Research IndicatorsGraph generated 25 June 2015 using data from PubMed using criteria.
Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic. Tag cloud generated 25 June, 2015 using data from PubMed, MeSH and CancerIndex
Specific Cancers (4)
Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.
Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).
OMIM, Johns Hopkin University
Referenced article focusing on the relationship between phenotype and genotype.
International Cancer Genome Consortium.
Summary of gene and mutations by cancer type from ICGC
Cancer Genome Anatomy Project, NCI
COSMIC, Sanger Institute
Somatic mutation information and related details
Search the Epigenomics database and view relevant gene tracks of samples.
Latest Publications: CUL3 (cancer-related)
van der Wijst MG, Brown R, Rots MGNrf2, the master redox switch: the Achilles' heel of ovarian cancer?
Biochim Biophys Acta. 2014; 1846(2):494-509 [PubMed
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Ovarian cancer is the most lethal gynecological tumor type in the world due to late stage detection, and resistance to chemotherapy. Therefore, alternative additional therapies are required. The etiology of ovarian cancer remains largely unknown, but risk factors point toward an important role for oxidative stress. Both healthy and tumor cells can cope with oxidative stress by activating the transcription factor Nrf2 (also known as Nfe2l2), the master regulator of antioxidant and cytoprotective genes. Indeed, for most ovarian cancers, aberrant activation of Nrf2 is observed, which is often associated with a copy number loss within the Nrf2-inhibitory complex KEAP1-CUL3-RBX1. A key role for Nrf2 in ovarian carcinogenesis has been validated by siRNA studies. However, to exploit the Nrf2 pathway for therapeutic interventions, potential side-effects should be minimized. In this review, we explore ovarian cancer specific factors with links to aberrant activity of Nrf2, to be exploited in future combination strategies, synergistic with direct Nrf2 inhibitory drugs. Particularly, we propose to stratify patients based on common ovarian cancer mutations (KRAS, BRAF, ERBB2, BRCA1 and its link with estradiol, TP53) for future NRF2 targeting strategies.
The NFE2-related factor 2 (NRF2) pathway is critical to initiate responses to oxidative stress; however, constitutive activation occurs in different cancer types, including serous ovarian carcinomas (OVCA). The KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex is a regulator of NRF2 levels. Hence, we investigated the DNA-level mechanisms affecting these genes in OVCA. DNA copy-number loss (CNL), promoter hypermethylation, mRNA expression, and sequence mutation for KEAP1, CUL3, and RBX1 were assessed in a cohort of 568 OVCA from The Cancer Genome Atlas. Almost 90% of cases exhibited loss-of-function alterations in any components of the NRF2 inhibitory complex. CNL is the most prominent mechanism of component disruption, with RBX1 being the most frequently disrupted component. These alterations were associated with reduced mRNA expression of complex components, and NRF2 target gene expression was positively enriched in 90% of samples harboring altered complex components. Disruption occurs through a unique DNA-level alteration pattern in OVCA. We conclude that a remarkably high frequency of DNA and mRNA alterations affects components of the KEAP1/CUL3/RBX1 complex, through a unique pattern of genetic mechanisms. Together, these results suggest a key role for the KEAP1/CUL3/RBX1 complex and NRF2 pathway deregulation in OVCA.
Zhang P, Gao K, Tang Y, et al.Destruction of DDIT3/CHOP protein by wild-type SPOP but not prostate cancer-associated mutants.
Hum Mutat. 2014; 35(9):1142-51 [PubMed
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Characterization of the exome and genome of prostate cancers by next-generation sequencing has identified numerous genetic alternations. SPOP (speckle-type POZ protein) was identified as one of the most frequently affected genes by somatic point mutations in prostate cancer, suggesting SPOP is potentially a key driver for prostate cancer development and progression. However, how SPOP mutations contribute to prostate cancer remains to be elucidated. SPOP acts as an adaptor protein of the CUL3-RBX1 E3 ubiquitin ligase complex and selectively recruits substrates for their ubiquitination and subsequent degradation. DDIT3 is an endoplasmic reticulum (ER) stress-responsive transcription factor playing an essential role in apoptotic execution pathways triggered by ER stress. Here, we identified DDIT3/CHOP as a bona fide substrate for the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP recognizes a Ser/Thr-rich degron in the transactivation domain of DDIT3 and triggers DDIT3 degradation via the ubiquitin-proteasome pathway. Strikingly, prostate cancer-associated mutants of SPOP are defective in promoting DDIT3 degradation. This study reveals novel molecular events underlying the regulation of DDIT3 protein homeostasis and provides insight in understanding the relationship between SPOP mutations and ER stress dysregulation in prostate cancer.
BACKGROUND: Family history of prostate cancer is a well-recognized risk factor. Previous linkage studies have reported a putative prostate cancer susceptibility locus at chromosome 17q21-22. SPOP (Speckle-type POZ protein) maps to the 17q21-22 candidate linkage region and is one of the most frequently mutated genes in sporadic prostate cancers.
METHODS: We performed targeted next generation sequencing to analyze 2009 exons from 202 genes in a candidate linkage region on chromosome 17q21-22 using 94 unrelated familial prostate cancer cases from the University of Michigan Prostate Cancer Genetics Project (n=54) and Johns Hopkins University (n=40) including the exons and UTRs of SPOP.
