Investigation Of EIF4E Phosphorylation In Translation And Potent Killing Of Colon Cancer Cells By Co-targeting Translation And Proteasome

BackgroundColorectal cancer(CRC)is a major cause of cancer mortality.Despite decades of cancer research,the survival rates for patients with colon cancer have improved only modestly.Many tumors are unresponsive to conventional therapy due to the resistance of tumor cells to apoptosis,or programmed cell death.Novel strategies for treatment are needed urgently.Translation initiation is an evolutionarily conserved cellular process that controls cell growth and metabolism in response to nutrients,growth factors,and cellular energy levels.Multiple oncogenic signaling pathways are activated in cancer cells to maintain its rapid growth and proliferation.All of these molecular pathways converge on m RNA translation.By being positioned as the nexus of translation initiation regulation,e IF4 E mRNA and protein levels are upregulated in various kinds of cancer cells.Besides transcriptional and translational regulation,eIF4 E is also regulated by post-translational modification.For example,only phosphorylated e IF4 E can induce tumorigenesis in some tumor models.On the basis of these research,we hypothesize that eIF4 E S209 phosphorylation may play an important role in colorectal tumorigenesis.In order to test this hypothesis,we will construct eIF4ES209 A KI HCT116 colon cancer cells and compare the colorectal tumorigenesis and cell metabolism between eIF4 EKI and WT(wild-type)HCT116 cells.We further investigate the regulation mechanism by using polysome experiments to test the molecular changes in the two kinds of cell types.So that we will clarify the role and mechanism of e IF4 E S209 phosphorylation in colorectal cancer,which will help develop more effective strategies for colon cancer treatment by targeting mRNA translation.Because numerous oncogenes and tumor suppressors control the activation of translation initiation,deregulation of this process is frequently observed in cancers and other diseases.Therefore,targeting the translation initiation became an attractive therapeutic approach.Preclinical and clinical studies have shown that the eIF4 A inhibi tor Episilvestrol and its analog Silvestrol are cytotoxic efficiently in multiple tumor types.However,one major pitfall with the translation-targeted therapeutics is that translation also participates in normal cellular physiology,and high doses of these compounds may result severe side effects.Thus,combination therapy with Episilvestrol and other drugs may be considered as an option for clinical treatment of colorectal cancer.Bortezomib is commonly used as a potentiating potent,we find that Bortezomin also synergizes with Episilvestrol to induce remarkable apoptosis in colon cancer cells.Objective:This study aims to investigate the role of e IF4 E phosphorylationin translation and targeting translation synergize with proteasome inhibitor to induce apoptosis in colon cancer cells.Methods and Materials:For the first part,firstly,we constructed eIF4 E phosphorylation KI cells and investigated its effects on mRNA translation.Secondly,we observed the phenomena of e IF4 E phosphorylation promoting colon cancer cell growth and glutamine addiction.Thirdly,mRNA CHIP and polysome experiments were performed to identify the molecular changes between WT and KI cells.Moreover,theidentified effects were verified in xenografts.For the second part,firstly,MTS,crystal violet,fragmented nuclear and colony formation assay were used to identify the effects of Episilvestrol inhibiting cell growth and inducing apoptosis in HCT116 cells.Secondly,the synergizing effects were verified by MTS,crystal violet,fragmented nuclear and colony formation assay.Thirdly,Western blot and RT-PCR were performed to identify the ER stress and CHOP-mediated DR5 induction.Subsequently,BAX,BAK,FADD knockout cell lines were employed to identify the possible apoptosis pathway leading to the cell death.Furthermore,several other colon cancer cell lines were tested for the synergy effects.Moreover,the identified combination effects were verified in xenografts.Results:In this study,we generate a HCT116 colon cancer cell model in which eIF4 E serine 209 was mutated to alanine as a genetic tool to define eIF4 E phosphorylation requirements in colon cancer cells.We show that eIF4 E phosphorylation promotes protein translation in colon cancers.Then we demonstrate that e IF4 E phosphorylation promotes colon cancer cell growth and cell metabolism.We further verified that eIF4 E phosphorylation and total eIF4 E are responsible for different groups of mRNA translation.Next,we found that only high concentration of Episilvestrol activates apoptosis in colon cancer cells.However,low concentration of Episilvestrol synergizes with Bortezomib to induce apoptosis in colon cancer cells.The mechanism of Episilvestrol strongly synergizing with Bortezomibwas they induce ER stress and extrinsic apo ptosis.And FADD-dependent apoptosis was required for the anti-tumor activities of Episilvestrol and Bortezomib combination.Morever,these effects were observed in several other colon cancer cell lines and xenografts.Conclusions:Collectively,these findings highlight that colon cancer cells have took full advantage use of eIF4 E phosphorylation that exists above the required threshold for normal development and physiology,which has been co-opted by tumor cells to weather the nutrient stress during their survival.And also provides a preclinical rationale for the use of combination therapies for CRC patients,suggesting that it may be a novel strategy by using Episilvestrol and Bortezomib for the killing of CRC cells.