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Genetic Disruption of KEAP1/CUL3 E3 Ubiquitin Ligase Complex Components Is a Key Mechanism of NF-Kappab Pathway Activation in Lung Cancer

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Genetic Disruption of KEAP1/CUL3 E3 Ubiquitin Ligase Complex Components Is a Key Mechanism of NF-Kappab Pathway Activation in Lung Cancer View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector ORIGINAL ARTICLE Genetic Disruption of KEAP1/CUL3 E3 Ubiquitin Ligase Complex Components is a Key Mechanism of NF-KappaB Pathway Activation in Lung Cancer Kelsie L. Thu, BSc,* Larissa A. Pikor, BSc,* Raj Chari, PhD,* Ian M. Wilson, PhD,* Calum E. MacAulay, PhD,* John C. English, MD,† Ming-Sound Tsao, MD,‡ Adi F. Gazdar, MD,§ Stephen Lam, MD,* Wan L. Lam, PhD,* and William W. Lockwood, PhD*ʈ pressed in samples with complex disruption, further demonstrating Introduction: Inhibitor of kappa light polypeptide gene enhancer in the effect of complex disruption on NF-␬B activity. B-cells, kinase beta (IKBKB) (IKK-␤/IKK-2), which activates NF-␬B, Conclusions: Gene dosage alteration is a prominent mechanism that is a substrate of the KEAP1-CUL3-RBX1 E3-ubiquitin ligase complex, disrupts each component of the KEAP1-CUL3-RBX1 complex and implicating this complex in NF-␬B pathway regulation. We investi- its NF-␬B stimulating substrate, IKBKB. Herein, we show that, gated complex component gene disruption as a novel genetic mecha- multiple component disruption of this complex represents a novel nism of NF-␬B activation in non-small cell lung cancer. mechanism of NF-␬B 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 Key Words: KEAP1, CUL3, RBX1, IKBKB, NF-␬B signaling, and IKBKB. Gene expression of these genes and NF-␬B target genes Genetic disruption. were analyzed in 48 tumors. IKBKB protein levels were assessed in (J Thorac Oncol. 2011;6: 1521–1529) tumors with and without complex or IKBKB genetic disruption. Com- plex component knockdown was performed to assess effects of the ␬ E3-ligase complex on IKBKB and NF- B levels, and phenotypic EAP1 is a substrate adaptor protein that binds substrates importance of IKBKB expression was measured by pharmacological Kto an E3-ubiquitin ligase complex, which also includes inhibition. CUL3 and RBX1 (Figure 1). Ubiquitination and degradation of Results: We observed strikingly frequent genetic disruption (42%) complex substrates prevents their accumulation and undesired and aberrant expression (63%) of the E3-ligase complex and IKBKB downstream effects. The best characterized substrate of KEAP1 in the samples examined. Although both adenocarcinomas and and the E3-complex is NRF2.1–3 In response to oxidative stress, squamous cell carcinomas showed complex disruption, the patterns NRF2 stimulates transcription of cytoprotective genes that scav- of gene disruption differed. IKBKB levels were elevated with enge harmful reactive molecules, preventing cellular damage.4 complex disruption, knockdown of complex components increased Interestingly, lung-specific KEAP1 knockout in mice was shown activated forms of IKBKB and NF-␬B proteins, and IKBKB inhi- to protect against cell damage caused by cigarette smoke by bition detriments cell viability, highlighting the biological signifi- enabling NRF2 accumulation and increased expression of its ␬ cance of complex disruption. NF- B target genes were overex- target genes.5 NRF2 has also been implicated in cancer cell resistance to chemotherapeutics by its activation of drug-metab- olizing and drug-efflux proteins.4 *Integrative Oncology Department, BC Cancer Research Centre, Vancouver; Recently, the KEAP1 E3-ligase complex has been im- †Department of Pathology, Vancouver General Hospital, Vancouver, ␬ 6 BC; ‡University Health Network, Ontario Cancer Institute/Princess Mar- plicated in the regulation of NF- B signaling, which pro- garet Hospital and University of Toronto, Toronto, Ontario, Canada; motes cell proliferation and survival.7–9 The NF-␬B pathway §Hamon Center for Therapeutic Oncology Research, University of Texas is activated in more than 60% of lung cancers; however, the Southwestern Medical Center, Dallas, Texas; and ʈCancer Genetics Branch, National Human Genome Research Institute, National Institutes genetic mechanisms underlying its activation remain largely of Health, Bethesda, Maryland. unknown.7,10–13 When inactive, NF-␬B is bound by inhibi- Disclosure: The authors declare no conflicts of interest. tory proteins (I␬B) in the cytoplasm. On stimulation, the Supplemental digital content is available for this article. Direct URL citations kinase inhibitor of kappa light polypeptide gene enhancer in appear in the printed text, and links to the digital files are provided in the ␬ HTML text of this article on the journal’s Web site. (www.jto.org). B-cells, kinase beta (IKBKB) phosphorylates I B, releasing Address for correspondence: Kelsie Thu, BSc, BC Cancer Research Centre, its inhibition, and enabling NF-␬B translocation to the nu- 675 W 10th Ave, Vancouver, Canada V5Z 1L3. E-mail: [email protected] cleus where it exerts its effects (Figure 1).14 Lee et al.6 The first two authors contributed equally to this work. demonstrated that KEAP1 binds IKBKB, drawing