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Title: 1,25(OH)2D3 in thyroid tumorigenesis and development
Other Titles: 1,25(OH)2D3 in tumorale en normale schildklier ontwikkeling
Authors: Clinckspoor, Isabelle
Issue Date: 20-Dec-2011
Abstract: This doctoral research project aimed to gain insight into the role of 1,25(OH)2D3, the active form of vitamin D, in the normal thyroid and in the pathogenesis, progression and treatment of thyroid cancer. As treatment modalities are very limited in case of poorly differentiated or anaplastic thyroid cancer, resulting in a very poor survival, the effect of treatment with 1,25(OH)2D3 in anaplastic thyroid cancer was investigated using different human thyroid cancer cell lines. In a second part of the study, the presence of the vitamin D receptor (VDR), the catabolizing enzyme CYP24A1 and the activating enzyme CYP27B1 was mapped in a spectrum of benign and malignant human thyroid tumour tissues. Finally, the role of 1,25(OH)2D3 in the normal thyroid was determined by investigating thyroid morphology and function in VDR knockout mice. In the first part of the study, the effect on cell growth and redifferentiation of supraphysiological doses of 1,25(OH)2D3 in a set of poorly differentiated to anaplastic thyroid cancer cell lines was investigated. Four human thyroid cancer cell lines (FTC-133, C643, 8505C and HTh74) were treated with 1,25(OH)2D3 or CD578, a superagonistic analogue, in monotherapy or in combination with paclitaxel, a taxane, or suberoylanilide hydroxamic acid (SAHA), a histone deacetylalase inhibitor. Clear effects on growth arrest were observed in a clonogenic assay, and absolute cell counts demonstrated a 24% to 36% reduction in all cell lines after 72h treatment with 1,25(OH)2D3 (10-6M) and a 60% inhibition after 120h in the most sensitive cell line HTh74. A similar growth inhibition was shown after treatment with a 1000-fold lower concentration of analogue CD578. The growth arrest was explained by antiproliferative effects, with an increased % of cells in the G0-G1 phase of the cell cycle and a decreased mRNA expression of transcription factor E2F1. Combination treatments of 1,25(OH)2D3 or CD578 with paclitaxel or SAHA (2 agents with known antineoplastic effect in thyroid cancer) resulted in an additive and in some conditions synergistic effect on the inhibition of proliferation. Redifferentiation analysis revealed only a modest increase in sodium iodide symporter (NIS) and thyroglobulin (Tg) mRNA expression after treatment with 1,25(OH)2D3, without additive effect after combination treatment. No effects were observed on thyroid stimulation hormone receptor (TSHR) or thyroid peroxidase (TPO) mRNA expression. These in vitro findings demonstrate that 1,25(OH)2D3, but especially superagonistic vitamin D analogues hold promise as adjuvant antiproliferative therapy in poorly differentiated to undifferentiated thyroid cancer, especially in combination with other drugs such as paclitaxel or SAHA.In the second part of the study, the expression of the VDR, CYP24A1 and CYP27B1 was studied in human benign (follicular adenoma) and malignant (anaplastic thyroid cancer and differentiated thyroid cancer, consisting of papillary thyroid cancer and follicular thyroid cancer) thyroid tumour tissues. As expected, differentiated thyroid cancer was characterized by clearly enhanced Ki67 and decreased NIS mRNA expression compared to normal thyroid tissue. Follicular adenoma had an intermediary profile with similar levels of Ki67, but decreased NIS mRNA expression compared to normal thyroid tissue. Both follicular adenoma and differentiated thyroid cancer demonstrated enhanced VDR, CYP24A1 and CYP27B1 expression compared to normal thyroid tissue. Papillary thyroid cancer - the most common thyroid cancer- with and without lymph node metastasis was further compared. Decreased VDR expression was found in metastasized papillary thyroid cancer tumours, implying reduced 1,25(OH)2D3 sensitivity. However at the same time decreased CYP24A1 and enhanced CYP27B1 expression were found in papillary thyroid cancer N1, suggesting enhanced local 1,25(OH)2D3 levels. Finally, in the highly aggressive anaplastic thyroid cancer, VDR and CYP24A1 expression was often lost, while CYP27B1 levels were similar to differentiated thyroid cancer. Moreover, tumour progression to highly proliferative and/or metastasizing anaplastic thyroid cancer was characterized by decreased sensitivity, since more cases with a negative staining for all three vitamin D metabolizing genes were found in these subgroups. Together, this part of the study showed an enhanced presence of key players in vitamin D metabolism and signalling in benign and differentiated malignant thyroid tumours, however decreasing in case of local nodal and especially distant metastasis, suggesting a local antitumor response of 1,25(OH)2D3 in early cancer stages, that is diminished in case of dedifferentiation and tumour progression. The enhanced expression of key players in vitamin D signalling in differentiated thyroid cancer and its correlation with cell cycle genes advocate the need for the further study of 1,25(OH)2D3 and especially super-agonistic analogues with a low calcaemic profile as a candidates for treatment in persistent and recurrent iodine refractory differentiated thyroid cancer.In the last part of the doctoral research project the role of the VDR in normal thyroid development and function was explored. Thyroid morphology was not altered in VDR KO mice, irrespective of the calcaemia. Also thyrocyte function, as measured by immunohistochemistry for thyroxine (T4) and iodinated thyroglobulin (Tg-I), was comparable. Dual oxidase (Duox) stained more cytoplasmic than apically in the VDR KO mice, but no evidence was present for increased oxidative stress or apoptosis. Still, serum TSH was lower in normocalcaemic VDR KO mice, suggesting a possible mild suppressive central (hypothalamic/pituitary) effect. Parafollicular C cell physiology was however clearly affected, with increased thyroidal calcitonin expression in VDR KO mice, irrespective of the diet. This observation was paralleled by increased serum calcitonin, however only in the normocalcaemic VDR KO mice. As the findings in mice suggested an inhibitory effect of 1,25(OH)2D3 via the VDR on calcitonin production in C cells, further investigations were performed to explore whether this also applied to humans. Therefore, serum calcitonin in normocalcaemic patients with low (<10ng/mL) and high (&gt;40ng/mL) serum levels of 25-OHD3 were compared. No difference was observed, indicating a difference between rodent and human C cell physiology without direct implications for the use and interpretation of serum calcitonin in patients.
Publication status: accepted
KU Leuven publication type: TH
Appears in Collections:Clinical and Experimental Endocrinology
Medical Clerkships Centers
Clinical Residents Medicine

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