Tomoko Morita

Dual Regulation of the Transcriptional Activity of Nrf1 by β-TrCP- and Hrd1-Dependent Degradation Mechanisms

ABSTRACT A growing body of evidence suggests that Nrf1 is an inducible transcription factor that maintains cellular homeostasis. Under physiological conditions, Nrf1 is targeted to the endoplasmic reticulum (ER), implying that it translocates into the nucleus in response to an activating signal. However, the molecular mechanisms by which the function of Nrf1 is modulated remain poorly understood. Here, we report that two distinct degradation mechanisms regulate Nrf1 activity and the expression of its target genes. In the nucleus, β-TrCP, an adaptor for the SCF (Skp1-Cul1-F-box protein) ubiquitin ligase, promotes the degradation of Nrf1 by catalyzing its polyubiquitination. This activity requires a DSGLS motif on Nrf1, which is similar to the canonical β-TrCP recognition motif. The short interfering RNA (siRNA)-mediated silencing of β-TrCP markedly augments the expression of Nrf1 target genes, such as the proteasome subunit PSMC4, indicating that β-TrCP represses Nrf1 activation. Meanwhile, in the cytoplasm, Nrf1 is degraded and suppressed by the ER-associated degradation (ERAD) ubiquitin ligase Hrd1 and valosin-containing protein (VCP) under normal conditions. We identified a cytoplasmic degradation motif on Nrf1 between the NHB1 and NHB2 domains that exhibited species conservation. Thus, these results clearly suggest that both β-TrCP- and Hrd1-dependent degradation mechanisms regulate the transcriptional activity of Nrf1 to maintain cellular homeostasis.

Central nervous system-specific deletion of transcription factor Nrf1 causes progressive motor neuronal dysfunction.

Cap’n’Collar (CNC) proteins heterodimerize with small Maf proteins and regulate the transcription of various genes. Small Maf‐deficient mice develop severe neurodegeneration, and it remains unclear whether CNC proteins are involved in this process. In this study, we examined the contribution of Nrf1, one of the CNC proteins, to neuronal homeostasis in vivo. As Nrf1 gene knockout mice are embryonic lethal, we developed a central nervous system (CNS)‐specific Nrf1 knockout (CKO) mouse line using mice bearing an Nrf1flox allele and Nestin‐Cre allele. At birth, the CKO mice appeared indistinguishable from control mice, but thereafter they showed progressive motor ataxia and severe weight loss. All Nrf1 CKO mice died within 3 weeks. These phenotypes are similar to those reported in small Maf‐deficient mice, suggesting the presence of collaboration between Nrf1 and small Maf proteins. We also found aberrant accumulation of polyubiquitinated proteins in various CNS regions and apparent neuronal loss in the hippocampus of Nrf1 CKO mice. An oxidative stress marker was accumulated in the spinal cords of the mice, but the expression patterns of oxidative stress response genes regulated by Nrf2 did not change substantially. These results show that Nrf1 sustains the CNS homeostasis through regulating target genes distinct from those regulated by Nrf2.

“Giant” colon lipoma: what kind of findings are necessary for the indication of endoscopic resection?

TO THE EDITOR: Gastroesophageal reflux disease (GERD) is a common problem in childhood and characterized by reflux of acidic gastric contents into the esophagus. Major pathophysiological factors for GERD include transient lower esophageal sphincter relaxation, decreased esophageal clearance, and delayed gastric emptying. Gastric acid hypersecretion has also been found in refractory GERD (1). The relationship between Helicobacter pyloriand GERD is not fully understood. It has been shown that there is an inverse correlation and H. pylori may have a protective role against GERD. Ammonia, a powerful neutralizing substance, and hypochlorhydria caused by severe corpus gastritis have been accepted as potential protective mechanisms (2–4). It has also been reported that GERD incidence increased after eradication of H. pylori infection (5). In this study, we wanted to determine the prevalence of H. pylori infection in patients with GERD. A total of 15 patients with GERD (eight boys, 53.3%), 1–8 yr of age (mean 3.3 6 2.5) were included. Diagnosis of GERD was established by 24-h esophageal pH monitoring. The patients had no symptoms of gastritis, and the most common symptoms were vomiting in 13 patients (86.7%), chronic cough in seven (46.7%), and wheezing in two (13.3%). The presence of H. pylori was defined by theH. pylori stool antigen test with a commercial kit (Premier Platinum, Meridian Diagnostics, Cincinnati, OH) using ELISA. The H. pylori stool antigen test is a noninvasive, simple, and fast method, especially in young patients, and has a sensitivity and specificity ranging from 93% to 100% (6, 7). Of the 15 patients, H. pylori was positive in one (6.7%). In our study, the prevalence of H. pylori infection was low, similar to other studies in which the prevalence has been found to be 8–16% (8, 9). Larger controlled studies need to be performed to examine the possible relationship between H. pylori infection and GERD.

