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

研究 SHP2-PLCG1 相互作用在 PDAC 钙信号传导和代谢中的作用

• 批准号: 10752562
• 负责人: Richard McKinnon Walsh
• 金额: $ 3.55万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752562
• 关键词: Ablation; Address; Affect; Aggressive behavior; Binding; Bioenergetics; Calcium; Calcium Signaling; Cancer Etiology; Catabolism; Cell Line; Cell Survival; Cells; Cessation of life; Citric Acid Cycle; Consumption; Data; Data Analyses; Dependence; Development; Disease; Disease Progression; Endoplasmic Reticulum; Enzymes; Genetic; Genus Hippocampus; Global Change; Glycolysis; Goals; Impairment; In Vitro; Individual; Ions; KRAS2 gene; Knock-out; Literature; MAP Kinase Gene; MEK inhibition; MEKs; Mediating; Metabolic; Metabolic stress; Metabolism; Methods; Mitochondria; Modeling; Molecular; Mus; Mutate; Mutation; Nodal; Oncogenes; Oncogenic; Outcome; Oxidoreductase; PLC gamma1; PTPN11 gene; Pancreatic Ductal Adenocarcinoma; Pathway Analysis; Pathway interactions; Patient-Focused Outcomes; Patients; Phenotype; Positioning Attribute; Process; Production; Proliferating; Proteins; Proteome; Regulation; Resistance; Role; Sampling; Signal Pathway; Signal Transduction; Source; Stress Tests; System; Testing; Tissues; Traction; Tumor Promotion; Tumorigenicity; Veins; calcium metabolism; cancer cell; cell type; design; enzyme activity; extracellular; genome-wide; improved; improved outcome; inhibitor; insight; interest; liquid chromatography mass spectrometry; mass spectrometer; migration; mitochondrial membrane; mouse model; mutant; novel; pancreas development; pancreatic cancer cells; pancreatic ductal adenocarcinoma cell; pharmacologic; phosphoproteomics; prevent; release of sequestered calcium ion into cytoplasm; resistance mechanism; response; targeted treatment; therapeutic development; tumor; tumor growth; tumor progression; tumorigenic; vector

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.

摘要 胰腺导管腺癌(PDAC)是一种高度致命性疾病，有望成为世界第二大恶性肿瘤。 到2025年，美国癌症相关死亡的主要原因。在这些案件中，90%以上 患者携带KRAS突变。不幸的是，针对PDAC的突变的直接靶向已经失败 以获得牵引力，但由于突变的KRAS对疾病无处不在，替代方法来减少下游 对MAPK信号转导进行了研究。最近，针对KRAS活性的上游调控因子SHP2 已经走到了这一领域的前列。Shp2在小鼠模型中对PDAC的发展是必不可少的， 尽管有突变的KRAS存在。然而，尽管SHP2在PDAC中的作用在很大程度上归因于其 对MAPK通路的调节，许多其他组织类型的证据表明SHP2应该起作用 通过其他机制促进肿瘤进展--最显著的是通过调节细胞内钙流量。 我们的初步数据显示，SHP2与磷脂酶-C-2相互作用于钙离子通量的上游。 Gamma-1(可编程控制器γ1)。最近的文献描述了这种相互作用的机制，正如我们已经开始定义的那样 干扰配对和下游钙流动的结果，提供了解偶联钙离子的证据 SHP2通过MAPK通路在PDAC中的典型作用。我们对新陈代谢功能的探讨 Shp2抑制的pDAC细胞导致我们假设shp2和plcγ1相互作用维持钙信号 在柠檬酸循环中激活线粒体酶所必需的。在这方面，我们将采取双管齐下的做法 研究SHP2基因S在新陈代谢中作用的方法：首先，采用安捷伦海马燃料弹性试验法 可以确定SHP2抑制细胞的代谢依赖性，以及代谢抵抗的机制 其次，我们将捕捉PDAC细胞对不同SHP_2的稳态代谢物图谱， MEK，或PLCγ1活性，采取无针对性的方法来定义每种蛋白质的代谢影响 单独和共同发挥作用。此外，我们认为，这种互动的结果是不受限制的。 线粒体功能的变化。因此，我们假设通过比较SHP2抑制和MEK 抑制PDAC细胞对其磷酸化蛋白质组的全局改变，我们将能够描绘MAPK- SHP2作为癌基因在PDAC信号转导中的独立功能。我们预计这项提议的结果将是 强调SHP2在肿瘤进展/MAPK通路外的癌细胞信号传递中的重要性，同时 仍然赞赏其在调节KRAS活动方面的作用。我们相信，这项研究的结论将产生一个新的 蛋白质相互作用网络将作为PDAC的目标进行研究，最终导致开发 改善患者预后的治疗方法。