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. 2022 Jun;41(25):3423-3432.
doi: 10.1038/s41388-022-02348-0. Epub 2022 May 16.

TP53, CDKN2A/P16, and NFE2L2/NRF2 regulate the incidence of pure- and combined-small cell lung cancer in mice

Samera H Hamad  1 , Stephanie A Montgomery  1   2 , Jeremy M Simon  1   3   4 , Brittany M Bowman  1 , Kyle B Spainhower  5 , Ryan M Murphy  6 , Erik S Knudsen  7 , Suzanne E Fenton  8 , Scott H Randell  9 , Jeremiah R Holt  10 , D Neil Hayes  10 , Agnieszka K Witkiewicz  7 , Trudy G Oliver  5 , M Ben Major  11   12 , Bernard E Weissman  13   14
Affiliations

TP53, CDKN2A/P16, and NFE2L2/NRF2 regulate the incidence of pure- and combined-small cell lung cancer in mice

Samera H Hamad et al. Oncogene. 2022 Jun.

Erratum in

Abstract

Studies have shown that Nrf2E79Q/+ is one of the most common mutations found in human tumors. To elucidate how this genetic change contributes to lung cancer, we compared lung tumor development in a genetically-engineered mouse model (GEMM) with dual Trp53/p16 loss, the most common mutations found in human lung tumors, in the presence or absence of Nrf2E79Q/+. Trp53/p16-deficient mice developed combined-small cell lung cancer (C-SCLC), a mixture of pure-SCLC (P-SCLC) and large cell neuroendocrine carcinoma. Mice possessing the LSL-Nrf2E79Q mutation showed no difference in the incidence or latency of C-SCLC compared with Nrf2+/+ mice. However, these tumors did not express NRF2 despite Cre-induced recombination of the LSL-Nrf2E79Q allele. Trp53/p16-deficient mice also developed P-SCLC, where activation of the NRF2E79Q mutation associated with a higher incidence of this tumor type. All C-SCLCs and P-SCLCs were positive for NE-markers, NKX1-2 (a lung cancer marker) and negative for P63 (a squamous cell marker), while only P-SCLC expressed NRF2 by immunohistochemistry. Analysis of a consensus NRF2 pathway signature in human NE+-lung tumors showed variable activation of NRF2 signaling. Our study characterizes the first GEMM that develops C-SCLC, a poorly-studied human cancer and implicates a role for NRF2 activation in SCLC development.

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Conflict of interest statement

Conflict of interest

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Figures

Fig. 1
Fig. 1. Invasive tumors developed by Trp53fl/fl;p16fl/fl;Nrf2+/+ and Trp53fl/fl;p16fl/fl;Nrf2E79Q/+ mice.
a An example of combined small cell lung cancer (C-SCLC) from a Trp53fl/fl;p16fl/fl;Nrf2E79Q/+ mouse, showing C-SCLC, scalebar=100μm with insets of 1) P-SCLC, scalebar=50μm, and 2) Large Cell Neuroendocrine Carcinoma (LCNEC), scalebar=50μm; b H&E and representative IHC for NKX2–1, P63 and NRF2 of a lung tumor metastasis in liver, scalebar=200μm. Images in a&b were taken using a BX61-Neville microscope; c Representative IF for ASCL1, CHG-A, SYP and PHH3 of a lung tumor metastasis in liver. Slides were imaged on the Vectra® Polaris Automated Quantitative Pathology Imaging System, scalebar=50μm.
Fig. 2
Fig. 2. Characterization of P-SCLC developed by Trp53fl/fl;p16fl/fl;Nrf2E79Q/+ mice.
a H&E of multiple P-SCLCs in Trp53fl/fl;p16fl/fl;Nrf2E79Q/+ mice, scalebar=200μm with insets showing P-SCLC lesions, scalebar=50μm. Images were taken using a BX61-Neville microscope; b Percentage of mice with P-SCLC in Trp53fl/fl;p16fl/fl;Nrf2+/+ and Trp53fl/fl;p16fl/fl;Nrf2E79Q/+ mice; c Total number of P-SCLC observed in each lung from Trp53fl/fl;p16fl/fl;Nrf2+/+ and Trp53fl/fl;p16fl/fl;Nrf2E79Q/+ mice (n = 9 per genotype) (See Supplementary Table 2 for more details). **P<0.01
Fig. 3
Fig. 3. Characterization and NRF2 genotyping of C-SCLC.
IHC for markers of lung differentiation and for tumor suppressor and NRF2 expression in C-SCLCs from Trp53fl/fl;p16fl/fl;Nrf2+/+ (a) and Trp53fl/fl;p16fl/fl;Nrf2E79Q/+mice (b), scalebar=200μm; IF staining for NE markers of the same tumors (c & d), scalebar=100μm; e Genotyping results of C-SCLC from (WT) Trp53fl/fl;p16fl/fl;Nrf2+/+ and (Het) Trp53fl/fl;p16fl/fl;Nrf2E79Q/+ mice, showing the presence of the recombined allele in tumors from Trp53fl/fl;p16fl/fl;Nrf2E79Q/+ mice; f Table summarizing the results of genotyping C-SCLCs from Trp53fl/fl;p16fl/fl;Nrf2+/+ and Trp53fl/fl;p16fl/fl;Nrf2E79Q/+ mice; a & b Images were taken using a BX61-Neville microscope; c & d Slides were imaged on the Vectra® Polaris Automated Quantitative Pathology Imaging System.
Fig. 4
Fig. 4. Characterization of P-SCLC developed by Trp53fl/fl;p16fl/fl;Nrf2+/+ and Trp53fl/fl;p16fl/fl;Nrf2E79Q/+ mice.
a IHC for markers of lung differentiation and for tumor suppressor and NRF2 expression in P-SCLCs from Trp53fl/fl;p16fl/fl;Nrf2+/+ and Trp53fl/fl;p16fl/fl;Nrf2E79Q/+mice,. Images were taken using a BX61-Neville microscope, scalebar=200μm; b IF staining for NE markers of the same tumors. Slides were imaged on the Vectra® Polaris Automated Quantitative Pathology Imaging System, scalebar=100μm.
Fig. 5
Fig. 5. Heatmap of NRF2 target gene expression in human neuroendocrine lung tumors.
Gene expression, histopathology, and tumor mutation data were mined from a previous meta-analysis of lung tumors. Gene expression data for NRF2 target genes were VST-normalized and median-centered, and hierarchically clustered separately within each tumor super-class. Sex, histopathological classifications, molecular clusters, and mutations in KEAP1/NRF2 are designated in colors at the top based on previous annotations. Higher expression values are designated in red, and lower expression values are designated in blue. Tumors carrying KEAP1/NRF2 tumors are designated in black.
Fig 6
Fig 6. Schematic representation of P-SCLC and C-SCLC development in Trp53fl/fl;p16fl/fl;Nrf2+/+ and Trp53fl/fl;p16fl/fl;Nrf2E79Q/+ GEMM.
The frequency of NRF2-negative P-SCLC lesions in Trp53fl/fl;p16fl/fl;Nrf2+/+ mice was ~ten-fold lower than the frequency of NRF2-positive P-SCLC observed in Trp53fl/fl;p16fl/fl;Nrf2E79Q/+ mice. Both genotypes developed NRF2-negative C-SCLC at similar frequencies. Whether the P-SCLC and C-SCLC develop independently of each other or sequentially remains an open question.
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