Regulation of P-Body Formation and mRNA Decapping in Cancer: Control by AKT and Pim Kinase Phosphorylation of EDC3

癌症中 P-Body 形成和 mRNA 脱帽的调控：AKT 和 Pim 激酶 EDC3 磷酸化的控制

基本信息

批准号：
10041072
负责人：
Koichi Okumura
金额：
$ 39.47万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10041072
关键词：
Affect Alanine Animal Model Antineoplastic Agents Aspartic Acid Breast Breast Cancer Cell Breast Cancer Model Breast Cancer Patient CRISPR/Cas technology Cancer Cell Growth Cancer Control Cell Culture Techniques Cell Line Cellular Stress Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Complex Consensus Data Enhancers Equilibrium Gene Expression Genes Genetic Transcription Goals Growth Human Hypoxia Knock-in Knock-out Malignant Neoplasms Mammary Neoplasms Measures Membrane Messenger RNA Methods Mutate Mutation Neoplasm Metastasis Oncogenes Oncogenic Organelles PC3 cell line Patient-derived xenograft models of breast cancer Patients Pharmaceutical Preparations Phenotype Phospho-Specific Antibodies Phosphorylation Phosphorylation Site Phosphotransferases Play Post-Translational Protein Processing Post-Translational Regulation Process Production Prostatic Neoplasms Protein Kinase Proteins Proto-Oncogene Proteins c-akt Reagent Regulation Repression Research Role Serine Serine Phosphorylation Site Signal Transduction Stress Techniques Tissues Transforming Growth Factors Translations Tumor Cell Invasion Tumor Cell Line Work Xenograft procedure anti-cancer base cancer cell castration resistant prostate cancer cell growth chemotherapy experimental study human tissue in vivo inhibitor/antagonist insight mRNA Decay mRNA Stability mRNA decapping malignant breast neoplasm migration mouse model mutant neoplastic cell new therapeutic target novel novel strategies novel therapeutic intervention nutrient deprivation prevent prostate cancer cell proto-oncogene protein pim response small molecule inhibitor spleen exonuclease stem cells theories triple-negative invasive breast carcinoma tumor tumor growth tumor progression tumorigenesis tumorigenic

项目摘要

PROJECT ABSTRACT Aberrant regulation of mRNA stability is a potential key regulator of cancer progression. Alterations in mRNA decapping efficiency can radically alter mRNA stability and thus the translation of specific driver proteins in cancer cells. Decapping and decay of mRNAs occurs in Processing bodies or P-bodies (PBs), which are membrane- less cytoplasmic assemblies that harbor the 5’-3’ mRNA decay machinery. The EDC3 protein, Enhancer of mRNA-DeCapping protein 3, plays a critical role in mRNA decay. EDC3 has also been implicated in PB assem- bly. Importantly, there is almost no understanding of how EDC3 function is regulated in cancer. Our preliminary results demonstrate that Pim and AKT protein kinases, which function as essential cancer drivers phosphorylate EDC3 on serine 161, which prevents EDC3 entry into PBs. EDC3 phosphorylation is highly elevated in tumor cells and cancer tissues compared to normal counterparts. Treatment of triple negative breast cancer and cas- trate resistant prostate cancer cells with small molecule inhibitors of Pim/AKT currently in human clinical trials blocks the phosphorylation of EDC3 and induces a 10-fold increase in P-body formation. Using a novel method to isolate PBs, our results identify significant changes in PB mRNA content including key cancer-regulatory mRNAs. Most importantly, mutation of the EDC3 phosphorylation site targeted by Pim and AKT to an alanine markedly inhibits prostate tumor growth and migration. Based on these results we hypothesize that EDC3 phos- phorylation by oncogenic protein kinases facilitates tumor growth by limiting the targeting, repression and decay of oncogenic mRNAs in PBs; inhibition of Pim and AKT kinases counteracts this thus inhibiting cancer cell growth. These results are significant because they reveal a new insight into post-translational regulation by EDC3, a key mRNA decay protein. More importantly they suggest that targeting PBs and mRNA decay proteins like EDC3 represent an entirely novel therapeutic approach to control tumor growth. This theory will be investi- gated with two Specific Aims- 1: Examine the effect of EDC3 phosphorylation on mRNA decay. Determine i) how EDC3-S161 phosphorylation affect its function in mRNA decay to regulate tumor growth, and ii) whether tumor growth factors change PB mRNA content, and are these changes controlled by EDC3 phosphorylation. 2: Examine whether EDC3 phosphorylation influences tumor growth by i) determining whether EDC3 phosphory- lation regulates the invasion, metastasis, and stem cell phenotype of breast cancer and ii) using PDX mouse models of breast cancer, determine whether the levels of Pim and AKT kinases regulate PB formation, a signal of the tumor’s response to these agents. Unique reagents to be used in these experiments include human breast PDXs with varying levels of Pim and AKT, and tumor cells that are knock-out for EDC3 and knock-in for EDC3 phospho-mutants. Novel techniques to isolate PBs from tumor cells, and measure invasion of tumor cells in animal models will be used. Developing an understanding of how oncogenic protein kinases control PB for- mation, EDC3 function, and mRNA decapping will enable novel approaches to inhibiting tumor growth.