RESULTS: We identified a novel SPOP missense mutation (N296I) in a man with prostate cancer diagnosed at age 43. This mutation completely segregates with prostate cancer affection status among the men in this family. The N296I mutation resides within the evolutionarily conserved Bric-a-brac, Tramtrack, Broad-complex (BTB) domain, involved in recruiting targets to Cul3 for degradation. Analysis of the prostate tumor from this individual verified the presence of heterozygous N296I as well as an ERG fusion.
CONCLUSIONS: We have discovered a novel mutation in SPOP that tracks with prostate cancer within a family and is predicted to be deleterious. Taken together, our results implicate SPOP as a candidate gene for hereditary prostate cancer.
The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.
Martinez VD, Vucic EA, Pikor LA, et al.Frequent concerted genetic mechanisms disrupt multiple components of the NRF2 inhibitor KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex in thyroid cancer.
Mol Cancer. 2013; 12(1):124 [PubMed
] Free Access to Full Article Related Publications
BACKGROUND: Reactive oxygen species contribute to normal thyroid function. The NRF2 oxidative response pathway is frequently and constitutively activated in multiple tumor types, including papillary thyroid carcinoma (PTC). Genetic mechanisms underlying NRF2 pathway activation in PTC are not fully understood. Thus, we aimed to determine whether inactivating patterns of DNA-level alterations affect genes encoding for individual NRF2 inhibitor complex components (CUL3/KEAP1/RBX1) occur in PTC.
FINDINGS: Combined patterns of epi/genetic alterations for KEAP1/CUL3/RBX1 E3 ubiquitin-ligase complex components were simultaneously interrogated for a panel of 310 PTC cases and 40 adjacent non-malignant tissues. Data were obtained from The Cancer Genome Atlas project. Enrichment of NRF2 pathway activation was assessed by gene-set enrichment analysis using transcriptome data. Our analyses revealed that PTC sustain a strikingly high frequency (80.6%) of disruption to multiple component genes of the NRF2 inhibitor complex. Hypermethylation is the predominant inactivating mechanism primarily affecting KEAP1 (70.6%) and CUL3 (20%), while copy number loss mostly affects RBX1 (16.8%). Concordantly, NRF2-associated gene expression signatures are positively and significantly enriched in PTC.
CONCLUSIONS: The KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex is almost ubiquitously affected by multiple DNA-level mechanisms and downstream NRF2 pathway targets are activated in PTC. Given the importance of this pathway to normal thyroid function as well as to cancer; targeted inhibition of NRF2 regulators may impact strategies for therapeutic intervention involving this pathway.
Glioblastoma is one of the most challenging forms of cancer to treat. Here we describe a computational platform that integrates the analysis of copy number variations and somatic mutations and unravels the landscape of in-frame gene fusions in glioblastoma. We found mutations with loss of heterozygosity in LZTR1, encoding an adaptor of CUL3-containing E3 ligase complexes. Mutations and deletions disrupt LZTR1 function, which restrains the self renewal and growth of glioma spheres that retain stem cell features. Loss-of-function mutations in CTNND2 target a neural-specific gene and are associated with the transformation of glioma cells along the very aggressive mesenchymal phenotype. We also report recurrent translocations that fuse the coding sequence of EGFR to several partners, with EGFR-SEPT14 being the most frequent functional gene fusion in human glioblastoma. EGFR-SEPT14 fusions activate STAT3 signaling and confer mitogen independence and sensitivity to EGFR inhibition. These results provide insights into the pathogenesis of glioblastoma and highlight new targets for therapeutic intervention.
It is already known that the Maitake (D-Fraction) mushroom is involved in stimulating the immune system and activating certain cells that attack cancer, including macrophages, T-cells, and natural killer cells. According to the U.S. National Cancer Institute, polysaccharide complexes present in Maitake mushrooms appear to have significant anticancer activity. However, the exact molecular mechanism of the Maitake antitumoral effect is still unclear. Previously, we have reported that Maitake (D-Fraction) induces apoptosis in breast cancer cells by activation of BCL2-antagonist/killer 1 (BAK1) gene expression. At the present work, we are identifying which genes are responsible for the suppression of the tumoral phenotype mechanism induced by Maitake (D-Fraction) in breast cancer cells. Human breast cancer MCF-7 cells were treated with and without increased concentrations of Maitake D-Fraction (36, 91, 183, 367 μg/mL) for 24 h. Total RNA were isolated and cDNA microarrays were hybridized containing 25,000 human genes. Employing the cDNA microarray analysis, we found that Maitake D-Fraction modified the expression of 4068 genes (2420 were upmodulated and 1648 were downmodulated) in MCF-7 breast cancer cells in a dose-dependent manner during 24 h of treatment. The present data shows that Maitake D-Fraction suppresses the breast tumoral phenotype through a putative molecular mechanism modifying the expression of certain genes (such as IGFBP-7, ITGA2, ICAM3, SOD2, CAV-1, Cul-3, NRF2, Cycline E, ST7, and SPARC) that are involved in apoptosis stimulation, inhibition of cell growth and proliferation, cell cycle arrest, blocking migration and metastasis of tumoral cells, and inducing multidrug sensitivity. Altogether, these results suggest that Maitake D-Fraction could be a potential new target for breast cancer chemoprevention and treatment.
Sato Y, Yoshizato T, Shiraishi Y, et al.Integrated molecular analysis of clear-cell renal cell carcinoma.
