Effect of inactivating heterozygous mutations in DNA repair genes on experimental lung carcinogenesis in mice

Background. Inactivating mutations in Chek2 and Gprc5a genes are known to be associated with cancer development. Experimental carcinogenesis studies in genetically modified mice generate new data on their influence on pathology development.

Aim. In the present study in a model of lung carcinogenesis, survival parameters as well as tumor multiplicity and size in mice with Chek2 and Gprc5a heterozygous inactivating mutations were evaluated.

Material and methods. F2 hybrid mice from crosses between CBAB6F1 males heterozygous for the studied mutations and wild-type BALB / c females were used: Chek2^dAA-carriers (76 males and 64 females) and Gprc5a^insA-carriers (60 males and 42 females). Starting at four months of age, mice received urethane (ethyl carbamate) intraperitoneally at a dose of 600 mg / kg weekly for 6 weeks. After genotyping by allele-specific PCR, animals were allocated to groups. Carcinogenesis parameters were evaluated 40 weeks after the beginning of the experiment.

Results. The proportion of mice with mutations surviving to the age of three months roughly followed the Mendelian distribution (35 / 41 males and 33 / 31 females) for the offspring of males heterozygous for Chek2^dAA and was significantly lower in the case of Gprc5a^insA (20 / 40 males and 17 / 25 females, p = 0.043). The death of Gprc5a^insA carriers during the experiment was also higher than in the control group (p = 0.0506 in females). Synchronous lung and thymus neoplasms were found in 2 out of 4 Gprc5a^insA females that died before the end of the experiment, which were not found in other groups. At the end of the experiment, no significant differences in tumor multiplicity, mean linear size, and volume were found between the groups of mice with and without mutations.

Conclusion. It was found that heterozygous inactivating mutation Chek2^dAA does not affect early age development and does not modify the parameters of induced lung carcinogenesis in mice. Heterozygous carriage of Gprc5a^insA mutation in mice increases the risk of early death and sensitivity to the toxic and carcinogenic effects of urethane.

1. Imyanitov E.N., Byrski T. Systemic treatment for hereditary cancers: A 2012 update. Hered Cancer Clin Pract 2013;11(1):2. DOI: 10.1186/1897-4287-11-2

2. De Castro D.G., Clarke P.A., Al-Lazikani B., Workman P. Personalized cancer medicine: Molecular diagnostics, predictive biomarkers, and drug resistance. Clin Pharmacol Ther 2013;93(3):252–9. DOI: 10.1038/clpt.2012.237

3. Pritchard C.C., Mateo J., Walsh M.F. et al. Inherited DNA-repair gene mutations in men with metastatic prostate cancer. N Engl J Med 2016;375(5):443–53. DOI: 10.1056/NEJMOA1603144

4. Cybulski C., Górski B., Huzarski T. et al. CHEK2 is a multiorgan cancer susceptibility gene. Am J Hum Genet 2004;75(6):1131–5. DOI: 10.1086/426403

5. Kilpivaara O., Vahteristo P., Falck J. et al. CHEK2 variant I157T may be associated with increased breast cancer risk. Int J Cancer 2004;111(4):543–7. DOI: 10.1002/IJC.20299

6. Cheng Y., Lotan R. Molecular cloning and characterization of a novel retinoic acid-inducible gene that encodes a putative G protein-coupled receptor. J Biol Chem 1998;273(52):35008–15. DOI: 10.1074/JBC.273.52.35008

7. Zhou H., Rigoutsos I. The emerging roles of GPRC5A in diseases. Oncoscience 2014;1(12):765–76. DOI: 10.18632/oncoscience.104

8. Kume H., Muraoka S., Kuga T. et al. Discovery of colorectal cancer biomarker candidates by membrane proteomic analysis and subsequent verification using selected reaction monitoring (SRM) and tissue microarray (TMA) analysis. Mol Cell Proteomics 2014;13(6):1471–84. DOI: 10.1074/mcp.M113.037093

