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FLI1 Promotes Protein Translation Via the Transcriptional Regulation of MKNK1 Expression

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FLI1 Promotes Protein Translation Via the Transcriptional Regulation of MKNK1 Expression INTERNATIONAL JOURNAL OF ONCOLOGY 56: 430-438, 2020 FLI1 promotes protein translation via the transcriptional regulation of MKNK1 expression CHUNLIN WANG1,2, JIALEI SONG1,2, WULING LIU1,2, YAO YAO1,2, PHILIPP KAPRANOV3, KLARKE M. SAMPLE4, BABU GAJENDRAN1,2, ELDAD ZACKSENHAUS5,6, XIAOJIANG HAO1,2 and YAACOV BEN-DAVID1,2* 1State Key Laboratory for Functions and Applications of Medicinal Plants, Guizhou Medical University; 2The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academic of Sciences, Province Science City, High Tech Zone, Baiyun, Guiyang, Guizhou 550014; 3Institute of Genomics, School of Biomedical Sciences, Huaqiao University, Xiamen, Fujian 361021; 4Central Laboratory, Guizhou Provincial People's Hospital, The Affiliated Hospital of Guizhou University Medical College, Guiyang, Guizhou 550002, P.R. China; 5Department of Medicine, University of Toronto, Toronto, ON M5S 1A8; 6Division of Advanced Diagnostics, Toronto General Research Institute, University Health Network, Toronto, ON M5G 2C4, Canada Received July 25, 2019; Accepted November 18, 2019 DOI: 10.3892/ijo.2019.4943 Abstract. The disruption of protein translation machinery is downregulated or upregulated MKNK1 expression, respec- a common feature of cancer initiation and progression, and tively. Promoter analysis identified a potent FLI1 binding site drugs that target protein translation offer new avenues for in the regulatory region of the MKNK1 promoter. In transient therapy. The translation initiation factor, eukaryotic initia- transfection experiments, FLI1 increased MKNK1 promoter tion factor 4E (eIF4E), is induced in a number of cancer cell activity, which was blocked by mutating the FLI1 binding lines and is one such candidate for therapeutic intervention. site. FLI1 specifically affected the expression of MKNK1, but Friend leukemia integration 1 (FLI1) is a potent oncogenic not that of MKNK2. The siRNA-mediated downregulation of transcription factor that promotes various types of cancer MKNK1 downregulated the expression of survivin (BIRC5) by promoting several hallmarks of cancer progression. FLI1 and significantly suppressed cell proliferation in culture. has recently been implicated in protein translation through FLI1 inhibitory compounds were shown to downregulate yet unknown mechanisms. This study identified a positive this oncogene through the suppression of MAPK/extracel- association between FLI1 expression and mitogen-activated lular-regulated kinase (ERK) signaling and the subsequent protein kinase (MAPK)-interacting serine/threonine kinase1 activation of miR-145, leading to a lower MKNK1 expression (MKNK1), the immediate upstream regulator of the eIF4E and the suppression of leukemic growth. These results uncover initiation factor. The short hairpin RNA (shRNA)-mediated a critical role for FLI1 in the control of protein translation silencing or overexpression of FLI1 in leukemic cell lines and the importance of targeting its function and downstream mediators, such as MKNK1, for cancer therapy. Introduction Correspondence to: Professor Yaacov Ben-David, The Key Laboratory of Chemistry for Natural Products of Guizhou Province The dysregulation of protein synthesis by oncogenes and and Chinese Academic of Sciences, Province Science City, High tumor suppressor genes is a common event in cancer (1) The Tech Zone, Baiyun, Guiyang, Guizhou 550014, P.R. China therapeutic targeting of the translational machinery has thus E-mail: [email protected] become an area of substantive interest for cancer therapy. In this respect, a number of small molecules/compounds have Abbreviations: MAPK, mitogen-activated protein kinase; FLI1, been discovered to target various components of the transla- friend leukemia integration 1, ERK, extracellular signal-regulated tional apparatus (2). kinase; eIF4E, eukaryotic initiation factor 4E; FMR1, fragile X mental retardation protein, MKNK, mitogen-activated protein Mitogen-activated protein kinase (MAPK)-interacting kinase (MAPK)-interacting serine/threonine kinase; ChIP, serine/threonine kinases (MKNKs) 1/2 (MKNK1/MKNK2) chromatin immunoprecipitation control translational initiation through the phosphorylation of eukaryotic initiation factor 4E (eIF4E) (3,4). MKNK1 is Key words: Friend leukemia integration 1, transcriptional regulation, recruited to eIF4E through the binding of eIF4G (5). When mitogen-activated protein kinase-interacting serine/threonine phosphorylated, eIF4E binds to other components within the kinase1, translation initiation, survivin, miR-145, proliferation initiation complex to begin translation (6). Of note, fragile X mental retardation protein (FMR1) has