Satoru Kondo

Exosomal HIF1α supports invasive potential of nasopharyngeal carcinoma-associated LMP1-positive exosomes.

It has emerged recently that exosomes are potential carriers of pro-tumorigenic factors that participate in oncogenesis. However, whether oncogenic transcription factors are transduced by exosomes is unknown. Hypoxia-inducible factor-1α (HIF1α) transcriptionally regulates numerous key aspects of tumor development and progression by promoting a more aggressive tumor phenotype, characterized by increased proliferation and invasiveness coupled with neoangiogenesis. It has been shown that the principal oncoprotein of Epstein–Barr virus (EBV), latent membrane protein 1 (LMP1), drives oncogenic processes and tumor progression of the highly invasive EBV malignancy, nasopharyngeal carcinoma (NPC). We now demonstrate that endogenous HIF1α is detectable in exosomes and that LMP1 significantly increases levels of HIF1α in exosomes. HIF1 recovered from exosomes retains DNA-binding activity and is transcriptionally active in recipient cells after exosome uptake. We also show that treatment of EBV-negative cells with LMP1-exosomes increases migration and invasiveness of NP cell lines in functional assays, which correlates with the phenotype associated with epithelial–mesenchymal transition (EMT). In addition, we provide evidence that HIF1α itself participates in exosome-mediated pro-metastatic effects in recipient cells, as exosome-mediated delivery of active and inactive forms of HIF1α results in reciprocal changes in the expression of E- and N-cadherins associated with EMT. Further, immunohistochemical analysis of NPC tumor tissues revealed direct correlation between protein levels of LMP1 and of the endosome/exosome marker tetraspanin, CD63, which suggests an increase in exosome formation in this EBV-positive malignancy. We hypothesize that exosome-mediated transfer of functional pro-metastatic factors by LMP1-positive NPC cells to surrounding tumor cells promotes cancer progression.

Pathogenic role of Epstein–Barr virus latent membrane protein-1 in the development of nasopharyngeal carcinoma

Undifferentiated nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-associated malignant tumor. A consistent elevation in EBV antibody titers is a well-established risk factor for the development of NPC. The pathophysiological relationship and molecular mechanisms of EBV-mediated carcinogenesis have not been fully elucidated. While NPC tumors are known to express three EBV-encoded proteins, EBNA1, LMP1, and LMP2, they also express a large number of virus-encoded small RNAs (EBERs) and microRNAs (miRNAs). Among them, LMP1 may be a central player in the development of NPC. LMP1, an EBV-encoded primary oncogene, functions as a viral mimic of the TNFR family member, CD40, and engages in a number of signaling pathways that induce morphological and phenotypic alterations in epithelial cells. LMP1 upregulates EMT, and contributes to the highly metastatic features of NPC. Moreover, LMP1-associated EMT is accompanied by the expression of cancer stem cell (CSC)/cancer progenitor cell (CPC) markers (CD44high/CD24low) and the acquisition of stem cell/progenitor cell-like properties. BART miRNAs, encoded from the BamHI-A region of the viral genome, are the most abundant transcripts. They modulate apoptosis and host innate immune defense mechanisms. Some BART1 miRNAs are considered to negatively regulate LMP1 protein expression. LMP1 is secreted via exosomes, is incorporated into EBV-uninfected cells by endocytosis, and affects the environment surrounding the tumor. Here we reviewed the contribution of EBV gene products to NPC pathogenesis in relation with LMP1.

Clustered MicroRNAs of the Epstein-Barr Virus Cooperatively Downregulate an Epithelial Cell-Specific Metastasis Suppressor

ABSTRACT The Epstein-Barr virus (EBV) encodes its own microRNAs (miRNAs); however, their biological roles remain elusive. The commonly used EBV B95-8 strain lacks a 12-kb genomic region, known as BamHI A rightward transcripts (BART) locus, where a number of BART miRNAs are encoded. Here, bacterial artificial chromosome (BAC) technology was used to generate an EBV B95-8 strain in which the 12-kb region was fully restored at its native locus [BART(+) virus]. Epithelial cells were stably infected with either the parental B95-8 virus or the BART(+) virus, and BART miRNA expression was successfully reconstituted in the BART(+) virus-infected cells. Microarray analyses of cellular gene expression identified N-myc downstream regulated gene 1 (NDRG1) as a putative target of BART miRNAs. The NDRG1 protein was barely expressed in B cells, highly expressed in epithelial cells, including primary epithelial cells, and strongly downregulated in the BART(+) virus-infected epithelial cells of various origins. Although in vitro reporter assays identified BART22 as being responsible for the NDRG1 downregulation, EBV genetic analyses revealed that BART22 was not solely responsible; rather, the entire BART miRNA cluster 2 was responsible for the downregulation. Immunohistochemical analyses revealed that the expression level of the NDRG1 protein was downregulated significantly in EBV-positive nasopharyngeal carcinoma specimens. Considering that NDRG1 encodes an epithelial differentiation marker and a suppressor of metastasis, these data implicate a causative relationship between BART miRNA expression and epithelial carcinogenesis in vivo. IMPORTANCE EBV-related epithelial cancers, such as nasopharyngeal carcinomas and EBV-positive gastric cancers, encompass more than 80% of EBV-related malignancies. Although it is known that they express high levels of virally encoded BART miRNAs, how these miRNAs contribute to EBV-mediated epithelial carcinogenesis remains unknown. Although a number of screenings have been performed to identify targets of viral miRNAs, many targets likely have not been identified, especially in case of epithelial cell infection. This is the first study to use EBV genetics to perform unbiased screens of cellular genes that are differentially expressed in viral miRNA-positive and -negative epithelial cells. The result indicates that multiple EBV-encoded miRNAs cooperatively downregulate NDRG1, an epithelial differentiation marker and suppressor of metastasis. The experimental system described in this study should be useful for further clarifying the mechanism of EBV-mediated epithelial carcinogenesis.

