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

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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.