Oral bacterium Streptococcus mutans promotes tumor metastasis through thrombosis formation.

Thrombosis is a well-known cardiovascular disease (CVD) complication that has caused death in many patients with cancer. Oral bacteria have been reported to contribute to systemic diseases, including CVDs, and tumor metastasis. However, whether oral bacteria-induced thrombosis induces tumor metastasis remains poorly understood. In this study, the cariogenic oral bacterium Streptococcus mutans was used to examine thrombosis in vitro and in vivo. Investigation of tumor metastasis to the lungs was undertaken by intravenous S. mutans implantation using a murine breast cancer metastasis model. The results indicated that platelet activation, aggregation, and coagulation were significantly altered in S. mutans-stimulated endothelial cells (ECs), with elevated neutrophil migration, thereby inducing thrombosis formation. Streptococcus mutans stimulation significantly enhances platelet and tumor cell adhesion to the inflamed ECs. Furthermore, S. mutans-induced pulmonary thrombosis promotes breast cancer cell metastasis to the lungs in vivo, which can be reduced by using aspirin, an antiplatelet drug. Our findings indicate that oral bacteria promote tumor metastasis through thrombosis formation. Oral health management is important to prevent CVDs, tumor metastasis, and their associated death.

Maximum standardized uptake value in 11C-methionine positron emission tomography may predict the prognosis of patients with oral squamous cell carcinoma.

The present study aimed to elucidate the correlation between the uptake of 11C-methionine (MET) by a primary tumor and the survival of patients with oral squamous cell carcinoma (OSCC). This study enrolled 31 patients who underwent radical surgery for OSCC. The patients underwent pretreatment MET-positron emission tomography (PET) scanning. We analyzed correlations between the maximum standardized uptake value (SUVmax) of MET-PET in a primary tumor and the clinicopathological features. Further, we compared overall survival (OS), disease-specific survival (DSS), and loco-regional recurrence (LRR) rates between the two groups according to SUVmax of MET-PET. SUVmax of MET-PET in a primary tumor was higher in patients with advanced T-classification and advanced clinical stage, with significant differences (P = 0.001 and P = 0.016, respectively). The patients with SUVmax of MET-PET ≥ 4.4 showed significantly lower DSS rates and higher LRR rates than those with SUVmax of < 4.4 (P = 0.015 and P = 0.016, respectively). SUVmax of MET-PET and OS rates showed no significant correlation (P = 0.073). The present study revealed that SUVmax of MET-PET may predict clinical outcomes and prognosis in patients with OSCC who underwent radical surgery.

Stability of standardized uptake values for quantitative bone SPECT for jawbone lesions: a single-center cross-sectional study.

BACKGROUND: The long time required for bone uptake of radiopharmaceutical material after injection for bone scintigraphy is a burden for patients with poor health. Thus, to assess whether the uptake time could be reduced for single-photon emission computed tomography (SPECT) of the jawbone, this study evaluated differences in maximum standardized uptake values (SUVmax) within patients using SPECT imaging at 2 and 3 hours after radiopharmaceutical injection. METHODS: A total of 33 patients undergoing treatment or in post-treatment follow-up for medication-related osteonecrosis of the jaw, who visited our hospital between July 2020 and August 2021 and could receive SPECT twice on the same day, were enrolled in the study. Patients were injected with technetium-99 m hydroxymethylene diphosphonate (Tc-99 m HMDP) intravenously. The SUVmax for healthy parietal bones and jawbone lesions were calculated from the SPECT images using quantitative analysis software, and the SUVmax were compared between 2- and 3-hour uptake times. RESULTS: After exclusion, 30 patients were included in the study. In the 2-hour and 3-hour images, the median SUVmax of the parietal bones were 1.90 and 1.81, respectively, and those of the jawbone lesions were 9.25 and 9.39, respectively. The limits of agreement (LOA) ranged from - 0.33 to 0.25 in the parietal bones, and the %LOA ranged from - 9.8 to 17.3% in the jawbone lesions, showing high equivalence between the two uptake durations. The SUVmax showed no clinical differences between the 2- and 3-hour uptake durations for Tc-99 m HMDP SPECT of the jawbone. CONCLUSIONS: The results of this study justify a 2-3-hour uptake window when performing quantitative SPECT of the jawbone. Therefore, the minimum uptake time can potentially be reduced to only 2 hours.

