Metastatic protein network in BRAFV600E positive human thyroid cancers

描述(由申请人提供)：一部分甲状腺癌患者颈部复发和转移，对目前的治疗方法无效，并死于这种疾病。BRAFV600E是甲状腺乳头状癌(PTC)和间变性甲状腺癌(ATC)中最常见的基因改变，与PTC向ATC的进展有关。我们的基因集浓缩分析显示，在BRAFV600E 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(例如凝血酶敏感蛋白-1(TSP-1))和受体(整合素)，包括整合素连接的激酶(即粘着斑激酶(FAK))和 转录因子(TF)HMGB1，它与黑色素瘤的进展和转移有关。TSP-1或BRAFV600E的敲除可抑制BRAFV600E阳性的PTC和ATC细胞的增殖、黏附、迁移/侵袭和转移，而TSP-1的敲除可显著降低磷酸化(P)-ERK1/2、pFAK和整合素的水平。新型选择性BRAFV600E抑制剂PLX4720在ATC原位小鼠模型中缩小肿瘤大小。然而，残留肿瘤的持续存在以及治疗3周后TSP-1、pERK1/2和pFAK蛋白的恢复表达表明，转移性甲状腺癌细胞通过上调这些蛋白的表达而获得对BRAFV600E抑制的抵抗力。我们的目标是通过确定BRAFV600E PTC细胞中转移的ECM蛋白触发的基本信号网络，识别BRAFV600E依赖和非依赖的侵袭性PTC的生物标志物。具体目标1：通过确定TSP-1在抑制BRAFV600E后刺激人PTC中ERK1/2和FAK磷酸化的分子级联，识别和评估PLX4720耐药PTC的潜在生物标志物。初步数据表明，TSP-1与BRAFV600E PTC的颈部复发有关。我们将确定TSP-1是否是PTC侵袭性的有效预后生物标志物，并建立适用于临床试验的基于免疫组织化学的筛查程序。为了在BRAFV600E阳性的PTC中确定更多潜在的新的预后生物标志物，我们计划确定TSP-1的相互作用。我们将评估其中一些指标与TSP-1表达及BRAFV600E阳性PTC的临床病理特征的相关性。为了更好地了解BRAFV600E在PTC进展中的功能，我们将应用无偏见的蛋白质组学分析结合功能分析来确定BRAFV600E PTC细胞中ECM蛋白的相互作用和细胞内信号级联。具体目的2：探讨HMGB1是否是PTC侵袭性的生物标志物。我们将HMGB1的表达与TSP-1的表达和BRAFV600E PTC的临床病理特征联系起来，探讨HMGB1是否调节TSP-1的表达或对TSP-1的表达至关重要的因子。这项研究的结果很可能(I)确定侵袭性BRAFV600E阳性PTC的新的预后生物标志物，可以在生物液和PTC组织中进行检测，以帮助监测正在接受靶向治疗的患者，并使这些甲状腺癌能够更早地得到诊断，(Ii)促进对当前治疗方案难以奏效的PTC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.