Investigating the role of SHP2-PLCG1 interaction in PDAC calcium signaling and metabolism

摘要 胰腺导管腺癌（PDAC）是一种高致死性疾病，预计将成为第二大 到2025年，它将成为美国癌症相关死亡的主要原因。在这些案例中，超过90%的 患者携带KRAS突变。不幸的是，直接靶向PDAC特异性突变已经失败， 获得牵引力，但由于突变KRAS的无处不在的疾病，替代方法，以减少下游 MAPK信号通路已被研究。最近，针对KRAS活性的上游调节因子SHP 2， 被带到了这个领域的前沿。SHP 2对于小鼠模型中PDAC的发展是必需的， 尽管存在突变的KRAS。然而，虽然SHP 2在PDAC中的作用主要归因于其在PDAC中的作用，但其在PDAC中的作用可能是由其自身的作用引起的。 尽管SHP 2对MAPK通路的调节起着重要作用，但许多其他组织类型的证据表明，SHP 2应该起着重要作用。 通过其他机制--最显著的是通过调节细胞内钙流--来影响肿瘤的进展。 我们的初步数据表明，SHP 2与磷脂酶-c-相互作用的钙流的上游， γ 1（PLCγ1）。最近的文献已经描述了这种相互作用的机制，因为我们已经开始定义 破坏配对和下游钙流的结果，提供了解偶联的证据。 SHP 2在来自MAPK途径的PDAC中的规范归因的作用。我们对代谢功能的探索 SHP 2抑制PDAC细胞导致我们假设SHP 2和PLCγ1相互作用以维持钙信号传导 柠檬酸循环中线粒体酶的激活所需的。在这种情况下，我们将采取双管齐下的措施， 研究SHP 2在代谢中作用的方法：首先，通过使用Agilent Seahorse Fuel-Flex测试， 可以精确定位SHP 2抑制细胞的代谢依赖性，以及代谢抵抗的机制。 应激;其次，我们将捕获PDAC细胞响应于差异SHP 2的稳态代谢物谱， MEK或PLCγ1活性，以采取非靶向方法来确定每种蛋白质的代谢影响。 单独和集体运作。此外，我们认为，这种互动的成果不仅限于 线粒体功能的改变因此，我们假设，通过比较SHP 2抑制和MEK 抑制PDAC细胞的磷酸化蛋白质组的整体变化，我们将能够描绘MAPK- SHP 2作为癌基因在PDAC信号传导中的独立功能。我们希望这项提案的结果 强调了SHP 2在MAPK途径之外的肿瘤进展/癌细胞信号传导中的重要性， 仍然认识到它在调节KRAS活性中的作用。我们相信，这项研究的结论将产生新的 蛋白质相互作用的网络作为PDAC的目标进行研究，最终导致开发 用于改善患者结果的治疗剂。

ABSTRACT Pancreatic Ductal Adenocarcinoma (PDAC) is a highly lethal disease and is expected to become the second leading cause of cancer-associated-death in the US by the year 2025. Of these cases, greater than 90% of patients harbor a mutation to KRAS. Unfortunately, direct targeting of the mutations specific to PDAC have failed to gain traction, but due to mutant KRAS’s ubiquity to the disease, alternative methods to reduce downstream MAPK signaling have been investigated. Recently, targeting of the upstream regulator of KRAS activity, SHP2 has been brought to the forefront of the field. SHP2 is essential for the development of PDAC in mouse models, despite mutant KRAS presence. However, while the role of SHP2 in PDAC has been largely attributed to its regulation of the MAPK pathway, evidence in many other tissue types argue that SHP2 should be contributing to tumor progression through other mechanisms – most notably, through regulation of intracellular calcium flux. Our preliminary data shows that SHP2 interacts upstream of calcium flux with the enzyme phospholipase-c- gamma-1 (PLCγ1). Recent literature has described the mechanism of this interaction as we have begun to define the outcomes of disrupting the pairing and downstream calcium flux, providing evidence that uncouples the canonically attributed role of SHP2 in PDAC from the MAPK pathway. Our probing of the metabolic functions of SHP2 inhibited PDAC cells leads us to hypothesize that SHP2 and PLCγ1 interact to sustain calcium signaling required for the activation of mitochondrial enzymes in the citric acid cycle. In this vein, we will take a two pronged approach to investigating SHP2’s role in metabolism: first, by using the Agilent Seahorse Fuel-Flex testing we can pinpoint the metabolic dependencies of SHP2 inhibited cells, and mechanisms of resistance to metabolic stress; second, we will capture the steady state metabolite profile of PDAC cells in response to differential SHP2, MEK, or PLCγ1 activity to take an untargeted approach to defining the metabolic implications of each protein’s function individually and collectively. Additionally, we believe that the outcomes of this interaction are not limited to changes in mitochondrial function. Therefore, we hypothesize that by comparing SHP2 inhibited and MEK inhibited PDAC cells’ global changes to their phospho-proteome, we will be able to delineate the MAPK- independent-functions of SHP2 as an oncogene in PDAC signaling. We expect the outcomes of this proposal to highlight the importance of SHP2 in tumor progression/cancer cell signaling outside of the MAPK pathway, while still appreciating its role in regulating KRAS activity. We believe that the conclusions of this study will yield a new network of protein interactions to be investigated as targets in PDAC, eventually leading to development of therapeutics for improved patient outcome.