Development of a risk prediction model for surgical site infection after lower third molar surgery.

BACKGROUND: There is little evidence regarding risk prediction for surgical site infection (SSI) after lower third molar (L3M) surgery. METHODS: We conducted a nested case-control study to develop a multivariable logistic model for predicting the risk of SSI after L3M surgery. Data were obtained from Hokkaido University Hospital from April 2013 to March 2020. Multiple imputation was applied for the missing values. We conducted decision tree (DT) analysis to evaluate the combinations of factors affecting SSI risk. RESULTS: We identified 648 patients. The final model retained the available distal space (Pell & Gregory II [p = 0.05], Pell & Gregory III [p < 0.01]), depth (Pell & Gregory B [p < 0.01], Pell & Gregory C [p < 0.01]), surgeon's experience (3-10 years [p = 0.25], <3 years [p < 0.01]), and simultaneous extraction of both L3M [p < 0.01]; the concordance-statistic was 0.72. The DT analysis demonstrated that patients with Pell and Gregory B or C and simultaneous extraction of both L3M had the highest risk of SSI. CONCLUSIONS: We developed a model for predicting SSI after L3M surgery with adequate predictive metrics in a single center. This model will make the SSI risk prediction more accessible.

Association between an Increased Serum CCL5 Level and Pathophysiology of Degenerative Joint Disease in the Temporomandibular Joint in Females.

Degenerative joint disease of the temporomandibular joints (DJD-TMJ) clinically manifests with symptoms such as orofacial pain, joint sounds and limited jaw movements. Our research group previously reported the functional necessity of a chemokine-chemokine receptor axis of CCL5-CCR5 in osteoclasts. Accumulated studies reported that this axis was involved in the pathogenesis of bone and joint destructive diseases, suggesting CCL5 as a potent biomarker. This study investigated whether or not the serum level of CCL5 can be a biomarker of DJD-TMJ and concomitantly analyzed changes in the serum and urine levels of bone markers to see whether or not changes in the rate of bone metabolism were predisposing. We enrolled 17 female subjects with diagnosed DJD-TMJ and sexually and age-matched 17 controls. The serum CCL5 level in DJD-TMJ subjects was significantly higher than that in the control subjects. Multivariate analyses indicated an association between an augmented CCL5 level and the rate of bone metabolism, especially in relatively young DJD-TMJ subjects without other systemic symptoms. A principal component analysis of serum markers and our pharmacological experiment using a postmenopausal model of ovariectomized rats suggested that an augmented serum CCL5 level specifically reflected DJD-TMJ and that covert changes in the rate of bone metabolism predisposed individuals to DJD-TMJ.

The oral bacterium Streptococcus mutans promotes tumor metastasis by inducing vascular inflammation.

Recent studies have demonstrated a relationship between oral bacteria and systemic inflammation. Endothelial cells (ECs), which line blood vessels, control the opening and closing of the vascular barrier and contribute to hematogenous metastasis; however, the role of oral bacteria-induced vascular inflammation in tumor metastasis remains unclear. In this study, we examined the phenotypic changes in vascular ECs following Streptococcus mutans (S. mutans) stimulation in vitro and in vivo. The expression of molecules associated with vascular inflammation and barrier-associated adhesion was analyzed. Tumor metastasis was evaluated after intravenous injection of S. mutans in murine breast cancer hematogenous metastasis model. The results indicated that S. mutans invaded the ECs accompanied by inflammation and NF-κB activation. S. mutans exposure potentially disrupts endothelial integrity by decreasing vascular endothelial (VE)-cadherin expression. The migration and adhesion of tumor cells were enhanced in S. mutans-stimulated ECs. Furthermore, S. mutans-induced lung vascular inflammation promoted breast cancer cell metastasis to the lungs in vivo. The results indicate that oral bacteria promote tumor metastasis through vascular inflammation and the disruption of vascular barrier function. Improving oral hygiene in patients with cancer is of great significance in preventing postoperative pneumonia and tumor metastasis.

