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

• 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
• 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

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剂。 mRNA的改变 脱皮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因素的蛋白质 细胞。 mRNA的脱酸和衰减发生在加工机构或p-bodies（PBS）中，这是膜 - 具有5'-3'mRNA衰变机械的细胞质组件较少。 EDC3蛋白，增强子 mRNA取代蛋白3在mRNA衰减中起着至关重要的作用。 EDC3在PB组件中也已浸渍 布莱。重要的是，几乎不了解在癌症中如何调节EDC3功能。我们的初步 结果表明，PIM和AKT蛋白激酶充当必需的癌症驱动因素 丝氨酸161上的EDC3，可防止EDC3进入PBS。 EDC3磷酸化在肿瘤中高度升高 细胞和癌症组织与正常的癌症组织相比。治疗三重阴性乳腺癌和cas- 当前在人类临床试验中的PIM/AKT的小分子抑制剂的抗性前列腺癌细胞 阻断EDC3的磷酸化并诱导P体形成10倍。使用新方法 为了分离PBS，我们的结果确定了PB mRNA含量（包括关键癌症调节）的重大变化 mrnas。最重要的是，PIM和AKT靶向的EDC3磷酸化位点的突变 明显抑制了前列腺肿瘤的生长和迁移。基于这些结果，我们假设EDC3 Phos- 致癌蛋白激酶的卵巢化通过限制靶向，表达和衰减来促进肿瘤的生长 PBS中的致癌mRNA；抑制PIM和Akt激酶会抵消这种抑制癌细胞 生长。这些结果很重要，因为它们揭示了对翻译后调节的新见解 EDC3，一种关键的mRNA衰减蛋白。更重要的是，他们建议靶向PBS和mRNA衰减蛋白 像EDC3一样，代表了一种完全新颖的热方法来控制肿瘤生长。该理论将是投资 带有两个特定的目标 - 1：检查EDC3磷酸化对mRNA衰变的影响。确定i） EDC3-S161磷酸化如何影响其在mRNA衰减中调节肿瘤生长的功能，以及ii）是否） 肿瘤生长因子改变了PB mRNA含量，并且这些变化是由EDC3磷酸化控制的。 2： 检查EDC3磷酸化是否通过i）确定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，并使用PDX小鼠 乳腺癌的模型，确定PIM和Akt激酶的水平是否调节PB形成，一个信号 肿瘤对这些药物的反应。这些实验中要使用的独特试剂包括人乳房 具有不同水平的PIM和AKT的PDX，EDC3敲除和EDC3敲门的肿瘤细胞 磷酸突。从肿瘤细胞中分离PBS的新技术，并测量肿瘤细胞中的侵袭 将使用动物模型。对致癌蛋白激酶如何控制Pb的了解 Mation，EDC3功能和mRNA脱甲将实现抑制肿瘤生长的新方法。