21P Urethane-induced lung carcinogenesis in genetically edited C57Bl/6 mice with CHEK2 and GPRC5A heterozygous inactivating mutations

Abstract

Some lung cancer (LC) patients carry germline truncating mutations in DNA repair or other cancer-related genes, however it is unclear whether these allelic variants play a causative role in LC predisposition, or, alternatively, are occasionally present in these subjects due to a chance. Case-control comparison is often not efficient due to rarity of gene-inactivating variants in population, therefore alternative approaches are required. We evaluated whether germ-line inactivation of GPRC5A and CHEK2 genes facilitates LC in mice. C57BL/6 mice with heterozygous inactivating mutations in the GPRC5A or CHEK2 genes were created using the CRISPR/Cas9 system. Donor eggs were obtained from the first generation of crossing mice of C57BL/6 (males) and CBA (females) inbred strains. Offspring mice F1 bearing inactivating mutations in GPRC5A and CHEK2 were used in further breeding with C57BL/6 wild-type strain for the study purposes. Altogether, 33 females and 21 males without mutations, 11 females and 19 males with mutations in CHEK2, and 13 females and 14 males with mutations in GPRC5A were analyzed. At the age of 3 months, mice were intraperitoneally injected with urethane (1 g/kg). The experiment was stopped after 45 weeks, all animals were autopsied and the lung tumor nodes were counted. In the wild-type (control) mice, the number of affected animals was 16/33 (48%) in females and 12/21 (57%) in males (pooled males/females: 28/54 (52%)). The rate of tumor development was not increased in CHEK2-heterozygous mice (females: 6/11 (55%); males: 7/19 (37%); pooled males/females: 13/30 (43%)). However, animals carrying GPRC5A heterozygous inactivating mutations showed a trend towards elevated occurrence of carcinogen-induced lung neoplasms (females: 9/13 (69%); males: 11/14 (79%); pooled males/females: 20/27 (74%); р = 0.06 when compared to controls). These data support evidences for the involvement of GPRC5A gene in pathogenesis of lung cancer.