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

Ral GTPase 激活上调 TTK/Mps-1 在胰腺肿瘤发生中的作用

• 批准号: 8398164
• 负责人: Jeran K Stratford
• 金额: $ 2.94万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8398164
• 关键词: Adenocarcinoma; Adenocarcinoma Cell; Agar; Amino Acid Sequence; Anchorage-Independent Growth; Antineoplastic Agents; Attention; Basic Science; Binding; Biological Assay; Catalytic Domain; Cell Line; Cell Survival; Cells; Characteristics; Clinical Trials; Data; Dependency; Disease; Ductal; Evaluation; Family; Frequencies; Gene Targeting; Genes; Growth; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; In Vitro; KRAS2 gene; MEKs; Malignant Neoplasms; Malignant neoplasm of pancreas; Measures; Mediating; Messenger RNA; Metastatic Lesion; Modeling; Molecular; Monomeric GTP-Binding Proteins; Mutation; Neoplasm Metastasis; Oncogenes; Paclitaxel; Pancreas; Pancreatic Ductal Adenocarcinoma; Pathway interactions; Patients; Peptide Sequence Determination; Phase; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Primary Neoplasm; Process; Property; Protein Isoforms; Protein Kinase; Protein Tyrosine Kinase; Proto-Oncogene Proteins c-akt; RNA Interference; Ras Inhibitor; Regulation; Role; Sampling; Signal Pathway; Signal Transduction; Structure; T-Cell Immunologic Specificity; TTK Protein Kinase; Techniques; Therapeutic; Tissues; Transcript; Translational Research; Ubiquitination; Up-Regulation; Validation; Xenograft procedure; cancer therapy; clinical application; drug discovery; human FRAP1 protein; in vivo; inhibitor/antagonist; interest; mRNA Expression; matrigel; mouse model; mutant; neoplastic cell; novel; overexpression; protein expression; protein protein interaction; ral Guanine Nucleotide Exchange Factor; ras Guanine Nucleotide Exchange Factors; small hairpin RNA; success; therapeutic development; therapeutic target; transcription factor; tumor; tumorigenesis

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.

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。虽然某些癌症的信号转导靶向疗法已取得成功，但尚未发现针对胰腺癌的显着靶向疗法。在约 100% 的胰腺癌导管腺癌 (PDAC) 中，癌基因 KRAS 因突变而被激活。然而，开发 K-Ras 直接抑制剂的努力却令人失望。因此，焦点已转移到抑制效应信号级联激活 K-Ras 的下游。经典的 K-Ras 效应通路 Raf-MEK-ERK 和 PI3K-AKT-mTOR 受到了最多的关注，目前有超过 40 种这两条通路的抑制剂正在临床试验中进行评估。然而，最近调查 KRAS 依赖性和合成致死率的研究认为，其他效应器是 K-Ras 调节转化的关键。 Ral 鸟嘌呤核苷酸交换因子 (RalGEF)-Ral 通路是一种非经典 K-Ras 效应通路，最近已验证其在 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 调节 PDAC 体外贴壁依赖性生长和体内肿瘤发生，而 RalB 调节体外侵袭和体内转移。转录因子激活是 Ral 效应信号传导的关键结果。因此，我们假设基因阵列分析将识别 Ral 信号传导的关键基因靶标，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，并鉴定出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 调节的转录本。与正常胰腺组织相比，我还发现 PDAC 原发性肿瘤和转移性病变中 TTK mRNA 过度表达。此外，我的初步研究已经验证了体外 PDAC 贴壁依赖性生长和基质胶侵袭中 TTK 表达的要求。因此，TTK 可能是抗 Ral 和 Ras 药物发现的一个有吸引力且易于处理的靶点。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 Ral 效应信号通路对 TTK 表达和 PDAC 生长的重要性。 RalBP1/RLIP76 以及 Sec5 和 Exo84 外囊组件是最具特征的 Ral 效应子，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，Aim 3 研究将利用 TTK 的错义和截短突变体来研究 PDAC 锚定独立生长和通过 Matrigel 的侵袭中 TTK 功能对激酶和非激酶序列的需求。总的来说，这些研究需要我掌握一系列实验技术，同时为我进行基础研究和转化研究做好准备。 公共卫生相关性：胰腺癌是一种致命疾病，治疗选择很少。 Ral GTPases 在癌基因 KRAS 下游的胰腺癌肿瘤发生中具有经过验证的作用。 TTK/Mps1 是一种受 Ral 信号传导调节的蛋白激酶，代表了胰腺癌药物发现中阻断 Ras 信号传导的新型抗 Ral 治疗靶点。