Abstract 4665: Lineage-tracing technology to understand the molecular events in a mouse model of tongue squamous cell carcinoma (SCC) carcinogenesis

Abstract

Oral cavity squamous cell carcinomas (OCSCCs) account for over 450,000 cases per year worldwide, present a high metastatic potential, and have a survival rate ≤ 50%. There is a high incidence of OCSCC, but the mechanisms of carcinogenesis remain unclear. Therefore, the aim of this research was to delineate the mutational landscape in developing tumors and OCSCCs by combining a lineage tracing approach and a mouse model of tongue carcinogenesis, previously established by our group. We used K14-CreERTAM; Rosa26LacZ mice, which allowed the long term basal stem cells of the epithelium to be permanently marked at the time of a two-day tamoxifen treatment. We then induced carcinogenesis by treating mice for 10 weeks with the carcinogen 4-nitroquinoline 1-oxide (4-NQO), which recapitulates key phenotypic and molecular features observed in human OCSCC. We performed Laser Capture Microdissection (LCM) to isolate and retrieve high-quality genomic DNA exclusively from each clone of permanently labeled, long-lived LacZ+ stem cells; each clone of cells arises from one LacZ+ stem cell. We then performed exome sequencing of 3 to 5 mice per group, sacrificed immediately after the 10-week treatment with 4-NQO (pre-neoplastic), and mice sacrificed >19 weeks after the end of 4-NQO treatment (SCC). As a reference genome for alignment and germline mutation exclusion, we included 3 mice of the same genetic background that did not receive 4-NQO. After 10 weeks of 4-NQO treatment the tongues were hyperplastic, with occasional necrosis and mild atypia, whereas all mice evaluated >19 weeks post 4-NQO treatment showed tongue SCCs. The total numbers of somatic mutations and LOH (loss of heterozygosity) identified using the mutation calling tool VarScan were 75% (4615±2170 vs 1148±76) higher in SCC compared to pre-neoplastic tongue samples. Among the mutations predicted as high and moderate impact, the levels of SNVs (single nucleotide variants) vs. indels were similar between SCCs and pre-neoplastic samples, 98.4% and 96.5%, respectively. By detailed analysis of the mutation types we showed that the pre-neoplastic tissues exhibited a much higher proportion of LOH events than the SCCs, 34.4% and 7.6%, respectively. Some of the genes affected by LOH in pre-neoplastic tissues were identified as tumor suppressors. Our findings suggest that a predominance of LOH in pre-neoplastic tongue tissue may result in loss of tumor suppressor genes, potentially defining the early steps of carcinogenesis in tongue SCCs. In conclusion, following the fate of long term tongue epithelial stem cells has allowed us to identify genomic perturbations occurring in the tongues of 4-NQO-treated mice and to find potential early biomarkers for OCSCC. Grant ID: R01CA205258; Funding Agency: NIH/NCI. Citation Format: Marta Melis, Tuo Zhang, Theresa Scognamiglio, Lorraine J. Gudas. Lineage-tracing technology to understand the molecular events in a mouse model of tongue squamous cell carcinoma (SCC) carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4665.