Nat Genet. 2013; 45(8):860-7 [PubMed
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Clear-cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer and its molecular pathogenesis is incompletely understood. Here we report an integrated molecular study of ccRCC in which ≥100 ccRCC cases were fully analyzed by whole-genome and/or whole-exome and RNA sequencing as well as by array-based gene expression, copy number and/or methylation analyses. We identified a full spectrum of genetic lesions and analyzed gene expression and DNA methylation signatures and determined their impact on tumor behavior. Defective VHL-mediated proteolysis was a common feature of ccRCC, which was caused not only by VHL inactivation but also by new hotspot TCEB1 mutations, which abolished Elongin C-VHL binding, leading to HIF accumulation. Other newly identified pathways and components recurrently mutated in ccRCC included PI3K-AKT-mTOR signaling, the KEAP1-NRF2-CUL3 apparatus, DNA methylation, p53-related pathways and mRNA processing. This integrated molecular analysis unmasked new correlations between DNA methylation, gene mutation and/or gene expression and copy number profiles, enabling the stratification of clinical risks for patients with ccRCC.
The NF1 tumor suppressor protein neurofibromin is a negative regulator of Ras. Neurofibromin is dynamically regulated by the proteasome, and its degradation and reexpression are essential for maintaining appropriate levels of Ras-GTP. Like p53, NF1/neurofibromin can be inactivated in cancer by both mutations and excessive proteasomal destruction; however, little is known about the mechanisms that underlie this latter process. Here, we show that a Cullin 3 (Cul3)/kelch repeat and BTB domain-containing 7 complex controls both the regulated proteasomal degradation of neurofibromin and the pathogenic destabilization of neurofibromin in glioblastomas. Importantly, RNAi-mediated Cul3 ablation and a dominant-negative Cul3 directly stabilize neurofibromin, suppress Ras and extracellular signal-regulated kinase, and inhibit proliferation in an NF1-dependent manner. Moreover, in glioblastomas where neurofibromin is chronically destabilized, Cul3 inhibition restabilizes the protein and suppresses tumor development. Collectively, these studies show a previously unrecognized role for Cul3 in regulating Ras and provide a molecular framework that can be exploited to develop potential cancer therapies.
Malloy MT, McIntosh DJ, Walters TS, et al.Trafficking of the transcription factor Nrf2 to promyelocytic leukemia-nuclear bodies: implications for degradation of NRF2 in the nucleus.
J Biol Chem. 2013; 288(20):14569-83 [PubMed
] Free Access to Full Article Related Publications
Ubiquitylation of Nrf2 by the Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase complex targets Nrf2 for proteasomal degradation in the cytoplasm and is an extensively studied mechanism for regulating the cellular level of Nrf2. Although mechanistic details are lacking, reports abound that Nrf2 can also be degraded in the nucleus. Here, we demonstrate that Nrf2 is a target for sumoylation by both SUMO-1 and SUMO-2. HepG2 cells treated with As2O3, which enhances attachment of SUMO-2/3 to target proteins, increased SUMO-2/3-modification (polysumoylation) of Nrf2. We show that Nrf2 traffics, in part, to promyelocytic leukemia-nuclear bodies (PML-NBs). Cell fractions harboring key components of PML-NBs did not contain biologically active Keap1 but contained modified Nrf2 as well as RING finger protein 4 (RNF4), a poly-SUMO-specific E3 ubiquitin ligase. Overexpression of wild-type RNF4, but not the catalytically inactive mutant, decreased the steady-state levels of Nrf2, measured in the PML-NB-enriched cell fraction. The proteasome inhibitor MG-132 interfered with this decrease, resulting in elevated levels of polysumoylated Nrf2 that was also ubiquitylated. Wild-type RNF4 accelerated the half-life (t½) of Nrf2, measured in PML-NB-enriched cell fractions. These results suggest that RNF4 mediates polyubiquitylation of polysumoylated Nrf2, leading to its subsequent degradation in PML-NBs. Overall, this work identifies Nrf2 as a target for sumoylation and provides a novel mechanism for its degradation in the nucleus, independent of Keap1.
Nrf2:INrf2 (Keap1) are cellular sensors of oxidative and electrophilic stress. Nrf2 is a nuclear factor that controls the expression and coordinated induction of a battery of genes that encode detoxifying enzymes, drug transporters, antiapoptotic proteins, and proteasomes. In the basal state, Nrf2 is constantly degraded in the cytoplasm by its inhibitor, INrf2. INrf2 functions as an adapter for Cul3/Rbx1 E3 ubiquitin ligase-mediated degradation of Nrf2. Chemicals, including antioxidants, tocopherols including α-tocopherol (vitamin E), and phytochemicals, and radiation antagonize the Nrf2:INrf2 interaction and lead to the stabilization and activation of Nrf2. The signaling events involve preinduction, induction, and postinduction responses that tightly control Nrf2 activation and repression back to the basal state. Oxidative/electrophilic signals activate unknown tyrosine kinases in a preinduction response that phosphorylates specific residues on Nrf2 negative regulators, INrf2, Fyn, and Bach1, leading to their nuclear export, ubiquitination, and degradation. This prepares nuclei for unhindered import of Nrf2. Oxidative/electrophilic modification of INrf2 cysteine 151 followed by PKC phosphorylation of Nrf2 serine 40 in the induction response results in the escape or release of Nrf2 from INrf2. Nrf2 is thus stabilized and translocates to the nucleus, resulting in a coordinated activation of gene expression. This is followed by a postinduction response that controls the "switching off" of Nrf2-activated gene expression. GSK3β, under the control of AKT and PI3K, phosphorylates Fyn, leading to Fyn nuclear localization. Fyn phosphorylates Nrf2 Y568, resulting in nuclear export and degradation of Nrf2. The activation and repression of Nrf2 provide protection against oxidative/electrophilic stress and associated diseases, including cancer. However, deregulation of INrf2 and Nrf2 due to mutations may lead to nuclear accumulation of Nrf2 that reduces apoptosis and promotes oncogenesis and drug resistance.
