Inhibition of oncogenic cap-dependent translation by 4EGI-1 reduces growth, enhances chemosensitivity and alters genome-wide translation in non-small cell lung cancer.

Hyperactivation of eIF4F-mediated translation occurs in many if not all cancers. As a consequence, cancer cells aberrantly enhance expression of malignancy-related proteins that are involved in cell cycle progression, angiogenesis, growth, and proliferation. With this in mind eIF4F is a promising molecular target for therapeutics that counteract pathological eIF4F activity. Here we used 4EGI-1, a small-molecule inhibitor of cap-mediated translation that disrupts formation of the eukaryotic initiation factor 4F (eIF4F) complex to treat non-small cell lung cancer (NSCLC). Treatment of cells with 4EGI-1 reduced cell proliferation, decreased cap-dependent complex formation, induced apoptosis, enhanced sensitivity to gemcitabine, and altered global cellular translation. Suppression of cap-dependent translation by 4EGI-1 resulted in diminished expression of oncogenic proteins c-Myc, Bcl-2, cyclin D1, and survivin, whereas ß-actin expression was left unchanged. In light of these results, small-molecule inhibitors like 4EGI-1 alone or with chemotherapy should be further evaluated in the treatment of NSCLC.

4EGI-1 represses cap-dependent translation and regulates genome-wide translation in malignant pleural mesothelioma.

Deregulation of cap-dependent translation has been implicated in the malignant transformation of numerous human tissues. 4EGI-1, a novel small-molecule inhibitor of cap-dependent translation, disrupts formation of the eukaryotic initiation factor 4F (eIF4F) complex. The effects of 4EGI-1-mediated inhibition of translation initiation in malignant pleural mesothelioma (MPM) were examined. 4EGI-1 preferentially inhibited cell viability and induced apoptosis in MPM cells compared to normal mesothelial (LP9) cells. This effect was associated with hypophosphorylation of 4E-binding protein 1 (4E-BP1) and decreased protein levels of the cancer-related genes, c-myc and osteopontin. 4EGI-1 showed enhanced cytotoxicity in combination with pemetrexed or gemcitabine. Translatome-wide polysome microarray analysis revealed a large cohort of genes that were translationally regulated upon treatment with 4EGI-1. The 4EGI-1-regulated translatome was negatively correlated to a previously published translatome regulated by eIF4E overexpression in human mammary epithelial cells, which is in agreement with the notion that 4EGI-1 inhibits the eIF4F complex. These data indicate that inhibition of the eIF4F complex by 4EGI-1 or similar translation inhibitors could be a strategy for treating mesothelioma. Genome wide translational profiling identified a large cohort of promising target genes that should be further evaluated for their potential significance in the treatment of MPM.

Cap-dependent translational control of oncolytic measles virus infection in malignant mesothelioma.

Malignant mesothelioma has a poor prognosis for which there remains an urgent need for successful treatment approaches. Infection with the Edmonston vaccine strain (MV-Edm) derivative of measles virus results in lysis of cancer cells and has been tested in clinical trials for numerous tumor types including mesothelioma. Many factors play a role in MV-Edm tumor cell selectivity and cytopathic activity while also sparing non-cancerous cells. The MV-Edm receptor CD46 (cluster of differentiation 46) was demonstrated to be significantly higher in mesothelioma cells than in control cells. In contrast, mesothelioma cells are not reliant upon the alternative MV-Edm receptor nectin-4 for entry. MV-Edm treatment of mesothelioma reduced cell viability and also invoked apoptotic cell death. Forced expression of eIF4E or translation stimulation following IGF-I (insulin-like growth factor 1) exposure strengthened the potency of measles virus oncolytic activity. It was also shown that repression of cap-dependent translation by treatment with agents [4EASO, 4EGI-1] that suppress host cell translation or by forcing cells to produce an activated repressor protein diminishes the strength of oncolytic viral efficacy.

Targeting Topoisomerase II Activity in NSCLC with 9-Aminoacridine Derivatives.

BACKGROUND: Etoposide and other type-II human topoisomerase (TOPOII) poisons are widely used for the treatment of many different cancer types, including non-small cell lung cancer (NSCLC). However, there is a risk for the development of therapy-related secondary leukemia following treatment with these TOPOII poisons. Five to seven years is the typical latency period for the development of secondary leukemia. One of the strategies to overcome this issue is to develop agents that do not act as poisons but still effectively inhibit topoisomerase activity. This has led to the development of acridine-based agents, which are catalytic TOPOII inhibitors, that do not generate DNA strand breaks that can lead to secondary malignancies in in vitro tests. MATERIALS AND METHODS: In this study, we showd antiproliferative activity of a series of acridine-based catalytic inhibitors of TOPOII using four NSCLC cell lines (H460, A549, H2009 and H2030). Cells were treated with four acridine-based compounds for 72 h. RESULTS: The results indicate that these compounds inhibit NSCLC cell proliferation with half-maximal effective concentration (EC50) ranging from 8.15 to 42.09 µM. Combination therapy with cisplatin resulted in increased potency. Poly (ADP-ribose) polymerase cleavage and Guava Nexin assays confirm that the primary mode of cell death was by apoptosis. CONCLUSION: This current work is part of a series of studies for this panel of acridine-based compounds bearing TOPOII-inhibitory activity against different solid tumor types. The acridine-based agents were found to substantially reduce NSCLC cell viability and induce apoptosis. In addition, the acridine-based compounds sensitized cells to cisplatin as measured by cell viability. The results are consistent with prior work on mesothelioma, small-cell lung cancer and pancreatic cancer with this same panel of 9-aminoacridine derivatives. These findings support further development of this type of catalytic TOPOII inhibitor as a novel agent for NSCLC therapy.

