RBX1

Gene Summary

Gene:RBX1; ring-box 1
Aliases: ROC1, RNF75, BA554C12.1
Location:22q13.2
Summary:This locus encodes a RING finger-like domain-containing protein. The encoded protein interacts with cullin proteins and likely plays a role in ubiquitination processes necessary for cell cycle progression. This protein may also affect protein turnover. Related pseudogenes exist on chromosomes 2 and 5.[provided by RefSeq, Sep 2010]
Databases:OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:E3 ubiquitin-protein ligase RBX1
Source:NCBIAccessed: 29 August, 2019

Ontology:

What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1994-2019)
Graph generated 29 August 2019 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

Tag cloud generated 29 August, 2019 using data from PubMed, MeSH and CancerIndex

Specific Cancers (6)

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).

Latest Publications: RBX1 (cancer-related)

Yang X, Liang R, Liu C, et al.
SOX9 is a dose-dependent metastatic fate determinant in melanoma.
J Exp Clin Cancer Res. 2019; 38(1):17 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: In this research, we aimed to resolve contradictory results whether SOX9 plays a positive or negative role in melanoma progression and determine whether SOX9 and its closely related member SOX10 share the same or distinct targets in mediating their functions in melanoma.
METHODS: Immunofluorescence, TCGA database and qPCR were used to analyze the correlation between the expression patterns and levels of SOX9, SOX10 and NEDD9 in melanoma patient samples. AlamarBlue, transwell invasion and colony formation assays in melanoma cell lines were conducted to investigate the epistatic relationship between SOX10 and NEDD9, as well as the effects of graded SOX9 expression levels. Lung metastasis was determined by tail vein injection assay. Live cell imaging was conducted to monitor dynamics of melanoma migratory behavior. RHOA and RAC1 activation assays measured the activity of Rho GTPases.
RESULTS: High SOX9 expression was predominantly detected in patients with distant melanoma metastases whereas SOX10 was present in the different stages of melanoma. Both SOX9 and SOX10 exhibited distinct but overlapping expression patterns with metastatic marker NEDD9. Accordingly, SOX10 was required for NEDD9 expression, which partly mediated its oncogenic functions in melanoma cells. Compensatory upregulation of SOX9 expression in SOX10-inhibited melanoma cells reduced growth and migratory capacity, partly due to elevated expression of cyclin-dependent kinase inhibitor p21 and lack of NEDD9 induction. Conversely, opposite phenomenon was observed when SOX9 expression was further elevated to a range of high SOX9 expression levels in metastatic melanoma specimens, and that high levels of SOX9 can restore melanoma progression in the absence of SOX10 both in vitro and in vivo. In addition, overexpression of SOX9 can also promote invasiveness of the parental melanoma cells by modulating the expression of various matrix metalloproteinases. SOX10 or high SOX9 expression regulates melanoma mesenchymal migration through the NEDD9-mediated focal adhesion dynamics and Rho GTPase signaling.
CONCLUSIONS: These results unravel NEDD9 as a common target for SOX10 or high SOX9 to partly mediate their oncogenic events, and most importantly, reconcile previous discrepancies that suboptimal level of SOX9 expression is anti-metastatic whereas high level of SOX9 is metastatic in a heterogeneous population of melanoma.

Tuli R, Shiao SL, Nissen N, et al.
A phase 1 study of veliparib, a PARP-1/2 inhibitor, with gemcitabine and radiotherapy in locally advanced pancreatic cancer.
EBioMedicine. 2019; 40:375-381 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Locally advanced pancreatic cancer (LAPC) has a dismal prognosis with current treatment modalities and one-third of patients die from local progression of disease. Preclinical studies with orthotopic PC demonstrated dramatic synergy between radiotherapy (RT) and the poly(ADP-ribose) polymerase-1/2 inhibitor (PARPi), veliparib. We conducted a phase I trial of gemcitabine, radiotherapy and dose-escalated veliparib in LAPC.
METHODS: This was a single institution investigator-initiated open-label, single-arm phase 1 clinical trial (NCT01908478). Weekly gemcitabine with daily IMRT and veliparib dose escalated using a Bayesian adaptive design were administered in treatment naïve LA or borderline resectable PC. The primary end point was identification of the MTD. Secondary endpoints included efficacy, characterization of PAR levels using ELISA, DDR alterations with targeted next generation sequencing and transcriptome analysis, tumor mutation burden (TMB) and microsatellite instability (MSI) status.
FINDINGS: Thirty patients were enrolled. The MTD of veliparib was 40 mg BID with gemcitabine 400 mg/m
INTERPRETATION: This is the first report of a PARPi-chemoradiotherapy combination in PC. The regimen was safe, tolerable at the RP2D, and clinically active as an upfront treatment strategy in patients biologically unselected by upfront chemotherapy. Expression of the DDR transcripts, PARP3 and RBX1, were associated with OS suggesting validation in a follow up phase 2 study. FUND: Phase One Foundation; National Institutes of Health [1R01CA188480-01A1, P01 CA098912]. Veliparib was provided by Abbvie.

