Fingerprinting Invasive Membrane Protrusions to Discover Metastatic Signatures

描述(申请人提供)：由于转移性胰腺癌细胞可以在癌症进展的最早阶段从原发肿瘤扩散，在患者出现症状之前很久(10年)，大多数患者在诊断时已经是晚期转移性疾病。这一点以及患者抵制一切形式的事实 在常规化疗中，胰腺癌的5年生存率不到5%，中位生存期为4-6个月。显然，为了对抗这种毁灭性的疾病，迫切需要新的生物标记物来进行早期检测，以及针对消除转移细胞的新的治疗靶点。因此，本研究的目的是寻找新的介导胰腺导管腺癌(PDAC)的蛋白质，作为治疗转移性PDAC的生物标志物和治疗靶点。为了实现这一目标，在目标1中，我们将通过对裸鼠胰腺中分离的转移细胞进行连续传代，建立高转移的基因工程小鼠和患者来源的PDAC细胞系。然后，我们将生化提纯他们的内向足突，驱动PDAC细胞的入侵和转移，用于蛋白质组和磷酸蛋白质组分析。为了识别转移特征，将使用已建立的亚磷酸盐鉴定方法、定量MudPIT质谱学和生物信息学来询问侵袭性内翻足膜总蛋白谱以及激酶激活网络的变化。在目标2中，我们将利用RNAi敲除技术和PDAC细胞转移的临床前动物模型，从功能上测试在目标1中确定的关键特征蛋白是否具有介导PDAC细胞转移的能力。我们使用体内选择的转移变异体的独特方法，结合跨足类纯化和全面的蛋白质组学，将揭示重要的新转移特征蛋白，作为识别和治疗转移PDAC细胞的“指纹”。识别新的转移蛋白是推动新的PDAC疗法和诊断方法发展的第一步，也是关键的一步，这是治疗这种毁灭性疾病迫切需要的。

DESCRIPTION (provided by applicant): Because metastatic pancreatic cancer cells can disseminate from the primary tumor during the earliest stages of cancer progression, long before (10 years) the patient becomes symptomatic, the majority of patients have advanced stage metastatic disease at the time of diagnosis. This and the fact that patients resist all forms of conventional chemotherapy, pancreatic cancer has a dismal 5 year survival rate of less than 5%, and a median survival of 4-6 months. Clearly, to combat this devastating disease new biomarkers for early detection and new therapeutic targets directed at eliminating metastatic cells are urgently needed. Therefore, the objective of work in this proposal is to identify new proteins that mediate pancreatic ductal adenocarcinoma (PDAC), which can be used as biomarkers at and therapeutic targets to treat metastatic PDAC. To achieve this goal, in Aim 1 we will generate highly metastatic genetically engineered mouse and patient-derived PDAC cell lines by serial passaging of isolated metastatic cells in the pancreas of athymic nude mice. We will then biochemically purify their invadopodia, which drive PDAC cell invasion and metastasis, for proteomic and phosphoproteomic analyses. To identify metastatic signatures, the invasive invadopodia membranes will be interrogated for changes in total protein profile as well as kinase activation networks using established phosphosite identification methods, quantitative MudPIT mass spectrometry, and bioinformatics. In Aim 2, we will functionally test selected key signature proteins identified in Aim 1 for their ability to mediate PDAC cell metastasis using RNAi knockdown technology and preclinical animal models of PDAC cell metastasis. Our unique approach of using in vivo selected metastatic variants combined with invadopodia purification and comprehensive proteomics will reveal important new metastatic signature proteins that serve as 'fingerprints' to identify and treat metastatic PDAC cells. The identification of novel metastatic proteins is the first, and critical step, in the process that drives development of new PDAC therapeutics and diagnostics, which are sorely needed to treat this devastating disease.