Genomic Association between Vitamin D3 Related Genes and Papillary Thyroid Carcinoma using The Cancer Genome Atlas and Nthy/BRAFV600E Cell Line

Abstract

Introduction: Tumor-suppressing effect of vitamin D3 has been reported in various carcinomas. However, the relevance between Vitamin D3 and thyroid carcinomas is not obvious. The purpose of this study was to evaluate vitamin D receptor (VDR) mRNA expression and prognosis in papillary thyroid cancer using The Cancer Genome Atlas (TCGA) analysis and to assess the relationship between vitamin D3 and papillary thyroid cancer cell growth and invasion using BRAFV600E cell line.

Methods: RNA sequencing and somatic mutation data from TCGA were analyzed. VDR mRNA expression was compared to clinicopathologic variables by linear regression. Tree-based classification was applied to find VDR cutoff and patients were split into low and high VDR group. To explain the association of high VDR mRNA expression and poor cancer progression, BRAFV600E-transfected Nthy-ori 3-1 cell line (Nthy/BRAFV600E) and BRAFWT-transfected Nthy cells (Nthy/WT) were treated with calcitriol (1,25(OH)2D3, an active form of Vitamin D3), and functional assays were performed to assess the differential effects of calcitriol on their tumorigenicity and invasiveness. CDNA microarray data analysis was performed to understand the results at a gene level.

Results: VDR mRNA expression was elevated in PTC than in normal thyroid tissue. VDR mRNA expressions were high in classic and tall cell variant PTC, extrathyroidal extension (ETE), and lateral neck node metastasis. High VDR group associated with PTC subtype, ETE and recurrence in logistic regression. In cell experiment, functional assays showed that calcitriol affects human thyroid cells differently depending on BRAF mutation status. The rate of proliferation was slightly reduced in Nthy and Nthy/WT, but it was not changed in Nthy/BRAFV600E. Anchorage-independent growth was inhibited by calcitriol in Nthy/BRAFV600E. In migration/invasion assay, calcitriol suppressed Matrigel invasion in both Nthy/WT and Nthy/BRAFV600E, but reduced cell migration only in Nthy/WT. In microarray data analysis, Nthy/BRAFV600E cells, CYP24A1 gene is highly expressed after calcitriol treatment. CYP24A1 upregulation is higher in Nthy/BRAFV600E than Nthy/WT.

Conclusion: Overexpression of VDR mRNA correlates with subtypes of papillary thyroid carcinoma that have the worse prognosis. Vitamin D3 has tumor-suppressing effect on human thyroid cells regardless of BRAF mutation status, but BRAF mutation makes them resistant to growth and migration-inhibitory effects of calcitriol. The BRAFV600E cells induced more CYP24A1 in high concentration of calcitriol which results in high VDR mRNA expression.