Fingerprinting Invasive Membrane Protrusions to Discover Metastatic Signatures

描述（由申请人提供）：由于转移性胰腺癌细胞可在癌症进展的最早阶段从原发性肿瘤扩散，早在患者出现症状之前（10年），因此大多数患者在诊断时患有晚期转移性疾病。这一点以及患者抵抗所有形式的事实 在常规化疗中，胰腺癌的5年生存率很低，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，迫切需要用于早期检测的新生物标志物和针对消除转移性细胞的新治疗靶点。因此，本提案中的工作目标是鉴定介导胰腺导管腺癌（PDAC）的新蛋白，其可用作治疗转移性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，我们将通过在无胸腺裸鼠的胰腺中连续传代分离的转移性细胞来产生高转移性基因工程小鼠和患者来源的PDAC细胞系。然后，我们将生物化学纯化其侵袭伪足，驱动PDAC细胞的侵袭和转移，用于蛋白质组学和磷酸化蛋白质组学分析。为了鉴定转移特征，将使用已建立的磷酸化位点鉴定方法、定量MudPIT质谱法和生物信息学来询问侵袭性侵袭伪足膜的总蛋白质谱以及激酶活化网络的变化。在目标2中，我们将使用RNAi敲低技术和PDAC细胞转移的临床前动物模型功能性地测试在目标1中鉴定的所选关键特征蛋白介导PDAC细胞转移的能力。我们使用体内选择的转移性变体结合侵袭伪足纯化和全面的蛋白质组学的独特方法将揭示重要的新转移性特征蛋白，其作为识别和治疗转移性PDAC细胞的“指纹”。新型转移性蛋白的鉴定是推动新的PDAC疗法和诊断的开发过程中的第一个关键步骤，这是治疗这种毁灭性疾病所迫切需要的。

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.