Phosphorylation-dependent regulation of GLI transcription factors

项目摘要 基底细胞癌（BCC）是一种局部浸润性上皮癌，在美国每年影响超过400万患者。 美国，并且仅由Hedgehog（HH）途径中的激活突变驱动。不适当 HH途径活化还驱动多种癌症的生长，包括脑癌、胰腺癌、前列腺癌和前列腺癌。 小细胞肺癌占所有人类癌症死亡的25%。HH拮抗剂，例如 维莫德吉是FDA批准用于治疗晚期和转移性BCC，然而，近60%的晚期BCC患者， 肿瘤显示出固有的维莫德吉耐药性，20%的应答肿瘤获得耐药性 每年这是一个高度相关的问题，因为晚期BCC病例估计接近40万例患者 这说明迫切需要确定SMO下游的治疗靶点以抑制HH 途径活性。不满足这一需求的后果可能是无法治疗那些 对目前批准的治疗具有耐药性，导致患BCC的患者的死亡率增加， 其他依赖于HH的癌症。 我们的长期目标是确定和开发靶向治疗药物，以治疗耐药HH驱动的 癌的本申请的总体目标是定义GLI如何在HH驱动的肿瘤细胞中被致癌激活。 癌我们的中心假设是，GLI磷酸化驱动转录激活和SMO 拮抗剂抗性BCC生长，靶向激活GLI的信号通路将抑制肿瘤生长。 增长我的两个具体目标将定义1）GLI锌指磷酸化和2）临床复发 GLI突变促进转录活性、肿瘤生长和耐药性。如何定义GLI 在癌症中激活的基因可能揭示新的治疗靶点，以治疗患有HH驱动的癌症的患者。 为了实现这些目标，我们将使用临床观察到过表达的BCC细胞系和同种异体移植物， 复发性GLI突变体，以在存在或不存在HH拮抗剂的情况下测定肿瘤生长。我们将使用 显示出增加的转录活性的GLI突变体，以定义GLI如何在癌症中被激活， 标准分子生物学、生物化学、细胞生物学和遗传技术。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，我们计划产生磷酸化特异性突变体， 定义每种激酶何时、何地以及如何作用于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的运作，我们将进行标准的增益-和 功能丧失研究，以分析肿瘤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.