. 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 Hamad¹, Stephanie A Montgomery^{1

2}, Jeremy M Simon^{1

3

4}, Brittany M Bowman¹, Kyle B Spainhower⁵, Ryan M Murphy⁶, Erik S Knudsen⁷, Suzanne E Fenton⁸, Scott H Randell⁹, Jeremiah R Holt¹⁰, D Neil Hayes¹⁰, Agnieszka K Witkiewicz⁷, Trudy G Oliver⁵, M Ben Major^{11

12}, Bernard E Weissman^{13

14}

Affiliations

¹ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
² Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
³ Department of Genetics, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
⁴ UNC Neuroscience Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
⁵ Department of Oncological Sciences, School of Medicine, University of Utah, Salt Lake City, UT, USA.
⁶ Department of Pharmacology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
⁷ Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA.
⁸ Division of National Toxicology Program, NIEHS/NIH, Research Triangle Park, NC, USA.
⁹ Marsico Lung Institute, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
¹⁰ University of Tennessee Health Science Center for Cancer Research, Department of Medicine, Division of Hematology and Oncology, University of Tennessee, Memphis, TN, USA.
¹¹ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA. bmajor@wustl.edu.
¹² Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA. bmajor@wustl.edu.
¹³ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA. weissman@med.unc.edu.
¹⁴ Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA. weissman@med.unc.edu.

PMID: 35577980
PMCID: PMC10039451
DOI: 10.1038/s41388-022-02348-0

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.

. 2022 Jun;41(25):3423-3432.

doi: 10.1038/s41388-022-02348-0. Epub 2022 May 16.

Authors

Affiliations

¹ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
² Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
³ Department of Genetics, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
⁴ UNC Neuroscience Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
⁵ Department of Oncological Sciences, School of Medicine, University of Utah, Salt Lake City, UT, USA.
⁶ Department of Pharmacology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
⁷ Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA.
⁸ Division of National Toxicology Program, NIEHS/NIH, Research Triangle Park, NC, USA.
⁹ Marsico Lung Institute, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
¹⁰ University of Tennessee Health Science Center for Cancer Research, Department of Medicine, Division of Hematology and Oncology, University of Tennessee, Memphis, TN, USA.
¹¹ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA. bmajor@wustl.edu.
¹² Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA. bmajor@wustl.edu.
¹³ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA. weissman@med.unc.edu.
¹⁴ Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA. weissman@med.unc.edu.

PMID: 35577980
PMCID: PMC10039451
DOI: 10.1038/s41388-022-02348-0

Erratum in

Correction: TP53, CDKN2A/P16, and NFE2L2/NRF2 regulate the incidence of pure- and combined-small cell lung cancer in mice.
Hamad SH, Montgomery SA, Simon JM, Bowman BM, Spainhower KB, Murphy RM, Knudsen ES, Fenton SE, Randell SH, Holt JR, Hayes DN, Witkiewicz AK, Oliver TG, Major MB, Weissman BE. Hamad SH, et al. Oncogene. 2022 Sep;41(39):4485. doi: 10.1038/s41388-022-02442-3. Oncogene. 2022. PMID: 36002660 No abstract available.

Abstract

Studies have shown that Nrf2^E79Q/+ 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 Nrf2^E79Q/+. 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-Nrf2^E79Q 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-Nrf2^E79Q allele. Trp53/p16-deficient mice also developed P-SCLC, where activation of the NRF2^E79Q 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.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest

No conflict of interest to report

Figures

**Fig. 1. Invasive tumors developed by *Trp53*^fl/fl;p16^fl/fl;Nrf2^+/+ and *Trp53*^fl/fl;p16^fl/fl;Nrf2^E79Q/+ mice.**
a An example of combined small cell lung cancer (C-SCLC) from a *Trp53*^fl/fl;p16^fl/fl;Nrf2^E79Q/+ 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. Characterization of P-SCLC developed by *Trp53*^fl/fl;p16^fl/fl;Nrf2^E79Q/+ mice.**
a H&E of multiple P-SCLCs in *Trp53*^fl/fl;p16^fl/fl;Nrf2^E79Q/+ 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 *Trp53*^fl/fl;p16^fl/fl;Nrf2^+/+ and *Trp53*^fl/fl;p16^fl/fl;Nrf2^E79Q/+ mice; c Total number of P-SCLC observed in each lung from *Trp53*^fl/fl;p16^fl/fl;Nrf2^+/+ and *Trp53*^fl/fl;p16^fl/fl;Nrf2^E79Q/+ mice (n = 9 per genotype) (See Supplementary Table 2 for more details). ***P<0.01*

**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 *Trp53*^fl/fl;p16^fl/fl;Nrf2^+/+ (a) and *Trp53*^fl/fl;p16^fl/fl;Nrf2^E79Q/+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) *Trp53*^fl/fl;p16^fl/fl;Nrf2^+/+ and (Het) *Trp53*^fl/fl;p16^fl/fl;Nrf2^E79Q/+ mice, showing the presence of the recombined allele in tumors from *Trp53*^fl/fl;p16^fl/fl;Nrf2^E79Q/+ mice; f Table summarizing the results of genotyping C-SCLCs from *Trp53*^fl/fl;p16^fl/fl;Nrf2^+/+ and *Trp53*^fl/fl;p16^fl/fl;Nrf2^E79Q/+ 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. Characterization of P-SCLC developed by *Trp53*^fl/fl;p16^fl/fl;Nrf2^+/+ and *Trp53*^fl/fl;p16^fl/fl;Nrf2^E79Q/+ mice.**
a IHC for markers of lung differentiation and for tumor suppressor and NRF2 expression in P-SCLCs from *Trp53*^fl/fl;p16^fl/fl;Nrf2^+/+ and *Trp53*^fl/fl;p16^fl/fl;Nrf2^E79Q/+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. 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. Schematic representation of P-SCLC and C-SCLC development in *Trp53*^fl/fl;p16^fl/fl;Nrf2^+/+ and *Trp53*^fl/fl;p16^fl/fl;Nrf2^E79Q/+ GEMM.**
The frequency of NRF2-negative P-SCLC lesions in *Trp53*^fl/fl;p16^fl/fl;Nrf2^+/+ mice was ~ten-fold lower than the frequency of NRF2-positive P-SCLC observed in *Trp53*^fl/fl;p16^fl/fl;Nrf2^E79Q/+ 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.

See this image and copyright information in PMC

References

1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin 2020; 70: 7–30. - PubMed
1. Society AC. Cancer Facts and Figures 2019. American Cancer Society Inc., 2019.
1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019; 69: 7–34. - PubMed
1. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004; 304: 1497–1500. - PubMed
1. Kodama T, Tsukaguchi T, Satoh Y, Yoshida M, Watanabe Y, Kondoh O et al. Alectinib shows potent antitumor activity against RET-rearranged non-small cell lung cancer. Mol Cancer Ther 2014; 13: 2910–2918. - PubMed

Publication types

Actions
Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Grants and funding

LinkOut - more resources

Full Text Sources
Medical
- MedlinePlus Health Information
Molecular Biology Databases
- Mouse Genome Informatics (MGI)
Research Materials
- NCI CPTC Antibody Characterization Program
Miscellaneous
- NCI CPTAC Assay Portal

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

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

Affiliations

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

Authors

Affiliations

Erratum in

Abstract

Conflict of interest statement

Figures

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Medical

Molecular Biology Databases

Research Materials

Miscellaneous