The Translation Factor eIF6 Is a Notch-Dependent Regulator of Cell Migration and Invasion

A growing body of evidence indicates that protein factors controlling translation play an important role in tumorigenesis. The protein known as eIF6 is a ribosome anti-association factor that has been implicated in translational initiation and in ribosome synthesis. Over-expression of eIF6 is observed in many natural tumours, and causes developmental and differentiation defects in certain animal models. Here we show that the transcription of the gene encoding eIF6 is modulated by the receptor Notch-1, a protein involved in embryonic development and cell differentiation, as well as in many neoplasms. Inhibition of Notch-1 signalling by γ-secretase inhibitors slowed down cell-cycle progression and reduced the amount of eIF6 in lymphoblastoid and ovarian cancer cell lines. Cultured ovarian cancer cell lines engineered to stably over-expressing eIF6 did not show significant changes in proliferation rate, but displayed an enhanced motility and invasive capacity. Inhibition of Notch-1 signalling in the cells over-expressing eIF6 was effective in slowing down the cell cycle, but did not reduce cell migration and invasion. On the whole, the results suggest that eIF6 is one of the downstream effectors of Notch-1 in the pathway that controls cell motility and invasiveness

Over-expression of eIF6 enhances migration and invasivity of ovarian cancer cells.

<p>(A, B) Migration assay: A2780/eIF6 and control cells were treated with DAPT 75 µM or DMSO for 36 h then seeded in the upper side of migration chambers. The cells migrated to the lower chambers after 36 h of incubation were stained with crystal violet dye. (C, D) Invasivity assay: cells were treated with DMSO or DAPT 75 µM for 36 hours and seeded in the upper side of invasion chambers. After 36 h, cells migrated in the lower chamber were stained. The total stained area in the lower chambers was estimated using the Image-J software. The histograms in (B) and (D) represent the average of three independent experiments. P values estimating the statistical significance of the observed experimental variations between different data sets (control cells with and without GSI; eIF6 cells with and without GSI; control and eIF6 cells without GSI; control and eIF6 cells with GSI) are shown for both cell migration and invasion experiments.</p

List of the primers used for the cloning of different constructs.

<p>The reaction sites used for the cloning are underlined.</p

Luciferase reporter assay.

<p>(A) Schematic representation of the region of the human eIF6 promoter containing two putative RBP-jk-binding sites (sites A-B). Different tracts of the promoter were cloned upstream of the luciferase gene in the pGL3 Basic luciferase plasmid obtaining the constructs indicated as FL, FR1, FR2 and FR3. (B) Luciferase assay. NIH-3T3 cells were cotransfected with <i>Renilla</i> luciferase plasmid and one of the reporter plasmids shown in A in the presence (black columns) or the absence (grey columns) of a plasmid expressing human Notch-1 (N1). (C) NIH-3T3 cells co-transfected with the FL plasmid and the N1 plasmid were further transfected with increasing amounts (0,25-0,5-1 µg) of a plasmid expressing a dominant-negative form of RBP-jk (RBP-jk DN).</p

eIF6 expression in stably-transfected A2780 ovarian cancer cells.

<p>eIF6 expression in a pool of A2780 cells stably transfected with the pcDNA3-eIF6 plasmid was analyzed by RT-PCR (A) and western blotting (B) as indicated. The intensity of the eIF6 RNA and protein bands was quantified relative to β-actin and β-tubulin, respectively, using the ImageJ software. The results represent the average of three independent experiments. (C) Analysis of the polysomal profiles of A2780/eIF6 and control cells by density gradient centrifugation. The areas under the polysomal peaks were quantified using the ImageJ software.</p

eIF6 over-expression does not significantly affect cell cycle.

<p>FACS analysis of cell-cycle distribution of control (A) and eIF6-over-expressing (B) A2780 cells grown for 72 h in the absence (top) or in the presence (bottom) of 75 µM DAPT.</p

EIF6 over-expression increases the motility and invasiveness of cancer cells by modulating the expression of a critical subset of membrane-bound proteins.

BACKGROUND: Eukaryotic Initiation factor 6 (eIF6) is a peculiar translation initiation factor that binds to the large 60S ribosomal subunits, controlling translation initiation and participating in ribosome biogenesis. In the past, knowledge about the mechanisms adopted by the cells for controlling protein synthesis by extracellular stimuli has focused on two translation initiation factors (eIF4E and eIF2), however, recent data suggest eIF6 as a newcomer in the control of downstream of signal transduction pathways. eIF6 is over-expressed in tumors and its decreased expression renders cells less prone to tumor growth. A previous work from our laboratory has disclosed that over-expression of eIF6 in transformed cell lines markedly increased cell migration and invasion. METHODS: Here, we performed a quantitative proteomic analysis of membrane-associated proteins in A2780 ovarian cancer cells over-expressing eIF6. Differentially expressed proteins upon eIF6 overproduction were further investigated in silico by Ingenuity Pathway Analysis (IPA). RT-qPCR and Western blot were performed in order to validate the proteomic data. Furthermore, the effects of a potent and selective inhibitor ML-141 in A2780 cells were evaluated using transwell migration assay. Finally, we explored the effects of eIF6 over-expression on WM793 primary melanoma cell lines. RESULTS: We demonstrated that: (i) the genes up-regulated upon eIF6 overproduction mapped to a functional network corresponding to cellular movements in a highly significant way; (ii) cdc42 plays a pivotal role as an effector of enhanced migratory phenotype induced upon eIF6 over-expression; (iii) the variations in abundance observed for cdc42 protein occur at a post-transcriptional level; (iv) the increased cell migration/invasion upon eIF6 over-expression was generalizable to other cell line models. CONCLUSIONS: Collectively, our data confirm and further extend the role of eIF6 in enhancing cell migration/invasion. We show that a number of membrane-associated proteins indeed vary in abundance upon eIF6 over-expression, and that the up-regulated proteins can be located within a functional network controlling cell motility and tumor metastasis. Full understanding of the role eIF6 plays in the metastatic process is important, also in view of the fact that this factor is a potentially druggable target to be exploited for new anti-cancer therapies