Ooi A, Dykema K, Ansari A, et al.CUL3 and NRF2 mutations confer an NRF2 activation phenotype in a sporadic form of papillary renal cell carcinoma.
Cancer Res. 2013; 73(7):2044-51 [PubMed
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Sustained activation of the stress-regulated transcription factor NRF2 (NFE2L2) is a prominent feature of many types of cancer, implying that mutations driving NRF2 may be important to tumor progression. In hereditary type 2 papillary renal cell carcinoma (PRCC2, also known as hereditary leiomyomatosis and renal cell cancer), NRF2 activation is a direct consequence of the accumulation of intracellular fumarate, a result of fumarate hydratase (FH) inactivation, but it is not clear how NRF2 may be activated in sporadic forms of PRCC2. Here we show that somatic mutations in NRF2, CUL3, and SIRT1 are responsible for driving the NRF2 activation phenotype in sporadic PRCC2. Transcriptome sequencing revealed the expression pattern of mutant alleles of NRF2, CUL3, and SIRT1 and also confirmed NRF2 activation in clinical specimens. Our results show a convergence in somatic mutations in sporadic PRCC2 with FH mutation in hereditary PRCC2.
To identify aggressiveness-associated molecular mechanisms and biomarker candidates in bladder cancer, we performed a SILAC (Stable Isotope Labelling by Amino acids in Cell culture) proteomic analysis comparing an invasive T24 and an aggressive metastatic derived T24T bladder cancer cell line. A total of 289 proteins were identified differentially expressed between these cells with high confidence. Complementary and validation analyses included comparison of protein SILAC data with mRNA expression ratios obtained from oligonucleotide microarrays, and immunoblotting. Cul3, an overexpressed protein in T24T, involved in the ubiquitination and subsequent proteasomal degradation of target proteins, was selected for further investigation. Functional analyses revealed that Cul3 silencing diminished proliferative, migration and invasive rates of T24T cells, and restored the expression of cytoskeleton proteins identified to be underexpressed in T24T cells by SILAC, such as ezrin, moesin, filamin or caveolin. Cul3 immunohistochemical protein patterns performed on bladder tumours spotted onto tissue microarrays (n = 284), were associated with tumor staging, lymph node metastasis and disease-specific survival. Thus, the SILAC approach identified that Cul3 modulated the aggressive phenotype of T24T cells by modifying the expression of cytoskeleton proteins involved in bladder cancer aggressiveness; and played a biomarker role for bladder cancer progression, nodal metastasis and clinical outcome assessment.
Kim MS, Je EM, Oh JE, et al.Mutational and expressional analyses of SPOP, a candidate tumor suppressor gene, in prostate, gastric and colorectal cancers.
APMIS. 2013; 121(7):626-33 [PubMed
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Mounting evidence exists that alterations of ubiquitination processes are involved in cancer pathogenesis. Speckle-type POZ protein (SPOP) is a key adaptor for Cul3-based ubiquitination process. Recent studies reported that SPOP may be a tumor suppressor gene (TSG) and somatic mutation of SPOP was detected in prostate cancer (PCA). The aim of this study was to see whether alterations of SPOP protein expression and somatic mutation of SPOP gene are features of cancers. In this study, we analyzed SPOP somatic mutation in 45 gastric (GC), 45 colorectal cancer (CRC) and 45 PCA by single-strand conformation polymorphism (SSCP). Also, we analyzed SPOP protein expression in 60 GC, 60 CRC and 60 PCA by immunohistochemistry. Overall, we detected three somatic missense mutations of SPOP gene in the coding sequences (p.Ser14Leu, p.Tyr87Cys and p.Phe133Leu). The mutations were observed in two PCA and one CRC. Of note, the p.Phe133Leu was a recurrent mutation reported in an earlier study. In the immunohistochemistry, SPOP protein was expressed in normal gastric, colonic and prostate epithelial cells, whereas it was lost in 30% of GC, 20% of CRC and 37% of PCA. Our data indicate that loss of SPOP expression was common in GC, CRC and PCA, but somatic mutation of SPOP in this study was rare in these tumors. Also, the data provide a possibility that loss of expression of SPOP gene might play a role in cancer pathogenesis by altering TSG functions of SPOP.
Wu Q, Wang C, Guo L, et al.Identification and characterization of novel microRNA candidates from deep sequencing.