Prognosis by cancer type and incidence of zoledronic acid-related osteonecrosis of the jaw: a single-center retrospective study.

PURPOSE: Survival time after bisphosphonate use has been increasingly recognized to be associated with the incidence of medication-related osteonecrosis of the jaw (MRONJ); however, this has not been elucidated sufficiently in the literature. This study aimed to clarify the incidence of MRONJ and the corresponding survival rate of patients treated with zoledronic acid (ZA) for each type of cancer and obtain useful information for the oral/dental supportive care of cancer patients. METHODS: We evaluated 988 patients who were administered ZA at our hospital; among them, 862 patients with metastatic bone tumors or myeloma were included. RESULTS: The median survival time (MST) after ZA initiation was 35, 34, 8, 41, 12, and 6 months for patients with breast, prostrate, lung, myeloma, renal, and other cancers, respectively. Patients with cancers that had a short survival time (lung and other cancers [MST = 8 and 6 months, respectively] and cancers with MST < 10 months) did not develop MRONJ; this could be attributed to the shorter duration of ZA administration. The cumulative incidence of MRONJ in breast cancer, prostate cancer, and multiple myeloma was related to the frequency of anti-resorptive drug use and the increased risk over time. In renal cancer, the cumulative incidence of MRONJ increased early, although the MST was 12 months. CONCLUSION: For the dentists in charge of dental management, it is essential to be aware of prognosis-related factors, predict MRONJ risk for each cancer treatment, and use risk prediction in dental management planning, particularly for cancers with non-poor prognosis.

Evaluation of the strategies to reduce third-generation oral cephalosporins in dentistry at a Japanese academic hospital: An interrupted time series analysis.

WHAT IS KNOWN AND OBJECTIVE: Third-generation oral cephalosporins, especially cefcapene-pivoxil (CFPN-PI), have been used frequently in the Japanese dental field. In December 2014 and April 2016, the newly published clinical guidelines recommended the use of amoxicillin (AMPC). Thus, it is important to evaluate the impact of these guidelines on the prescription profiles of prophylactic antibiotics, clinical outcomes and cost-effectiveness of antibiotics. METHODS: We conducted a retrospective study to analyse an interrupted time series analysis from April 2013 to March 2020 at the Department of Dentistry of Hokkaido University Hospital. A segmented regression model was used to estimate the changes in the incidence of infectious complications following tooth extraction. Prescribed antibiotic data were evaluated via days of therapy (DOT). Antibiotic costs were calculated in terms of the Japanese yen (JPY). RESULTS AND DISCUSSION: We identified 17,825 eligible patients. The incidence rates of infectious complications (SSI + dry socket) and SSI after tooth extraction were 3.2% and 2.2%, respectively, during the entire period. The extraction of impacted third molars corresponded to 5.0% and 3.4%, respectively. However, their incidence rates were not significantly different during this period. The use of prophylactic antibiotics and antibiotic cost showed consistent trends following the implementation of guidelines. The mean DOT of CFPN-PI decreased (ranging from 4893.6 DOTs/1000 patients [March 2013 to November 2014] to 3856.4 DOTs/1000 patients [December 2014 to March 2016]; p < 0.001, and from 3856.4 DOTs/1000 patients [December 2014 to March 2016] to 2293.9 DOTs/1000 patients [April 2016 to March 2020]; p < 0.001). In contrast, the mean DOT of AMPC was found to be increased (ranging from 1379.7 DOTs/1000 patients [March 2013 to November 2014] to 3236.3 DOTs/1000 patients [December 2014 to March 2016]; p < 0.001, and from 3236.3 DOTs/1000 patients [December 2014 to March 2016] to 4597.8 DOTs/1000 patients [April 2016 to March 2020]; p < 0.001). The mean monthly cost was decreased (ranging from 905.3 JPY [March 2013 to November 2014] to 788.7 JPY [December 2014 to March 2016]; p = 0.003, and from 788.7 JPY [December 2014 to March 2016] to 614.0 JPY [April 2016 to March 2020]; p < 0.001). WHAT IS NEW AND CONCLUSION: After December 2014, prophylactic antibiotics were switched from CFPN-PI to AMPC, and the incidence rate of infectious complications was not significantly different over time. However, changing antibiotics is useful from a cost-effectiveness perspective.