Li Y, Liu Y, Xu H, et al.
Heterozygous deletion of chromosome 17p renders prostate cancer vulnerable to inhibition of RNA polymerase II.
Nat Commun. 2018; 9(1):4394 [PubMed] Free Access to Full Article Related Publications
Heterozygous deletion of chromosome 17p (17p) is one of the most frequent genomic events in human cancers. Beyond the tumor suppressor TP53, the POLR2A gene encoding the catalytic subunit of RNA polymerase II (RNAP2) is also included in a ~20-megabase deletion region of 17p in 63% of metastatic castration-resistant prostate cancer (CRPC). Using a focused CRISPR-Cas9 screen, we discovered that heterozygous loss of 17p confers a selective dependence of CRPC cells on the ubiquitin E3 ligase Ring-Box 1 (RBX1). RBX1 activates POLR2A by the K63-linked ubiquitination and thus elevates the RNAP2-mediated mRNA synthesis. Combined inhibition of RNAP2 and RBX1 profoundly suppress the growth of CRPC in a synergistic manner, which potentiates the therapeutic effectivity of the RNAP2 inhibitor, α-amanitin-based antibody drug conjugate (ADC). Given the limited therapeutic options for CRPC, our findings identify RBX1 as a potentially therapeutic target for treating human CRPC harboring heterozygous deletion of 17p.

Ma J, Chang K, Peng J, et al.
SPOP promotes ATF2 ubiquitination and degradation to suppress prostate cancer progression.
J Exp Clin Cancer Res. 2018; 37(1):145 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Next-generation sequencing of the exome and genome of prostate cancers has identified numerous genetic alterations. SPOP (Speckle-type POZ Protein) is one of the most frequently mutated genes in primary prostate cancer, suggesting that SPOP may be a potential driver of prostate cancer. The aim of this work was to investigate how SPOP mutations contribute to prostate cancer development and progression.
METHODS: To identify molecular mediators of the tumor suppressive function of SPOP, we performed a yeast two-hybrid screen in a HeLa cDNA library using the full-length SPOP as bait. Immunoprecipitation and Western Blotting were used to analyze the interaction between SPOP and ATF2. Cell migration and invasion were determined by Transwell assays. Immunohistochemistry were used to analyze protein levels in patients' tumor samples.
RESULTS: Here we identified ATF2 as a bona fide substrate of the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP recognizes multiple Ser/Thr (S/T)-rich degrons in ATF2 and triggers ATF2 degradation via the ubiquitin-proteasome pathway. Strikingly, prostate cancer-associated mutants of SPOP are defective in promoting ATF2 degradation in prostate cancer cells and contribute to facilitating prostate cancer cell proliferation, migration and invasion.
CONCLUSION: SPOP promotes ATF2 ubiquitination and degradation, and ATF2 is an important mediator of SPOP inactivation-induced cell proliferation, migration and invasion.

Kumar A, Shaha C
RBX1-mediated ubiquitination of SESN2 promotes cell death upon prolonged mitochondrial damage in SH-SY5Y neuroblastoma cells.
Mol Cell Biochem. 2018; 446(1-2):1-9 [PubMed] Related Publications
Sestrins are evolutionary conserved stress-inducible genes which regulate the axis of cell survival and cell death. Suppression of Sestrin 2 (SESN2) has been linked with increase in oxidative stress and cell death but mechanistic details related to regulation of SESN2 during mitochondrial damage remain unknown. Our study shows that prolonged CCCP-induced mitochondrial damage decreases SESN2 levels and viability of SH-SY5Y cells while overexpression of SESN2 significantly rescues the viability of cells. Further, we demonstrate that Ring box protein 1 (RBX1) is a novel interactive partner and E3 ligase for SESN2 which mediates its K-48-linked ubiquitination upon extensive mitochondrial damage. Downregulation of RBX1 causes stabilization in levels of SESN2. Notably, silencing of RBX1 expression substantially declines cell death and generation of mitochondrial ROS in response to prolonged mitochondrial damage. Taken together, we suggest that SESN2 is critical to protect cells against detrimental effect of mitochondrial damage and RBX1 is a negative regulator of SESN2 which hampers its stabilization.

Celik ZE, Kaynar M, Karabagli P, et al.
The relation between Ring Box-1 protein overexpression and tumor grade and stage in bladder urothelial cell carcinoma.
Cancer Biomark. 2017; 20(4):389-394 [PubMed] Related Publications
BACKGROUND: Ring Box Protein-1 (RBX-1), a component of SCF E3 ubiquitin ligases, has a crucial role in bladder urothelial cell carcinoma (UCC) carcinogenesis and progression.
OBJECTIVES: In the present study, it is aimed to determine the expression of RBX-1 protein in bladder UCC and the association between tumor grade, stage and RBX-1 expression.
METHODS: Ninety UCC samples and 20 samples containing foci of normal bladder urothelium were recruited and analyzed immunohistochemically in terms of RBX-1 expression. Immuno-reactivity scoring system (IRS) was used to determine RBX-1 expression levels.
RESULTS: RBX-1 overexpression was associated with high tumor grade (p= 0.001) and advanced stage (p= 0.001). pT1 tumors showed higher RBX-1 expression than pTa tumors. pT2 tumors showed not only higher expression than pTa tumors but also higher expression than the total of pTa and pT1 groups combined. There was no statistically significant relation between RBX-1 expression and patient gender (p= 0.116) or age (p= 0.191).
CONCLUSIONS: In bladder UCC, RBX-1 overexpression is associated with high tumor grade and advanced stage and represents biological potential of invasiveness and aggressive disease. Results of the present study have to be supported with further studies to reveal clinical and therapeutic implications of RBX-1 overexpression in bladder UCC.