Clin Chim Acta. 2013; 415:239-44 [PubMed
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In our previous study, we screened a candidate new microRNA (miRNA) based on the deep sequencing and bioinformatics analysis. In this paper, we evaluated the novel miRNA in the following experiment: 1) the secondary structure of the precursor of novel-miR has the characteristic of a stem-loop hairpin structure, and mature miRNA is far from loops and bulges. 2) we used BLAST (Basic Local Alignment Search Tool) to compare the novel-miR sequence to that found in the GenBank. Novel-miR sequence existed in Mus musculus, Drosophila grimshawi, Rattus norvegicus, Xenopus laevis, Spodoptera frugiperda, Papio anubis, Salmo salar and so on. Then multiple sequence alignment (MSA) showed that sequence from 5 to 11 bp and 13 to 17 bp exhibited 100% similarity, where there is significant sequence conservation. Novel-miR showed similarity in the seed region with the known miR-3675-3p, indicating that these miRNAs are likely to belong to the same family and thus may share common biology. 3) novel-miR from MCF-7 and MB-MDA-231 was validated by Northern blot and detected in the serum and tissue samples of BC patients, respectively, by real-time PCR. The data showed that novel-miR was downregulated in the BC cancerous tissues and serum of breast cancer patients (P<0.05). 4) transfection of novel-miR mimics into MCF-7 cell significantly inhibited cell growth detected by CCK-8 assay (P<0.05). 5) to identify the mRNA targets of novel-miR, we performed a computational screen for genes with novel-miR complementary sites in their 3'-UTR using several open access databases. In addition, we used the CapitalBio® Molecule Annotation System V3.0 to perform gene ontology (GO) analysis on the target genes of novel-miR and specific biological process categories were enriched. 7 genes (CUL3, KRAS, ETS1, MNT, CNTN3, CCNK and FOXO3) which have a high prediction score and are associated with cell proliferation, apoptosis and cell cycle were chosen. 3'-UTR luciferase report assay suggested that miR-BS1 negatively regulated CNTN3. In the conclusion, novel-miR, named miR-3675b, is a true, functional and novel miRNA. Candidate novel miRNA from deep sequencing which will be qualified as a "real" miRNA must be validated by a series of functional experiments.
Tian H, Zhang B, Di J, et al.Keap1: one stone kills three birds Nrf2, IKKβ and Bcl-2/Bcl-xL.
Cancer Lett. 2012; 325(1):26-34 [PubMed
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Oxidative stress, implicated in the etiology of cancer, results from an imbalance in the production of Reactive Oxygen Species (ROS) and cell's own antioxidant defenses. As a oxidative stress sensor, Keap1 functions as both an adaptor for Cul3⋅Rbx1 E3 ligase complex mediated degradation of the transcription factor Nrf2, and a master regulator of cytoprotective gene expression. Although Nrf2 is a well known substrate for Keap1, the DGR domain of Keap1 has been reported also to bind other proteins directly or indirectly. IKKβ as positive regulator of NF-κB is also destabilized by Keap1, which resulted in inhibiting NF-κB-derived tumor promotion. In addition, anti-apoptotic Bcl-2/Bcl-xL protein was identified as another substrate for the Keap1-Cul3-E3 ligase complex. Keap1 led to the repression and destabilization of Bcl-2, decreased Bcl-2:Bax heterodimers and facilitated cancer cells apoptosis. Given that Keap1 might function as a tumor suppressor protein to mitigate tumor progression, the different kinds of Keap1 somatic mutations were detected in numerous cancer cells. Therefore, it is important to understand the Keap1-involved signaling cascades. This review primarily focuses on the prevention of tumorigenesis role of Keap1 through negative regulation of three substrates Nrf2, IKKβ and Bcl-2/Bcl-xL, with emphasis on the recent findings indicating the cancer guarder function of Keap1.
Toyama T, Yoshida E, Shinkai Y, Kumagai YDNA microarray analysis of human neuroblastoma SH-SY5Y cells exposed to methylmercury.
J Toxicol Sci. 2011; 36(6):843-5 [PubMed
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To investigate the adaptive response to the environmental electrophile methylmercury (MeHg), we performed DNA microarray analysis of human neuroblastoma SH-SY5Y cells exposed to a sub-cytotoxic dose of MeHg (1 µM) for 6 hr. The expression of 15 genes increased 10-fold or more in response to MeHg. Four of these genes are associated with detoxification and excretion of MeHg into the extracellular space, and are regulated by transcription factor Nrf2 through the electrophile response element. Interestingly, Cullin3, a negative regulator of Nrf2, was identified as a down-regulated gene during MeHg exposure.
Kim B, Nam HJ, Pyo KE, et al.Breast cancer metastasis suppressor 1 (BRMS1) is destabilized by the Cul3-SPOP E3 ubiquitin ligase complex.
Biochem Biophys Res Commun. 2011; 415(4):720-6 [PubMed
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Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis without affecting primary tumorigenesis. The regulatory mechanism of BRMS1 at the protein level has not been revealed until recently. Here, we found that cullin 3 (Cul3), a component of E3 ubiquitin ligase, is a new binding partner of BRMS1 and the interaction between BRMS1 and Cul3 is mediated by the SPOP adaptor protein. Intriguingly, BRMS1 turns out to be a potent substrate that is ubiquitinated by the Cul3-SPOP complex. Knockdown of SPOP increases the level of BRMS1 protein and represses the expression of BRMS1 repressive target genes such as OPN and uPA in breast cancer cells. These results suggest that the novel regulatory mechanism of BRMS1 by Cul3-SPOP complex is important for breast cancer progression.
INTRODUCTION: Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB) (IKK-β/IKK-2), which activates NF-κB, is a substrate of the KEAP1-CUL3-RBX1 E3-ubiquitin ligase complex, implicating this complex in NF-κB pathway regulation. We investigated complex component gene disruption as a novel genetic mechanism of NF-κB activation in non-small cell lung cancer.
