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

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。基因表达的改变 去包裹效率可以从根本上改变mRNA的稳定性，从而改变癌症中特定驱动蛋白的翻译 细胞。MRNAs的解帽和衰变发生在处理体或P-体(PBS)中，它们是膜- 含有5‘-3’信使核糖核酸衰变机制的细胞质组件较少。EDC3蛋白，其增强子 信使核糖核酸解捕获蛋白3在信使核糖核酸的降解过程中起着关键作用。EDC3还卷入了PB Assem- 布莱。重要的是，几乎没有人了解EDC3在癌症中的功能是如何调节的。我们的预赛 结果表明，作为基本癌症驱动因素的Pim和AKT蛋白激酶具有磷酸化作用 丝氨酸161上的EDC3，阻止EDC3进入PBS。EDC3磷酸化在肿瘤中高度升高 细胞和癌组织与正常对应物相比。三阴性乳腺癌和乳腺癌的治疗 具有Pim/AKT小分子抑制剂的前列腺癌耐药细胞目前正在进行临床试验 阻断EDC3的磷酸化，并诱导P小体形成增加10倍。使用一种新的方法 为了分离PBS，我们的结果确定了PB mRNA含量的显著变化，包括关键的癌症调节 MRNAs。最重要的是，Pim和AKT靶向的EDC3磷酸化位点突变为丙氨酸 显著抑制前列腺癌的生长和迁移。根据这些结果，我们假设EDC3PHO- 癌基因蛋白激酶的磷酸化通过限制靶向、抑制和衰退来促进肿瘤的生长 在PBS中抑制致癌的mRNAs；抑制Pim和AKT激酶抵消这种作用，从而抑制癌细胞 成长。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 EDC3，一个关键的信使核糖核酸衰退蛋白。更重要的是，他们建议靶向PBS和mRNA衰变蛋白 像EDC3一样，它代表了一种控制肿瘤生长的全新治疗方法。这一理论将被研究-- 门控有两个特定的目标-1：检查EDC3磷酸化对mRNA衰退的影响。确定i) EDC3-S161的磷酸化如何影响其在调节肿瘤生长的mRNA衰退中的功能，以及ii)是否 肿瘤生长因子改变了PB的mRNA含量，并且这些变化是由EDC3磷酸化控制的。2： 检查EDC3磷酸化是否通过以下方式影响肿瘤生长：i)确定EDC3磷酸化是否- LATA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激酶水平是否调节PB的形成，这是一个信号 肿瘤对这些药物的反应。在这些实验中使用的独特试剂包括人类乳房 具有不同水平的Pim和AKT的PDX，以及EDC3基因敲除和EDC3基因敲除的肿瘤细胞 磷酸盐突变体。从肿瘤细胞中分离PBS并测量肿瘤细胞侵袭力的新技术 将使用动物模型。加深对致癌蛋白激酶如何控制PB的理解 信息、EDC3功能和信使核糖核酸解离将使抑制肿瘤生长的新方法成为可能。

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.