BRAFV600E and VEGFR2 synergize to trigger papillary thyroid cancer progression

描述(申请人提供)：乳头状甲状腺癌(PTC)通常预后良好；然而，携带BRAFV600E突变的PTC患者对放射性碘治疗表现出抵抗，转移率高，存活率低。BRAFV600E的抑制剂，包括维莫拉非尼，最近被测试用于治疗BRAFV600E-黑色素瘤，结果非常有希望。然而，对这些化合物产生继发性肿瘤耐药性的研究已有报道。PTC侵袭性的另一个潜在参与者是血管内皮生长因子受体2(VEGFR2)，它是血管生成的关键调节因子。以VEGFR2为靶点的索拉非尼对转移性PTC只有部分疗效，提示该药也存在二级耐药机制。我们的初步结果显示，转移性原代培养的人BRAFV600E阳性PTC细胞对高剂量的维莫拉非尼或索拉非尼具有耐药性，但两种药物联合处理后细胞存活率下降了90%以上。BRAFV600E上调PTC细胞中VEGFR2和pERK1/2的表达，触发内皮细胞和周细胞的募集。我们还观察到acac�(代谢基因)的上调，表明BRAFV600E利用促进PTC转移的代谢反应，这是一种新的机制。我们的初步数据还表明，VEGFR2与BRAFV600E PTC的颈部复发有关。我们的目标是确定BRAFV600E和VEGFR2如何适应控制PTC细胞进程的自分泌和旁分泌信号通路。我们的中心假设是BRAFV600E和VEGFR2通路在PTC细胞中协同作用，增加细胞外基质(ECM)蛋白的分泌，导致PTC细胞黏附、迁移/侵袭和血管生成增加。我们提出了BRAFV600E/ERK1/2信号通路在PTC细胞中触发VEGFR2高表达的第二途径。在维莫拉非尼存在的情况下，交替的VEGFR2途径与残留的BRAFV600E活性协同作用，导致PTC细胞产生继发性耐药。我们将通过操作VEGFR2和BRAFV600E的功能并在我们的新模型中评估肿瘤特性来测试这一点：概述PTC微环境的3D共培养系统，具有杂合BRAFWT/V600E的原代人类PTC细胞的原位小鼠，以及芯片序列分析。目的1：阐明BRAFV600E和VEGFR2促进PTC侵袭性的自分泌和旁分泌途径。这些研究将描绘VEGFR2相互作用的ECM蛋白，促进BRAFV600E PTC中ERK1/2的磷酸化，并为了解维莫拉非尼和索拉非尼的继发性耐药提供新的视角。目的：探讨血管内皮生长因子受体2(VEGFR2)蛋白表达是否可作为预测BRAFV600E PTC侵袭性的长期生物标志物。目的：评价抗BRAFV600E和抗VEGFR2联合治疗临床前人类杂合子BRAFWT/V600E PTC小鼠模型的疗效。这些拟议的研究很有可能为BRAFV600E-PTC联合靶向治疗的临床试验提供证据，并确定侵袭性PTC的药物开发和生物标记物的新靶点。

DESCRIPTION (provided by applicant): Papillary thyroid cancer (PTC) typically has a favorable prognosis; however, patients with PTC carrying the BRAFV600E mutation show resistance to radioiodine treatment and have high rates of metastases and low survival rates. Inhibitors of BRAFV600E, including vemurafenib, have been tested recently as treatments for BRAFV600E-melanomas, with very promising results. However, development of secondary tumor resistance to these compounds has been reported. Another potential player in PTC aggressiveness is vascular endothelial growth factor receptor 2 (VEGFR2), a crucial regulator of angiogenesis. Sorafenib, which targets VEGFR2, was only partially effective against metastatic PTC, suggesting secondary resistance mechanisms to this drug as well. Our preliminary results show that metastatic primary human BRAFV600E positive PTC cells in vitro were resistant to high doses of either vemurafenib or sorafenib alone, but combined treatment with both drugs decreased cell viability by greater than 90%. BRAFV600E up-regulates VEGFR2 and pERK1/2 in PTC cells, triggering recruitment of endothelial cells and pericytes. We also observed upregulation of ACAC� (metabolic gene) suggesting that BRAFV600E harnesses a metabolic response that promotes PTC metastasis, a novel mechanism. Our preliminary data also indicate that VEGFR2 is associated with neck recurrence in BRAFV600E PTC. Our objective is to determine how BRAFV600E and VEGFR2 fit into the autocrine and paracrine signaling pathways that control PTC cell progression. Our central hypothesis is that BRAFV600E and VEGFR2 pathways synergize in PTC cells, increasing secretion of extracellular matrix (ECM) proteins and leading to increased PTC cell adhesion, migration/invasion, and angiogenesis. We propose a secondary pathway in which the BRAFV600E/ERK1/2 signaling cascade triggers hyper-expression of VEGFR2 in PTC cells. The alternate VEGFR2 pathway synergizes with residual BRAFV600E activity in the presence of vemurafenib, conferring secondary drug resistance in PTC cells. We will test this by manipulating VEGFR2 and BRAFV600E function and assessing tumorogenic properties in our novel models: a 3D co-culture system that recapitulates the PTC microenvironment, an orthotopic mouse with primary human PTC cells with heterozygous BRAFWT/V600E, and ChIp-seq analysis. Aim 1: To elucidate the autocrine and paracrine pathways by which BRAFV600E and VEGFR2 promote PTC aggressiveness. These studies will delineate VEGFR2-interacting ECM proteins that enhance ERK1/2 phosphorylation in BRAFV600E PTC and provide new insight into vemurafenib and sorafenib secondary drug resistance. Aim 2: To assess whether VEGFR2 protein expression is a long-term prognostic biomarker for PTC aggressiveness in BRAFV600E PTC. Aim 3: To assess efficacy of anti-BRAFV600E and anti-VEGFR2 combined therapy in a pre-clinical mouse model of human heterozygous BRAFWT/V600E PTC. These proposed studies are highly likely to provide evidence supporting clinical trials of combined targeted therapy for BRAFV600E-PTC and identify novel targets for drug development and biomarkers for aggressive PTC.