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，治疗选择有限。这种疾病的特点是激活 KRAS 突变的频率很高（95%），这是 PDAC 进展的已知驱动因素。然而，迄今为止，尚未开发出成功的抗 K-Ras 疗法。目前的工作重点是抑制 K-Ras 信号传导的效应子，特别是 Raf 和 PI3K 信号传导途径。然而，针对这些途径的抑制剂，当用作单一疗法或联合疗法时，对于 KRAS 突变癌症的长期治疗是无效的。这些抑制剂未能取得成功的部分原因是其他效应器在 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性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 生长的其他效应信号事件的作用，以便设计有效的组合靶向疗法。小 GTP 酶 Rac1 在驱动 K-Ras 突变癌症中具有已知的作用，但 Rac1 促进肿瘤生长的具体效应器尚未确定。在我的研究中，我们假设 PAK1 丝氨酸/苏氨酸激酶以及相关亚型 PAK2 和 PAK3 是突变型 K-​​Ras PDAC 中 Rac1 下游的关键组件。为了支持这一点，我的初步结果发现，与正常组织相比，Pak1 蛋白水平在胰腺癌细胞系和患者肿瘤样本中过度表达。此外，我确定 shRNA 介导的 Pak1 蛋白表达的稳定抑制可在体外抑制 PDAC 贴壁依赖性和贴壁依赖性生长以及基质胶侵袭。此外，PDAC 细胞系中 K-Ras 的敲低会导致磷酸-PAK1 (T423) 水平降低，表明 PAK1 活性降低。最近，PAK3 的基因抑制被确定为对 ERK1/2 药物抑制敏感的细胞。这项研究表明，其他 I 组 PAK 同工型 PAK2 和 PAK3 可能有助于 PAK1 驱动 PDAC 生长。这些结果为我提出的研究提供了理论依据和基础，以进一步验证 I 组 PAK 同工型在 PDAC 中的作用。此外，由于我观察到 PDAC 细胞和患者肿瘤样本中正常细胞质 Pak1 存在核隔离，因此我推测 Rac1-Pak1 活性对于通过独特的亚细胞定位特异性功能支持突变型 K-​​Ras 胰腺癌的生长和侵袭至关重要。为了验证这一假设，我将应用最近设计的诱导型 Pak1 构建体来对 Pak1 底物进行时空测定，这对于 Pak1 依赖性 PDAC 生长非常重要。这些研究将产生有关 PAK 功能的新颖的基础和转化信息，并且需要我应用一系列生化、分子和细胞技术。