METHODS: A total of 644 tumor- and 90 cell-line genomes were analyzed for gene dosage status of the individual complex components and IKBKB. Gene expression of these genes and NF-κB target genes were analyzed in 48 tumors. IKBKB protein levels were assessed in tumors with and without complex or IKBKB genetic disruption. Complex component knockdown was performed to assess effects of the E3-ligase complex on IKBKB and NF-κB levels, and phenotypic importance of IKBKB expression was measured by pharmacological inhibition.
RESULTS: We observed strikingly frequent genetic disruption (42%) and aberrant expression (63%) of the E3-ligase complex and IKBKB in the samples examined. Although both adenocarcinomas and squamous cell carcinomas showed complex disruption, the patterns of gene disruption differed. IKBKB levels were elevated with complex disruption, knockdown of complex components increased activated forms of IKBKB and NF-κB proteins, and IKBKB inhibition detriments cell viability, highlighting the biological significance of complex disruption. NF-κB target genes were overexpressed in samples with complex disruption, further demonstrating the effect of complex disruption on NF-κB activity.
CONCLUSIONS: Gene dosage alteration is a prominent mechanism that disrupts each component of the KEAP1-CUL3-RBX1 complex and its NF-κB stimulating substrate, IKBKB. Herein, we show that, multiple component disruption of this complex represents a novel mechanism of NF-κB activation in non-small cell lung cancer.
Steroid receptor co-activator-3 (SRC-3/AIB1) is an oncogene that is amplified and overexpressed in many human cancers. However, the molecular mechanisms that regulate 'activated SRC-3 oncoprotein' turnover during tumorigenesis remain to be elucidated. Here, we report that speckle-type POZ protein (SPOP), a cullin 3 (CUL3)-based ubiquitin ligase, is responsible for SRC-3 ubiquitination and proteolysis. SPOP interacts directly with an SRC-3 phospho-degron in a phosphorylation-dependent manner. Casein kinase Iɛ phosphorylates the S102 in this degron and promotes SPOP-dependent turnover of SRC-3. Short hairpin RNA knockdown and overexpression experiments substantiated that the SPOP/CUL3/Rbx1 ubiquitin ligase complex promotes SRC-3 turnover. A systematic analysis of the SPOP genomic locus revealed that a high percentage of genomic loss or loss of heterozygosity occurs at this locus in breast cancers. Furthermore, we demonstrate that restoration of SPOP expression inhibited SRC-3-mediated oncogenic signaling and tumorigenesis, thus positioning SPOP as a tumor suppressor.
De Smaele E, Di Marcotullio L, Moretti M, et al.Identification and characterization of KCASH2 and KCASH3, 2 novel Cullin3 adaptors suppressing histone deacetylase and Hedgehog activity in medulloblastoma.
Neoplasia. 2011; 13(4):374-85 [PubMed
] Free Access to Full Article Related Publications
Medulloblastoma is the most common pediatric malignant brain tumor, arising from aberrant cerebellar precursors' development, a process mainly controlled by Hedgehog (Hh) signaling pathway. Histone deacetylase HDAC1 has been recently shown to modulate Hh signaling, deacetylating its effectors Gli1/2 and enhancing their transcriptional activity. Therefore, HDAC may represent a potential therapeutic target for Hh-dependent tumors, but still little information is available on the physiological mechanisms of HDAC regulation. The putative tumor suppressor REN(KCTD11) acts through ubiquitination-dependent degradation of HDAC1, thereby affecting Hh activity and medulloblastoma growth. We identify and characterize here two REN(KCTD11) homologues, defining a new family of proteins named KCASH, as "KCTD containing, Cullin3 adaptor, suppressor of Hedgehog." Indeed, the novel genes (KCASH2(KCTD21) and KCASH3(KCTD6)) share with REN(KCTD11) a number of features, such as a BTB domain required for the formation of a Cullin3 ubiquitin ligase complex and HDAC1 ubiquitination and degradation capability, suppressing the acetylation-dependent Hh/Gli signaling. Expression of KCASH2 and -3 is observed in cerebellum, whereas epigenetic silencing and allelic deletion are observed in human medulloblastoma. Rescuing KCASHs expression reduces the Hedgehog-dependent medulloblastoma growth, suggesting that loss of members of this novel family of native HDAC inhibitors is crucial in sustaining Hh pathway-mediated tumorigenesis. Accordingly, they might represent a promising class of endogenous "agents" through which this pathway may be targeted.
Uruno A, Motohashi HThe Keap1-Nrf2 system as an in vivo sensor for electrophiles.
Nitric Oxide. 2011; 25(2):153-60 [PubMed
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The Keap1-Nrf2 regulatory system plays a central role in cytoprotection from electrophilic and oxidative stress. In unstressed conditions, Nrf2 is constantly ubiquitinated by the Cul3-Keap1 ubiquitin E3 ligase complex and is degraded in the proteasome. Upon the exposure to electrophilic and oxidative stress, reactive cysteine residues in Keap1 are covalently modified, which abrogates the E3 ligase activity of the Cul3-Keap1 complex. Consequently Nrf2 is stabilized and induces the transcription of various cytoprotective genes. Structural analyses have revealed the overall structure of the Keap1 homodimer as well as structural features of the association between Keap1 and Nrf2, which has greatly enhanced our understanding of the molecular mechanisms involved in the regulation of the Keap1-Nrf2 system. Recently nitric oxide signaling has been shown to activate Nrf2, suggesting that Nrf2 is a mediator of the cytoprotective effect of nitric oxide. Analyses of Nrf2-null mice have revealed a critical contribution of Nrf2 to the protection from various diseases caused by electrophilic and oxidative stress. In contrast, constitutive activation of Nrf2 has been found in many cancers, resulting in resistance against chemotherapy and radiotherapy in cancer cells. Thus, Nrf2 is a promising target for drug development. The development of Nrf2 inducers and inhibitors is an important challenge for enhancing therapies for stress-induced diseases and cancers, respectively.
