Because there is no surface marker known for thyroid stem/progenitor cells, and thyroid is a very small organ, particularly of mouse, which is the most frequently used animal model in research, the progress has been very slow. In order to circumvent the problem, we previously used Hoechst dye-resistant side population (SP) cells isolated from mouse thyroids by fluorescence-activated cell sorting (FACS). Thyroid SP cells demonstrated stem/progenitor-like characteristics based on gene expression patterns and cell culture studies, with a half of them expressing stem cell antigen 1 (SCA1), a candidate stem cell marker for various tissues. Using these mouse thyroid-derived SP cells, a cell line named SPTL (side population cells-derived thyroid cell line) was established. This continuous source has allowed us to more easily study and characterize thyroid stem/progenitor cells. SPTL cells are undifferentiated as determined by electron microscope, and express SCA1, but not thyroid differentiation markers by western blotting including NKX2-1 and PAX8, critical transcription factors for thyroid differentiation, homeostasis, and function. However, qRT-PCR results showed that Nkx2-1 is slightly expressed in SPTL cells at the levels higher than that of embryonic stem (ES) cells. SPTL cells do not express ES cell markers such as Oct4, Nanog and Rex1, while they express Gata4 and Gata6, the endoderm lineage marker genes at higher levels than those of ES cells or differentiated thyroid cells. The results suggest that SPTL cells may be partially thyroid-specified progenitor-like cells. Nk2-1 expression was increased when SPTL cells were cultured in low serum conditions, and SPTL cells formed follicle-like structures in 3-dimensional Matrigel cultures in vitro, while other thyroid differentiation marker genes than Nxk2-1 were not expressed in either 2D or 3D cultured SPTL cells. This suggests that there may be some factors still lacking in the culture media. Or SPTL cells lack the capacity to fully differentiate into thyroid. However, when SPTL cells were orthotopically or intravenously injected to mice after partial thyroidectomy or partial cut, several SPTL cells were found in part of follicles and they expressed NKX2-1. The results altogether suggest that NKX2-1 may be required for thyroid follicle formation and PAX8 may be required for follicle maturation to become functional thyroid. SPTL cells highly express genes involved in epithelial-mesenchymal transition, as demonstrated by RNA seq analysis, and exhibit a gene-expression pattern similar to anaplastic thyroid carcinoma. These results demonstrate that SPTL cells have the capacity to differentiate into thyroid to a limited degree. SPTL cells may provide an excellent tool to study stem cells, including cancer stem cells of the thyroid. In collaboration, we also study the mechanisms of thyroid carcinogenesis and molecules/genes that influence this process. CYP24A1, the primary inactivating enzyme for vitamin D, is often overexpressed in human cancers, potentially neutralizing the antitumor effects of calcitriol, the active form of vitamin D. However, it is unclear whether CYP24A1 expression serves as a functional contributor versus only a biomarker for tumor progression. In order to address this question, the role of CYP24A1 on malignant progression of a murine model of BrafV600E-induced papillary thyroid cancer (PTC) was investigated. Mice harboring wild-type Cyp24a1 (BVECyp24a1-wt) developed PTC at 5 weeks of age. Mice harboring a homozygous deletion of Cyp24a1 (BVECyp24a1-null) exhibited a 4-fold reduction in tumor growth. Notably, the tumorigenic potential of BVECyp24a1-null-derived tumor cells was nearly abolished in immunocompromised nude mice. This phenotype was associated with downregulation of the MAPK, PI3K/Akt, and TGFb signaling pathways and a loss of epithelial-mesenchymal transition (EMT) in BVECyp24a1-null cells, associated with downregulation of genes involved in EMT, tumor invasion, and metastasis. While calcitriol treatment did not decrease cell proliferation in BVECyp24a1-null cells, it strengthened antitumor responses to the BRAFV600E inhibitor PLX4720 in both BVECyp24a1-null and BVECyp24a1-wt cells. Our findings offer direct evidence that Cyp24a1 functions as an oncogene in PTC, where its overexpression activates multiple signaling cascades to promote malignant progression and resistance to PLX4720 treatment.
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