Tsutomu Ohta

Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy

The nuclear factor E2-related factor 2 (Nrf2) is a master transcriptional activator of genes encoding numerous cytoprotective enzymes that are induced in response to environmental and endogenously derived oxidative/electrophilic agents. Under normal, nonstressed circumstances, low cellular concentrations of Nrf2 are maintained by proteasomal degradation through a Keap1-Cul3-Roc1-dependent mechanism. A model for Nrf2 activation has been proposed in which two amino-terminal motifs, DLG and ETGE, promote efficient ubiquitination and rapid turnover; known as the two-site substrate recognition/hinge and latch model. Here, we show that in human cancer, somatic mutations occur in the coding region of NRF2, especially among patients with a history of smoking or suffering from squamous cell carcinoma; in the latter case, this leads to poor prognosis. These mutations specifically alter amino acids in the DLG or ETGE motifs, resulting in aberrant cellular accumulation of Nrf2. Mutant Nrf2 cells display constitutive induction of cytoprotective enzymes and drug efflux pumps, which are insensitive to Keap1-mediated regulation. Suppression of Nrf2 protein levels by siRNA knockdown sensitized cancer cells to oxidative stress and chemotherapeutic reagents. Our results strongly support the contention that constitutive Nrf2 activation affords cancer cells with undue protection from their inherently stressed microenvironment and anti-cancer treatments. Hence, inactivation of the Nrf2 pathway may represent a therapeutic strategy to reinforce current treatments for malignancy. Congruously, the present study also provides in vivo validation of the two-site substrate recognition model for Nrf2 activation by the Keap1-Cul3-based E3 ligase.

Loss of Keap1 Function Activates Nrf2 and Provides Advantages for Lung Cancer Cell Growth

Oxidative and electrophilic stresses are sensed by Keap1, which activates Nrf2 to achieve cytoprotection by regulating the expression of drug-metabolizing and antioxidative stress enzymes/proteins. Because oxidative and electrophilic stresses cause many diseases, including cancer, we hypothesized that an abnormality in the Nrf2-Keap1 system may facilitate the growth of cancer cells. We sequenced the KEAP1 gene of 65 Japanese patients with lung cancer and identified five nonsynonymous somatic mutations at a frequency of 8%. We also identified two nonsynonymous somatic KEAP1 gene mutations and two lung cancer cell lines expressing KEAP1 at reduced levels. In lung cancer cells, low Keap1 activity (due to mutations or low-level expression) led to nuclear localization and constitutive activation of Nrf2. The latter resulted in constitutive expression of cytoprotective genes encoding multidrug resistance pumps, phase II detoxifying enzymes, and antioxidative stress enzymes/proteins. Up-regulation of these target genes in lung cancer cells led to cisplatin resistance. Nrf2 activation also stimulated growth of lung cancer-derived cell lines expressing KEAP1 at low levels and in mutant cell lines and in Keap1-null mouse embryonic fibroblasts under homeostatic conditions. Thus, inhibition of NRF2 may provide new therapeutic approaches in lung cancers with activation of Nrf2.

Complex Formation and Functional Versatility of Mre11 of Budding Yeast in Recombination

Meiotic recombination of S. cerevisiae contains two temporally coupled processes, formation and processing of double-strand breaks (DSBs). Mre11 forms a complex with Rad50 and Xrs2, acting as the binding core, and participates in DSB processing. Although these proteins are also involved in DSB formation, Mre11 is not necessarily holding them. The C-terminal region of Mre11 is required only for DSB formation and binds to some meiotic proteins. The N-terminal half specifies nuclease activities that are collectively required for DSB processing. Mre11 has a DNA-binding site for DSB formation and another site for DSB processing. It has two regions to bind to Rad50. Mre11 repairs methyl methanesulfonate-induced DSBs by reactions that require the nuclease activities and those that do not.