Metastatic basal cell carcinoma of buccal mucosa: a report of a rare case.

BACKGROUND: Basal cell carcinoma (BCC) is the most common cancer worldwide. Most of BCCs can be detected in the early stages and are generally well controlled with local resection. Despite the high incidence of BCC, metastasis is rarely observed. Metastatic BCCs generally have an aggressive phenotype and are refractory to conventional treatment. CASE PRESENTATION: We describe a rare case of BCC in which a series of local relapses culminated in metastasis into the oral cavity 10 years after the first diagnosis of cutaneous BCC. We performed surgical resection and postoperative radiotherapy in this patient; 11 months after the final course of radiotherapy, the BCC remains stable, and the patient continues to be monitored regularly. CONCLUSIONS: Because metastatic BCC is refractory to current treatment and difficult to control, his treatment history and the pathohistological features of BCC had to be considered in posttreatment planning.

Oral adverse events due to zinc deficiency after pancreaticoduodenectomy requiring continuous intravenous zinc supplementation: a case report and literature review.

BACKGROUND: Zinc is mainly absorbed in the duodenum and proximal jejunum, which are removed during pancreaticoduodenectomy (PD). Little is known about the adverse oral events and skin disorders caused by zinc deficiency after PD. Herein, we reviewed studies on the development of zinc deficiency after PD and reported about a patient with zinc deficiency after PD who required home intravenous zinc replacement. CASE PRESENTATION: A 73-year-old woman with glossitis, taste disorder, and acrodermatitis enteropathica-like eruption on her fingers presented to the Division of Dentistry and Oral Surgery 69 days after PD. Her serum zinc level markedly decreased to 30 µg/dL. Oral zinc administration was inadequate to treat hypozincemia after PD; therefore, multi-trace elements were injected intravenously during readmission. Her serum zinc levels recovered, and her lesions gradually improved. Furthermore, a central venous port was implanted to maintain normal serum zinc levels, and she continued self-injecting zinc at home. CONCLUSIONS: Zinc deficiency after PD rarely occurs. The clinical oncologist community, including dentists responsible for the oral care of cancer patients, should be aware of the oral adverse events, such as dysgeusia, glossitis, and oral pain, associated with zinc deficiency after cancer surgery and that induced by chemotherapy or head and neck radiation therapy.

Novel mutation in the ALPL gene with a dominant negative effect in a Japanese family.

INTRODUCTION: Hypophosphatasia (HPP) is caused by mutations in the ALPL gene encoding tissue nonspecific alkaline phosphatase (TNSALP) and inherited in either an autosomal recessive or autosomal dominant manner. It is characterized clinically by defective mineralization of bone, dental problems, and low serum ALP levels. In the current report, we demonstrate a novel mutation in the ALPL gene (c.244G > A p.Gly82Arg) in a Japanese family with low serum ALP levels. MATERIALS AND METHODS: The ALPL gene analysis using hybridization capture-based next-generation sequencing was performed. The expression plasmids of the wild type and mutated TNSALP were introduced into COS-7 cells. The enzymatic activity of ALP in the cell lysates was measured using p-nitrophenylphosphate as a substrate. RESULTS: TNSALP with the novel ALPL mutation (c.244G > A p.Gly82Arg) completely lost its enzymatic activity and suppressed that of wild-type TNSALP, corroborating its dominant negative effect. The diagnosis of autosomal dominant HPP was confirmed in three members of the family. CONCLUSION: Our approach would help to avoid the inappropriate use of bone resorption inhibitors for currently mis- or under-diagnosed HPP, given that the presence of further, yet undetected mutations of the ALPL gene are plausible.