Taguchi K, Motohashi H, Yamamoto MMolecular mechanisms of the Keap1–Nrf2 pathway in stress response and cancer evolution.
Genes Cells. 2011; 16(2):123-40 [PubMed
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The Keap1–Nrf2 regulatory pathway plays a central role in the protection of cells against oxidative and xenobiotic damage. Under unstressed conditions, Nrf2 is constantly ubiquitinated by the Cul3–Keap1 ubiquitin E3 ligase complex and rapidly degraded in proteasomes. Upon exposure to electrophilic and oxidative stresses, reactive cysteine residues of Keap1 become modified, leading to a decline in the E3 ligase activity, stabilization of Nrf2 and robust induction of a battery of cytoprotective genes. Biochemical and structural analyses have revealed that the intact Keap1 homodimer forms a cherry-bob structure in which one molecule of Nrf2 associates with two molecules of Keap1 by using two binding sites within the Neh2 domain of Nrf2. This two-site binding appears critical for Nrf2 ubiquitination. In many human cancers, missense mutations in KEAP1 and NRF2 genes have been identified. These mutations disrupt the Keap1–Nrf2 complex activity involved in ubiquitination and degradation of Nrf2 and result in constitutive activation of Nrf2. Elevated expression of Nrf2 target genes confers advantages in terms of stress resistance and cell proliferation in normal and cancer cells. Discovery and development of selective Nrf2 inhibitors should make a critical contribution to improved cancer therapy.
Cytosolic inhibitor of Nrf2 (INrf2) is an adaptor protein that mediates ubiquitination/degradation of NF-E2-related factor 2 (Nrf2), a master regulator of cytoprotective gene expression. In this paper, we demonstrate that INrf2 degrades endogenous antiapoptotic B-cell CLL/lymphoma 2 (Bcl-2) protein and controls cellular apoptosis. The DGR domain of INrf2 interacts with the BH2 domain of Bcl-2 and facilitates INrf2:Cul3-Rbx1-mediated ubiquitination of Bcl-2 by the conjugation of ubiquitin molecules to lysine17 of Bcl-2. Further studies showed that INrf2 enhanced etoposide-mediated accumulation of Bax, increased release of cytochrome c from mitochondria, activated caspase-3/7, and enhanced DNA fragmentation and apoptosis. Antioxidants antagonized Bcl-2:INrf2 interaction, led to the release and stabilization of Bcl-2, increased Bcl-2:Bax heterodimers and reduced apoptosis. Moreover, dysfunctional/mutant INrf2 in human lung cancer cells failed to degrade Bcl-2, resulting in decreased etoposide and UV/γ radiation-mediated DNA fragmentation. These data provide the first evidence of INrf2 control of Bcl-2 and apoptotic cell death, with implications in antioxidant protection, survival of cancer cells containing dysfunctional INrf2, and drug resistance.
DNA methylation and histone modifications play an important role in transcription regulation. In cancer cells, many promoters become aberrantly methylated through the activity of the de novo DNA methyltransferases DNMT3a and DNMT3b and acquire repressive chromatin marks. NEDD8 is a ubiquitin-like protein modifier that is conjugated to target proteins, such as cullins, to regulate their activity, and cullin 4A (CUL4A) in its NEDD8-modified form is essential for repressive chromatin formation. We found that DNMT3b associates with NEDD8-modified proteins. Whereas DNMT3b interacts directly in vitro with NEDD8, conjugation of NEDD8 to target proteins enhances this interaction in vivo. DNMT3b immunoprecipitated two major bands of endogenously NEDDylated proteins at the size of NEDDylated cullins, and indeed DNMT3b interacted with CUL1, CUL2, CUL3, CUL4A, and CUL5. Moreover, DNMT3b preferentially immunoprecipitated the NEDDylated form of endogenous CUL4A. NEDD8 enhanced DNMT3b-dependent DNA methylation. Chromatin immunoprecipitation assays suggest that DNMT3b recruits CUL4A and NEDD8 to chromatin, whereas deletion of Dnmt3b reduces the association of CUL4A and NEDD8 at a repressed promoter in a cancer cell line.
Hur W, Sun Z, Jiang T, et al.A small-molecule inducer of the antioxidant response element.
Chem Biol. 2010; 17(5):537-47 [PubMed
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Eukaryotic cells counteract oxidative and other environmental stress through the activation of Nrf2, the transcription factor that controls the expression of a host of protective enzymes by binding to the antioxidant response element (ARE). The electrophilic molecules that are able to activate Nrf2 and its downstream target genes have demonstrated therapeutic potential in carcinogen-induced tumor models. Using a high-throughput cellular screen, we discovered a class of ARE activator, which we named AI-1, that activates Nrf2 by covalently modifying Keap1, the negative regulator of Nrf2. Biochemical studies indicated that modification of Cys151 of Keap1 by AI-1 disrupted the ability of Keap1 to serve as an adaptor for Cul3-Keap1 ubiquitin ligase complex, thereby causing stabilization and transcriptional activation of Nrf2. AI-1 and its biotinylated derivative are useful pharmacological probes for investigating the molecular details of the cellular antioxidant response.