An J, Ponthier CM, Sack R, et al.
pSILAC mass spectrometry reveals ZFP91 as IMiD-dependent substrate of the CRL4
Nat Commun. 2017; 8:15398 [PubMed] Free Access to Full Article Related Publications
Thalidomide and its derivatives lenalidomide and pomalidomide (IMiDs) are effective treatments of haematologic malignancies. It was shown that IMiDs impart gain-of-function properties to the CUL4-RBX1-DDB1-CRBN (CRL4

Jin X, Wang J, Gao K, et al.
Dysregulation of INF2-mediated mitochondrial fission in SPOP-mutated prostate cancer.
PLoS Genet. 2017; 13(4):e1006748 [PubMed] Free Access to Full Article Related Publications
Next-generation sequencing of the exome and genome of prostate cancers has identified numerous genetic alternations. SPOP (Speckle-type POZ Protein) was one of the most frequently mutated genes in primary prostate cancer, suggesting SPOP is a potential driver of prostate cancer development and progression. However, how SPOP mutations contribute to prostate cancer pathogenesis remains poorly understood. SPOP acts as an adaptor protein of the CUL3-RBX1 E3 ubiquitin ligase complex that generally recruits substrates for ubiquitination and subsequent degradation. ER-localized isoform of the formin protein inverted formin 2 (INF2) mediates actin polymerization at ER-mitochondria intersections and facilitates DRP1 recruitment to mitochondria, which is a critical step in mitochondrial fission. Here, we revealed that SPOP recognizes a Ser/Thr (S/T)-rich motif in the C-terminal region of INF2 and triggers atypical polyubiquitination of INF2. These ubiquitination modifications do not lead to INF2 instability, but rather reduces INF2 localization in ER and mitochondrially associated DRP1 puncta formation, therefore abrogates its ability to facilitate mitochondrial fission. INF2 mutant escaping from SPOP-mediated ubiquitination is more potent in prompting mitochondrial fission. Moreover, prostate cancer-associated SPOP mutants increase INF2 localization in ER and promote mitochondrial fission, probably through a dominant-negative effect to inhibit endogenous SPOP. Moreover, INF2 is important for SPOP inactivation-induced prostate cancer cell migration and invasion. These findings reveal novel molecular events underlying the regulation of INF2 function and localization, and provided insights in understanding the relationship between SPOP mutations and dysregulation of mitochondrial dynamics in prostate cancer.

Chen Z, Wang K, Hou C, et al.
CRL4B
Sci Rep. 2017; 7(1):1175 [PubMed] Free Access to Full Article Related Publications
Cell cycle progression in mammals is strictly controlled by a number of cyclin-dependent kinases (CDKs) and CDK inhibitors (CKIs), the expression of which is often dysregulated in cancer cells. Our previous work revealed that Cullin 4B (CUL4B), a critical component of the Cullin4B-RING E3 ligase complex (CRL4B), is overexpressed in human osteosarcoma cells through an unknown mechanism. Here, we demonstrated that CUL4B forms an E3 ligase with RBX1 (RING-box 1), DDB1 (DNA damage binding protein 1), and DCAF11 (DDB1 and CUL4 associated factor 11) in human osteosarcoma cells. In vitro and in vivo ubiquitination analyses indicated that CRL4B

Zhang J, Li S, Shang Z, et al.
Targeting the overexpressed ROC1 induces G2 cell cycle arrest and apoptosis in esophageal cancer cells.
Oncotarget. 2017; 8(17):29125-29137 [PubMed] Free Access to Full Article Related Publications
Recent reports showed that regulator of Cullins-1 (ROC1) play an important role in tumor progression in a tumor-specific manner. However, the role and mechanism of ROC1 in esophageal cancer remains elusive. Here we demonstrated that ROC1 was overexpressed in esophageal squamous cell carcinomas, which was positive associated with poor prognosis of esophageal cancer patients. ROC1 knockdown significantly inhibited the growth of esophageal cancer cells in vitro and in vivo. Mechanistically, ROC1 silencing induced G2 cell cycle arrest and triggered apoptosis by accumulating the pro-apoptotic protein NOXA. Consistently, the downregulation of NOXA expression via siRNA substantially attenuated apoptosis induced by ROC1 silencing. These findings suggest that ROC1 is an appealing drug target for esophageal cancer.

