Defining the Role and Mechanism of Pak1 in Supporting Pancreatic Cancer

描述(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。这种疾病的特点是高频率激活KRAS突变(95%)，这是PDAC进展的已知驱动因素。然而，到目前为止，还没有成功的抗K-RAS疗法。目前的努力主要集中在抑制K-RAS信号的效应子，特别是Raf和PI3K信号通路。然而，针对这些途径的抑制剂，当作为单一疗法或联合使用时，对KRAS突变癌症的长期治疗无效。这些抑制剂缺乏成功的部分原因是，其他效应器在K-RAS依赖的肿瘤生长中的重要性，以及克服抑制剂活性的代偿信号程序的上调。因此，迫切需要更好地了解支持突变K-RAS驱动的PDAC生长的其他效应器信号事件的作用，以便设计有效的联合靶向治疗。小的GTPase rac1在驱动K-ras突变的癌症中具有已知的作用，但rac1促进肿瘤生长的特定效应因子尚未确定。在我的研究中，我们假设PAK1丝氨酸/苏氨酸激酶及其相关的亚型PAK2和PAK3是突变的K-RAS PDAC中rac1下游的关键成分。为了支持这一点，我的初步结果发现，与正常组织相比，Ak1蛋白在胰腺癌细胞系和患者肿瘤样本中过表达。此外，我还确定稳定的shRNA介导的对Pak1蛋白表达的抑制抑制了PDAC在体外的锚定非依赖性生长和Matrigel侵袭。此外，PDAC细胞系中K-RAS的敲除导致磷酸化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的药物抑制敏感的细胞。这项研究表明，其他I组PAK亚型，PAK2和PAK3，可能有助于PAK1推动PDAC的生长。这些结果为我提出的进一步验证第一组PAK亚型在PDAC中的作用的研究提供了理论基础和基础。此外，由于我观察到在PDAC细胞和患者肿瘤样本中正常的细胞质pak1的核隔离，我假设rac1-pak1的活性对于支持突变的K-ras胰腺癌的生长和侵袭至关重要，这是通过不同的亚细胞定位特异性功能来实现的。为了验证这一假设，我将应用最近设计的可诱导的PAC1结构来时空确定对依赖于PAC1的PDAC生长至关重要的PAC1底物。这些研究将产生有关PAK功能的新的基本和翻译信息，并需要我应用一系列生化、分子和细胞技术。

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.