The coordinated actions of TIM-3 on cancer and myeloid cells in the regulation of tumorigenicity and clinical prognosis in clear cell renal cell carcinomas

TIM-3 interferes with tumor immunosurveillance. Expression of TIM-3 in patients, on both tumor and immune cells, was found to correlate with poorer outcomes. Mouse and in vitro studies showed that TIM-3 increases tumorigenic properties and treatment resistance. Clear cell renal cell carcinoma (ccRCC) is one of most common cancers in urogenital organs. Although recent experimental and clinical studies have shown the immunogenic properties of ccRCC as illustrated by the clinical sensitivities to various immunotherapies, the detailed immunoregulatory machineries governing the tumorigenicity of human ccRCC remain largely obscure. In this study, we demonstrated the clinical significance and functional relevance of T-cell immunoglobulin and mucin domain-containing molecule-3 (TIM-3) expressed on tumor cells and myeloid cells in patients with ccRCC. TIM-3 expression was detected on cancer cells and CD204+ tumor-associated macrophages (TAM), and higher expression level of TIM-3 was positively correlated with shorter progression-free survival (PFS) in patients with ccRCC. We found that TIM-3 expression was detected on a large number of tumors, and there was significant correlation between an increased number of TAMs and high expression level of TIM-3 in patients with ccRCC. Furthermore, TIM-3 rendered RCC cells with the ability to induce resistance to sunitinib and mTOR inhibitors, the standard regimen for patients with ccRCC, as well as stem cell activities. TIM-3 expression was induced on CD14+ monocytes upon long-term stimulation with RCC cells, and TIM-3–expressing myeloid cells play a critical role in augmenting tumorigenic activities of TIM-3-negative RCC cells. More importantly, treatment with anti–TIM-3 mAb suppressed its tumorigenic effects in in vitro and in vivo settings. These findings indicate the coordinated action of TIM-3 in cancer cells and in myeloid cells regulates the tumorigenicity of human RCC. Cancer Immunol Res; 3(9); 999–1007. ©2015 AACR.

The Casein Kinase 2-Nrf1 Axis Controls the Clearance of Ubiquitinated Proteins by Regulating Proteasome Gene Expression

ABSTRACT Impairment of the ubiquitin-proteasome system (UPS) has been implicated in the pathogenesis of human diseases, including neurodegenerative disorders. Thus, stimulating proteasome activity is a promising strategy to ameliorate these age-related diseases. Here we show that the protein kinase casein kinase 2 (CK2) regulates the transcriptional activity of Nrf1 to control the expression of the proteasome genes and thus the clearance of ubiquitinated proteins. We identify CK2 as an Nrf1-binding protein and find that the knockdown of CK2 enhances the Nrf1-dependent expression of the proteasome subunit genes. Real-time monitoring of proteasome activity reveals that CK2 knockdown alleviates the accumulation of ubiquitinated proteins upon proteasome inhibition. Furthermore, we identify Ser 497 of Nrf1 as the CK2 phosphorylation site and demonstrate that its alanine substitution (S497A) augments the transcriptional activity of Nrf1 and mitigates proteasome dysfunction and the formation of p62-positive juxtanuclear inclusion bodies upon proteasome inhibition. These results indicate that the CK2-mediated phosphorylation of Nrf1 suppresses the proteasome gene expression and activity and thus suggest that the CK2-Nrf1 axis is a potential therapeutic target for diseases associated with UPS impairment.

Analysis of K-ras Codon 12 Mutation in Flat and Nodular Variants of Serrated Adenoma in the Colon

AbstractPURPOSE: The developmental process of serrated adenomas is obscure, and the importance of genetic alterations has not been elucidated clearly. The possibility that the developmental process and genetic alterations of serrated adenomas could differ from those of ordinary tubular adenomas was explored in this work. METHODS: Serrated adenomas were obtained by endoscopic resection (n = 57) and divided into two groups: flat (n = 10) and nodular (n = 47). Mutation of the K-ras gene was analyzed by enriched polymerase chain reaction–enzyme-linked mini-sequence assay, which can detect not only the presence of a mutation but also the mutation type of K-ras codon 12 with high sensitivity. Methylation-specific polymerase chain reaction was performed with specific primers for the DNA repair gene O6-methylguanine-DNA methyltransferase. RESULTS: Serrated adenomas located in the rectum were more likely to have a K-ras mutation (9/12, 75 percent), whereas serrated adenomas of the flat type were less likely to have one (1/10, 10 percent). Furthermore, nodular serrated adenomas that occurred in the rectum possessed a high frequency of K-ras gene codon 12 point mutation (8/10, 80 percent) despite an overall frequency of 46.8 percent (22/47). A mutation of the K-ras codon 12 gene was detected in 23 (40.4 percent) of 57 serrated adenomas. Three types of point mutations of codon 12 were detected, with the mutation of GAT being observed most frequently. CONCLUSIONS: This study shows that development of nodular serrated adenomas may depend on the mutation of the K-ras codon 12 gene, whereas development of flat serrated adenomas may not. Additionally, serrated adenomas that occur in the rectum are closely related to the mutation of the K-ras codon 12 gene. K-ras mutations in serrated adenomas may be unaffected by the epigenetic silencing of O6-methylguanine-DNA methyltransferase by promoter hypermethylation.

