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. 2014 Jan 30;5(2):530-43.
doi: 10.18632/oncotarget.1528.

First MNKs degrading agents block phosphorylation of eIF4E, induce apoptosis, inhibit cell growth, migration and invasion in triple negative and Her2-overexpressing breast cancer cell lines

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First MNKs degrading agents block phosphorylation of eIF4E, induce apoptosis, inhibit cell growth, migration and invasion in triple negative and Her2-overexpressing breast cancer cell lines

Senthilmurugan Ramalingam et al. Oncotarget. .

Abstract

Some retinoic acid metabolism blocking agents (RAMBAs) are known to exhibit a wide range of anticancer activities by mechanisms that are still not completely resolved. This study investigated the anticancer efficacy and mechanism(s) of novel RAMBA retinamides (RRs) in triple negative and Her-2 overexpressing breast cancer cells. Specifically, we examined the possibility that RRs affect the translational machinery in these breast cancer (BC) cells. Recent findings suggest that overexpression of eukaryotic translation initiation factor 4E (eIF4E) in breast cancers critically augments CAP-dependent mRNA translation and synthesis of proteins involved in cell growth, cell proliferation, invasion and apoptosis evasion. The oncogenic potential of eIF4E is strictly dependent on serine209 phosphorylation by upstream MAPK-interacting kinases (Mnks). Targeting Mnk/eIF4E pathway for blocking Mnk function and eIF4E phosphorylation is therefore a novel approach for treating BCs, particularly for Her2-positive and triple negative breast cancers that have no indications for endocrine therapy or effective treatment regimes. We report for the first time that the degradation of Mnk1 by RRs in BC cells blocks eIF4E phosphorylation and subsequently inhibits cell growth, colonization, invasion, and migration and induce apoptosis. Most importantly, the anticancer efficacy of RRs was mediated via degrading Mnk rather than inhibiting its kinase activity like Mnk inhibitors (cercosporamide and CGP57380). Furthermore, RRs potencies on peIF4E down-regulation and growth inhibition were superior to those of two clinically relevant retinoids and the Mnk inhibitors. Together our findings provide the first preclinical proof-of-concept of novel Mnk degrading agents for Mnk/eIF4E based therapeutic treatment of breast cancers.