Correlation between endothelial CXCR7 expression and clinicopathological factors in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) impairs functionality and sensuousness resulting in poor quality of life. Biomarkers can predict disease trajectory and lead to effective treatments. Transcriptomics have identified genes that are upregulated in tumor endothelial cells (TECs) compared with normal endothelial cells (NECs). Among them, chemokine receptor 7 (CXCR7) is highly expressed in TECs of several cancers and involved in angiogenesis of TECs. However, levels of CXCR7 in OSCC blood vessels have not been fully investigated. In this study, we analyzed the correlation between CXCR7 expression in TECs and clinicopathological factors in OSCC. Immunohistochemistry for CXCR7 and CD34 was performed on 59 OSCC tissue specimens resected between 1996 and 2008 at Hokkaido University Hospital. CXCR7 expression in blood vessels was evaluated by the ratio of CXCR7+/CD34+ blood vessels. CXCR7 expression was 42% and 19% in tumor and non-tumor parts, respectively, suggesting that CXCR7 expression is higher in TECs than in NECs. CXCR7 expression in TECs correlated with advanced T-stage and cancer stage. Overall survival and disease-free survival rates were higher in low-expressing CXCR7 patients than in high-expressing. These results suggest that CXCR7 expression in blood vessels may be a useful diagnostic and prognostic marker for OSCC patients.

Incidence and risk of antiresorptive agent-related osteonecrosis of the jaw (ARONJ) after tooth extraction in patients with autoimmune disease.

INTRODUCTION: Antiresorptive agent-related osteonecrosis of the jaw (ARONJ) is a rare but serious complication in patients receiving antiresorprtive agents (AR). However, the incidence of ARONJ after tooth extraction in patients with autoimmune disease (AID) remains unclear. The present study aimed to clarify the high-risk population of ARONJ in patients with AID. MATERIALS AND METHODS: The study population comprised 232 patients treated with AR, AID or non-AID, who had undergone dental extraction from January 2011 to September 2017. The incidence and risk factors of ARONJ were analysed retrospectively. Additionally, the relationship between ARONJ and osteoporotic fracture (OF) and AR discontinuation during dental procedures was investigated. RESULTS: Of 232 patients, 10 developed ARONJ within 1 year of dental extraction. The incidence of ARONJ in patients with AID was higher than that in non-AID patients (2.0/100 person-year vs 0.5/100 person-year; p = 0.03). Among the AID patients, RA patients had strikingly high incidence of ARONJ (3.6/100 person-year). The incidence of neither ARONJ nor OF significantly differed between patients who continued and discontinued AR in the perioperative period. CONCLUSION: Patients with AID who undergo dental extraction are at high risk of ARONJ. Discontinuation of AR would not significantly contribute to reduce the incidence of ARONJ in those patients.

Prevalence of and risk factors for postoperative hemorrhage after lower third molar extraction on warfarin therapy: a multicenter retrospective study in Japan.

Postoperative hemorrhage after tooth extraction is a critical and clinically important issue for clinicians and patients receiving anticoagulants. The purpose of the present study was to investigate the prevalence of and risk factors for postoperative hemorrhage after lower third molar extraction in Japanese patients receiving warfarin therapy. A total of 142 patients who underwent lower third molar extraction between January 2010 and December 2016 were included, and their medical records were retrospectively reviewed. The prevalence of and risk factors for postoperative hemorrhage were investigated. The prevalence of postoperative hemorrhage after lower third molar extraction was significantly higher in patients receiving warfarin than in healthy subjects (21.8% vs 0.7%, P < 0.001). The cutoff value for PT-INR was 2.11 based on a receiver-operating characteristic analysis. A multivariate analysis indicated that an elevated PT-INR value [hazard ratio (HR) 3.798, 95% confidence interval (CI) 1.400-10.467, P < 0.01], preoperative antibiotic administration (HR 4.434, 95% CI 1.591-14.775, P < 0.01), difficulties with intraoperative hemostasis (HR 16.298, 95% CI 2.986-110.677, P < 0.01), and higher serum creatinine levels (HR 7.465, 95% CI 1.616-39.576, P < 0.05) are significant predictors of postoperative hemorrhage after lower third molar extraction. Multivariate correlations were observed between risk factors including an elevated PT-INR value, preoperative antibiotic administration, and higher serum creatinine levels, and postoperative hemorrhage after lower third molar extraction in patients receiving warfarin therapy. Clinicians need to consider these risk factors for postoperative hemorrhage after the lower third molar extraction and monitor PT-INR in patients receiving warfarin therapy.

