Defining the Role and Mechanism of Pak1 in Supporting Pancreatic Cancer

描述（由申请方提供）：胰腺导管腺癌（PDAC）是一种极致命的癌症，治疗选择有限。这种疾病的特征是高频率的激活KRAS突变（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。目前的努力集中在抑制K-Ras信号传导的效应物，特别是Raf和PI 3 K信号传导途径。然而，靶向这些途径的抑制剂，当作为单一疗法或组合使用时，对于KRAS突变型癌症的长期治疗无效。这些抑制剂缺乏成功的部分原因是其他效应物在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，迫切需要更好地理解支持突变K-Ras驱动的PDAC生长的其他效应信号事件的作用，以设计有效的组合靶向治疗。小的GTribal Rac 1在驱动K-Ras突变型癌症中具有已知的作用，但Rac 1促进肿瘤生长的特定效应物尚未确定。在我的研究中，我们假设PAK 1丝氨酸/苏氨酸激酶和相关的亚型PAK 2和PAK 3是突变型K-Ras PDAC中Rac 1下游的关键组分。为了支持这一点，我的初步结果发现，与正常组织相比，Pak 1蛋白水平在胰腺癌细胞系和患者肿瘤样本中过表达。此外，我确定了稳定的shRNA介导的Pak 1蛋白表达抑制PDAC锚定非依赖性和依赖性的生长和Matrigel入侵在体外。此外，PDAC细胞系中K-Ras的敲低导致磷酸-PAK 1（T423）水平降低，表明PAK 1活性降低。最近，PAK 3的遗传抑制被确定为对ERK 1/2的药理学抑制敏感的细胞。这项研究表明，其他组I PAK亚型，PAK 2和PAK 3，可能有助于PAK 1驱动PDAC生长。这些结果为我提出的进一步验证I组PAK亚型在PDAC中的作用的研究提供了理论依据和基础。此外，因为我已经观察到PDAC细胞和患者肿瘤样本中正常细胞质Pak 1的核隔离，我假设Rac 1-Pak 1活性对于支持突变K-Ras胰腺癌生长和侵袭是至关重要的，通过不同的亚细胞定位特异性功能。为了验证这一假设，我将应用最近设计的诱导型Pak 1构建Pak 1基板的时空测定是重要的Pak 1依赖PDAC的增长。这些研究将产生关于PAK功能的新的基础和翻译信息，并将需要我应用一系列生物化学，分子和细胞技术。

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.