Thyroid Cancer Resistance to Vitamin D Receptor Activation Is Associated with 24-Hydroxylase Levels But Not theffFokI Polymorphism

Abstract

Background: The vitamin D receptor (VDR) has been studied as a novel target for cancer therapy in many tissue types as VDR ligands decrease cell proliferation in vitro and decrease tumor growth in vivo in sensitive cells. The objective of this study was to analyze the response to VDR agonist therapy in a panel of validated thyroid cancer cells and assess genetic markers predicting sensitivity and resistance to calcitriol and the noncalcemic analog DP006. Methods: Thyroid cancer cell lines were analyzed for VDR and RXR protein by Western blot. Cell growth after VDR agonist treatment (calcitriol or DP006) was assessed after 6 days of treatment by viable cell assay. To investigate calcitriol/DP006 resistance in VDR-expressing cells, the VDR was sequenced and 1-a and 24-hydroxylase mRNA expression was assessed. Results: VDR protein was variably expressed in the thyroid cancer cell lines and its presence was not sufficient for decreased viable cell count in response to calcitriol or DP006. The most sensitive cells (TPC1) have an ff FokI VDR polymorphism and the most resistant cells (HTh7 and 8505C) have an FF FokI VDR. This is a unique finding given that the balance of the literature of VDR polymorphisms describes an association of the ff FokI polymorphism with cancer risk and/or decreased VDR transactivation. 1-a and 24-hydroxylase mRNA expression before and after VDR agonist therapy was examined. 1-a-Hydroxylase levels did not change after calcitriol treatment. However, we found that higher baseline 24-hydroxylase levels and/or lower stimulation of 24-hydroxylase levels after calcitriol treatment were associated with relative resistance to calcitriol/DP006. Conclusions: The VDR represents a novel therapeutic target in poorly differentiated thyroid cancer; however, the efficacy of VDR agonist therapy to decrease viable thyroid cancer cell count cannot be predicted solely on the presence of the VDR. The FF FokI VDR genotype and high baseline 24-hydroxylase levels were associated with relative resistance to calcitriol and DP006. Therefore, identifiable markers of sensitivity or resistance to VDR agonist therapy may allow for a personalized use of these agents in poorly differentiated thyroid cancer.

Gene Expression of Vitamin D Metabolic Enzymes at Baseline and in Response to Vitamin D Treatment in Thyroid Cancer Cell Lines

“…These results are similar to those seen by Sharma et al, who reported that the TPC1 and C643 thyroid cancer cells are sensitive to vitamin D receptor activation and have lower baseline levels of CYP24A1 and CYP27B1 as compared with resistant thyroid cancer cell lines. They also reported that thyroid cancer cells with the FF FokI variant of the VDR were relatively resistant to the effects of 1,25(OH) 2 D 3 (8). What is unknown is whether one can overcome the relative resistance to vitamin D in specific cell lines by administering higher doses of either D 3 or 1,25(OH) 2 D 3 .…”

“…It is already known that thyroid cells have VDRs and CYP27B1 and CYP24A1 gene expression (8). Additionally, Sharma and colleagues have already reported VDR expression for TPC1, C643, Hth7(8), BCPAP, and KTC1 (16) thyroid cancer cell lines and all cells have VDR expression with Hth7 having the least. The presence of CYP27A1 and CYP2R1 gene expression indicates there is potential for thyroid cells to generate active vitamin D (1,25(OH) 2 D 3 ) in the presence of adequate levels of D 3 .…”

“…It is known that CYP24A1 gene expression is directly regulated by 1,25(OH) 2 D 3 , so it is logical that expression would increase with treatment with 1,25(OH) 2 D 3 and potentially D 3 (if there was adequate conversion to 25OHD 3 ). CYP24A1 gene expression is also regulated in response to activation of the vitamin D receptor (3) and can be variable depending on whether the cells are relatively sensitive or resistant to vitamin D (8). TPC1 papillary thyroid cancer cells had nearly undetectable baseline levels of CYP24A1, but showed a significant increase in gene expression after treatment with both D 3 and 1,25(OH) 2 D 3 .…”

“…It is already known that thyroid and thyroid cancer cells express vitamin D receptors (VDRs) and CYP27B1 (8, 9). However, VDR expression is variable in distinct thyroid cancers cell lines (C643, BCPAP, Hth7, Hth74, K1, KAT18, SW1736 and TPC1), and the presence of VDR does not predict reduction in cell viability in response to treatment with calcitriol (1,25(OH) 2 D 3 ) or a non-calcemic vitamin D analog (8).…”

“…However, VDR expression is variable in distinct thyroid cancers cell lines (C643, BCPAP, Hth7, Hth74, K1, KAT18, SW1736 and TPC1), and the presence of VDR does not predict reduction in cell viability in response to treatment with calcitriol (1,25(OH) 2 D 3 ) or a non-calcemic vitamin D analog (8). One study showed that in vitro administration of calcitriol increased expression of the tumor suppressor protein p27 and decreased cell proliferation in the WRO follicular thyroid carcinoma cell line (10).…”