The direct binding of collagen XVII and collagen IV is disrupted by pemphigoid autoantibodies.

The basement membrane zone (BMZ) is framed by hemidesmosomes and extracellular matrix (ECM) including collagen IV (COL4). Hemidesmosomes are multiprotein complexes that include collagen XVII (COL17). BMZ proteins can be targeted in autoimmune subepidermal blistering diseases, e.g., pemphigoid targeting COL17. The blistering mechanisms in pemphigoid have not been fully elucidated, especially in mucous membrane pemphigoid (MMP), which mainly affects the mucosa. In this study, we showed that oral lesions in pemphigoid may be attributed to the inhibition of protein-protein interactions by autoantibodies. Using immunoprecipitation, we revealed that COL17 directly binds to COL4 in normal human keratinocytes and normal human oral keratinocytes. In particular, the C-terminus of COL17 is binding site to COL4 in oral keratinocytes. The precise COL4-binding region on COL17 was determined by protein-protein binding assay to be from amino acid Gly1175 to Asp1340 on the C-terminus. MMP-IgG or mAb recognizing the C-terminus hindered the interaction of COL17 with COL4 in oral keratinocytes. Furthermore, keratinocyte adhesion strength to COL4-coated plates was significantly reduced by the treatment of mAb against the C-terminus. In addition, the inflammatory infiltrates around perilesions were significantly less in MMP compared to BP. These results indicate that pemphigoid IgG targeting the C-terminus plays a pathogenic role in blister formation in the oral mucosa to inhibit protein interactions with less inflammation.

The identification of autoantigens in mucous membrane pemphigoid using immortalized oral mucosal keratinocytes.

BACKGROUND: Mucous membrane pemphigoid (MMP) is a rare chronic autoimmune subepithelial blistering disorder, targeting multiple basement membrane zone (BMZ) proteins including collagen XVII (COL17). Circulating autoantibodies of MMP are often undetected due to their lower titers. The oral mucosa is a valuable substrate for the detection of autoantibodies in MMP patients. However, obtaining normal human oral mucosa is more difficult than obtaining normal human skin. We established immortalized normal human oral mucosal keratinocytes (OMKs) and performed immunoblotting using immortalized OMK lysate for detecting autoantigens in MMP. METHODS: Immortalized OMKs were generated from primary OMKs using E6/E7 proteins of HPV. We compared the protein expression levels of major BMZ proteins between primary OMKs and immortalized OMKs. We performed immunoblotting to detect autoantigens using cell lysates from immortalized OMKs in 30 MMP patients. RESULTS: There were no significant differences between primary OMKs and immortalized OMKs in terms of protein expression levels of the BMZ proteins, including COL17, laminin 332, integrin α6/ß4, collagen VII, and collagen IV. Cell lysates of immortalized OMKs effectively identified MMP autoantigens in 60% (18/30) of MMP sera. We found an interesting case of MMP whose autoantibodies preferentially reacted to the 120-kD protein that is an ectodomain of COL17. CONCLUSION: We demonstrated that a cell lysate of immortalized OMKs is a reliable substrate for the detection of MMP autoantigens. This newly developed immunoblotting analysis method promises to contribute to the diagnosis of MMP.

HuR translocation to the cytoplasm of cancer cells in actin-independent manner.

