTPs and CCs. Cell survival rate assays were performed after 6, 12, and 24 h. After 6 h, DNA double-strand breaks (DSBs) were evaluated using anti-γH2AX antibodies with immunohistochemistry. mRNAs expressions of mediator of DNA damage checkpoint 1 (MDC1) and ataxia telangiectasia and Rad3-related protein (ATR), were analyzed. Expressions of miR-22 and miR-185 were analyzed because miR-22 targets MDC1 and miR-185, ATR.METHODSCigarette smoke extract (CSE) was obtained from HTPs and CCs. Primary human oral keratinocytes (HOKs) were treated with 5% and 20% CSE from HTPs and CCs. Cell survival rate assays were performed after 6, 12, and 24 h. After 6 h, DNA double-strand breaks (DSBs) were evaluated using anti-γH2AX antibodies with immunohistochemistry. mRNAs expressions of mediator of DNA damage checkpoint 1 (MDC1) and ataxia telangiectasia and Rad3-related protein (ATR), were analyzed. Expressions of miR-22 and miR-185 were analyzed because miR-22 targets MDC1 and miR-185, ATR.The HOKs had equivalent survival rates after exposure to the same concentrations of CSE from CCs and HTPs. HTPs increased foci formation of γH2AX in HOKs, as did CCs (without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of MDC1 and ATR decreased in cells exposed to CSE from CCs and HTPs (MDC1: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05; ATR: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of miR-22 and miR-185 were not significantly increased when exposed to CSE from CCs or HTPs.RESULTSThe HOKs had equivalent survival rates after exposure to the same concentrations of CSE from CCs and HTPs. HTPs increased foci formation of γH2AX in HOKs, as did CCs (without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of MDC1 and ATR decreased in cells exposed to CSE from CCs and HTPs (MDC1: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05; ATR: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of miR-22 and miR-185 were not significantly increased when exposed to CSE from CCs or HTPs.HTPs and CCs had similar cytotoxic effects. HTPs are genotoxic, can cause DSBs, and have toxicogenomic damage because they inhibit the MDC1 and ATR-CHK1 DNA repair pathways in the oral mucosa. The miRNA-mRNA axis was not related to these inhibitions.CONCLUSIONSHTPs and CCs had similar cytotoxic effects. HTPs are genotoxic, can cause DSBs, and have toxicogenomic damage because they inhibit the MDC1 and ATR-CHK1 DNA repair pathways in the oral mucosa. The miRNA-mRNA axis was not related to these inhibitions.

Congenital tooth agenesis is caused by the impairment of crucial genes related to tooth development, such as Wnt signaling pathway genes. Here, we investigated the genetic causes of sporadic congenital tooth agenesis. Exome sequencing, followed by Sanger sequencing, identified a novel single-nucleotide deletion in WNT10A (NC_000002.12(NM_025216.3):c.802del), which was not found in the healthy parents of the patient. Thus, we concluded that the variant was the genetic cause of the patient’s agenesis.