Chou CK, Chang YT, Korinek M, et al.
The Regulations of Deubiquitinase USP15 and Its Pathophysiological Mechanisms in Diseases.
Int J Mol Sci. 2017; 18(3) [PubMed] Free Access to Full Article Related Publications
Deubiquitinases (DUBs) play a critical role in ubiquitin-directed signaling by catalytically removing the ubiquitin from substrate proteins. Ubiquitin-specific protease 15 (USP15), a member of the largest subfamily of cysteine protease DUBs, contains two conservative cysteine (Cys) and histidine (His) boxes. USP15 harbors two zinc-binding motifs that are essential for recognition of poly-ubiquitin chains. USP15 is grouped into the same category with USP4 and USP11 due to high degree of homology in an N-terminal region consisting of domains present in ubiquitin-specific proteases (DUSP) domain and ubiquitin-like (UBL) domain. USP15 cooperates with COP9 signalosome complex (CSN) to maintain the stability of cullin-ring ligase (CRL) adaptor proteins by removing the conjugated ubiquitin chains from RBX1 subunit of CRL. USP15 is also implicated in the stabilization of the human papillomavirus type 16 E6 oncoprotein, adenomatous polyposis coli, and IκBα. Recently, reports have suggested that USP15 acts as a key regulator of TGF-β receptor-signaling pathways by deubiquitinating the TGF-β receptor itself and its downstream transducers receptor-regulated SMADs (R-SMADs), including SMAD1, SMAD2, and SMAD3, thus activating the TGF-β target genes. Although the importance of USP15 in pathologic processes remains ambiguous so far, in this review, we endeavor to summarize the literature regarding the relationship of the deubiquitinating action of USP15 with the proteins involved in the regulation of Parkinson's disease, virus infection, and cancer-related signaling networks.

Mi J, Zou Y, Lin X, et al.
Dysregulation of the miR-194-CUL4B negative feedback loop drives tumorigenesis in non-small-cell lung carcinoma.
Mol Oncol. 2017; 11(3):305-319 [PubMed] Free Access to Full Article Related Publications
Cullin 4B (CUL4B), a scaffold protein that assembles CRL4B ubiquitin ligase complexes, is overexpressed in many types of cancers and represses many tumor suppressors through epigenetic mechanisms. However, the mechanisms by which CUL4B is upregulated remain to be elucidated. Here, we show that CUL4B is upregulated in non-small-cell lung carcinoma (NSCLC) tissues and is critically required for cell proliferation and migration in vitro and for xenograft tumor formation in vivo. We found that microRNA-194 (miR-194) and CUL4B protein were inversely correlated in cancer specimens and demonstrated that miR-194 could downregulate CUL4B by directly targeting its 3'-UTR. We also showed that CUL4B could be negatively regulated by p53 in a miR-194-dependent manner. miR-194 was further shown to attenuate the malignant phenotype of lung cancer cells by downregulating CUL4B. Interestingly, CRL4B also epigenetically represses miR-194 by catalyzing monoubiquitination at H2AK119 and by coordinating with PRC2 to promote trimethylation at H3K27 at the gene clusters encoding miR-194. RBX1, another component in CRL4B complex, is also targeted by miR-194 in NSCLC cells. Our results thus establish a double-negative feedback loop between miR-194 and CRL4B, dysregulation of which contributes to tumorigenesis. The function of miR-194 as a negative regulator of CUL4B has therapeutic implications in lung cancer.

Rulina AV, Mittler F, Obeid P, et al.
Distinct outcomes of CRL-Nedd8 pathway inhibition reveal cancer cell plasticity.
Cell Death Dis. 2016; 7(12):e2505 [PubMed] Free Access to Full Article Related Publications
Inhibition of protein degradation by blocking Cullin-RING E3 ligases (CRLs) is a new approach in cancer therapy though of unknown risk because CRL inhibition may stabilize both oncoproteins and tumor suppressors. Probing CRLs in prostate cancer cells revealed a remarkable plasticity of cells with TMPRSS2-ERG translocation. CRL suppression by chemical inhibition or knockdown of RING component RBX1 led to reversible G0/G1 cell cycle arrest that prevented cell apoptosis. Conversely, complete blocking of CRLs at a higher inhibitor dose-induced cytotoxicity that was amplified by knockdown of CRL regulator Cand1. We analyzed cell signaling to understand how varying degrees of CRL inhibition translated to distinct cell fates. Both tumor suppressor and oncogenic cell signaling pathways and transcriptional activities were affected, with pro-metastatic Wnt/β-catenin as the most upregulated. Suppression of the NF-κB pathway contributed to anti-apoptotic effect, and androgen receptor (AR) and ERG played decisive, though opposite, roles: AR was involved in protective quiescence, whereas ERG promoted apoptosis. These data define AR-ERG interaction as a key plasticity and survival determinant in prostate cancer and suggest supplementary treatments that may overcome drug resistance mechanisms regulated by AR-ERG interaction.

Huber AL, Papp SJ, Chan AB, et al.
CRY2 and FBXL3 Cooperatively Degrade c-MYC.
Mol Cell. 2016; 64(4):774-789 [PubMed] Free Access to Full Article Related Publications
For many years, a connection between circadian clocks and cancer has been postulated. Here we describe an unexpected function for the circadian repressor CRY2 as a component of an FBXL3-containing E3 ligase that recruits T58-phosphorylated c-MYC for ubiquitylation. c-MYC is a critical regulator of cell proliferation; T58 is central in a phosphodegron long recognized as a hotspot for mutation in cancer. This site is also targeted by FBXW7, although the full machinery responsible for its turnover has remained obscure. CRY1 cannot substitute for CRY2 in promoting c-MYC degradation. Their unique functions may explain prior conflicting reports that have fueled uncertainty about the relationship between clocks and cancer. We demonstrate that c-MYC is a target of CRY2-dependent protein turnover, suggesting a molecular mechanism for circadian control of cell growth and a new paradigm for circadian protein degradation.