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Figures

Figure 1
Figure 1. Effect of VNLG-152 on anchorage-dependent growth and cell cycle distribution
MDA-MB-231 (A) and MDA-MB-468 (B) cells were treated with VNLG-152, ATRA, 4-HPR, VN/14-1 and Mnk inhibitor (10 μmol/L) for 14 days. Colonies were fixed with methanol and stained with crystal violet. (C) Data represents the mean ± S.E from three independent experiments. *, P< 0.01; ♣, P<0.001 compared with vehicle treated control. (D) MDA-MB-231 and MDA-MB-468 cells were treated with indicated compound 15 μmol/L for 24 h. Total cell lysates were separated by SDS-PAGE and probed with cyclin D1 and D3 antibodies. (E) MDA-MB-231 cells were treated with VNLG-152 (2 μmol/L) for indicated time period, stained with PI and analysed with a FACScalibur flow cytometer. (F) Western blotting for cell cycle related proteins. Vehicle treated cells were included as a control and all blots were reprobed for β-actin for equal protein loading and transfer.
Figure 2
Figure 2. RRs induces apoptosis in TNBC cells
(A) The individual cell lines as indicated were seeded in 24 well plate, and treated with 10 μmol/L of VNLG-152 the next day. After 72 h the plates were analyzed for early apoptotic and viable cells using acridine orange/ethidium bromide staining. (B & C) Indicated cells were treated with VNLG-147, -152, -153 ATRA, 4-HPR and VN/14-1 (10 μmol/L) for 72 h and apoptosis induction was examined by oligonucleosomal fragmentation. Data are shown relative to vehicle treated control and the bars are means of three replicate determinations plus standard deviations. *, P< 0.05; ♣, P<0.01 compared with vehicle treated control. (D) Apoptosis induction in TNBC cells by VNLG-152 (1–10 μmol/L) was assessed by oligonucleosomal fragmentation after a 72 h incubation. (E) Apoptosis induction in triple negative breast cancer cells by VNLG-152 (10 μmol/L) was evaluated with or without the caspase inhibitor ZVAD (5 μmol/L). Data are shown relative to vehicle treated control and the bars are means of three replicate determinations plus standard deviations. *, P< 0.01, control versus VNLG-152; ♣, P<.01, ZVAD versus ZVAD plus VNLG-152. (F) Expression of PARP and cleaved PARP was investigated after treatment with VNLG-152 (10 μmol/L) and with or without the caspase inhibitor ZVAD (5 μmol/L) by western blot.
Figure 3
Figure 3. VNLG-152 inhibits migratory and invasive potential of TNBC cells
(A & B) Effect of the treatment of VNLG-152 (2.5 μmol/L) and indicated compounds (10 μmol/L) on breast cancer cell migration by wound healing assay for 24 h. (C) Cells were seeded on matrigel coated boyden chamber and treated with VNLG-152 (2 μmol/L) and indicated compounds (10 μmol/L). Dose selection was based on dose-dependent studies. Representative photomicrographs of initial and final wounds and membrane invasion are shown at 100x magnification. (D) Quantification of the number of invaded cells and the data are shown relative to vehicle treated control and the bars are means of three replicate determinations plus standard deviations. *, P< 0.01; ♣, P<0.001 compared with vehicle treated control.
Figure 4
Figure 4. RRs reduce Mnk expression and eIF4E phosphorylation in breast cancer cells
Equal protein concentrations from MDA-MB-231 (A) and MDA-MB-468 (B) and SKBR-3 (C) cells treated for 24 h with RRs, CGP57380 and cercosporamide (15 μmol/L) were separated by SDS-PAGE and western blots probed with antibodies to Mnk1 and serine 209 phosphorylated eIF4E. Companion blots were probed for total eIF4E. Dose response analysis for the inhibition of Mnkl and 2, peIF4E and eIF4E protein by RRs on MDA-MB-231 (D, F) and MDA-MB-468 (E, F) cells treated with VNLG-152 at indicated concentration and CGP 57380 or cercosporamide (15 μmol/L) for 24 h. Vehicle treated cells were included as a control and all blots were reprobed for β-actin for loading control.
Figure 5
Figure 5. VNLG-152 induced degradation/polyubiquitination of Mnk protein and schematic model of MNK mediated eIF4E activation and inhibition by RAMBA retinamides (VNLG-152)
MDA-MB-231 (A) and MDA-MB-468 (B) cells were treated with 20 μmol/L of VNLG-152, 5 μmol/L of MG-132, and combinations for 24 h. Mnk protein was immunoprecipitated with Mnk antibody (mouse) and the precipitated protein was subjected to western blot analysis with anti-ubiquitin antibody (Ub) (C, upper panel). The same blot was used to detect Mnk protein with anti-Mnk (rabbit) antibody after stripping (C, lower panel). Knockdown of Mnk1 by transfection with siRNA against (Mnk1) or its scramble control blocks eIF4E phosphorylation in MDA-MB-231 (A) and MDA-MB-468 (B) cells. All blots were reprobed with β-actin for equal protein loading and transfer. The data are representatives of two independent experiments. D: Activation of Ras/Erk pathway or p38 MAPK downstream of cytokine or stress stimuli results in activation of Mnk1 via phosphorylation. Activated Mnk subsequently binds to eIF4G and phosphorylates eIF4E within the eIF4F complex, resulting in the translation of certain mRNAs vital for oncogenic transformation. Conversely, PP2A can directly dephosphorylate Mnk and eIF4E proteins, leading to increased association of eIF4E with 4E-BP1 protein and reduced eIF4E translational machinery. RAMBA retinamides exert their inhibitory mode of action by inducing Mnk1 degradation subsequently reducing eIF4E phosphorylation and eIF4E driven cap dependent mRNA translation.
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