Metastatic protein network in BRAFV600E positive human thyroid cancers

描述（由申请人提供）：甲状腺癌患者的一个子集有颈部复发和转移，对目前的治疗难治，并死于这种疾病。BRAFV 600 E是乳头状癌（PTC）和甲状腺间变性癌（ATC）中最常见的遗传变异，与PTC向ATC的进展有关。我们的基因集富集分析显示，在BRAFV 600 E PTC中上调的17个基因集中有7个富含促转移细胞外基质（ECM）蛋白（例如血小板反应蛋白-1（TSP-1））和受体（整联蛋白），包括整联蛋白连接的激酶（即粘着斑激酶（FAK））， 转录因子（TF）HMGB 1，其与黑色素瘤进展和转移有关。TSP-1或BRAFV 600 E的敲低抑制BRAFV 600 E阳性PTC和ATC细胞中的细胞增殖、粘附、迁移/侵袭和转移，并且TSP-1的敲低显著降低磷酸（p）-ERK 1/2、pFAK和整联蛋白的水平。新型选择性BRAFV 600 E抑制剂PLX 4720在ATC原位小鼠模型中缩小肿瘤大小。然而，治疗3周后残留肿瘤的持续存在和TSP-1、pERK 1/2和pFAK蛋白表达的恢复表明，转移性甲状腺癌细胞通过上调这些蛋白获得对BRAFV 600 E抑制的抗性。我们的目标是通过确定BRAFV 600 E PTC细胞中转移性ECM蛋白触发的基本信号传导网络，鉴定侵袭性PTC的BRAFV 600 E依赖性和非依赖性生物标志物。具体目标1：通过定义抑制BRAFV 600 E后TSP-1刺激人BRAFV 600 E PTC中ERK 1/2和FAK磷酸化的分子级联，鉴定和评估PLX 4720耐药PTC的潜在生物标志物。初步数据表明，TSP-1与BRAFV 600 E PTC的颈部复发相关。我们将确定TSP-1是否是PTC侵袭性的有效预后生物标志物，并建立一个适合临床试验的基于免疫化学的筛查过程。为了在BRAFV 600 E阳性PTC中鉴定更多潜在的新预后生物标志物，我们计划鉴定TSP-1相互作用物。我们将评估其中一些与TSP-1表达和BRAFV 600 E阳性PTC的临床病理特征的相关性。为了更好地了解BRAFV 600 E在PTC进展中的功能，我们将应用无偏蛋白质组学分析结合功能测定来确定BRAFV 600 E PTC细胞中ECM蛋白相互作用和细胞内信号级联。具体目的2：探讨HMGB 1是否是PTC侵袭性的生物标志物。我们将HMGB 1表达与TSP-1表达和BRAFV 600 E PTC的临床病理特征相关联，并探讨HMGB 1是否调节TSP-1或对TSP-1表达至关重要的TF的表达。这项研究的结果可能（i）确定侵袭性BRAFV 600 E阳性PTC的新预后生物标志物，这些生物标志物可以在生物液体和PTC组织中进行测定，以帮助监测接受靶向治疗的患者，并使这些甲状腺癌的早期诊断成为可能，以及（ii）促进对目前治疗难治性PTC的创新疗法的开发。 公共卫生相关性：甲状腺癌患者的一个子集具有复发和转移，对当前的治疗是难治的，并且死于该疾病。BRAF基因中的V600 E突变是乳头状癌（PTC）和甲状腺间变性癌（ATC）中最常见的遗传改变，并与这些疾病的进展有关，因此需要早期诊断和新的治疗方法。这项研究的结果（将整合临床病理学和转化基础内分泌研究）将能够常规评估可在生物体液和甲状腺癌组织中测定的新的预后转移性生物标志物，并促进对目前治疗难治的转移性甲状腺癌的创新疗法的开发。

DESCRIPTION (provided by applicant): A subset of patients with thyroid cancer has neck recurrences and metastases, is refractory to current treatments, and dies of the disease. BRAFV600E, the most frequent genetic alteration in both papillary (PTC) and anaplastic thyroid cancers (ATC), is implicated in progression from PTC to ATC. Our Gene Set Enrichment Analysis revealed that 7 of 17 gene sets up-regulated in BRAFV600E PTC were enriched in pro-metastatic extracellular matrix (ECM) proteins (e.g. thrombospondin-1 (TSP-1)) and receptors (integrins), including the integrin-linked kinases (i.e. focal adhesion kinase (FAK)) and the transcription factor (TF) HMGB1, which has been implicated in melanoma progression and metastasis. Knockdown of either TSP-1 or BRAFV600E inhibits cell proliferation, adhesion, migration/invasion, and metastasis in BRAFV600E-positive PTC and ATC cells and knockdown of TSP-1 significantly decreases levels of phospho(p)-ERK1/2, pFAK, and integrins. The novel selective BRAFV600E inhibitor PLX4720 shrinks tumor size in an orthotopic mouse model of ATC. However, persistence of the residual tumor and resumption of TSP-1, pERK1/2, and pFAK protein expression after 3 weeks of treatment suggest that metastatic thyroid cancer cells acquire resistance to BRAFV600E inhibition by up-regulating these proteins. Our objective is to identify BRAFV600E-dependent and -independent biomarkers for aggressive PTC by determining the essential signaling networks triggered by metastatic ECM proteins in BRAFV600E PTC cells. Specific Aim 1: To identify and assess potential biomarkers for PLX4720-resistant PTC by defining the molecular cascades by which TSP-1 stimulates ERK1/2 and FAK phosphorylation in human BRAFV600E PTC following inhibition of BRAFV600E. Preliminary data suggest that TSP-1 is associated with neck recurrence in BRAFV600E PTC. We will determine whether TSP-1 is a valid prognostic biomarker for PTC aggressiveness and establish an immunohistochemistry-based screening process suitable for clinical trials. To identify a larger panel of potential new prognostic biomarkers in BRAFV600E positive PTC, we plan to identify TSP-1 interactors. We will assess the correlation of some of these with TSP-1 expression and with clinico-pathological features of BRAFV600E positive PTC. To obtain a better understanding of BRAFV600E function in PTC progression, we will apply an unbiased proteomic analysis combined with functional assays to determine ECM protein interactions and intracellular signaling cascades in BRAFV600E PTC cells. Specific Aim 2: To explore whether HMGB1 is a biomarker for PTC aggressiveness. We will correlate HMGB1 expression with TSP-1 expression and clinico-pathological features of BRAFV600E PTC and explore whether HMGB1 regulates expression of TSP-1 or of TFs crucial for TSP-1 expression. The results of this study are likely to (i) identify new prognostic biomarkers of aggressive BRAFV600E positive PTC that can be assayed in biological fluids and PTC tissues to help monitor patients undergoing targeted therapies and enable earlier diagnosis of these thyroid cancers, and (ii) foster development of innovative therapies for PTC refractory to current treatments. PUBLIC HEALTH RELEVANCE: A subset of patients with thyroid cancer has recurrences and metastases, is refractory to current treatments, and dies of the disease. The V600E mutation in the BRAF gene is the most frequent genetic alteration in both papillary (PTC) and anaplastic thyroid cancers (ATC), and is implicated in progression of these diseases, for which early diagnosis and new therapies are needed. Results from this study (which will integrate clinical-pathologic and translational basic endocrine investigations) will enable routine assessment of new prognostic metastatic biomarkers that could be assayed in biological fluids and thyroid carcinoma tissues, and foster the development of innovative therapies for metastatic thyroid cancers that are refractory to current treatments.