BackgroundAberrant cap-dependent translation is implicated in tumorigenesis in multiple tumor types including mesothelioma. In this study, disabling the eIF4F complex by targeting eIF4E with eIF4E-specific antisense oligonucleotide (4EASO) is assessed as a therapy for mesothelioma.MethodsMesothelioma cells were transfected with 4EASO, designed to target eIF4E mRNA, or mismatch-ASO control. Cell survival was measured in mesothelioma treated with 4EASO alone or combined with either gemcitabine or pemetrexed. Levels of eIF4E, ODC, Bcl-2 and β-actin were assessed following treatment. Binding to a synthetic cap-analogue was used to study the strength of eIF4F complex activation following treatment.ResultseIF4E level and the formation of eIF4F cap-complex decreased in response to 4EASO, but not mismatch control ASO, resulting in cleavage of PARP indicating apoptosis. 4EASO treatment resulted in dose dependent decrease in eIF4E levels, which corresponded to cytotoxicity of mesothelioma cells. 4EASO resulted in decreased levels of eIF4E in non-malignant LP9 cells, but this did not correspond to increased cytotoxicity. Proteins thought to be regulated by cap-dependent translation, Bcl-2 and ODC, were decreased upon treatment with 4EASO. Combination therapy of 4EASO with pemetrexed or gemcitabine further reduced cell number.Conclusion4EASO is a novel drug that causes apoptosis and selectively reduces eIF4E levels, eIF4F complex formation, and proliferation of mesothelioma cells. eIF4E knockdown results in decreased expression of anti-apoptotic and pro-growth proteins and enhances chemosensitivity.

Introduction Oncolytic virus therapy is a promising therapy for numerous tumor types. Edmonston-strain measles virus (MV) has been tested in clinical trials for ovarian cancer, glioma, and myeloma. Therefore, the antitumor activity of MV against NSCLC was assessed. Methods Human NSCLC cells and immortalized lung epithelial cell lines, Beas2B, were infected with either MV producing GFP (MV-GFP) or MV producing carcinoembryonic antigen (MV-CEA). Cells were assessed for viability, induction of apoptosis by caspase and PARP cleavage, and for viral transgene production. The dependency of MV entry on CD46 and nectin-4 were determined using blocking antibodies. The role of host translational activity on viral replication was assessed by overexpression of eIF4E and translation inhibition. Antitumor activity was assessed by measuring treated NSCLC xenografts from flanks of nude mice. Results MV infection of NSCLC cells results in potent cell killing in most of the cell lines compared to immortalized Beas2B cells and induces apoptosis. MV infection was prevented by blocking of CD46, however was independent of nectin-4 blockade. Tumor weights are diminished following intratumoral injections of MV-CEA in 1 of 2 cell lines and result in detectable viral transgene in serum of mice. Conclusions These data indicate that MV is oncolytic for human NSCLC and this was independent of nectin-4 expression. Dysregulated protein translational machinery may play a role in determining tumor tropism in NSCLC. MV combined with gemcitabine could be explored further as chemovirotherapy for NSCLC.

BACKGROUND: Insulin-like growth factor (IGF)-I signalling stimulates proliferation, survival, and invasion in malignant mesothelioma and other tumour types. Studies have found that tumourigenesis is linked to dysregulation of cap-dependent protein translation. METHODS: The effect of IGF stimulation on cap-mediated translation activation in mesothelioma cell lines was studied using binding assays to a synthetic 7-methyl GTP-cap analogue. In addition, cap-mediated translation was genetically repressed in these cells with a dominant active motive of 4E-BP1. RESULTS: In most mesothelioma cell lines, IGF-I stimulation resulted in a hyperphosphorylation-mediated inactivation of 4E-BP1 compared with that in normal mesothelial cells. An inhibitor of Akt diminished IGF-I-mediated phosphorylation of 4E-BP1, whereas inhibiting MAPK signalling had no such effect. IGF-I stimulation resulted in the activation of the cap-mediated translation complex as indicated by an increased eIF4G/eIF4E ratio in cap-affinity assays. Akt inhibition reversed the eIF4G/eIF4E ratio. Mesothelioma cells transfected with an activated 4E-BP1 protein (4E-BP1

Elevated levels of eukaryotic translation initiation factor 4E (eIF4E) enhance translation of many malignancy-related proteins, such as vascular endothelial growth factor (VEGF), c-Myc and osteopontin. In non-small-cell lung cancer (NSCLC), levels of eIF4E are significantly increased compared with normal lung tissue. Here, we used an antisense oligonucleotide (ASO) to inhibit the expression of eIF4E in NSCLC cell lines. eIF4E levels were significantly reduced in a dose-dependent manner in NSCLC cells treated with eIF4E-specific ASO (4EASO) compared with control ASO. Treatment of NSCLC cells with the 4EASO resulted in decreased cap-dependent complex formation, decreased cell proliferation and increased sensitivity to gemcitabine. At the molecular level, repression of eIF4E with ASO resulted in decreased expression of the oncogenic proteins VEGF, c-Myc and osteopontin, whereas expression of β-actin was unaffected. Based on these findings, we conclude that eIF4E-silencing therapy alone or in conjunction with chemotherapy represents a promising approach deserving of further investigation in future NSCLC clinical trials.

Malignant mesothelioma is a devastating disease with a poor prognosis for which there is a clear need for more successful therapeutic approaches. Triptolide, a diterpenoid triepoxide, is a highly effective agent against several cancer types in animal models. Owing to triptolide's poor solubility in water, a water-soluble analog, minnelide, was synthesized. Minnelide is a prodrug of triptolide and is activated by exposure to phosphatases that are found in all body tissues, including blood. Mesothelioma cells were treated in vitro with minnelide or its parent compound, triptolide. Minnelide and triptolide were both found to significantly reduce mesothelioma cell viability and induce apoptosis. The level of Hsp70, a protein that promotes cancer cell survival, was measured in mesothelioma cells before and after treatment with triptolide. Hsp70 levels were decreased in a dose-dependent manner. In addition, triptolide sensitized cells to gemcitabine and pemetrexed as measured by cell viability. Mice bearing mesothelioma flank tumors were treated with daily injections (28 d) of minnelide or saline solution and xenograft tumor growth recorded. Mice displayed significantly reduced tumor burden. These findings support the clinical evaluation of minnelide therapy for mesothelioma.