APOBEC3A and 3C decrease human papillomavirus 16 pseudovirion infectivity.

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) proteins are cellular DNA/RNA-editing enzymes that play pivotal roles in the innate immune response to viral infection. APOBEC3 (A3) proteins were reported to hypermutate the genome of human papillomavirus 16 (HPV16), the causative agent of cervical cancer. However, hypermutation did not affect viral DNA maintenance, leaving the exact role of A3 against HPV infection elusive. Here we examine whether A3 proteins affect the virion assembly using an HPV16 pseudovirion (PsV) production system, in which PsVs are assembled from its capsid proteins L1/L2 encapsidating a reporter plasmid in 293FT cells. We found that co-expression of A3A or A3C in 293FT cells greatly reduced the infectivity of PsV. The reduced infectivity of PsV assembled in the presence of A3A, but not A3C, was attributed to the decreased copy number of the encapsidated reporter plasmid. On the other hand, A3C, but not A3A, efficiently bound to L1 in co-immunoprecipitation assays, which suggests that this physical interaction may lead to reduced infectivity of PsV assembled in the presence of A3C. These results provide mechanistic insights into A3s inhibitory effects on the assembly phase of the HPV16 virion.

Evaluation of dysphagia at the initial diagnosis of amyotrophic lateral sclerosis

OBJECTIVEnDysphagia eventually occurs in amyotrophic lateral sclerosis (ALS). Swallowing in patients with ALS at their initial diagnosis was evaluated using videofluoroscopy (VF).nnnMETHODSnNineteen consecutive patients with ALS, 14 with bulbar symptoms, and 5 without them, underwent VF. Fourteen physiologic components, 6 oral and 8 pharyngeal components, were assessed during the examination.nnnRESULTSnSignificantly poorer scores were observed in three of the 6 oral components and 3 of the 8 pharyngeal components in patients with bulbar symptoms. Furthermore, bolus transport from the oral cavity to pharynx, pharyngeal constriction, oral residue and pharyngeal residue were impaired in patients even without bulbar symptoms. On the other hand, pharyngoesophageal segment opening was preserved in patients even with bulbar symptoms. Bolus transport and initiation of pharyngeal swallow were correlated with the swallowing category of the ALS severity scale.nnnCONCLUSIONnDefining types of impairment in patients with or without bulbar symptoms is useful for evaluating dysphagia in this disease. Although VF showed impairment of oral and pharyngeal phases of swallowing, the oral phase affected the eating habit in ALS at the initial diagnosis.

Role of activation-induced cytidine deaminase in the development of oral squamous cell carcinoma

Purpose In humans, activation-induced cytidine deaminase (AID) expression results due to inflammation and this deaminase activity is also involved in carcinogenesis. The aim of this study is to investigate the correlation between AID expression and the clinical classification of oral cancer tissues. Experimental Design The current study investigated the correlation between AID expression and the clinical classification of oral cancer tissues from 27 patients who underwent surgical resection using immunohistochemistry. Specific AID expression and its induction by cytokine stimulation were investigated in cultured HSC oral cancer cell lines by reverse transcriptase PCR. Results AID expression was detected in 10 of 27 specimens (37.0%). AID expression was more frequently detected in early-stage cancer, especially in early stage T, than in late-stage cancer (T1/T2 vs. T3/4; Pu200a=u200a0.0493, N0 vs. N1/2/3; Pu200a=u200a0.0793). HSC-2, a nonmetastatic oral cancer cell line, abundantly expressed endogenous AID, whereas no such expression was observed in HSC-3, a metastatic oral cancer cell line. Moreover, AID expression was substantially induced in HSC-2 cells by stimulation of an inflammation-related cytokine, TNF-α. Conclusions Aberrant AID expression in the oral epithelium would contribute to the initiation of oral squamous cell carcinoma. Avoiding persistent AID inducible condition such as frequent cleaning of oral cavity would play an important role for the prevention of developing oral cancer.