Hayes JD, McMahon M, Chowdhry S, Dinkova-Kostova ATCancer chemoprevention mechanisms mediated through the Keap1-Nrf2 pathway.
Antioxid Redox Signal. 2010; 13(11):1713-48 [PubMed
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The cap'n'collar (CNC) bZIP transcription factor Nrf2 controls expression of genes for antioxidant enzymes, metal-binding proteins, drug-metabolising enzymes, drug transporters, and molecular chaperones. Many chemicals that protect against carcinogenesis induce Nrf2-target genes. These compounds are all thiol-reactive and stimulate an adaptive response to redox stress in cells. Such agents induce the expression of genes that posses an antioxidant response element (ARE) in their regulatory regions. Under normal homeostatic conditions, Nrf2 activity is restricted through a Keap1-dependent ubiquitylation by Cul3-Rbx1, which targets the CNC-bZIP transcription factor for proteasomal degradation. However, as the substrate adaptor function of Keap1 is redox-sensitive, Nrf2 protein evades ubiquitylation by Cul3-Rbx1 when cells are treated with chemopreventive agents. As a consequence, Nrf2 accumulates in the nucleus where it heterodimerizes with small Maf proteins and transactivates genes regulated through an ARE. In this review, we describe synthetic compounds and phytochemicals from edible plants that induce Nrf2-target genes. We also discuss evidence for the existence of different classes of ARE (a 16-bp 5'-TMAnnRTGABnnnGCR-3' versus an 11-bp 5'-RTGABnnnGCR-3', with or without the embedded activator protein 1-binding site 5'-TGASTCA-3'), species differences in the ARE-gene battery, and the identity of critical Cys residues in Keap1 required for de-repression of Nrf2 by chemopreventive agents.
Death-associated protein kinase (DAPK) was identified as a mediator of interferon (IFN)-induced cell death. How IFN controls DAPK activation remains largely unknown. Here, we identify the BTB-Kelch protein KLHL20 as a negative regulator of DAPK. KLHL20 binds DAPK and Cullin 3 (Cul3) via its Kelch-repeat domain and BTB domain, respectively. The KLHL20-Cul3-ROC1 E3 ligase complex promotes DAPK polyubiquitination, thereby inducing the proteasomal degradation of DAPK. Accordingly, depletion of KLHL20 diminishes DAPK ubiquitination and degradation. The KLHL20-mediated DAPK ubiquitination is suppressed in cells receiving IFN-alpha or IFN-gamma, which induces an enrichment/sequestration of KLHL20 in the PML nuclear bodies, thereby separating KLHL20 from DAPK. Consequently, IFN triggers the stabilization of DAPK. This mechanism of DAPK stabilization is crucial for determining IFN responsiveness of tumor cells and contributes to IFN-induced autophagy. This study identifies KLHL20-Cul3-ROC1 as an E3 ligase for DAPK ubiquitination and reveals a regulatory mechanism of DAPK, through blocking its accessibility to this E3 ligase, in IFN-induced apoptotic and autophagic death. Our findings may be relevant to the problem of IFN resistance in cancer therapy.
Hu L, Miao W, Loignon M, et al.Putative chemopreventive molecules can increase Nrf2-regulated cell defense in some human cancer cell lines, resulting in resistance to common cytotoxic therapies.
Cancer Chemother Pharmacol. 2010; 66(3):467-74 [PubMed
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Nrf2 is a key transcription factor, which induces a cytoprotective gene array. Nrf2 is regulated at the posttranslational level through proteasomal degradation through an interaction with the adapter protein Keap1. High levels of Nrf2, resulting from a loss of function mutation in Keap1, were reported in chemoresistant non-small cell lung cancer. We observed very low levels of Nrf2 and of Nrf2-regulated detoxification proteins as a frequent phenotype in the more chemosensitive breast cancer, and when engineering increased Nrf2 levels, we found resistance to both doxorubicin and paclitaxel. We here show that basal Nrf2 levels in different cell lines correlate with their respective sensitivity to a common cytotoxic chemotherapy. Nrf2 and its regulated genes and proteins are the targets of a major strategy in cancer prevention. Molecules that interfere with the Nrf2-Keap1-Cul3 protein-protein interactions result in higher levels of Nrf2. Both naturally occurring and synthetic molecules with this effect have been suggested as clinical chemopreventive agents, including molecules derived from cruciferous vegetables such as the isothiocyanate sulforaphane and even green tea polyphenols. Here, we determine the impact of these putative chemopreventive agents on the sensitivity of established cancer cell lines to chemotherapy. We confirmed that these molecules do increase Nrf2 and detoxification enzyme levels in breast cancer cell lines with very low basal Nrf2 levels, and this is associated with significant chemoresistance to cytotoxic drugs. Both effects are less in another breast cancer cell line with intermediate Nrf2, and in lung cancer cells with high Nrf2, these same molecules have no effect on Nrf2 but do actually enhance chemoresistance. While the details of dose and schedule of these agents require further study in in vivo models, these data sound a cautionary note for the use of these agents in patients with established cancers who are undergoing chemotherapy treatment.