Role of newly identified, thyroid-specific LincRNA in BRAFV600E thyroid carcinoma

brafv600e -乳头状甲状腺癌（V600E-PTC）侵袭性特别强，由于其碘代谢失调，其受害者对放射性碘治疗无反应，转移和复发率高，生存率低。BRAFV600E抑制剂，如vemurafenib (vemu)，最初显示出临床前景，是唯一的治疗选择，但对这些药物的耐药性被广泛报道。我们的假设是，首个发现的甲状腺特异性长基因间非编码RNA (lincRNA) Xloc001313 （Xloc13）的表观遗传沉默解除了对碘代谢的调节，维持了肿瘤细胞的存活，并促进了肿瘤的进展和耐药性。Xloc13位于碘代谢关键酶TPO的编码基因下游，其在V600E-PTC中的表达量远低于正常组织。我们的项目旨在确定这些效应背后的细胞机制，并利用这些发现更好地针对新疗法进行临床试验。Vemu处理诱导Xloc13表达，表明它被BRAFV600E沉默，在正常甲状腺细胞中，CRISPR使其失活，降低TPO水平和碘摄取，同时增加TSP-1水平（细胞外基质蛋白）和细胞增殖。Xloc13 RNA增强子映射到TSP-1启动子上的dna结合基序，导致TSP-1的下调，从而使TGFβ1失活，抑制PTC细胞和周细胞的活力。我们最近证明，V600E-PTC样品富含共同表达TSP-1和tgf - β1的周细胞，以维持V600E-PTC细胞的存活。Xloc13沉默与高周细胞相关

BRAFV600E-papillary thyroid carcinoma (V600E-PTC) is particularly aggressive, and as it deregulates iodine metabolism, its victims are unresponsive to radioiodine treatment, and suffer high rates of metastasis and recurrence, and low survival rates. BRAFV600E inhibitors such as vemurafenib (vemu), which at first showed clinical promise, are the only therapeutic option, but resistance to these drugs is widely reported. Our premise is that the epigenetic silencing of the first identified thyroid-specific long intergenic non-coding RNA (lincRNA), Xloc001313 (Xloc13) deregulates iodine metabolism, sustains tumor cell survival, and promotes progression and drug resistance. Xloc13 is active downstream of the coding gene for TPO, a key enzyme for iodine metabolism, and its expression is far lower in V600E-PTC than normal tissue. Our project is designed to determine the cellular mechanisms behind these effects, and use these findings to better target clinical trials of new treatments. Vemu treatment induces Xloc13 expression, indicating it is silenced by BRAFV600E, and its inactivation by CRISPR in normal thyroid cells reduces TPO levels and iodine uptake, while increasing TSP-1 levels (extracellular matrix protein) and cell proliferation. Xloc13 RNA enhancer maps to DNA-binding motifs on the TSP-1 promoter, leading to down-regulation of TSP-1, which inactivates TGFβ1 to inhibit PTC cell and pericyte viability. We recently demonstrated that V600E-PTC samples are enriched in pericytes that co- express TSP-1 and TGFβ1 to sustain V600E-PTC cell survival. Xloc13 silencing correlates with high pericyte density, linking its action to pericyte biology. We also identified regulatory motifs in the Xloc13 promoter for EZH2 and E2F1 as transcriptional repressors up-regulated in vemu-resistant V600E-PTC cells and ChIP- enriched at these DNA-binding motifs. Critically, combining inhibitors of BRAFV600E+EZH2+CDK4/6 durably rescued Xloc13, and reduced PTC cell and pericyte viability. Xloc13 down-regulation therefore plays a key role in V600E-PTC, and our central objective is to determine its mechanisms of regulation and their impact on tumor progression and resistance. We hypothesize that BRAFV600E synergizes with EZH2 and the CDK4/6 (upstream of E2F1) pathway to down-regulate Xloc13, which deregulates iodine metabolism, enhances tumor viability, and increases invasion signaling through pericyte action, which leads to secondary drug resistance and tumor progression. Using cutting-edge technologies (CRISPR, ChIP, ATAC, RAP) and new cancer mouse model, our specific Aims are: 1) To identify the mechanisms regulating Xloc13 in V600E-PTC and their impact on tumor aggressiveness; 2) To determine the role of Xloc13 in the regulation of TPO in V600E-PTC and its impact on iodine metabolism and PTC cell survival; 3) To assess the efficacy of anti-BRAFV600E-EZH2-CDK4/6 combined therapy on human V600E-PTC in vivo. Our studies will have high clinical significance by supporting new clinical trials of trimodal combined therapies for advanced V600E-PTC patients, and identifying new mechanisms in PTC and its microenvironment which could serve as drug development targets for this tumor.