项目摘要 mRNA 稳定性的异常调节是癌症进展的潜在关键调节因子。脱帽效率可以从根本上改变 mRNA 稳定性，从而改变癌症中特定驱动蛋白的翻译 mRNA 的脱帽和衰变发生在加工体或 P 体 (PB) 中，它们是膜-体。含有 5'-3' mRNA 衰变机制的细胞质组件较少。 EDC3 蛋白，增强子。 mRNA-DeCapping 蛋白 3 在 mRNA 降解中发挥着关键作用，EDC3 也与 PB 组装有关。重要的是，我们对 EDC3 功能在癌症中的调控方式几乎一无所知。结果表明，作为重要癌症驱动因素的 Pim 和 AKT 蛋白激酶可磷酸化 EDC3 丝氨酸 161 可以阻止 EDC3 进入 PB，EDC3 磷酸化在肿瘤中高度升高。与正常省份相比，三阴性乳腺癌和癌组织的细胞和癌组织的治疗。使用 Pim/AKT 小分子抑制剂治疗耐药性前列腺癌细胞目前正在进行人体临床试验使用一种新方法阻断 EDC3 的磷酸化并诱导 P-body 形成增加 10 倍。为了分离 PB，我们的结果确定了 PB mRNA 含量的显着变化，包括关键的癌症调节最重要的是，Pim 和 AKT 靶向的 EDC3 磷酸化位点突变为丙氨酸。显着抑制前列腺肿瘤的生长和迁移基于这些结果，我们发现 EDC3 磷酸化。致癌蛋白激酶的磷酸化通过限制靶向、抑制和衰变促进肿瘤生长 PB 中致癌 mRNA 的表达；抑制 Pim 和 AKT 激酶可抵消这一作用，从而抑制癌细胞这些结果意义重大，因为它们揭示了对翻译后调控的新见解。 EDC3，一种关键的 mRNA 衰减蛋白，更重要的是，他们建议靶向 PB 和 mRNA 衰减蛋白。像 EDC3 代表了一种控制肿瘤生长的全新治疗方法。门控有两个具体目标 - 1：检查 EDC3 磷酸化对 mRNA 衰减的影响，确定 i)。 EDC3-S161 磷酸化如何影响其在 mRNA 衰变中调节肿瘤生长的功能，以及 ii) 是否肿瘤生长因子改变 PB mRNA 含量，这些变化是由 EDC3 磷酸化控制的 2：通过 i) 确定 EDC3 磷酸化是否影响肿瘤生长，检查 EDC3 磷酸化是否影响肿瘤生长。调节乳腺癌的侵袭、转移和干细胞表型，ii) 使用 PDX 小鼠乳腺癌模型，确定 Pim 和 AKT 激酶的水平是否调节 PB 形成（信号）这些实验中使用的独特试剂包括人类乳房。具有不同水平 Pim 和 AKT 的 PDX，以及 EDC3 敲除和 EDC3 敲入的肿瘤细胞从肿瘤细胞中分离 PB 并测量肿瘤细胞侵袭的新技术。将使用动物模型来了解致癌蛋白激酶如何控制 PB- 化、EDC3 功能和 mRNA 脱帽将使抑制肿瘤生长的新方法成为可能。