Genetic Alteration of Keap1 Confers Constitutive Nrf2 Activation and Resistance to Chemotherapy in Gallbladder Cancer

BACKGROUND & AIMS Biliary tract cancer (BTC) is a highly malignant tumor, and identification of effective therapeutic targets to improve prognosis is urgently required. Oncogenic activation of survival genes is important for cancer cells to overcome oxidative stresses induced by their microenvironments that include chronic inflammation or exposure to anticancer drugs. We attempted to examine whether deregulation of Nrf2, a master transcriptional factor of various cytoprotective genes against oxidative stress, plays a role in the carcinogenesis of BTC. METHODS We screened genetic alteration of Keap1, a negative regulator of Nrf2, in BTC including tumors originated from gallbladder and extra- and intrahepatic bile ducts. Functional analysis of cancer-related mutant Keap1 in Nrf2 repression and the association between Nrf2 activation and resistance to 5-fluorouracil (5-FU) were investigated. RESULTS Recurrent (in 1/11 cell lines and 6/53 primary tumors) Keap1 gene alterations were observed in BTC and were especially frequent (4/13, 30.7%) in gallbladder cancer (GBC). These alterations led to a considerable loss of Nrf2 repression activity, caused constitutive activation of Nrf2, and promoted cell proliferation. Down-regulation of Nrf2 activity by either Keap1 complementation or Nrf2 short interference RNA increased sensitivity to 5-FU in Keap1-altered BTC cells. CONCLUSIONS Keap1 mutation occurs frequently in GBC. Aberrant Nrf2 activation provoked by Keap1 alteration is one of the molecular mechanisms for chemotherapeutic resistance in GBC and will be a novel therapeutic target as an enhancer of sensitivity to 5-FU-based regimens.

Rad52 forms ring structures and co-operates with RPA in single-strand DNA annealing.

The RAD52 epistasis group in Saccharomyces cerevisiae is involved in various types of homologous recombination including recombinational double‐strand break (DSB) repair and meiotic recombination. A RecA homologue, Rad51, plays a pivotal role in homology search and strand exchange. Genetic analysis has shown that among members of its epistasis group, RAD52 alone is required for recombination between direct repeats yielding deletions. Very little has been discovered about the biochemical roles and structure of the Rad52 protein.

Expression profiling in ovarian clear cell carcinoma: Identification of hepatocyte nuclear factor-1β as a molecular marker and a possible molecular target for therapy of ovarian clear cell carcinoma

Of all of the epithelial ovarian cancers, clear cell carcinoma (CCC) of the ovary has the worst prognosis. We applied the oligonucleotide array technique to identify genes generally involved in CCC. Of the approximately 12,600 genes that were analyzed, 28 were expressed significantly differently between four CCC and seven non-CCC cell lines. Among 16 up-regulated genes in CCC, we further investigated a transcription factor, hepatocyte nuclear factor-1 beta (HNF-1 beta). We validated up-regulation of HNF-1 beta in CCC in terms of both mRNA and protein level using real-time quantitative reverse transcriptase-polymerase chain reaction and immunoblotting. Immunohistochemical analysis of 83 surgically resected ovarian cancers showed that almost all CCC specimens (21 of 22 cases) had nuclear staining for HNF-1 beta, whereas most non-CCC specimens (60 of 61 cases) showed no immunostaining or only focal and faint staining in the nucleus. Furthermore, we investigated the significance of HNF-1 beta expression in CCC using RNA interference. The reduction of HNF-1 beta expression by RNA interference induced apoptotic cell death in ovarian CCC cells, which was confirmed by terminal dUTP nick-end labeling and fluorescence-activated cell-sorting analyses. Our results suggest that HNF-1 beta is not only an excellent CCC-specific molecular marker but also a molecular target for therapy of ovarian CCC.

Unique function of the Nrf2-Keap1 pathway in the inducible expression of antioxidant and detoxifying enzymes.

Publisher Summary This chapter examines Nrf2 and other cap “n” collar (CNC) family factors—namely Nrf1 and Nrf3, which cross-talk with the Nrf2–Keap1 regulatory pathway. The emergence of the Nrf2–Keap1 regulatory system attracts a wide range of interests from a variety of fields in medical and biological sciences. The chapter reveals, through a consensus binding sequence comparison with antioxidant responsive element (ARE), that transcription factor Nrf2 (NF-E2–related factor 2) might be an important regulatory factor, which activates transcription of these cytoprotective genes. Cellular homeostasis against oxidative stress and xenobiotic insults is accomplished by the coordinate expression of antioxidant and drug detoxifying genes. These cytoprotective genes include the phase 2 detoxification enzyme genes, such as NAD(P)H:quinone oxidoreductase (NQO1) and glutathione S-transferases (GSTs); and antioxidant genes, such as heme oxygenase 1 (HO-1) and γ-glutamylcysteine synthetase (γ-GCS). Extensive analyses of the regulatory mechanisms for the phase 2 enzyme genes revealed that the inducible expression of these enzymes is attained at the transcriptional level through a cis-acting element called ARE or electrophile responsive elements (EpRE). Examination of Nrf3-deficient mice also supports the contention that Nrf3 does not contribute to the inducible expression of the nqo1 gene in the liver and intestine. These results, taken together, argue that the Nrf2–Keap1 regulatory system is the mainstream pathway for the regulation of antioxidant and phase 2 detoxifying enzyme gene expressions.

