Inhibition of UCH-L1 Deubiquitinating Activity with Two Forms of LDN-57444 Has Anti-Invasive Effects in Metastatic Carcinoma Cells.

Normally ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed in the central nervous and reproductive systems of adults, but its de novo expression has been detected in many human cancers. There is a growing body of evidence that UCH-L1 de-ubiquitinating (DUB) activity plays a major pro-metastatic role in certain carcinomas. Here we tested anti-metastatic effects of the small-molecule inhibitor of UCH-L1 DUB activity, LDN-57444, in cell lines from advanced oral squamous cell carcinoma (OSCC) as well as invasive nasopharyngeal (NP) cell lines expressing the major pro-metastatic gene product of Epstein-Barr virus (EBV) tumor virus, LMP1. To overcome the limited aqueous solubility of LDN-57444 we developed a nanoparticle formulation of LDN-57444 by incorporation of the compound in polyoxazoline micellear nanoparticles (LDN-POx). LDN-POx nanoparticles were equal in effects as the native compound in vitro. Our results demonstrate that inhibition of UCH-L1 DUB activity with LDN or LDN-POx inhibits secretion of exosomes and reduces levels of the pro-metastatic factor in exosomal fractions. Both forms of UCH-L1 DUB inhibitor suppress motility of metastatic squamous carcinoma cells as well as nasopharyngeal cells expressing EBV pro-metastatic Latent membrane protein 1 (LMP1) in physiological assays. Moreover, treatment with LDN and LDN-POx resulted in reduced levels of pro-metastatic markers, a decrease of carcinoma cell adhesion, as well as inhibition of extra-cellular vesicle (ECV)-mediated transfer of viral invasive factor LMP1. We suggest that soluble inhibitors of UCH-L1 such as LDN-POx offer potential forms of treatment for invasive carcinomas including EBV-positive malignancies.

Modulation of the tumor microenvironment by Epstein-Barr virus latent membrane protein 1 in nasopharyngeal carcinoma.

Latent membrane protein 1 (LMP1) is a primary oncogene encoded by the Epstein-Barr virus, and various portions of LMP1 are detected in nasopharyngeal carcinoma (NPC) tumor cells. LMP1 has been extensively studied since the discovery of its transforming property in 1985. LMP1 promotes cancer cell growth during NPC development and facilitates the interaction of cancer cells with surrounding stromal cells for invasion, angiogenesis, and immune modulation. LMP1 is detected in 100% of pre-invasive NPC tumors and in approximately 50% of advanced NPC tumors. Moreover, a small population of LMP1-expressing cells in advanced NPC tumor tissue is proposed to orchestrate NPC tumor tissue maintenance and development through cancer stem cells and progenitor cells. Recent studies suggest that LMP1 activity shifts according to tumor development stage, but it still has a pivotal role during all stages of NPC development.

Expression of interferon regulatory factor 7 correlates with the expression of Epstein-Barr Virus latent membrane protein 1 and cervical lymph node metastasis in nasopharyngeal cancer.

Interferon regulatory factor 7 (IRF7) has oncogenic properties in several malignancies such as Epstein-Barr virus (EBV)-associated lymphoma. However, there is no evidence whether IRF7 is associated with the oncogenesis of nasopharyngeal cancer (NPC), the pathogenesis of which is closely associated with EBV. Herein, we report that expression of IRF7 was increased in normal nasopharyngeal cells that expressed the EBV principal oncoprotein, latent membrane protein 1 (LMP1). In addition, IRF7 was mainly expressed in the nucleus in both normal nasopharyngeal cells and nasopharyngeal cancer cells that expresses LMP1. On immunohistochemical analysis, IRF7 was predominantly localized in the nucleus in biopsy samples of NPC tissues. In total, IRF7 expression was detected with 36 of 49 specimens of these tissues. Furthermore, the expression score of IRF7 correlated with the expression score of LMP1. Moreover, the expression score of IRF7 is associated with cervical lymph-node metastasis, which reflects the highly metastatic nature of this cancer. Taken together, our results suggest that expression of IRF7 is one of the metastatic effectors of LMP1 signalling in EBV-associated NPC.

Olfactory Dysfunction in IgG4-Related Disease.

