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 转移造成90％的癌症相关死亡和针对此目标的新药物 迫切需要疾病阶段。转移性癌细胞利用发育途径 获得间充质干细胞状状态。这个上皮间质转变（EMT） 增强癌细胞运动，侵袭和生存。抑制EMT，反过来可以阻止 转移，令人惊讶的是，可以使癌细胞对化学疗法敏感。癌症的关键目标 因此，药物发现是鉴定促进EMT的可药物蛋白。详细 肝细胞癌（HCC）的药物蛋白质组学研究，我们证明了蛋白质 激酶是EMT和耐药性的关键驱动因素。值得注意的是，我们的研究预测了71 促进EMT的激酶，其中六个研究了IDG靶标。我们将评估 这六种IDG激酶的翻译潜力，即NUAK2，CAMK1D，STK17A，STK17B， STK32B和CDK10，通过仔细检查其在EMT和耐药性中的功能。我们将使用 用CRISPR/CAS9的靶向基因破坏以淘汰（KO）在间充质中的这些激酶 HCC细胞系，并异位地在上皮HCC系中表达它们。对EMT国家和 耐药性将通过QPCR测试（1），并对15个可靠EMT进行蛋白质印迹分析 标记，（2）在强大的透明入侵测定中，以及（3）小规模的药物筛查。 将有效的KO与这些正交分析相结合将确保明确的规范 激酶在HCC细胞EMT和药物反应中的作用。