9. Liu S., Zhong S., Ye D. et al. Repression of G protein-coupled receptor family C group 5 member A is associated with pathologic differentiation grade of oral squamous cell carcinoma. J Oral Pathol Med 2013;42(10):761–8. DOI: 10.1111/jop.12077

10. Subrungruang I., Thawornkuno C., Porntip C.P. et al. Gene expression profiling of intrahepatic cholangiocarcinoma. Asian Pacific J Cancer Prev 2013;14(1):557–63. DOI: 10.7314/APJCP.2013.14.1.557

11. Imyanitov E., Panchenko A., Permyakov O. et al. 21P Urethaneinduced lung carcinogenesis in genetically edited C57Bl/6 mice with CHEK2 and GPRC5A heterozygous inactivating mutations. Ann Oncol 2020;31:S251. DOI: 10.1016/j.annonc.2020.08.173

12. Bahassi E.M., Robbins S.B., Moying Y. et al. Mice with the CHEK2*1100delC SNP are predisposed to cancer with a strong gender bias. Proc Natl Acad Sci USA 2009;106(40):17111–6. DOI: 10.1073/PNAS.0909237106

13. Tao Q., Fujimoto J., Men T. et al. Identification of the retinoic acid-inducible Gprc5a as a new lung tumor suppressor gene. J Natl Cancer Inst 2007;99(22):1668–82. DOI: 10.1093/jnci/djm208

14. Xu J., Tian J., Shapiro S.D. Normal lung development in RAIG1-deficient mice despite unique lung epithelium-specific expression. Am J Respir Cell Mol Biol 2005;32(5):381–7. DOI: 10.1165/RCMB.2004-0343OC

15. Panchenko A.V., Pigarev S.E., Fedoros E.I. et al. Urethaneinduced transgenerational carcinogenesis in the offspring of male BALB/c mice exposed to general uniform gamma irradiation. Voprosy onkologii = Oncology issues 2023;69(2):246–52. (In Russ.). DOI: 10.37469/0507-3758-2023-69-2-246-252

16. Takai H., Naka K., Okada Y. et al. Chk2-deficient mice exhibit radioresistance and defective p53-mediated transcription. EMBO J 2002;21(19):5195. DOI: 10.1093/EMBOJ/CDF506

17. Stolarova L., Kleiblova P., Janatova M. et al. CHEK2 germline variants in cancer predisposition: Stalemate rather than checkmate. Cells 2020;9(12):2675. DOI: 10.3390/CELLS9122675

18. Niida H., Murata K., Shimada M. et al. Cooperative functions of Chk1 and Chk2 reduce tumour susceptibility in vivo. EMBO J 2010;29(20):3558. DOI: 10.1038/EMBOJ.2010.218

19. Wang J., Farris A.B., Xu K. et al. GPRC5A suppresses protein synthesis at the endoplasmic reticulum to prevent radiation-induced lung tumorigenesis. Nat Commun 2016;7:11795. DOI: 10.1038/ncomms11795

20. Fujimoto J., Nunomura-Nakamura S., Liu Y. et al. Development of Kras mutant lung adenocarcinoma in mice with knockout of the airway lineage-specific gene Gprc5a. Int J Cancer 2017;141(8):1589–99. DOI: 10.1002/ijc.30851

21. Deng J., Fujimoto J., Ye X.F. et al. Knockout of the tumor suppressor gene Gprc5a in mice leads to NF-kappa B activation in airway epithelium and promotes lung inflammation and tumorigenesis. Cancer Prev Res (Phila) 2010;3(4):424–37. DOI: 10.1158/1940-6207.CAPR-10-0032

22. Wang T., Jing B., Xu D. et al. PTGES/PGE2 signaling links immunosuppression and lung metastasis in Gprc5a-knockout mouse model. Oncogene 2020;39(15):3179–94. DOI: 10.1038/s41388-020-1207-6