Role of TTK/Mps-1 upregulation by Ral GTPase activation in pancreatic oncogenesis

描述（由申请人提供）：胰腺癌是一种致命的疾病，几乎没有可用的治疗选择。虽然在针对某些癌症的信号转导靶向疗法中已经取得了成功，但尚未确定针对胰腺癌的显著靶向疗法。癌基因KRAS在约100%的胰腺癌导管腺癌（PDAC）中被突变激活。然而，开发K-Ras的直接抑制剂的努力令人失望。因此，焦点已经转移到抑制效应信号级联激活 K-Ras的下游经典的K-Ras效应子途径Raf-MEK-ERK和PI 3 K-AKT-mTOR受到了最多的关注，这两种途径的>40种抑制剂目前正在临床试验中进行评估。然而，最近研究KRAS依赖性和合成致死性的研究认为，其他效应子是K-Ras调节转化的关键。Ral鸟嘌呤-核苷酸交换因子（RalGEF）-Ral途径是一种非经典的K-Ras效应途径，最近已证实其在K-Ras介导的肿瘤发生中的重要性。RalGEF激活RalA和RalB Ras样小GTP酶。RalA在体外调节PDAC锚定非依赖性生长和体内肿瘤发生，而RalB在体外调节侵袭和体内转移。转录因子活化是Ral效应子信号传导的关键结果。因此，我们假设基因阵列分析将确定Ral信号传导的关键基因靶点，为药物发现提供治疗上有用的途径。最近，我进行了基因阵列分析，并确定了编码双特异性蛋白激酶TTK（也称为Mps-1）的基因作为PDAC中受RalA和RalB调控的转录本。与正常胰腺组织相比，我还发现PDAC原发肿瘤和转移性病变中TTK mRNA过表达。此外，我的初步研究已经验证了TTK表达在PDAC锚定非依赖性生长和Matrigel体外侵袭中的需要。因此，TTK可能是抗Ral和Ras药物发现的一个有吸引力和易处理的靶标。在目标1中，我将进一步验证TTK在PDAC肿瘤发生中的作用，通过使用原位肿瘤小鼠模型，更好地模拟患者的侵袭性和转移性疾病。在目标2中，我将确定特异性Ral效应物信号传导途径对TTK表达和PDAC生长的重要性。RalBP 1/RLIP 76和Sec 5和Exo 84外囊组分是最佳表征的Ral效应子，并且我将确定将Ral与这些效应子差异解偶联的Ral突变体是否可以挽救由于内源性Ral沉默而受损的TTK表达。最后，目标3研究将利用TTK的错义和截短突变体来研究PDAC锚定非依赖性生长和通过Matrigel的侵袭中TTK功能中对激酶和非激酶序列的需求。总的来说，这些研究将要求我掌握一系列实验技术，同时为我做基础和转化研究做准备。

DESCRIPTION (provided by applicant): Pancreatic cancer is a deadly disease with few therapeutic options available. While success has been made in signal transduction-targeted therapies for some cancers, no significant targeted therapies have been identified for pancreatic cancer. The oncogene KRAS is activated by mutation in ~100% of pancreatic cancer ductal adenocarcinomas (PDAC). However, efforts to develop direct inhibitors of K-Ras have been disappointing. Therefore, focus has shifted to inhibiting the effector signaling cascades activated downstream of K-Ras. The canonical K-Ras effector pathways, Raf-MEK-ERK and PI3K-AKT-mTOR have received the most attention with >40 inhibitors of these two pathways currently under evaluation in clinical trials. However, recent studies investigating KRAS dependency and synthetic lethality argue that other effectors are key to K-Ras regulated transformation. The Ral guanine-nucleotide exchange factor (RalGEF)-Ral pathway is a non-canonical K-Ras effector pathway that have recently been validated for its importance in K-Ras-mediated oncogenesis. RalGEF activates the RalA and RalB Ras-like small GTPases. RalA regulates PDAC anchorage-independent growth in vitro and tumorigenesis in vivo, whereas RalB regulates invasion in vitro and metastasis in vivo. Transcription factor activation is a key consequence of Ral effector signaling. Therefore, we hypothesize that gene array profiling will identify key gene targets of Ral signaling, providing therapeutically useful avenues for drug discovery. Recently I performed gene array analyses and identified the gene encoding the dual specificity protein kinase TTK (also called Mps-1) as a transcript regulated by both RalA and RalB in PDAC. I also identified TTK mRNA overexpression in PDAC primary tumors and metastatic lesions compared to normal pancreas tissue. Furthermore, my preliminary studies have validated the requirement for TTK expression in PDAC anchorage-independent growth and Matrigel invasion in vitro. Thus, TTK may be an attractive and tractable target for anti-Ral and Ras drug discovery. In Aim 1, I will further validate the role of TTK in PDAC oncogenesis by using orthotopic tumor mouse models that better model the invasive and metastatic disease of the patient. In Aim 2, I will establish the importance of specific Ral effector signaling pathways for TTK expression and PDAC growth. RalBP1/RLIP76 and the Sec5 and Exo84 exocyst components are the best- characterized Ral effectors and I will determine if Ral mutants that differentially uncouple Ral from these effectors can rescue impaired TTK expression due to endogenous Ral silencing. Finally, Aim 3 studies will utilize missense and truncation mutants of TTK to investigate the requirement for kinase and non-kinase sequences in TTK function in PDAC anchorage-independent growth and invasion through Matrigel. Taken together, these studies will require me to master an array of experimental techniques, while preparing me to do both basic and translational research.