Targeting understudied kinases in cancer cell plasticity and drug resistance

针对癌细胞可塑性和耐药性中尚未研究的激酶

• 批准号: 10045760
• 负责人: Martin Golkowski
• 金额: $ 15.55万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-08 至 2022-09-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10045760
• 关键词: Antineoplastic Agents; BAY 54-9085; Biological Assay; CDK10 gene; CRISPR/Cas technology; Cell Survival; Cells; Cessation of life; Clinical Trials; Data; Development; Disease; Disseminated Malignant Neoplasm; Dose; Drug Screening; Drug Targeting; Drug resistance; Ensure; Enzymes; Epithelial; Epithelial Cells; Epithelium; Follow-Up Studies; Genes; Goals; Knock-out; Malignant Neoplasms; Measures; Mesenchymal; Mesenchymal Stem Cells; Messenger RNA; NUAK1 gene; Neoplasm Metastasis; Pathway interactions; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Primary carcinoma of the liver cells; Protein Kinase; Proteins; Reagent; Role; Testing; Transcript; Western Blotting; cancer cell; cell motility; chemotherapy; clinically relevant; drug development; drug discovery; drug-sensitive; established cell line; experimental study; hepatocellular carcinoma cell line; kinase inhibitor; knock-down; new therapeutic target; novel anticancer drug; overexpression; protein biomarkers; protein expression; response; stable cell line; stem-like cell

Project Summary Metastasis is responsible for 90% of cancer-related deaths and novel drugs that target this disease stage are urgently needed. Metastatic cancer cells exploit developmental pathways to acquire a mesenchymal stem cell-like state. This epithelial-mesenchymal transition (EMT) enhances cancer cell motility, invasion and survival. Inhibition of EMT, in turn, can block metastasis and, strikingly, can sensitize cancer cells to chemotherapy. A critical goal of cancer drug discovery, therefore, is to identify druggable proteins that promote the EMT. In a detailed pharmacoproteomics study of hepatocellular carcinoma (HCC), we demonstrated that protein kinases are critical drivers of EMT and drug resistance. Remarkably, our study predicted 71 kinases to promote EMT, among them six understudied IDG targets. We will evaluate the translational potential of these six IDG kinases, i.e. NUAK2, CAMK1D, STK17A, STK17B, STK32B and CDK10, by scrutinizing their function in EMT and drug resistance. We will use targeted gene disruption with CRISPR/Cas9 to knock-out (KO) these kinases in mesenchymal HCC cell lines and ectopically express them in epithelial HCC lines. Impact on EMT state and drug resistance will be tested (1) by qPCR and western blot analysis of 15 reliable EMT markers, (2) in a robust trans-well invasion assay, and (3) a small-scale drug screen. Combining efficient KO with these orthogonal assays will ensure unambiguous specification of kinase roles in HCC cell EMT and drug response.

项目摘要 90%的癌症相关死亡与转移有关，针对这一点的新药也是如此 疾病阶段是迫切需要的。转移性癌细胞利用发育途径 获得间充质干细胞样状态。这种上皮-间充质转化(EMT) 增强癌细胞的运动性、侵袭性和存活率。抑制EMT，反过来又可以阻断 而且，引人注目的是，它可以使癌细胞对化疗敏感。癌症的关键目标 因此，药物发现就是识别促进EMT的可用药蛋白质。在一个详细的 肝细胞癌的药物蛋白质组学研究，我们证明了该蛋白 激酶是EMT和耐药的关键驱动因素。值得注意的是，我们的研究预测了71 其中6个未被研究的IDG靶点。我们将评估 这六种IDG激酶即NUAK2、CAMK1D、STK17A、STK17B、 STK32B和CDK10在EMT和耐药中的作用。我们将使用 用CRISPR/Cas9靶向基因干扰敲除(KO)间充质细胞中的这些激酶 并在上皮性肝癌细胞系中异位表达。对EMT状态和 耐药性检测：(1)对15例可靠的EMT进行定量聚合酶链式反应和蛋白质印迹分析。 标记物，(2)强大的跨井侵袭试验，和(3)小规模药物筛选。 将有效的KO与这些正交分析相结合将确保明确的规格 激酶在肝癌细胞EMT和药物反应中的作用。