Human antigen R (HuR) is a RNA-binding protein, which binds to the AU-rich element (ARE) in the 3'-untranslated region (3'-UTR) of certain mRNA and is involved in the export and stabilization of ARE-mRNA. HuR constitutively relocates to the cytoplasm in many cancer cells, however the mechanism of intracellular HuR trafficking is poorly understood. To address this question, we examined the functional role of the cytoskeleton in HuR relocalization. We tested the effect of actin depolymerizing macrolide latrunculin A or myosin II ATPase activity inhibitor blebbistatin for HuR relocalization induced by the vasoactive hormone Angiotensin II in cancer and control normal cells. Western blot and confocal imaging data revealed that both inhibitors attenuated the cytoplasmic HuR in normal cells but no such alteration was observed in cancer cells. Concomitant with changes in intracellular HuR localization, both inhibitors markedly decreased the accumulation and half-lives of HuR target ARE-mRNAs in normal cells, whereas no change was observed in cancer cells. Furthermore, co-immunoprecipitation experiments with HuR proteins revealed clear physical interaction with ß-actin only in normal cells. The current study is the first to verify that cancer cells can implicate a microfilament independent HuR transport. We hypothesized that when cytoskeleton structure is impaired, cancer cells can acquire an alternative HuR trafficking strategy.

EWS-FLI1 regulates a transcriptional program in cooperation with Foxq1 in mouse Ewing sarcoma.

EWS-FLI1 constitutes an oncogenic transcription factor that plays key roles in Ewing sarcoma development and maintenance. We have recently succeeded in generating an ex vivo mouse model for Ewing sarcoma by introducing EWS-FLI1 into embryonic osteochondrogenic progenitors. The model well recapitulates the biological characteristics, small round cell morphology, and gene expression profiles of human Ewing sarcoma. Here, we clarified the global DNA binding properties of EWS-FLI1 in mouse Ewing sarcoma. GGAA microsatellites were found to serve as binding sites of EWS-FLI1 albeit with less frequency than that in human Ewing sarcoma; moreover, genomic distribution was not conserved between human and mouse. Nevertheless, EWS-FLI1 binding sites within GGAA microsatellites were frequently associated with the histone H3K27Ac enhancer mark, suggesting that EWS-FLI1 could affect global gene expression by binding its target sites. In particular, the Fox transcription factor binding motif was frequently observed within EWS-FLI1 peaks and Foxq1 was identified as the cooperative partner that interacts with the EWS portion of EWS-FLI1. Trib1 and Nrg1 were demonstrated as target genes that are co-regulated by EWS-FLI1 and Foxq1, and are important for cell proliferation and survival of Ewing sarcoma. Collectively, our findings present novel aspects of EWS-FLI1 function as well as the importance of GGAA microsatellites.

Adenovirus infection induces HuR relocalization to facilitate virus replication.

HuR is an RNA-binding protein of the embryonic lethal abnormal vision (ELAV) family, which binds to the AU-rich element (ARE) in the 3'-untranslated region (UTR) of certain mRNAs and is involved in the nucleo-cytoplasmic export and stabilization of ARE-mRNAs. The cytoplasmic relocalization of ARE-mRNAs with several proteins such as HuR and pp32 increases in cells transformed by the adenovirus oncogene product E4orf6. Additionally, these ARE-mRNAs were stabilized and acquired the potential to transform cells. Although, the relocalization of HuR and the stabilization of ARE-mRNAs are crucial for cell transformation, evidence regarding the relationship of HuR and ARE-mRNAs with adenovirus replication is lacking. In this report, we demonstrate that adenovirus infection induces the relocation of HuR to the cytoplasm of host cells. Analysis using the luciferase-ARE fusion gene and the tetracycline (tet)-off system revealed that the process of stabilizing ARE-mRNAs is activated in adenovirus-infected cells. Heat shock treatment or knockdown-mediated depletion of HuR reduced adenovirus production. Furthermore, expression of ARE-including viral IVa2 mRNA, decreased in HuR-depleted infected cells. These results indicate that HuR plays an important role in adenovirus replication, at least in part, by up-regulating IVa2 mRNA expression and that ARE-mRNA stabilization is required for both transformation and virus replication.