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

描述(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。虽然针对某些癌症的信号转导靶向治疗已取得成功，但尚未确定针对胰腺癌的重大靶向治疗。癌基因KRAS在~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一直令人失望。因此，重点已经转移到抑制激活的效应器信号级联上 在K-RAS的下游。经典的K-RAS效应通路，Raf-MEK-ERK和PI3K-AKT-mTOR受到了最多的关注，这两个通路中的&gt；40抑制剂目前正在进行临床试验。然而，最近研究KRAS依赖性和合成致死性的研究认为，其他效应器是K-RAS调控转化的关键。Ral鸟嘌呤核苷酸交换因子(Ralgef)-Ral途径是一种非典型的K-RAS效应途径，最近被证实在K-RAS介导的肿瘤发生中具有重要作用。Ralgef激活Rala和RalB Ras样小GTP酶。RalA在体外调节PDAC的非黏附生长和体内成瘤，而RalB在体外调节侵袭和体内转移。转录因子的激活是RAL效应信号转导的关键结果。因此，我们假设基因阵列图谱将识别Ral信号的关键基因靶点，为药物开发提供治疗上有用的途径。最近，我进行了基因芯片分析，发现编码双特异性蛋白激酶TTK(也称为MPS-1)的基因是PDAC中受Rala和RalB共同调控的转录本。我还发现，与正常胰腺组织相比，PDAC原发肿瘤和转移性病变中TTK mRNA过表达。此外，我的初步研究证实了TTK在PDAC体外锚定非依赖性生长和Matrigel侵袭中的表达需求。因此，TTK可能是抗RAL和RAS药物开发的一个有吸引力和易操作的靶点。在目标1中，我将通过使用原位肿瘤小鼠模型进一步验证TTK在PDAC肿瘤发生中的作用，该模型可以更好地模拟患者的侵袭性和转移性疾病。在目标2中，我将确定特定的Ral效应信号通路对TTK表达和PDAC生长的重要性。RalBP1/RLIP76和Sec5和Exo84外囊成分是最具特征性的Ral效应器，我将确定与这些效应器不同的Ral突变体是否能够挽救由于内源性Ral沉默而受损的TTK表达。最后，Aim 3研究将利用TTK的错义和截断突变来研究在PDAC非锚定生长和通过Matrigel侵袭过程中对TTK功能中的激酶和非激酶序列的需求。综上所述，这些研究需要我掌握一系列的实验技术，同时为我做基础研究和翻译研究做准备。 公共卫生相关性：胰腺癌是一种致命的疾病，几乎没有治疗选择。Ral GTP酶在癌基因KRAS下游的胰腺癌发生中起着被证实的作用。TTK/Mps1是一种受RAL信号调节的蛋白激酶，在胰腺癌药物开发中代表着阻断RAS信号的一个新的抗RAL治疗靶点。

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. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is a deadly disease with few therapeutic options. Ral GTPases have a validated role in pancreatic cancer oncogenesis downstream of the oncogene KRAS. TTK/Mps1 is a protein kinase regulated by Ral signaling and represents a novel anti-Ral therapeutic target for blocking Ras signaling in pancreatic cancer drug discovery.