Phosphorylation-dependent regulation of GLI transcription factors

项目摘要 基底细胞癌是一种局部浸润性上皮癌，每年影响超过400万患者。 在美国，Hedgehog(HH)途径中的突变是唯一被激活的。不适当 HH途径的激活也推动了各种癌症的生长，包括脑、胰腺、前列腺癌和 小细胞肺癌占所有人类癌症死亡人数的25%。HH拮抗剂，如 Vismodegib被FDA批准用于治疗晚期和转移性基底细胞癌，然而，近60%的晚期 肿瘤表现出固有的粘液耐药，有反应的肿瘤中有20%获得抗药性 每年都有。这是一个高度相关的问题，因为晚期BCC病例估计接近400,000名患者 每年，说明迫切需要确定SMO下游的治疗靶点以抑制HH 途径活性。不能满足这种需求的后果很可能是无法治疗那些 对目前批准的治疗方法产生抵抗力，导致基底细胞癌患者和 其他依赖HH的癌症。 我们的长期目标是确定和开发靶向疗法来治疗耐药的HH驱动的 癌症。该应用程序的总体目标是定义在HH驱动的情况下GLI是如何被致癌基因激活的 癌症。我们的中心假设是GLI磷酸化驱动转录激活和SMO 拮抗剂耐药BCC生长，靶向激活GLI的信号通路将抑制肿瘤 成长。我的两个具体目标将定义1)Gli锌指如何磷酸化和2)临床复发 Gli突变促进转录活性、肿瘤生长和耐药性。定义GLI是如何 在癌症中被激活可能为治疗HH驱动的癌症患者提供新的治疗靶点。 为了实现这些目标，我们将使用BCC细胞系和同种异体移植物，这些细胞株和同种异体移植物过度表达临床观察和 在存在或不存在HH拮抗剂的情况下检测复发的GLI突变以检测肿瘤生长。我们将使用 GLI突变体表现出增强的转录活性，以定义GLI如何在癌症中被激活 标准分子生物学、生物化学、细胞生物学和基因技术。我们的初步数据显示 我们已经确定了三种调节GLI活性的激酶，我们计划产生磷酸特异性突变体来 定义何时、何地以及每一种激酶如何作用于GLI。我们将对人类肿瘤进行免疫荧光染色 适当的抗体来验证这些途径在人类中发挥作用，我们将执行标准增益-和 功能丧失研究，以分析肿瘤整个生命周期中涉及的途径。

Project Summary Basal cell carcinomas (BCCs) are locally invasive epithelial cancers that affect over 4 million patients a year in the United States and are solely driven by activating mutations in the Hedgehog (HH) pathway. Inappropriate HH pathway activation also drives growth of a variety of cancers including brain, pancreatic, prostate, and small cell lung cancer that account for up to 25% of all human cancer deaths. HH antagonists such as vismodegib are FDA-approved to treat advanced and metastatic BCCs, however, nearly 60% of advanced tumors display inherent vismodegib resistance and 20% of tumors that do respond acquire drug resistance every year. This is a highly relevant issue as advanced BCC cases are estimated to approach 400,000 patients each year, illustrating a critical need to identify therapeutic targets downstream of SMO to suppress HH pathway activity. The consequence of not meeting this need will likely be the inability to treat patients who are resistant to current approved therapies, leading to an increase in mortality for patients inflicted with BCC and other HH-dependent cancers. Our long-term objective is to identify and develop targeted therapeutics to treat drug-resistant HH-driven cancers. The overall objective of this application is to define how GLI is oncogenically activated in HH-driven cancer. Our central hypothesis is that GLI phosphorylation drives transcriptional activation and SMO antagonist-resistant BCC growth, and targeting the signaling pathways that activate GLI will suppress tumor growth. My two specific aims will define 1) how GLI zinc finger phosphorylation and 2) how clinically recurrent GLI mutations promote transcriptional activity, tumor growth, and drug resistance. Defining how GLI is activated in cancer may reveal novel therapeutic targets to treat patients with HH-driven cancers. To achieve these aims, we will use BCC cell lines and allografts that overexpress clinically observed and recurrent GLI mutants to assay for tumor growth in the presence or absence of HH antagonists. We will use the GLI mutants that show increased transcriptional activity to define how GLI is activated in cancer using standard molecular biology, biochemistry, cell biology, and genetic techniques. Our preliminary data has already identified three kinases that regulate GLI activity, and we plan to generate phospho-specific mutants to define when, where, and how each kinase acts on GLI. We will immunofluorescently stain human tumors with appropriate antibodies to verify these pathways operate in humans, and we will perform standard gain- and loss-of-function studies to analyze the pathways involved over the lifetime of the tumor.