Yang Y, He S, Wang Q, et al.
Autophagic UVRAG Promotes UV-Induced Photolesion Repair by Activation of the CRL4(DDB2) E3 Ligase.
Mol Cell. 2016; 62(4):507-19 [PubMed] Free Access to Full Article Related Publications
UV-induced DNA damage, a major risk factor for skin cancers, is primarily repaired by nucleotide excision repair (NER). UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy. It was initially isolated as a cDNA partially complementing UV sensitivity in xeroderma pigmentosum (XP), but this was not explored further. Here we show that UVRAG plays an integral role in UV-induced DNA damage repair. It localizes to photolesions and associates with DDB1 to promote the assembly and activity of the DDB2-DDB1-Cul4A-Roc1 (CRL4(DDB2)) ubiquitin ligase complex, leading to efficient XPC recruitment and global genomic NER. UVRAG depletion decreased substrate handover to XPC and conferred UV-damage hypersensitivity. We confirmed the importance of UVRAG for UV-damage tolerance using a Drosophila model. Furthermore, increased UV-signature mutations in melanoma correlate with reduced expression of UVRAG. Our results identify UVRAG as a regulator of CRL4(DDB2)-mediated NER and suggest that its expression levels may influence melanoma predisposition.

Wang W, Chen H, Liu Z, et al.
Regulator of cullins-1 expression knockdown suppresses the malignant progression of muscle-invasive transitional cell carcinoma by regulating mTOR/DEPTOR pathway.
Br J Cancer. 2016; 114(3):305-13 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Regulator of cullins-1 (ROC1) is a key subunit in the cullin-RING ligase (CRL) protein complex. Our previous study indicated that ROC1 was essential for bladder cancer cell survival and that ROC1 knockdown inhibited CRL activity, triggering G2 phase arrest and senescence. However, the role of ROC1 in the malignant progression of bladder cancer remained unknown.
METHODS: ROC1 expression in cancer cells was knocked down by siRNA silencing. The effects of ROC1 silencing were evaluated by in vitro assays for cell migration and by an in vivo mouse metastasis model. Epithelial-mesenchymal transition (EMT) induction was evaluated by immunofluorescence staining and western blotting of EMT-associated proteins. ROC1 expression in human tumours was further evaluated by immunohistochemical analysis.
RESULTS: ROC1 knockdown suppresses bladder cancer cell migration by inhibiting EMT. ROC1 knockdown inhibited EMT by inhibiting mammalian target of rapamycin (mTOR) activity via the accumulation of the mTOR-inhibitory protein DEPTOR, a CRL substrate. DEPTOR knockdown partially rescued ROC1 knockdown-inhibited EMT and the ROC1-induced inhibition of cancer cell migration. Furthermore, in vivo studies using a nude mouse metastasis model confirmed the in vitro data. Finally, tissue microarray analysis of clinical bladder cancer specimens indicated a positive correlation between ROC1 expression and EMT.
CONCLUSIONS: ROC1 has an important role in the malignant progression of bladder cancer via the mTOR/DEPTOR pathway. ROC1 may serve as a novel therapeutic target for the treatment of muscle-invasive transitional cell carcinoma.

Harder B, Jiang T, Wu T, et al.
Molecular mechanisms of Nrf2 regulation and how these influence chemical modulation for disease intervention.
Biochem Soc Trans. 2015; 43(4):680-6 [PubMed] Free Access to Full Article Related Publications
Nrf2 (nuclear factor erytheroid-derived-2-like 2) transcriptional programmes are activated by a variety of cellular stress conditions to maintain cellular homoeostasis. Under non-stress conditions, Nrf2 is under tight regulation by the ubiquitin proteasome system (UPS). Detailed mechanistic investigations have shown the Kelch-like ECH-associated protein 1 (Keap1)-cullin3 (Cul3)-ring-box1 (Rbx1) E3-ligase to be the primary Nrf2 regulatory system. Recently, both beta-transducin repeat-containing E3 ubiquitin protein ligase (β-TrCP) and E3 ubiquitin-protein ligase synoviolin (Hrd1) have been identified as novel E3 ubiquitin ligases that negatively regulate Nrf2 through Keap1-independent mechanisms. In addition to UPS-mediated regulation of Nrf2, investigations have revealed a cross-talk between Nrf2 and the autophagic pathway resulting in activation of Nrf2 in a non-canonical manner. In addition to regulation at the protein level, Nrf2 was recently shown to be regulated at the transcriptional level by oncogenic K-rat sarcoma (Ras). A consequence of these differential regulatory mechanisms is the dual role of Nrf2 in cancer: the canonical, protective role and the non-canonical 'dark-side' of Nrf2. Based on the protective role of Nrf2, a vast effort has been dedicated towards identifying novel chemical inducers of Nrf2 for the purpose of chemoprevention. On the other hand, upon malignant transformation, some cancer cells have a constitutively high level of Nrf2 offering a growth advantage, as well as rendering cancer cells resistant to chemotherapeutics. This discovery has led to a new paradigm in cancer treatment; the initially counterintuitive use of Nrf2 inhibitors as adjuvants in chemotherapy. Herein, we will discuss the mechanisms of Nrf2 regulation and how this detailed molecular understanding can be leveraged to develop Nrf2 modulators to prevent diseases, mitigate disease progression or overcome chemoresistance.

