Cancer Gene Therapy (2015) 22, 396–401 © 2015 Nature America, Inc. All rights reserved 0929-1903/15 www.nature.com/cgt ORIGINAL ARTICLE Antisense oligonucleotide targeting eukaryotic translation initiation factor 4E reduces growth and enhances chemosensitivity of non-small-cell lung cancer cells SC Thumma1, BA Jacobson1, MR Patel1, BW Konicek2, MJ Franklin1, J Jay-Dixon1, A Sadiq1,ADe1, JR Graff2 and RA Kratzke1 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. Cancer Gene Therapy (2015) 22, 396–401; doi:10.1038/cgt.2015.34; published online 31 July 2015 INTRODUCTION eIF4E. These structural differences account for why mRNAs differ In 2012, worldwide, an estimated 1.8 million new lung cancer in their dependence on eIF4E. A recent report has also shown that cases occurred and an estimated 1.5 million patients succumbed eIF4E promotes nuclear export of mRNAs containing an eIF4E- 6 to the disease. Lung cancer was the most frequently diagnosed sensitivity element (4E-SE) in their 3′ UTR. These observations cancer and caused the most deaths in 2012.1 New therapeutic indicate that eIF4E enhances the translation of a specific group of agents are needed for this fatal disease. mRNAs by increasing their cytoplasmic levels and the efficiency Deregulation of translation initiation has been implicated in the with which they associate with the eIF4F complex. Importantly, pathogenesis of several cancer types.2 Under normal conditions, these mRNAs encode proteins involved in cellular growth and translation initiation is tightly regulated by the availability of survival, including many proto-oncogenes, such as vascular eukaryotic initiation factor 4E (eIF4E). Free eIF4E binds mRNA via endothelial growth factor (VEGF), c-Myc, survivin, Bcl-2 and cyclin the 5′ 7-methyl-GTP-cap structure, after which it brings these D1.7 It has been shown that eIF4E overexpression causes mRNAs to the eIF4F complex for unwinding of mRNA secondary malignant transformation in several in vitro and in vivo models. structure, which allows the 40S ribosomal subunit to scan through For example, NIH-3T3 cells overexpressing eIF4E develop a the 5′ untranslated region (UTR) until it recognizes the start codon malignant phenotype.8 In addition, overexpression of eIF4E using AUG. The availability of free eIF4E is controlled by eIF4E-binding a β-actin promoter produces tumors in many tissues.9 Also, eIF4E proteins (4E-BPs), which sequester eIF4E and block its association levels have been shown to be elevated in surveys of different with the eIF4F complex. 4E-BP1 binding to eIF4E is regulated by human malignancies of the prostate,10 breast,11 stomach,12 several pathways including the Ras/extracellular signal regulated colon13 and skin14 as well as cancers of the hematopoietic kinase (ERK) and phosphatidylinositol-4,5-bisphosphate 3-kinase system.15 In non-small-cell lung cancer (NSCLC), levels of eIF4E are (PI3 kinase)/AKT/mammalian target of rapamycin (mTOR) path- substantially increased compared with normal lung tissue,16 with ways. Phosphorylation of 4E-BP1 by these signaling pathways increased eIF4E expression being associated with tumor inactivates 4E-BP1 freeing eIF4E to interact with eIF4G to form the invasiveness.17 Repression of eIF4E activity, however, decreases eIF4F complex.3 the tumorigencity of NSCLC cells in xenograft models.18 These Specific RNA motifs related to eIF4E have led to the notion that results suggest that targeting eIF4E and decreasing its availability eIF4E coordinates the translation of proteins that function in cell will lead to decreased eIF4F function and hence suppress NSCLC growth and survival pathways.4 The translation of mRNAs with tumor growth. complex 5′UTRs are particularly dependent on eIF4E because of Given that eIF4E acts as an oncogene when it is overexpressed the inefficiency of scanning for the start codon in long, highly or aberrantly activated, investigators are in the process of structured 5′UTRs.5 In contrast, mRNAs with short, unstructured developing drugs that specifically target eIF4E in an attempt to 5′UTRs are translated efficiently in the presence of low levels of decrease its level or affect its hyperactivity in translation initiation. 