OR09-2 The RCAN 1.4 Metastasis Progression Suppressor Gene is Hypermethylated at Intron 1 and Downregulated in Papillary Thyroid Cancer

Abstract

Abstract Progressive metastasis is the proximate cause of cancer-related mortality for the majority of patients with solid tumors. Identifying both drivers, and gatekeepers of late stage progression (metastasis progression suppressors; MPS) is crucial to improve biomarkers and treatments. We previously identified Regulator of Calcineurin 1.4 (RCAN1.4) as a MPS gene in thyroid cancer that functions in part through overexpression of the cap-n-collar transcription factor NFE2L3 (NRF3). However, the mechanisms for RCAN1.4 loss in thyroid cancer, and whether or not this is reversible, had not be addressed. Because the RCAN 1.4 promoter contains CG rich regions reported to be hypermethyalated in other tissues, we hypothesized that RCAN 1.4 downregulation in thyroid cancer was similarly regulated. Studies were performed in both cell lines and de-identified human papillary thyroid cancer (PTC) tumor and normal tissue pairs from our Endocrine Neoplasia Repository tumor bank. Five thyroid cancer cell lines (TPC1, FTC133, BCPAP, C643, and 8505C) with different genetic drivers were selected based on low expression of RCAN 1.4. To determine if lines display methylation-regulated expression of RCAN 1.4, cells were treated with 1-10uM decitabine, a DNA methyl transferase inhibitor. In all cell lines, RCAN1.4 mRNA and protein levels increased with decitabine treatment, consistent with hypermethylation. Analysis of the literature and in silico analysis of the RCAN 1.4 gene identified CG rich regions as putative methylation targets in the proximal promoter and in intron 1. Using two different methods; 1) isolation of MseI-digestion DNA fragments using CpG MethylQuest beads followed by qPCR and 2) methylation-specific PCR (MSP) of bisulfite-modified DNA, hypermethylation of the intron 1 CG rich region was identified. There was no evidence of hypermethylation of the CG rich region of the proximal promoter. Methylation of the two regions also was assessed on 18 pairs PTC and adjacent normal tissues. Similar to the cell lines, hypermethylation was identified at intron 1 of RCAN 1.4 in PTC versus normal tissues. QRT-PCR results confirmed lower levels of RCAN1-4 mRNA in the PTC vs normal tissue. These samples also demonstrated increased NRF3 expression versus normal tissue. Taken together, these data demonstrate that RCAN 1.4 is downregulated in thyroid cancer cells and human PTCs by hypermethylation of CG rich regions in intron 1 and that this effect is reversible using demethylating agents. Presentation: Saturday, June 11, 2022 11:45 a.m. - 12:00 p.m.