Uddin S, Bhat AA, Krishnankutty R, et al.
Involvement of F-BOX proteins in progression and development of human malignancies.
Semin Cancer Biol. 2016; 36:18-32 [PubMed] Related Publications
The Ubiquitin Proteasome System (UPS) is a core regulator with various protein components (ubiquitin-activating E1 enzymes, ubiquitin-conjugating E2 enzymes, ubiquitin-protein E3 ligases, and the 26S proteasome) which work together in a coordinated fashion to ensure the appropriate and efficient proteolysis of target substrates. E3 ubiquitin ligases are essential components of the UPS machinery, working with E1 and E2 enzymes to bind substrates and assist the transport of ubiquitin molecules onto the target protein. As the UPS controls the degradation of several oncogenes and tumor suppressors, dysregulation of this pathway leads to several human malignancies. A major category of E3 Ub ligases, the SCF (Skp-Cullin-F-box) complex, is composed of four principal components: Skp1, Cul1/Cdc53, Roc1/Rbx1/Hrt1, and an F-box protein (FBP). FBPs are the substrate recognition components of SCF complexes and function as adaptors that bring substrates into physical proximity with the rest of the SCF. Besides acting as a component of SCF complexes, FBPs are involved in DNA replication, transcription, cell differentiation and cell death. This review will highlight the recent literature on three well characterized FBPs SKP2, Fbw7, and beta-TRCP. In particular, we will focus on the involvement of these deregulated FBPs in the progression and development of various human cancers. We will also highlight some novel substrates recently identified for these FBPs.

Khatri R, Shah P, Guha R, et al.
Aromatase Inhibitor-Mediated Downregulation of INrf2 (Keap1) Leads to Increased Nrf2 and Resistance in Breast Cancer.
Mol Cancer Ther. 2015; 14(7):1728-37 [PubMed] Free Access to Full Article Related Publications
Aromatase inhibitors are effective drugs that reduce or eliminate hormone-sensitive breast cancer. However, despite their efficacy, resistance to these drugs can occur in some patients. The INrf2 (Keap1):Nrf2 complex serves as a sensor of drug/radiation-induced oxidative/electrophilic stress. INrf2 constitutively suppresses Nrf2 by functioning as an adapter protein for the Cul3/Rbx1-mediated ubiquitination/degradation of Nrf2. Upon stress, Nrf2 dissociates from INrf2, is stabilized, translocates to the nucleus, and coordinately induces a battery of cytoprotective gene expression. Current studies investigated the role of Nrf2 in aromatase inhibitor resistance. RT-PCR and immunoblot assays showed that aromatase inhibitor-resistant breast cancer LTLTCa and AnaR cells express lower INrf2 and higher Nrf2 protein levels, as compared with drug-sensitive MCF-7Ca and AC1 cells, respectively. The increase in Nrf2 was due to lower ubiquitination/degradation of Nrf2 in aromatase inhibitor-resistant cells. Higher Nrf2-mediated levels of biotransformation enzymes, drug transporters, and antiapoptotic proteins contributed to reduced efficacy of drugs and aversion to apoptosis that led to drug resistance. shRNA inhibition of Nrf2 in LTLTCa (LTLTCa-Nrf2KD) cells reduced resistance and sensitized cells to aromatase inhibitor exemestane. Interestingly, LTLTCa-Nrf2KD cells also showed reduced levels of aldehyde dehydrogenase, a marker of tumor-initiating cells and significantly decreased mammosphere formation, as compared with LTLTCa-Vector control cells. The results together suggest that persistent aromatase inhibitor treatment downregulated INrf2 leading to higher expression of Nrf2 and Nrf2-regulated cytoprotective proteins that resulted in increased aromatase inhibitor drug resistance. These findings provide a rationale for the development of Nrf2 inhibitors to overcome resistance and increase efficacy of aromatase inhibitors.

Díaz-Rodríguez E, Pandiella A
Modulation of cereblon levels by anti-myeloma agents.
Leuk Lymphoma. 2016; 57(1):167-76 [PubMed] Related Publications
The use of thalidomide derivatives (IMIDs) has improved multiple myeloma prognosis, through an unknown mechanism of action. Recently one molecular target, the cereblon (CRBN) protein, has been identified. CRBN acts by binding to DDB1-CUL4-ROC1 forming a ubiquitin ligase multiprotein complex. We have generated antibodies to different regions of CRBN protein, and analyzed the biological consequences of augmenting or decreasing CRBN levels. CRBN was expressed in all the myeloma cell lines tested, independently of their sensitivity to IMIDs, and the CRBN-DDB1-CUL4 complex was efficiently formed. At the molecular level, long-term treatment with IMIDs induced a slight decrease in CRBN levels and a reduction in the CRBN-DDB1-CUL4 complex. Interestingly, treatment with other anti-myeloma drugs downregulated cellular contents of CRBN, and in a much faster fashion. These results suggest that CRBN is an important mediator of the cellular response to IMIDs, but also critical in the maintenance of cell viability and/or proliferation.

