Thyroid regeneration and carcinogenesis

Division Of Basic Sciences - Nci

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Abstract

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 cells in thin and elongated shapes were found near the tracheal cartilage and muscle in both Tshr-null and wild-type mouse thyroids that were positive for NKX2-1. Some of these cells were also positive for Pax8, another transcription factor, critical for differentiation and function of thyroid. The thin and elongated cells were clearly different from NKX2-1-positive round-shaped thyroid follicular cells. These cells were similar to those previously found in Nkx2-1;TPO-Cre mice, a natural thyroid regeneration mode, in which Nxk2-1 gene was thyroid follicular cells specifically deleted. The results suggested that a cluster of thin elongated cells may be a precursor to thyroid follicular cells that are present in both genotype mouse thyroids, which may become more visible after partial thyroidectomy, particularly in wild-type thyroids, to participate in regeneration of follicular cells. Levels of mRNA expression for various thyroid-specific genes, growth factors, their receptors, and related signaling molecules were not particularly different between thyroids of Tshr-null and wild-type mice although endogenous levels and the degrees of increase/decrease after partial thyroidectomy were different for most of the mRNAs between the two genotypes. These results suggested that a cluster of thin elongated cells present near the tracheal cartilage and muscle are a source of newly formed follicles, and this type of regeneration process of thyroid follicular cells is TSH signaling-independent. Whether the thin elongated cells can be considered as thyroid stem/progenitor cells and what are the origin of these cells remain to be understood. In order to study the role of NKX2-1 in thyroid carcinogenesis, mouse thyroid adenoma cell lines were newly established from mice that were fed a diet containing amitrole (3-amino-1,2,4-triazole). Amitrole is a non-food herbicide, and is known to induce thyroid tumors by inhibiting thyroid peroxidase activity and iodide uptake. Adenomas developed within 6-12 months, which were collected, pooled and subjected to cell culture. Five cell lines each derived from thyroid adenomas of individual mouse were successfully established, which were named CAT (cells from amitrole treated thyroids). Two lines, CAT458 and 459 showed epithelial characteristics, expressing E-cadherin, while other 3 CAT lines expressed vimentin, a mesenchymal marker. CAT458 cells had almost no expression of NKX2-1, while CAT459 cells expressed NKX2-1. We chose these two cell lines for further studies. When constitutively active myristoylated AKT (caAKT) was introduced, they showed multilayer growth, and CAT459s (subclone of CAT459) cells lost NKX2-1 expression. These results suggested a possible relationship between the expression of NKX2-1 and pAKT, and the involvement of pAKT in contact inhibition-free cell growth, a sign of malignancy. CAT458s and CAT459s cells are manipulated to express or suppress NKX2-1 and/or caAKT in tetracycline-inducible and/or non-inducible fashion to understand whether and/or how NKX2-1 or caAKT affects the natures ofcells and expression patterns of genes in these cells. We plan to carry out RNAseq and ChIPseq using CAT458 and/or 459 cells with and without NKX2-1 or caAKT expression to understand the molecular changes occurring under the expression of these genes that may confer CAT458 and/or 459 cells more differentiated or more malignant natures.