been shown to regu- late the translation of specific mRNAs through its binding WANG et al: FLI1 REGULATES PROTEIN SYNTHESIS IN PART THROUGH MKNK1 431 to eIF4E (7). Higher levels of phosphorylated eIF4E have MigR1 (1 µg) or MigR1-FLI1 (1 µg) were transfected into 293T also been reported to increase the rate of development and cells (ATCC-CRL-3216) using Lipofectamine 2000 (Life progression of various types of cancer (6). Upstream events Technologies; Thermo Fisher Scientific) following the manu- that activate ERK/MAPK and p38 kinases have been shown facturer's protocol. Renilla luciferase was used in transfection to phosphorylate residues Thr209 and Thr214 in MKNK1 and as an internal control to examine the transfection efficiency, Thr244 and Thr249 in MKNK2 (4,8,9). While the phosphory- according to the manufacturer's recommendations (Promega). lation and activation of eIF4E are necessary for oncogenic The transfected cells were then plated 8x103 cells/well into transformation, knockout experiments have revealed that both 96-well plates and luciferase activity was determined, as MKNK1 and MKNK2 are dispensable for normal develop- previously described (21). ment (10). Targeting eIF4E may therefore be an effective The DP17 (1x106) cells were transfected with MigR1-FLI1 strategy for cancer therapy with minimal side-effects (11). (2 µg) and MigR1 (2 µG) vector, using Lipofectamine 2000, Friend virus-induced Leukemia-1 (FLI1) transcription and cells were then selected for neomycin resistance by factor (TF) is a potent oncogene that drives the initiation and growing the cells in medium containing G418 (0.8 mg/ml; progression of leukemia and other types of cancer (12-15). Gibco; Thermo‑Fisher Scientific). During oncogenesis, FLI1 alters a number of hallmarks of cancer, including proliferation, apoptosis, differentiation, Chromatin immunoprecipitation (ChIP) analysis. For ChIP angiogenesis, genomic stability and inflammation (15-17). assay, CB7 cells were washed, crosslinked with formaldehyde, Thus, the therapeutic targeting of FLI1 is an attractive resuspended in lysis buffer Magna Chip G kits (Millipore) and strategy. Accordingly, several classes of compounds with a fixed cells sonicated using the Sonics Vibra VCX150 (Scientz potent anti‑FLI1 activity have recently been identified (18‑22). Biotechnology). At this stage, a portion of chromatin aliquot Among these, two flavagline‑like compounds exhibiting potent was removed for input control. To the isolated chromatin protein FLI1 inhibition were also identified (22). These compounds G sepharose beads was added followed by incubation for 1 h suppress c-Raf-MEK-MAPK/ERK signaling, resulting in the at room temperature. Immunoprecipitations were performed reduced phosphorylation of eIF4E and the inhibition of FLI1 overnight at 4̊C with 1 µg of FLI1 antibody (Cat. no. ab15289, protein synthesis. Abcam) or non-specific normal rabbit immunoglobulin G The present study, at least to the best of our knowledge, (IgG; Cat. no. 2729, Cell Signaling Technology) antibodies. demonstrates for the first time that FLI1 is capable of binding Precipitates were then washed and reverse crosslinked, to the promoter of MKNK1, directly activating MKNK1 using the instructions provided with the Magna Chip G kits transcription and subsequently, the phosphorylation of eIF4E. (Millipore). Precipitated chromatin was then incubated with Thus, these results highlight the use of FLI1 inhibitors for the proteinase K at 50̊C for 2 h, DNA‑purified with phenol chlo- suppression of protein translation for the treatment of cancers roform extraction and resuspended in TE buffer. RT-qPCR overexpressing this TF. was performed to amplify the MKNK1 promoter regions containing FLI1 binding site 1 (position -482 to -205) and for Materials and methods negative ChI control (position -730 to -453). The sequences of the ChIP primers are presented in Table SII. The percentage of Cell lines and drug treatment. Mycoplasma negative erythro- input was calculated by RT-qPCR based upon the intensity of leukemia cell lines (human HEL [ATCC-TIB-180] and K562 the amplifiedFLI1 DNA divided by the amplified input DNA. [ATCC-CCL-243]; mouse KH16, CB7 and DP17 (generated by Amplified DNA was also resolved on a 2% agarose gel and the authors and others) (13,18) were cultured and maintained illustrated in Fig. 3E (right panel). in Dulbecco's Modified Eagle Medium supplemented with 5% fetal bovine serum (HyClone, GE Healthcare). RNA preparations and RT‑qPCR. Total RNA was extracted For drug treatment, the HEL cells were treated with 3 µM of from the growing culture of HEL cells using TRIzol reagent A1544, A1545 (22) and CGP57380 and 24 h later subjected to (Life Technologies; Thermo Fisher Scientific) according to either western blot analysis or RT-qPCR. The drug CGP57380 the manufacturer's protocol. A NanoDrop 2000 spectropho-
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