“Keaping” a lid on lung cancer: the Keap1-Nrf2 pathway

Figures & data

Figure 1. The KEAP1-NRF2 pathway is altered in human lung cancers.

Schematic of KEAP1-NRF2 pathway under homeostatic conditions (left), oxidative conditions (center) or synergistic conditions, for example in a cancer cell (right). Transcriptional activity of NRF2 is highlighted in the green energy bar. Common mutation hotspots in adenocarcinoma (ADC; black circles), squamous cell carcinoma (SqCC; pink circles) or common to both (half black/pink circles) are highlighted in the protein constructs of KEAP1 and NRF2 (NFE2L2). KEAP1, Kelch-like ECH-associated protein 1; NRF2/NFE2L2, Nuclear factor erythoid-2-related factor 2; CUL3, Cullin 3; D, DLG domain; E, ETGE domain; ARE, antioxidant response element; MAF, v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog; AKT, RAC-alpha serine/threonine-protein kinase; AMPK, AMP-activated protein kinase; GSK3β, Glycogen synthase kinase 3β.

Table 1. Mouse models engineered to harbor KEAP1-NRF2 pathway alterations.

Mouse mutant	Features of the GEMM	Histopathology	Reference
Keap1^f/f	Adenoviral Cre-dependent deletion of Keap1 (floxed exons 2 – 5) sporadically in lung epithelium.	Normal lung morphology, no tumor development (12–15 months post-infection)	Best et al., 2018
Keap1^f/f;Pten^f/f (K1P)	Adenoviral Cre-dependent deletion of Keap1 (floxed exons 2 – 3) and Pten (floxed exon 5) sporadically in lung epithelium	Multifocal bronchiolocentric ADC with a papillary-predominant pattern; latency 12 months. Tumors were sensitive to anti-PD-1/anti-CTLA4 inhibition.	Best et al., 2018
Kras^LSL-G12^D/+;Nrf2^−/-	Adenoviral Cre-dependent expression of Kras^G^12^D on a background of Nrf2 germline loss.	Nrf2 loss inhibits Kras^G^12^D-induced lung tumorigenesis.	DeNicola et al., 2012
Kras^LSL-G12D/+;p53^f/f (KP) with Keap1 LOF	Lentiviral sgKeap1-pSECC inhalation: Cre-mediated expression of Kras^G12^D, and deletion of p53 and CRISPR-induced mutation of Keap1.	Keap1 mutation promoted Kras^G12^D;p53^Δ/Δ tumorigenesis by Nrf2 stabilization. Tumors were sensitive to glutaminase inhibition.	Romero et al., 2017
Kras^LSL-G12^D/+;Roas26^LSL-tdTomato;H11^LSL-Cas9 (KT;Cas9)	Inhalation of a pool of barcoded Lenti-sgTS-Pool/Cre vectors: Cre-dependent expression of Kras^G12^D, dtTomato, Cas9 and an sgRNA targeting known/putative tumor suppressor genes^a.	sgRNAs targeting the Keap1 gene were not enriched in Kras^G12^D-induced lung tumors.	Rogers et al., 2017; Rogers et al., 2018
Kras^LSL-G12D/+;p53^f/f; Roas26^LSL-tdTomato;H11^LSL-Cas9 (KPT;Cas9)	Inhalation of a pool of barcoded Lenti-sgTS-Pool/Cre vectors: Cre-dependent expression of Kras^G12D, dtTomato, Cas9 and inactivation of p53 and CRISPR-Cas9-mediated mutation of known/putative tumor suppressor genes^a.	sgRNAs targeting the Keap1 gene were not enriched in Kras^G12D;p53^Δ/Δ lung tumors.	Rogers et al., 2017; Rogers et al., 2018
Kras^LSL-G12D/+;Lkb1^f/f; Roas26^LSL-tdTomato;H11^LSL-Cas (KLT;Cas9)	Inhalation of a pool of barcoded Lenti-sgTS-Pool/Cre vectors: Cre-dependent expression of Kras^G12D, dtTomato, Cas9 and inactivation of Lkb1 and CRISPR-Cas9-mediated mutation of known/putative tumor suppressor genes^a.	sgRNAs targeting the Keap1 gene were not enriched in Kras^G12D;Lkb1^Δ/Δ lung tumors.	Rogers et al., 2017; Rogers et al., 2018
Kras^LSL-G12D/+;Lkb1^f/f	Lentiviral Nrf2-Cre inhalation: expression of Nrf2, oncogenic Kras^G12D and concomitant deletion of Lkb1.	Nrf2 overexpression decreased the incidence of SqCC and increased ADC formation.	Li et al., 2015

^aLenti-sgTS-Pool/Cre contains 4 vectors with inert sgRNAs and 11 vectors targeting known and candidate tumor suppressor genes (Setd2, Lkb1, Rb1, Apc, Rbm10, Cdkn2a, Trp53, Keap1, Smad4, Arid1a and Atm)

Figure 2. Directed loss of Keap1 and Pten in lung basal progenitor cells promotes lung SqCC formation.

(A) Timeline of polidocanol (POL) administration and adenovirus infection in Keap1^f/f/Pten^f/f mice. Briefly, mice were intra-tracheally (i.t.) injected with 10 µl of 2% w/v polidocanol (POL) one day prior to intra-nasal infection of Ad5-K14-Cre virus. Mice were sacrificed, and lung tissue analyzed 12 months following adenoviral infection. (B) Timeline of Ad5-CMV-Cre virus infection in Keap1^f/f/Pten^f/f/LSL-Sox2 (K1PS) mice. (C) Quantification of histological phenotype of lung tumors in Ad5-CMV-Cre-infected Keap1^f/f/Pten^f/f (K1P) mice [6] (n = 6), Ad5-K14-Cre-infected K1P mice (n = 5) and Ad5-CMV-Cre-infected K1PS mice (n = 3). (D) Representative Hematoxylin & Eosin (H&E) and immunostained sections of lung ADC and SqCC tumors detected in Keap1^f/f/Pten^f/f mice 12 months following Ad5-K14-Cre infection. Scale bars; H&E-stained sections 200 µM; Nkx2.1, Nqo1, K5 and Sox2 50 µM. (E) Schematic representation of the consquences of combined loss of Keap1 and Pten in distinct cell types or when combined with enforced Sox2 expression. Combined loss of Keap1 and Pten in bronchiolar and/or alveolar type 2 (AT2) epithelium results in the formation of ADC tumors, while restricted loss of Keap1 and Pten to basal progenitor cells promotes SqCC formation. Interestingly, overexpression of Sox2 in K1P mice promoted the ADC to SqCC transdifferentiation, likely initiated from a “switched” Club or AT2 cell.

Best SA, De Souza DP, Kersbergen A, et al. Synergy between the KEAP1/NRF2 and PI3K Pathways Drives Non-Small-Cell Lung Cancer with an Altered Immune Microenvironment. Cell Metab. 2018;27:935–43 e4.

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