Gene expression analysis of soft tissue sarcomas: characterization and reclassification of malignant fibrous histiocytoma

In soft tissue sarcomas, the diagnosis of malignant fibrous histiocytoma (MFH) has been a very controversial issue, and MFH is now considered to be reclassified into pleomorphic subtypes of other sarcomas. To characterize MFH genetically, we used an oligonucleotide microarray to analyze gene expression in 105 samples from 10 types of soft tissue tumors. Spindle cell and pleomorphic sarcomas, such as dedifferentiated liposarcoma, myxofibrosarcoma, leiomyosarcoma, malignant peripheral nerve sheath tumor (MPNST), fibrosarcoma and MFH, showed similar gene expression patterns compared to other tumors. Samples from those five sarcoma types could be classified into respective clusters based on gene expression by excluding MFH samples. We calculated distances between MFH samples and other five sarcoma types (dedifferentiated liposarcoma, myxofibrosarcoma, leiomyosarcoma, MPNST and fibrosarcoma) based on differentially expressed genes and evaluated similarities. Three of the 21 MFH samples showed marked similarities to one of the five sarcoma types, which were supported by histological findings. Although most of the remaining 18 MFH samples showed little or no histological resemblance to one of the five sarcoma types, 12 of them showed moderate similarities in terms of gene expression. These results explain the heterogeneity of MFH and show that the majority of MFHs could be reclassified into pleomorphic subtypes of other sarcomas. Taken together, gene expression profiling could be a useful tool to unveil the difference in the underlying molecular backgrounds, which leads to a rational taxonomy and diagnosis of a diverse group of soft tissue sarcomas.

Overexpression of KIT in chromophobe renal cell carcinoma

We analysed gene-expression profiles in 15 surgical specimens of conventional, papillary, and chromophobe renal cell carcinomas (RCCs) using high-density oligonucleotide arrays. From about 12 000 genes targeted by the array, 67 were upregulated specifically in each histological type of RCC. The oncogene KIT was one of the genes whose expression was upregulated specifically in chromophobe RCCs. Immunohistochemical analysis demonstrated the KIT gene product on the cell membrane of chromophobe RCC in all cases, although it was not detected in conventional RCCs or non-neoplastic kidneys except for weak staining in the cytoplasm of renal tubules. These results suggest that each histological subtype of RCC has a unique gene-expression profile, and in particular indicates for the first time that KIT could be a useful marker for chromophobe RCC. As overexpression of KIT might be involved in tumor growth, KIT could be a new therapeutic target in this special type of RCC.

BRG1 Interacts with Nrf2 To Selectively Mediate HO-1 Induction in Response to Oxidative Stress

ABSTRACT NF-E2-related factor 2 (Nrf2) regulates antioxidant-responsive element-mediated induction of cytoprotective genes in response to oxidative stress. The purpose of this study was to determine the role of BRG1, a catalytic subunit of SWI2/SNF2-like chromatin-remodeling complexes, in Nrf2-mediated gene expression. Small interfering RNA knockdown of BRG1 in SW480 cells selectively decreased inducible expression of the heme oxygenase 1 (HO-1) gene after diethylmaleate treatment but did not affect other Nrf2 target genes, such as the gene encoding NADPH:quinone oxidoreductase 1 (NQO1). Chromatin immunoprecipitation analysis revealed that Nrf2 recruits BRG1 to both HO-1 and NQO1 regulatory regions. However, BRG1 knockdown selectively decreased the recruitment of RNA polymerase II to the HO-1 promoter but not to the NQO1 promoter. HO-1, but not other Nrf2-regulated genes, harbors a sequence of TG repeats capable of forming Z-DNA with BRG1 assistance. Similarly, replacement of the TG repeats with an alternative Z-DNA-forming sequence led to BRG1-mediated activation of HO-1. These results thus demonstrate that BRG1, through the facilitation of Z-DNA formation and subsequent recruitment of RNA polymerase II, is critical in Nrf2-mediated inducible expression of HO-1.