it to the Copyright © 2011 by the International Association for the Study of Lung Cancer E3-ligase complex for ubiquitination and degradation, pro- ISSN: 1556-0864/11/0609-1521 viding a mechanism of NF-␬B regulation. These findings Journal of Thoracic Oncology • Volume 6, Number 9, September 2011 1521 Thu et al. Journal of Thoracic Oncology • Volume 6, Number 9, September 2011 A Funconal Ub complex NRF2 Degradaon of NRF2 NRF2 KEAP1 and inacvaon of RBX1 cytoprotecve genes KEAP1 RBX1 CUL3 CUL3 Ub IKBKB IKBKB degradaon IKBKB KEAP1 and control of RBX1 NF-kappaB signaling Ub Ubiquin CUL3 B Disrupted, IKBKB Accumulaon of Acvaon of non-funconal IKBKB NF-kappaB complex KEAP1 NFκB NFκB RBX1 IKBKB NFκB IKBKB NFκB NFκB CUL3 IKBKB FIGURE 1. The KEAP1 Cullin-3 E3-ubiquitin ligase complex and its roles in maintaining cellular levels of NRF2 and IKBKB. A, When complex components are intact, KEAP1 facilitates binding of NRF2 or IKBKB, which promotes their ubiquitination. This complex prevents accumulation of IKBKB and subsequent NF-␬B activation. B, Disruption of any complex component com- promises function leading to stabilization and accumulation of IKBKB and aberrant activation of NF-␬B. provide significant evidence of the integral role of KEAP1 mechanism that may be responsible for IKBKB accumulation and the E3-ligase complex in NF-␬B signaling. and stimulation of NF-␬B. In this study, we (1) investigated The ability of the E3-ligase complex to ubiquitinate whether these complex components and IKBKB (8p11.21) IKBKB was most efficient when all three complex compo- exhibit gene dosage and expression alterations and the fre- nents were expressed and intact, suggesting disruption of quencies at which they occur in a large cohort of clinical lung even a single component compromises function. Although tumors, (2) investigated whether the complex components somatic DNA alterations have been observed in the genes display subtype-specific disruption, and (3) assessed the func- encoding some of these complex components, it is not known tional consequence of complex disruption on NF-␬B activity, whether complex component gene disruptions are a key as these genetic events may be significant contributors to the mechanism of NF-␬B activation in lung cancer.6 In lung NF-␬B activation commonly observed in lung cancer. cancer, only KEAP1 has been thoroughly investigated at the DNA level, and a recent study reported that low KEAP1 MATERIALS AND METHODS expression was associated with poor patient outcome.15 KEAP1 deletions, mutations, and DNA hypermethylation Non-small Cell Lung Cancer Samples have been reported in 3 to 41% of lung tumors16–21; however, A total of 261 lung tumors (169 adenocarcinomas the moderate frequency of KEAP1 gene disruption alone is [ACs] and 92 squamous cell carcinomas [SCC]) were accrued not sufficient to explain the high (Ͼ60%) frequency of from Vancouver General Hospital (Vancouver, Canada) and NF-␬B activation in lung cancer.11 Princess Margaret Hospital (Toronto, Canada) following We propose that genetic disruption of any one E3- ethics approval with patient consent (see Table, Supple- ubiquitin ligase complex component may be sufficient to mental Digital Content 1, which lists patient demograph- result in tumorigenic NF-␬B activation due to loss of com- ics, http://links.lww.com/JTO/A112). Tissue sections were plex function and subsequent accumulation of IKBKB.6,14,22 microdissected with the guidance of lung pathologists. We hypothesize that somatic disruptions of CUL3 (2q36.2) Matched nonmalignant lung tissue was also obtained for a and RBX1 (22q13.2), in addition to KEAP1 (19p13.2) occur subset of the primary tumors collected. DNA for all 261 frequently in lung tumors, representing a prominent genetic samples was extracted using standard phenol-chloroform pro- 1522 Copyright © 2011 by the International Association for the Study of Lung Cancer Journal of Thoracic Oncology • Volume 6, Number 9, September 2011 KEAP1/CUL3 E3 Ubiquitin Ligase Complex cedures. RNA was extracted from tumor and matched non- HapMap individuals as a reference.29 In total, 90 unique malignant tissues using RNeasy Mini Kits (Qiagen Inc., NSCLC cell lines were analyzed, and genetic disruption was Mississauga, ON). Non-small cell lung cancer (NSCLC) cell noted if either platform (SNP or SMRT array) detected a copy lines (H1650, HCC827, H3255, H358, H23, HCC95, H2347, number alteration. The Broad Institute’s Tumorscape data- and H2122) were obtained from American Type Culture base (www.broadinstitute.org/tumorscape) was also accessed Collection or the laboratory of Adi F. Gazdar and cultivated as to investigate copy number status at these four gene loci.30 described previously.23 These cell lines were fingerprinted to confirm their identity.24 Human bronchial epithelial cells Western Blot Assessment of Total and (HBEC-KT) were provided by Dr. John Minna (University of Phospho-IKBKB and Total and Phospho-NF-␬B Texas Southwestern) and maintained as described previously.25 Protein Levels Primary nonmalignant human bronchial epithelial (NHBE) lung IKBKB protein levels were assessed in eight NSCLC cell cells were obtained from Lonza (Walkersville, MD).
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