IgG4-related disease is a newly recognized systemic disease, and its elucidation is progressing. However, little is known about its sinonasal manifestations. The aim of this study was to assess the olfaction of patients with IgG4-related disease. Twenty-five patients with IgG4-related disease underwent T&T olfactometry to measure olfactory function. We analyzed the clinical features, including serum IgG4 and IgE levels, involved organs, and sinonasal computed tomography scores to explore the etiology of olfactory dysfunction. Thirteen patients with IgG4-related disease were found to have moderate to severe olfactory dysfunction (52%). There were no differences in the clinical features between the olfactory dysfunction group and the normal group. In 7 patients, the inferior turbinate was biopsied to study the correlation between olfaction score and the number of IgG4-positive cells, but no such correlation was found. Six hyposmia patients recovered to a normal state. Five patients recovered after corticosteroid treatment and 1 recovered spontaneously. We found that the prevalence of olfactory dysfunction was high in patients with IgG4-related disease and that it could be reversed. Olfactory dysfunction appears to be a novel important manifestation of IgG4-related disease.

Daily self-reported behavioural efficacy records on hypertension digital therapeutics as digital metrics associated with the reduction in morning home blood pressure: post-hoc analysis of HERB-DH1 trial.

Digital therapeutics for hypertension, proven effective in the HERB-DH1 trial, enable patients to record and track their daily actions and achievements to improve their lifestyles using an app. However, the association between recording daily behaviours and blood pressure (BP) reduction has not yet been investigated. We conducted a post-hoc analysis to investigate the relationship between them in the HERB-DH1 trial. We defined the counts of daily records of antihypertensive behaviour taken by the patient into the app as daily self-reported behavioural efficacy records (SER). SER was categorised into quartiles, and the trend of changes from baseline to week 12 in morning home systolic blood pressure (SBP), salt intake checklist score, and body weight was assessed. A total of 156 patients with hypertension were included in the analysis. A higher total count of SER was associated with greater SBP reduction (P for trend: 0.049). Patients with a higher SER for salt intake and weight reduction showed reductions in SBP (P for trend: 0.034 and 0.027, respectively). Furthermore, patients with higher salt intake SER exhibited a decrease in the salt intake checklist scores, and patients with greater weight reduction SER experienced a reduction in body weight (P for trend: 0.001 and 0.007, respectively). SER during digital therapeutics is associated with a reduction in morning home SBP in patients with hypertension. Enhancing patients' intrinsic motivation and self-efficacy, as evaluated by the SER, can play an important role in reducing BP by promoting lifestyle improvement. Daily self-reported behavioural efficacy records (SER) defined as the number of patient's app inputs of recall of day-by-day activity of behaviours at the end of the day, is partially affected by self-efficacy and affinity of app, resulting in the effectiveness of digital therapeutics.

Upregulation of special AT-rich-binding protein 1 by Epstein-Barr virus latent membrane protein 1 in human nasopharyngeal cells and nasopharyngeal cancer.

A global regulator of chromatin remodelling and gene expression, special AT-rich-binding protein 1 (SATB1) has been implicated in promotion of growth and metastasis of a number of cancers. Here, we demonstrate that the principal oncogene of Epstein-Barr virus (EBV), latent membrane protein 1 (LMP1) upregulates SATB1 RNA and protein expression in human nasopharyngeal cell lines. Silencing of endogenously expressed SATB1 with specific short hairpin RNA decreases cell proliferation and resistance to apoptosis induced by growth factor withdrawal. Additionally, we provide evidence that LMP1-mediated expression of Survivin, a multifunctional protein involved in promoting cell growth and survival, is mediated at least in part by SATB1 in human nasopharyngeal cells. Finally, we show that SATB1 protein levels are elevated in tissue samples from patients with nasopharyngeal carcinoma (NPC), and are directly correlated with the expression of LMP1. Taken together, our results suggest that SATB1 functions as a pro-metastatic effector of LMP1 signalling in EBV-positive NPC.

Inhibition of autophagy by chloroquine makes chemotherapy in nasopharyngeal carcinoma more efficient.

OBJECTIVES: A combination of platinum-based chemotherapy and radiotherapy is the standard treatment for nasopharyngeal carcinoma (NPC). However, the efficacy of chemotherapy has reached a plateau. Many autophagy studies suggest that autophagy can either promote or suppress to cancer progression. Thus, a role of autophagy in the acquisition of chemoradioresistance has recently been a notable event. Therefore, we examined the relationship between autophagy and chemotherapy in NPC. METHODS: The expression of Beclin 1 and microtubule-associated protein light chain 3 (LC3), a marker of autophagy, was determined by immunohistochemistry in the biopsy samples of patients with NPC before and after the first course of chemotherapy. Additionally, to investigate in the effect of autophagy suppression in chemotherapy, NPC cell line C666-1 cells were treated with cisplatin and/or chloroquine, an inhibitor of autophagy. RESULTS: The expression of Beclin 1 increased after chemotherapy in all patients. In NPC cell line C666-1, compared to cisplatin alone, combination therapy (cisplatin and chloroquine) reduced cell viability, and promoted cell apoptosis. CONCLUSIONS: These results suggest that autophagy, represented by Beclin 1, is upregulated after chemotherapy in both in vitro and in vivo NPC studies. Inhibition of autophagy could therefore be new strategy for NPC treatment.