Triptolide and its prodrug minnelide suppress Hsp70 and inhibit in vivo growth in a xenograft model of mesothelioma.

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.

Resistance to EGFR-TKI can be mediated through multiple signaling pathways converging upon cap-dependent translation in EGFR-wild type NSCLC.

INTRODUCTION: For the majority of patients with non-small-cell lung cancer (NSCLC), response to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is suboptimal. In models of acquired resistance to EGFR-TKI, activation of Akt phosphorylation is frequently observed. Because Akt activation results in downstream initiation of cap-dependent protein translation, we hypothesized that a strategy of targeting cap-dependent translation in combination with erlotinib might enhance therapy. METHODS: NSCLC cells that are wild type for EGFR were assayed for sensitivity to erlotinib. Serum-starved NSCLC cells were assayed for EGFR signaling and downstream pathway activation by immunoblot after stimulation with epidermal growth factor. EGFR signaling and signaling mediators of cap-dependent translation were assayed by immunoblot under serum-replete conditions 24 hours after treatment with erlotinib. Finally, combination treatment with erlotinib and two different cap-dependent translation inhibitors were done to assess the effect on cell viability. RESULTS: EGFR signaling is coupled to activation of cap-dependent translation in EGFR wild-type cells. Erlotinib inhibits EGFR phosphorylation in EGFR-TKI resistant cells, however, results in activation of downstream signaling molecules including Akt and extracellular regulated kinase, ERK 1/2, resulting in maintenance of eukaryotic initiation factor 4F (eIF4F) activation. eIF4F cap-complex formation is maintained in erlotinib-resistant cells, but not in erlotinib-sensitive cells. Finally, using an antisense oligonucleotide against eukaryotic translation initiation factor 4E and a small-molecule inhibitor to disrupt eIF4F formation, we show that cap-dependent translation inhibition can enhance sensitivity to erlotinib. CONCLUSION: The results of these studies support further clinical development of translation inhibitors for treatment of NSCLC in combination with erlotinib.

Novel acridine-based agents with topoisomerase II inhibitor activity suppress mesothelioma cell proliferation and induce apoptosis.

Human topoisomerase II (hTopoII) inhibitors are important chemotherapeutic agents in many different settings including treatment of malignant mesothelioma. Topoisomerase poisons, such as etoposide and doxorubicin, function by trapping the DNA-enzyme covalent complex producing DNA strand breaks which can ultimately lead to cancer cell death, as well as development of secondary malignancies. While these compounds have been used successfully in treating a wide variety of cancers, their use against mesothelioma has been limited. This study evaluates the anti-proliferative activity of series of acridine-based catalytic inhibitors of hTopoII using four mesothelioma cell lines (H513, H2372, H2461, and H2596). The results indicate these compounds inhibit malignant cell proliferation with EC(50) values ranging from 6.9 to 32 µM. Experiments are also performed that show that combination therapies may be used to increase potency. Based on the results of PARP cleavage and Guava Nexin assay, it is concluded that the primary mode of cell death is by apoptosis. The results are consistent with prior work involving pancreatic cancer and hTopoII catalytic inhibitors and suggest substituted acridines may hold promise in treating malignant mesothelioma.

Novel role of c-jun N-terminal kinase in regulating the initiation of cap-dependent translation.

Initiation of protein translation by the 5' mRNA cap is a tightly regulated step in cell growth and proliferation. Aberrant activation of cap-dependent translation is a hallmark of many cancers including non-small cell lung cancer. The canonical signaling mechanisms leading to translation initiation include activation of the Akt/mTOR pathway in response to the presence of nutrients and growth factors. We have previously observed that inhibition of c-jun N-terminal kinase (JNK) leads to inactivation of cap-dependent translation in mesothelioma cells. Since JNK is involved in the genesis of non-small cell lung cancer (NSCLC), we hypothesized that JNK could also be involved in activating cap-dependent translation in NSCLC cells and could represent an alternative pathway regulating translation. In a series of NSCLC cell lines, inhibition of JNK using SP600125 resulted in inhibition of 4E-BP1 phosphorylation and a decrease in formation of the cap-dependent translation complex, eIF4F. Furthermore, we show that JNK-mediated inhibition of translation is independent of mTOR. Our data provide evidence that JNK is involved in the regulation of translation and has potential as a therapeutic target in NSCLC.