Chen X, Wang Y, Zang W, et al.
miR-194 targets RBX1 gene to modulate proliferation and migration of gastric cancer cells.
Tumour Biol. 2015; 36(4):2393-401 [PubMed] Related Publications
RING box protein1 (RBX1), an essential component of SCF E3 ubiquitin ligases, plays an important role in gastric cancer. In the study, miR-194 and RBX1 expression was evaluated in 76 pairs of gastric tumor and non-tumor tissue samples by qRT-PCR, and clinicopathological characteristics were analyzed. CCK8, transwell assay, wound healing assay, and flow cytometry assay were performed to evaluate the effect of miR-194 on gastric cancer (GC) cellular proliferation, invasion, migration, apoptosis, and cell cycle, respectively. Luciferase reporter assays and Western blotting were used to evaluate whether RBX1 is a direct target of miR-194. The Kaplan-Meier method and log-rank test were used to evaluate the correlation between miR-194 or RBX1 expression and patient survival. Then, we found that miR-194 was significantly downregulated and RBX1 upregulated in GC tissues; both of which showed significant association with tumor size, location, invasion, and tumor node metastasis. Cell proliferation, invasion, and migration were significantly restricted with miR-194 overexpression. miR-194 downregulated RBX1 protein expression, and luciferase assays showed that binding sites in the RBX1 3'UTR were required for miR-194-mediated repression of RBX1, indicating that RBX1 was a direct target of miR-194. Transfection of RBX1 without the 3'UTR restored the miR-194-inhibiting migration function. miR-194 overexpression or RBX1 lowexpression was associated with prolonged survival of GC patients. In conclusion, upregulation of miR-194 can inhibit proliferation, migration, and invasion of GC cells, possibly by targeting RBX1. Aberrant expression of miR-194 and RBX1 is correlated to GC patient survival time.

Huang G, Stock C, Bommeljé CC, et al.
SCCRO3 (DCUN1D3) antagonizes the neddylation and oncogenic activity of SCCRO (DCUN1D1).
J Biol Chem. 2014; 289(50):34728-42 [PubMed] Free Access to Full Article Related Publications
The activity of cullin-RING type ubiquitination E3 ligases is regulated by neddylation, a process analogous to ubiquitination that culminates in covalent attachment of the ubiquitin-like protein Nedd8 to cullins. As a component of the E3 for neddylation, SCCRO/DCUN1D1 plays a key regulatory role in neddylation and, consequently, cullin-RING ligase activity. The essential contribution of SCCRO to neddylation is to promote nuclear translocation of the cullin-ROC1 complex. The presence of a myristoyl sequence in SCCRO3, one of four SCCRO paralogues present in humans that localizes to the membrane, raises questions about its function in neddylation. We found that although SCCRO3 binds to CAND1, cullins, and ROC1, it does not efficiently bind to Ubc12, promote cullin neddylation, or conform to the reaction processivity paradigms, suggesting that SCCRO3 does not have E3 activity. Expression of SCCRO3 inhibits SCCRO-promoted neddylation by sequestering cullins to the membrane, thereby blocking its nuclear translocation. Moreover, SCCRO3 inhibits SCCRO transforming activity. The inhibitory effects of SCCRO3 on SCCRO-promoted neddylation and transformation require both an intact myristoyl sequence and PONY domain, confirming that membrane localization and binding to cullins are required for in vivo functions. Taken together, our findings suggest that SCCRO3 functions as a tumor suppressor by antagonizing the neddylation activity of SCCRO.

van der Wijst MG, Brown R, Rots MG
Nrf2, the master redox switch: the Achilles' heel of ovarian cancer?
Biochim Biophys Acta. 2014; 1846(2):494-509 [PubMed] Related Publications
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.

Martinez VD, Vucic EA, Thu KL, et al.
Unique pattern of component gene disruption in the NRF2 inhibitor KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex in serous ovarian cancer.
Biomed Res Int. 2014; 2014:159459 [PubMed] Free Access to Full Article Related Publications
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.

Chen J, Ruan X, Wang S, et al.
Antitumor effect and biological pathways of a recombinant adeno-associated virus as a human renal cell carcinoma suppressor.
Tumour Biol. 2014; 35(11):10993-1003 [PubMed] Free Access to Full Article Related Publications
The aims of this work are to study the antitumor effect of the adeno-associated virus on the xenografted tumors of chick embryo chorioallantoic membrane and predict potential genes and biological pathways which are associated with renal cell carcinoma. The adeno-associated virus NT4-TAT-6 × His-VHLbeta was constructed and identified. Then, chick embryos with xenografted tumor were divided into three groups and respectively inoculated with rAAV/NT4-TAT-6 × His-VHLbeta (group A), empty virus (group B), and phosphate-buffered saline (group C, the control subject). Antitumor effect in each group was investigated by means of immunofluorescence observation. Genes interacted with von Hippel-Lindau were screened by Search Tool for the Retrieval of Interacting Genes/Proteins database, while pathway analysis were performed based on Kyoto Encyclopedia of Genes and Genomes. The growth of xenografted tumors inoculated with recombinant adeno-associated virus was slower than the control subjects. The tumor volumes of group A showed significant difference compared with group B and group C (P < 0.05). Growth of xenografted tumors which administered with the recombinant adeno-associated virus was inhibited. Among the protein-protein interaction network, TCEB2, HIF1A, TCEB1, CUL2, RBX1, and PHF17 were hub genes which might be involved in the development of renal cell carcinoma. The most significant signaling pathway was renal cell carcinoma. In this paper, we constructed and identified the recombinant adeno-associated virus NT4-TAT-6 × His-VHLbeta and studied the antitumor effect of the adeno-associated virus on xenografted tumors of chicken embryo chorioallantoic membrane. In addition, genes in the protein-protein interaction network which are associated with renal cell carcinoma were revealed and the biological pathway of renal cell carcinoma was identified. Our results provide a gene-therapeutic agent for the treatment of human renal cell carcinoma.