Detection of hypermutated human papillomavirus type 16 genome by Next-Generation Sequencing

Human papillomavirus type 16 (HPV16) is a major cause of cervical cancer. We previously demonstrated that C-to-T and G-to-A hypermutations accumulated in the HPV16 genome by APOBEC3 expression in vitro. To investigate in vivo characteristics of hypermutation, differential DNA denaturation-PCR (3D-PCR) was performed using three clinical specimens obtained from HPV16-positive cervical dysplasia, and detected hypermutation from two out of three specimens. One sample accumulating hypermutations in both E2 and the long control region (LCR) was further subjected to Next-Generation Sequencing, revealing that hypermutations spread across the LCR and all early genes. Notably, hypermutation was more frequently observed in the LCR, which contains a viral replication origin and the early promoter. APOBEC3 expressed abundantly in an HPV16-positive cervix, suggesting that single-stranded DNA exposed during viral replication and transcription may be efficient targets for deamination. The results further strengthen a role of APOBEC3 in introducing HPV16 hypermutation in vivo.

Factors Affecting Outcomes of Alternating Chemoradiotherapy for Nasopharyngeal Cancer

Objective: Nasopharyngeal cancer (NPC) is radiosensitive and chemosensitive. We evaluated the efficacy of alternating chemoradiotherapy in patients with advanced NPC. Methods: Alternating chemoradiotherapy was initiated in 30 patients with NPC, and 27 patients with cancer stages II (n = 6), III (n = 8), IVA (n = 9), and IVB (n = 4) were retrospectively analyzed. Chemotherapy was initially administered followed by radiotherapy, and chemotherapy, radiotherapy, and chemotherapy were alternately administered. Of the 27 patients, 22 patients received cisplatin (50 mg/m2/day, days 6 and 7) and 5-fluorouracil (5-FU; 800 mg/m2/day, days 1-5), whereas 5 patients received carboplatin (AUC 4-5, day 6) and 5-FU. Results: Of the 27 patients, 19 (70%) received 3 chemotherapy courses. The total duration of alternating chemoradiotherapy was 81 to 101 days (median, 90 days). At a median follow-up of 53 months, the 5-year progression-free survival (PFS) was 71%. Multivariate analysis showed that weight loss and the number of chemotherapy courses had a significant effect on PFS. Conclusion: Alternating chemoradiotherapy led to similar or higher survival rates compared with concurrent chemoradiotherapy, which was characterized by good compliance and adaptable intensity.

IL-18 is highly expressed in inflammatory infiltrates of submandibular glands in patients with immunoglobulin G4–related disease

Immunoglobulin (Ig) G4-related disease (IgG4-RD) is a new disease entity characterized by high serum IgG4 concentrations, infiltration of IgG4-positive plasmacytes, and fibrosis of various organs. Several groups have reported that IgG4-RD is a unique inflammatory disorder characterized by an immune reaction predominantly mediated by T helper (Th) 2 and regulatory T cells. Meanwhile, recent studies have demonstrated that interleukin (IL) 18 has a potential to trigger the production of Th2 cytokines by Th1 cells. We analyzed IL-18 expression in submandibular glands of patients with IgG4-RD (20 cases) and controls (19 cases) by immunohistochemical analysis and quantitative real-time reverse-transcription polymerase chain reaction. We found that IL-18 was highly expressed in submandibular glands of patients with IgG4-RD than in controls with both protein (P < .05, χ(2) test) and messenger RNA levels (P < .05, Mann-Whitney U test). In addition, the expression of IL-18 and IL-13 was correlated in submandibular glands of patients with IgG4-RD. Moreover, by analyzing dual immunofluorescence staining, a few numbers of cells were double positive for IL-13 and interferon γ at the inflammatory infiltrates of submandibular glands of patients with IgG4-RD. These data suggest a possibility that IL-13 is produced by Th1 cells. We speculated that IL-18 stimulates Th1 cells producing Th2 cytokines and enhances the immune reaction of Th2 cytokines in pathogenesis of IgG4-RD.

Intraoperative free jejunum flap monitoring with indocyanine green near-infrared angiography

The free jejunum flap technique has been regarded as the optimal approach during circumferential pharyngolaryngectomy reconstruction. Although classical patency tests are available, an intraoperative guarantee of the patency of anastomoses and microcirculations is inevitable. Indocyanine green near-infrared angiography (ICGA) was intraoperatively performed in six patients after reconstruction using the free jejunum flap. An adequate arterial as well as venous phase was observed. In addition to classical patency tests and doppler, we have successfully monitored the flap after total pharyngolalyngectomy intraoperatively using the ICGA. Our preliminary results implicate that this novel technique offers secure intraoperative monitoring of a free jejunum graft. This technique will provide us with advantages over regular patency test in selected cases.