Thyroid regeneration and carcinogenesis

Division Of Basic Sciences - Nci

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Abstract

To understand whether TSH signaling plays a role in thyroid regeneration, Tshr (TSH receptor)-null and littermate wild-type mice were subjected to partial thyroidectomy (acute thyroid injury model) that activates thyroid regeneration. A cluster of thin and elongated shaped cells, different from round shaped thyroid follicular cells, were found near the tracheal cartilage and muscle in both Tshr-null and wild-type mouse thyroids that were positive for NKX2-1, with some cells also positive for Pax8. The cluster of elongated cells were found connected to a newly formed follicle-like structure with NKX2-1 positive cells at the end or in the middle of the cluster. NXK2-1 and PAX8 are the transcription factors critical for development, differentiation, and function of the thyroid, and are considered thyroid differentiation markers. These results suggested that a cluster of thin elongated cells may be a precursor to thyroid follicular cells. These clusters of thin elongated cells were present in both Tshr-null and wild-type mouse thyroids, which may become more visible after partial thyroidectomy, particularly in wild-type thyroids. When the levels of mRNAs for various thyroid-specific genes, growth factors, their receptors, and related signaling molecules were measured using control Tshr-null and wild-type mice, different expression levels were found in some of these genes between the two genotypes, however, changes in their expression after partial thyroidectomy followed similar pattern in both genotypes. These results suggested that at least this type of thyroid regeneration does not require TSH signaling. In order to address questions whether the thin elongated cells can be considered as thyroid stem/progenitor cells and what are the origin of these cells, we carried out Nanostring GeoMX analysis, where cells from the area of a cluster of thin elongated cells, the surrounding tissues, and normal thyroid tissues were captured by laser microdissection, followed by RNAseq analysis of each area of cells. Fifty-one differentially expressed genes were obtained in the NKX2-1 cell cluster vs normal thyroid that overlapped between two separate sets of RNAs. Heat maps of these differentially expressed genes showed that many immune-related genes such as Igha, Igkc, H2-Aa, H2-Ab1, H2-Eb1, Cd74, and Cxcl12 were highly expressed in the NKX2-1 cell cluster. This suggests that immune cells may be the origin of thyroid follicular cells, at least in the regeneration process taking place after partial thyroidectomy. This is in agreement with our previously obtained results using bone marrow transplantation studies with GFP transgenic mice; GFP-positive cells were found in the elongated cluster of cells, suggesting that this cell cluster could be at least partly derived from bone marrow cells. We are currently carrying out in situ hybridization using RNAscope (Bio-Techne) and immunohistochemistry to validate the results we obtained by GeoMx. If indeed validated, the results demonstrate that immune cells may be the origin of thyroid follicular cells, at least in the regeneration process taking place after partial thyroidectomy. This is a completely unexpected new finding. The results will provide a different way to look at the physiology and diseases of the thyroid, and to restore thyroid functions in humans after thyroidectomy due to various thyroid diseases including cancer. The results will help in the development of novel treatment options for thyroid diseases. Carcinogenesis occurs in multi-steps in many tissues, including the thyroid. In order to understand multi-step carcinogenesis and its relation to NKX2-1 expression, we have established mouse thyroid adenomatous nodule-derived cell lines. Nkx2-1fl/fl mice were fed a diet containing amitrole (3-amino-1,2,4-triazole), starting at the age of 5 weeks. Amitrole is a non-food herbicide, known to induce thyroid tumors by inhibiting thyroid peroxidase activity. Adenomatous nodules developed within 6-12 months in their thyroids, which were dissected, digested with collagenase I and displace I, and cultured for 5 passages. Five cell lines were eventually successfully established, which were named CAT (cells from amitrole treated thyroids). Among them, two cell lines, CAT458 and 459 showed epithelial characteristics with E-cadherin expression, while other cell lines CAT411, 413, and 427 expressed vimentin, a mesenchymal marker. CAT459 cells expressed NKX2-1, while CAT458 cells had almost no expression of NKX2-1. NKX2-1-positive CAT459 cells showed higher mRNA expression of some thyroid differentiation markers than did NKX2-1-negative CAT458s (sub-line of CAT458) cells, and NKX2-1 overexpression increased and/or induced their expression. IGF-I signaling was transduced in thyroid stimulating hormone receptor (TSHR)-negative CAT458s and 459 cells, and NVP-ADW72, a selective IGF-I receptor (IGF-IR) inhibitor suppressed their proliferation. No tumors developed in immunocompromised mice after subcutaneous injection of CAT458s or 459 cells. Whole Genome Sequencing analysis revealed that there were no prominent gene mutations involved in thyroid cancers such as Braf, Trp53, and Tert, but several missense mutations were found in tumor suppressor genes in these cell lines, consistent with their being benign adenoma cells. CAT458s and 459 cells provide a tool to further clarify the process of thyroid multi-step carcinogenesis and differentiation.