Siah-1 is associated with expression of hypoxia-inducible factor-1α in oral squamous cell carcinoma.

OBJECTIVE: Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of the HIF-1α and HIF-1ß subunits. HIF-1 is a central regulator of responses to hypoxia; it enhances metastasis-related factors such as matrix metalloproteinases and vascular endothelial growth factor (VEGF). We have reported critical roles for HIF-1α in tumor microenvironments, and oncogenic properties of HIF-1α have been suggested in malignancies. Seven in absentia homologue (Siah) appeared to upregulate HIF-1 production, which prompted us to investigate the Siah association with HIF-1α expression in oral squamous cell carcinoma (OSCC). METHODS: Samples from fifty-five patients with OSCC were evaluated by immunohistochemistry for the protein expressions of Siah-1 and -2, HIF-1α, and VEGF. The expression levels of each protein and clinicopathological data were statistically analyzed. RESULTS: Siah-1 and, Siah-2, HIF-1α, and VEGF were immunolocalized on the cell membranes and cytoplasm of the tumor cells. The expression of Siah-1 showed a linear dependence on the expression of HIF-1α (r=0.627, p<0.001). In 17 cases of the large tumor size category (T3 and 4), the mean Siah-1 expression score was significantly higher than in 41 cases of the small tumor size category (T1 and 2; p=0.001). In addition, in 16 cases of the lymph node metastasis-positive category (N1-3), the mean Siah-1 expression score was significantly higher than that in 42 cases of the lymph node metastasis-negative category (N0, p=0.001). CONCLUSION: These results suggested that the expressions of Siah-1 and HIF-1α were clearly correlated in OSCC. Moreover, Siah-1 appears to be correlated with clinicopathological data, particularly tumor size.

T-status and an oral fluoropyrimidine, S-1, adjuvant chemotherapy are prognostic factors in reduced-RADPLAT for resectable hypopharyngeal cancer.

CONCLUSION: Reduced-RADPLAT for HPC achieved comparative survival and locoregional control rates with lower toxicities compared with concurrent chemoradiotherapies including original RADPLAT. S-1 adjuvant chemotherapy showed a survival benefit. OBJECTIVES: To evaluate the efficacy and toxicities of targeted intra-arterial (IA) infusion of cisplatin with concurrent radiotherapy with a reduced dose (reduced-RADPLAT) for resectable hypopharyngeal cancer (HPC). METHODS: Between 1999-2012, 50 patients with stage II-IVA HPC primarily treated by reduced-RADPLAT were analyzed. They were treated by 2-5 courses of IA cisplatin infusion (100 mg per body) with simultaneous systemic infusion of sodium thiosulfate concurrent with conventional radiotherapy (66-70 Gy). After 2003, S-1, an oral fluoropyrimidine, adjuvant chemotherapy was administered to all eligible patients. RESULTS: During a median follow-up of 48.6 months, the estimated 3- and 5-year overall survival (OS), progression-free survival (PFS), locoregional control, and laryngoesophageal dysfunction-free survival (LEDFS) rates were 76.0% and 62.0%, 58.0% and 50.0%, 66.0% and 62.0%, and 56.0% and 54.0%, respectively. Grade 3 toxicities were observed in 30.0%. No patient had grade 4 or higher toxicities. No patient required tube feeding or tracheotomy at 3 months after treatment. T4-lesions and S-1 administration were significant factors predicting poor and good OS, PFS, and LEDFS, respectively.

Potential Interest in Circulating miR-BART17-5p As a Post-Treatment Biomarker for Prediction of Recurrence in Epstein-Barr Virus-Related Nasopharyngeal Carcinoma.

