Abstract 1219: Establishment and characterization of amitrole-induced mouse thyroid adenoma-derived cell lines
Authors: Shirai Yo-Taro , Hoshi Nobuo , Liu Huaitian , Lee Maxwell P. , Kimura Shioko 
Published: April 2023

Abstract
Some types of thyroid cancers are known to arise from thyroid adenomas, as a malignant transition from benign adenoma, as proposed in the multi-step carcinogenesis model. Thyroid cancer studies using human thyroid cancer cell lines have been intensely performed by many researchers. However, mouse thyroid adenoma cell lines have not been well established and characterized. In order to obtain further insights into thyroid carcinogenesis process, we established five cell lines from thyroid adenomas developed in mice treated with goitrogen amitrole. Among them, two cell lines, CAT458/458s (CAT: cells from amitrole treated thyroids; CAT458s: a sub-line from CAT458) and CAT459 were found to be positive for E-cadherin and Claudin-1, representative epithelial markers, and negative for Vimentin, a mesenchymal marker, suggesting that they originated from thyroid epithelial cells. We found higher expression of DUOX1, a member of NADPH oxidase involved in thyroid hormone synthesis, in both CAT458s and 459 cells than normal mouse thyroids, presumably due to an unknown mechanism caused by amitrole treatment. NKX2-1, a master regulator of thyroid development, is not expressed in CAT458s cells while NKX2-1 is abundantly expressed in CAT459 cells. Consistent with this finding, NKX2-1-positive CAT459 cells showed higher mRNA expression of thyroid differentiation markers including Tg, Duox1, and Duox2. NKX2-1 overexpression in NKX2-1-negative CAT458s cells using a lentiviral infection system induced mRNA expressions of these thyroid differentiation marker genes, suggesting that partial thyroid differentiation of the adenoma cells is induced by NKX2-1. TSH and IGF-I are known to work synergistically for thyroid growth. However, Tshr mRNA expression is not detected in either CAT458s or 459 cells, suggesting that TSH signal is not responsible for their growth. On the other hand, IGF-IR protein is expressed and IGF-I induces AKT and S6 phosphorylation in both cells. We further tried to assess somatic gene mutations involved in adenoma development by whole-genome sequencing (WGS). WGS analysis revealed that somatic missense mutations are not found in genes known to be involved in thyroid carcinogenesis and cancer progression, such as Braf, Trp53, Ret, Tert, Hras, Kras, and Nras. This finding suggests that prominent oncogenic mutations are not required for thyroid adenoma development that may later progress to carcinoma. Currently we are validating several missense mutations, including mutations in tumor suppressor genes Atm and Tgfb1, found in these cell lines. In conclusion, we established mouse thyroid adenoma cell lines with different differentiation and molecular profiles. CAT458s and 459 cells would provide a tool to further clarify the process of thyroid adenoma development and thyroid differentiation.
Citation Format: Yo-Taro Shirai, Nobuo Hoshi, Huaitian Liu, Maxwell P. Lee, Shioko Kimura. Establishment and characterization of amitrole-induced mouse thyroid adenoma-derived cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1219.