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] Related Publications
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.

Xu H, Choe C, Shin SH, et al.
Silencing of KIF14 interferes with cell cycle progression and cytokinesis by blocking the p27(Kip1) ubiquitination pathway in hepatocellular carcinoma.
Exp Mol Med. 2014; 46:e97 [PubMed] Free Access to Full Article Related Publications
Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27(Kip1) protein level specifically increased after KIF14 knockdown. The increase in p27(Kip1) was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27(Kip1) accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27(Kip1) for degradation by the 26S proteasome, leading to accumulation of p27(Kip1). The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.

Martinez VD, Vucic EA, Thu KL, et al.
Disruption of KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex components by multiple genetic mechanisms: Association with poor prognosis in head and neck cancer.
Head Neck. 2015; 37(5):727-34 [PubMed] Related Publications
BACKGROUND: The NRF2 pathway has multiple pro-tumorigenic functions, and Nrf2 levels are increased in head and neck squamous cell carcinoma (HNSCC). The KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex is a negative regulator of NRF2. In this study, we investigated mechanisms of disruption of individual complex components.
METHODS: Clinical and genomic profiles for 302 patients with HNSCC were obtained from The Cancer Genome Atlas. Combined pattern of epi/genetic alterations for individual components revealed frequent of complex disruption. Gene-set enrichment analysis was performed on expression data to identify affected pathways.
RESULTS: DNA loss is the main mechanism of alteration for all component genes, whereas hypermethylation largely affects only KEAP1. Combined analysis revealed that 64% of patients with HNSCC have disruption in this protein complex. Concordantly, NRF2-associated gene signature is enriched in HNSCC. Survival was significantly diminished among patients with one or more disrupted components.
CONCLUSION: The KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex is frequently disrupted in HNSCC by multiple mechanisms. NRF2-based prognostics will benefit from integrated analysis of component genes.

Wu HC, Lin YC, Liu CH, et al.
USP11 regulates PML stability to control Notch-induced malignancy in brain tumours.
Nat Commun. 2014; 5:3214 [PubMed] Free Access to Full Article Related Publications
The promyelocytic leukaemia (PML) protein controls multiple tumour suppressive functions and is downregulated in diverse types of human cancers through incompletely characterized post-translational mechanisms. Here we identify USP11 as a PML regulator by RNAi screening. USP11 deubiquitinates and stabilizes PML, thereby counteracting the functions of PML ubiquitin ligases RNF4 and the KLHL20-Cul3 (Cullin 3)-Roc1 complex. We find that USP11 is transcriptionally repressed through a Notch/Hey1-dependent mechanism, leading to PML destabilization. In human glioma, Hey1 upregulation correlates with USP11 and PML downregulation and with high-grade malignancy. The Notch/Hey1-induced downregulation of USP11 and PML not only confers multiple malignant characteristics of aggressive glioma, including proliferation, invasiveness and tumour growth in an orthotopic mouse model, but also potentiates self-renewal, tumour-forming capacity and therapeutic resistance of patient-derived glioma-initiating cells. Our study uncovers a PML degradation mechanism through Notch/Hey1-induced repression of the PML deubiquitinase USP11 and suggests an important role for this pathway in brain tumour pathogenesis.

Li H, Tan M, Jia L, et al.
Inactivation of SAG/RBX2 E3 ubiquitin ligase suppresses KrasG12D-driven lung tumorigenesis.
J Clin Invest. 2014; 124(2):835-46 [PubMed] Free Access to Full Article Related Publications
Cullin-RING ligases (CRLs) are a family of E3 ubiquitin ligase complexes that rely on either RING-box 1 (RBX1) or sensitive to apoptosis gene (SAG), also known as RBX2, for activity. RBX1 and SAG are both overexpressed in human lung cancer; however, their contribution to patient survival and lung tumorigenesis is unknown. Here, we report that overexpression of SAG, but not RBX1, correlates with poor patient prognosis and more advanced disease. We found that SAG is overexpressed in murine KrasG12D-driven lung tumors and that Sag deletion suppressed lung tumorigenesis and extended murine life span. Using cultured lung cancer cells, we showed that SAG knockdown suppressed growth and survival, inactivated both NF-κB and mTOR pathways, and resulted in accumulation of tumor suppressor substrates, including p21, p27, NOXA, and BIM. Importantly, growth suppression by SAG knockdown was partially rescued by simultaneous knockdown of p21 or the mTOR inhibitor DEPTOR. Treatment with MLN4924, a small molecule inhibitor of CRL E3s, also inhibited the formation of KrasG12D-induced lung tumors through a similar mechanism involving inactivation of NF-κB and mTOR and accumulation of tumor suppressor substrates. Together, our results demonstrate that Sag is a Kras-cooperating oncogene that promotes lung tumorigenesis and suggest that targeting SAG-CRL E3 ligases may be an effective therapeutic approach for Kras-driven lung cancers.

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