OBJECTIVES: Epstein-Barr virus (EBV)-related micoRNAs (miRNAs), BamHI-A rightward transcripts (BART)-miRNAs, are released in a stable form from viable cells, which are abundant in patients with EBV-positive nasopharyngeal carcinoma (NPC). We estimated copy numbers of circulating miR-BART2-5p, miR-BART17-5p, and miR-BART18-5p as well as BamHI-W DNA as biomarkers. MATERIALS AND METHODS: Serums from 31 EBV-positive (confirmed by in situ hybridization for EBV-encoded small RNAs) NPC patients and 40 non-NPC controls were analyzed. Among the 31 NPC patients, serums at the initial diagnosis and three months after treatment were obtained from 20 patients, and serums only at three months after treatment were obtained from 11 patients. RESULTS: The sensitivity/specificity of circulating BamHI-W DNA, miR-BART2-5p, miR-BART17-5p, and miR-BART18-5p for the diagnosis of NPC before treatment were 100 / 100, 85 / 85, 60 / 95, and 25 / 100%, respectively. For BamHI-W DNA, NPC patients with stage IV disease had significantly higher copy numbers than those with I-III. Copy numbers decreased significantly post-treatment. In contrast, copy numbers of the three BART-miRNAs showed no significant correlation with the clinical stage at diagnosis or any significant post-treatment change. After treatment, BamHI-W DNA and miR-BART17-5p were detected in 5 and 6 cases out of 11 patients with recurrent or residual tumors, respectively. However, BamHI-W DNA and miR-BART17-5p were absent in all 20 patients without relapse or residual tumors. CONCLUSION: The copy number of circulating BamHI-W DNA is a more useful biomarker for the initial diagnosis of NPC than the three BART-miRNAs examined. Post-treatment detection of miR-BART17-5p is a potential biomarker of a poor prognosis.

Immunoglobulin class switching to IgG4 in Warthin tumor and analysis of serum IgG4 levels and IgG4-positive plasma cells in the tumor.

We previously reported a case of immunoglobulin (Ig)G4-related immune inflammation in Warthin tumor. Increased serum IgG4 levels and tissue infiltration of IgG4-positive plasma cells are characteristics of IgG4-related disease (IgG4-RD), a newly emerging clinicopathological entity. However, the relationship between IgG4-RD and Warthin tumor remains to be elucidated. We aimed to investigate the involvement of systemic and local IgG4 production and class-switch recombination in Warthin tumor. We examined serum IgG4 levels and also analyzed the involvement of IgG4-positive plasma cells in Warthin tumors (18 cases) compared with those of pleomorphic adenomas (19 cases) as controls. Furthermore, in specimens of Warthin tumors (3 cases), pleomorphic adenomas (2 cases), and IgG4-RDs (2 cases), we examined messenger RNA expression of activation-induced cytidine deaminase, IgG4 germline transcripts and productive IgG4 by reverse transcription polymerase chain reaction. Serum IgG4 levels were increased in 5 of 18 Warthin tumors and not in any of the 19 pleomorphic adenomas. Infiltration of IgG4-positive plasma cells was detected in 4 Warthin tumors and none in the pleomorphic adenomas. Moreover, activation-induced cytidine deaminase, IgG4 germline transcripts, and productive IgG4 messenger RNA were found to be expressed in 2 of 3 Warthin tumors as well as IgG4-RDs by reverse transcription polymerase chain reaction, but not in pleomorphic adenomas. In conclusion, immunoglobulin class switching to IgG4 may be involved in the pathogenesis of Warthin tumor, and it is possible that certain inflammatory background with an immune reaction is involved in the pathogenesis of Warthin tumor.

Warthin's tumor associated with IgG4-related disease.

Immunoglobulin G4-related disease (IgG4-RD) is an inflammatory condition associated with elevated serum IgG4 levels and tissue infiltration by IgG4-expressing plasma cells. Several inflammatory conditions associated with IgG4-RD have been reported. Warthin's tumor is a benign parotid gland tumor consisting of oncocytic epithelial cells and lymphoid stroma containing lymphoid follicles with reactive germinal centers. This is the first report describing a case of Warthin's tumor with possible involvement of IgG4-RD. The patient was a 71-year-old man presenting with swollen right parotid and bilateral submandibular glands. He had a history of a Warthin's tumor of the left parotid gland, autoimmune pancreatitis, and an inflammatory abdominal aortic aneurysm. Laboratory tests revealed a high serum IgG4 level. Histological examination of the resected parotid gland showed a Warthin's tumor and a nodule showing severe lymphocytic infiltration containing many IgG4-positive plasma cells. This case shows the possible involvement of Warthin's tumor with IgG4-RD.