Defining the Role and Mechanism of Pak1 in Supporting Pancreatic Cancer

定义 Pak1 在支持胰腺癌中的作用和机制

• 批准号: 8718319
• 负责人: Nicole Marie Baker
• 金额: $ 3.05万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8718319
• 关键词: Ablation; Abnormal Cell; Adenocarcinoma Cell; Anchorage-Independent Growth; Automobile Driving; Binding; Biochemical; Biological; Cancer Etiology; Cancer cell line; Cell Line; Cells; Cellular Morphology; Cellular biology; Cessation of life; Colon Carcinoma; Coupled; Development; Diagnosis; Disease; Drug Targeting; Epithelial Cells; Estrogen Receptors; Evaluation; Event; Exclusion; Exhibits; Family member; Foundations; Frequencies; Genes; Genetic Suppression; Growth; Human; In Vitro; Invaded; KRAS2 gene; Libraries; Location; MAPK3 gene; MEKs; Malignant Neoplasms; Malignant neoplasm of pancreas; Mediating; Mitogen-Activated Protein Kinases; Molecular; Monomeric GTP-Binding Proteins; Mutate; Mutation; Neoplasm Metastasis; Normal tissue morphology; Nuclear; Oncogenes; Pancreas; Pancreatic Ductal Adenocarcinoma; Pathway interactions; Patients; Phosphorylation; Phosphotransferases; Protein Isoforms; Protein Kinase; Protein Overexpression; Protein-Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins c-akt; RNA Interference; Role; Sampling; Signal Pathway; Signal Transduction; Small Interfering RNA; Survival Rate; Techniques; Testing; Therapeutic; Up-Regulation; anticancer research; cancer therapy; combinatorial; design; gemcitabine; human FRAP1 protein; in vivo; inhibitor/antagonist; malignant breast neoplasm; matrigel; mutant; neoplastic cell; novel; overexpression; pancreatic cancer cells; pancreatic neoplasm; programs; protein expression; public health relevance; small hairpin RNA; small molecule; standard of care; success; tumor; tumor growth; tumor progression; zebrafish development

DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDAC) is an extremely lethal cancer with limited treatment options. This disease is characterized by a high frequency of activating KRAS mutations (95%), which is a known driver of PDAC progression. However, to date, no successful anti-K-Ras therapies have been developed. Current efforts have focused on inhibition of effectors of K-Ras signaling, in particular the Raf and PI3K signaling pathways. However, inhibitors targeting these pathways, when used as monotherapy or in combination, have been ineffectual for long-term treatment of KRAS mutant cancers. The lack of success of these inhibitors is due, in part, to the importance of other effectors in K-Ras-dependent cancer growth and the upregulation of compensatory signaling programs that overcome inhibitor activity. Consequently, there is a pressing need to better understand the role of other effector signaling events that support mutant K-Ras-driven PDAC growth in order to design effective combinatorial-targeted therapies. The small GTPase Rac1 has a known role in driving K-Ras mutant cancers, but the specific effectors through which Rac1 promotes tumor growth have not been defined. For my studies, we hypothesize that the PAK1 serine/threonine kinase, and related isoforms, PAK2 and PAK3, are key components downstream of Rac1 in mutant K-Ras PDAC. In support of this, my preliminary results found that Pak1 protein levels are overexpressed in pancreatic cancer cell lines and in patient tumor samples when compared to normal tissues. Additionally I determined that stable shRNA-mediated suppression of Pak1 protein expression inhibited PDAC anchorage-independent and -dependent growth and Matrigel invasion in vitro. Further, knockdown of K-Ras in PDAC cell lines results in reduced phospho-PAK1 (T423) levels, indicating a decrease in PAK1 activity. Recently, genetic suppression of PAK3 was determined to sensitive cells to pharmacologic inhibition of ERK1/2. This study suggests that other Group I PAK isoforms, PAK2 and PAK3, may be contributing to PAK1 driving PDAC growth. These results provide the rationale and foundation for my proposed studies to further validate the role of Group I PAK isoforms in PDAC. Additionally, because I have observed a nuclear sequestration of the normally cytoplasmic Pak1 in PDAC cells and patient tumor samples, I hypothesize that a Rac1-Pak1 activity is critical for supporting mutant K-Ras pancreatic cancer growth and invasion through distinct, subcellular localization-specific functions. To test this hypothesis, I will apply a recently designed inducible Pak1 construct for the spatio-temporal determination of Pak1 substrates that are important for Pak1-dependent PDAC growth. These studies will generate novel basic and translational information regarding PAK function and will require my application of a spectrum of biochemical, molecular and cellular techniques.

描述（由申请人提供）：胰腺导管腺癌（PDAC）是一种极其致命的癌症，治疗方案有限。该疾病的特征是激活KRAS突变的高频（95％），这是PDAC进展的已知驱动因素。但是，迄今为止，尚未开发成功的抗K-RAS疗法。当前的努力集中在抑制K-RAS信号传导的效应子上，尤其是RAF和PI3K信号通路。但是，当用作单一疗法或组合的靶向这些途径的抑制剂对KRAS突变癌的长期治疗无效。这些抑制剂缺乏成功的部分原因是其他效应子在K-RAS依赖性癌症生长中的重要性以及克服抑制剂活性的补偿信号传导程序的上调。因此，迫切需要更好地了解支持突变K-RAS驱动的PDAC生长的其他效应子信号传导事件的作用，以设计有效的组合靶向疗法。小型GTPase Rac1在驱动K-RAS突变体癌症中具有已知作用，但是尚未定义Rac1促进肿瘤生长的特定效应子。在我的研究中，我们假设PAK1丝氨酸/苏氨酸激酶以及相关的同工型PAK2和PAK3是突变K-RAS PDAC中Rac1下游的关键成分。为此，我的初步结果发现，与正常组织相比，胰腺癌细胞系和患者肿瘤样品中的PAK1蛋白水平过表达。另外，我确定稳定的shRNA介导的PAK1蛋白表达抑制抑制了PDAC锚定非依赖性和依赖性生长以及体外依赖性生长和矩阵侵袭。此外，PDAC细胞系中K-RAS的敲低导致磷酸化-PAK1（T423）水平降低，表明PAK1活性的降低。最近，确定对PAK3的遗传抑制对ERK1/2的药理抑制敏感细胞。这项研究表明，其他I组PAK同工型PAK2和PAK3可能会导致PAK1驱动PDAC生长。这些结果为我的拟议研究提供了基础，以进一步验证I组在PDAC中的作用。此外，由于我已经观察到PDAC细胞和患者肿瘤样品中正常细胞质PAK1的核次数，因此我假设Rac1-Pak1活性对于支持突变的K-RAS胰腺癌生长和通过不同细胞细胞局部定位特异性功能的侵袭至关重要。为了检验这一假设，我将应用最近设计的诱导PAK1构建体，以确定对PAK1依赖性PDAC生长很重要的PAK1底物的时空测定。这些研究将产生有关PAK功能的新型基本和翻译信息，并需要我应用一系列生化，分子和细胞技术。