A case of early-stage gastric plasmacytoma.

Extramedullary plasmacytomas are rare, and mostly occur in the upper respiratory tract.1 Those in the gastrointestinal tract2 and stomach constitute approximately 10% and 5%, respectively, of all extramedullary plasmacytomas.2 In particular, cases of early-stage gastric plasmacytoma in which neoplastic cells are confined to the mucosa or submucosa have been extremely rare.3 We report here a case of an early stage, superficially spreading type of gastric plasmacytoma in which the endoscopic features changed after a few months’ interval. Helicobacter pylori were found histopathologically in biopsy specimens. As the mucosal lesion was markedly improved endoscopically, but histologically unchanged after H. pylori eradication, a subtotal gastrectomy was performed. The eradication of H. pylori may improve the endoscopic findings but not affect the histological findings of atypical plasma cell infiltration in gastric plasmacytoma. A 77-year-old man was referred to the medical clinic with nausea that had begun 2 months earlier. In an upper endoscopic study performed on July 7, 1999, irregular reddish mucosa with a shallow ulcer was detected in the anterior wall of the antrum (Fig. 1A). In biopsy specimens, plasmacytoma was suspected. The patient was then referred to our hospital for further evaluation of the gastric lesion, on August 5, 1999. Physical examination on admission showed no remarkable findings. Neither an abdominal mass nor peripheral lymph nodule swelling was noted. Urinalysis, blood analysis, proteinogram, and blood chemistry showed no unusual findings. Bence Jones’ protein was also not detectable in a concentrated urine sample. M protein was not detected. Serum immunoglobulin values were within normal limits. Letter to the editor

Endoscopic Resection of a Duodenal Gangliocytic Paraganglioma

A gangliocytic paraganglioma (GP) is an extremely rare neurogenic tumor nearly located in the second portion of the duodenum and has been regarded as benign. We report a case of duodenal GP in a 53-year-old man. The GP arose in the second portion of the duodenum and was shown by histological examination to consist of epithelioid cells, spindle cells, and ganglion-like cells. An endoscopic ultrasonography showed a round, well-demarcated, inhomogeneous, submucosal tumor in the 3rd-4th layer. We resected it by endoscopic mucosal resection.

Novel chemoimmunotherapeutic strategy for hepatocellular carcinoma based on a genome-wide association study

Pharmacotherapeutic options are limited for hepatocellular carcinoma (HCC). Recently, we identified the anti-tumor ligand MHC class I polypeptide-related sequence A (MICA) gene as a susceptibility gene for hepatitis C virus-induced HCC in a genome-wide association study (GWAS). To prove the concept of HCC immunotherapy based on the results of a GWAS, in the present study, we searched for drugs that could restore MICA expression. A screen of the FDA-approved drug library identified the anti-cancer agent vorinostat as the strongest hit, suggesting histone deacetylase inhibitors (HDACis) as potent candidates. Indeed, the HDACi-induced expression of MICA specific to HCC cells enhanced natural killer (NK) cell-mediated cytotoxicity in co-culture, which was further reinforced by treatment with an inhibitor of MICA sheddase. Similarly augmented anti-tumor activity of NK cells via NK group 2D was observed in vivo. Metabolomics analysis revealed HDACi-mediated alterations in energy supply and stresses for MICA induction and HCC inhibition, providing a mechanism for the chemoimmunotherapeutic actions. These data are indicative of promising strategies for selective HCC innate immunotherapy.

The cell competition-based high-throughput screening identifies small compounds that promote the elimination of RasV12-transformed cells from epithelia.

Recent studies have revealed that cell competition can occur between normal and transformed epithelial cells; normal epithelial cells recognize the presence of the neighboring transformed cells and actively eliminate them from epithelial tissues. Here, we have established a brand-new high-throughput screening platform that targets cell competition. By using this platform, we have identified Rebeccamycin as a hit compound that specifically promotes elimination of RasV12-transformed cells from the epithelium, though after longer treatment it shows substantial cytotoxic effect against normal epithelial cells. Among several Rebeccamycin-derivative compounds, we have found that VC1-8 has least cytotoxicity against normal cells but shows the comparable effect on the elimination of transformed cells. This cell competition-promoting activity of VC1-8 is observed both in vitro and ex vivo. These data demonstrate that the cell competition-based screening is a promising tool for the establishment of a novel type of cancer preventive medicine.