1Division of Hematology, Department of Medicine, Oncology and Transplantation, University of Minnesota Medical School, Minneapolis, MN, USA and 2Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA. Correspondence: Dr RA Kratzke, Division of Hematology, Oncology and Transplantation, University of Minnesota Medical School, 420 Delaware Avenue SE, MMC 480, Minneapolis, MN 55455, USA. E-mail: [email protected] Received 17 April 2015; revised 30 June 2015; accepted 10 July 2015; published online 31 July 2015 4EASO as a therapy for non-small-cell lung cancer SC Thumma et al 397 Disruption of the eIF4E-to-eIF4G interaction is one approach eIF4E-specific ASO (4EASO) targets the 3′ UTR of human and to attenuate eIF4E-mediated tumorigenicity. A compound desig- murine eIF4E mRNA using the sequence, 5′-TGTCATATTCCTGGA nated as 4EGI-1, which mimicked the inhibitory properties of 4E- TCCTT-3′, where the underlined nucleotides are 2’-methoxyethyl BP1, was identified following a high-through put screen.19 4EGI-1 modified. The ASO control was designed with the same base was found to suppress cancer cell growth along with attenuation composition as the 4EASO but with a mismatched sequence of of c-Myc and Bcl-XL oncoprotein expression. Another tactic to 5′-TCTTATGTTTCCGAACCGTT-3′. target the eIF4E-to-eIF4G association is to design 4EBP-based For ASO cell treatment, Oligofectamine (Invitrogen, Life Techno- eIF4E-binding peptides that block eIF4E from binding eIF4G. logies, Waltham, MA, USA) was mixed 1:16 with Opti-MEM I (Gibco, A compound based upon a 19-residue fragment of 4E-BP1 was Life Technologies) and incubated at room temperature for 5 min. generated and shown to inhibit xenograft tumor growth in an The appropriate concentration of ASO was then diluted in Opti- ovarian cancer model.20 4E1RCat, a small-molecule inhibitor of the MEM I and mixed with the Oligofectamine-Opti-MEM I mixture eIF4E-to-eIF4G interaction was developed recently and shown to and incubated for 45 min. Cells were washed with RPMI 1640 impede eIF4E-to-eIF4G interaction by binding to a region on eIF4E (Gibco, Life Technologies) followed by addition of fresh RPMI 1640 that also binds both eIF4G and 4EBP1. This compound was shown on adherent cells. This transfection solution was added to cells to to reverse tumor chemoresistance in a genetically engineered achieve a 1:4 ratio between the transfection solution and RPMI lymphoma mouse model.21 1640 followed by incubation of this mixture for 4 h at 37 °C in a Another approach to directly target eIF4E hyperactivity is to CO2 incubator. After transfection, RPMI 1640 containing sufficient antagonize the eIF4E-mRNA–cap interaction. Therapies that either serum was added to cells until a final serum concentration of decrease eIF4E abundance or hinder the association of eIF4E with 10% was reached. Cells were harvested or counted after 72 h the 5’ mRNA cap would accomplish this objective. Two teams of of incubation. For cell proliferation analysis, cells were seeded investigators have developed cap-analog libraries that function to onto six-well plates at 125 000 cells per well 1 day before ASO either prevent binding of eIF4E to capped mRNAs or dislodge transfection. For all remaining studies, 10-cm plates were seeded capped mRNAs from eIF4E and therefore the eIF4E complex.22,23 with 1 × 106 cells per plate one day before ASO transfection. One promising cap-analog, 4Ei1,24 is a prodrug that is converted to its functionally active derivative, 7-BnGMP (7-benzyl guanosi- Cell viability assay with ASO and gemcitabine nemonophosphate) in human breast carcinoma cells where it Cells seeded onto six-well plates for 1 day were then transfected has been shown to downregulate cap-dependent translation. with ASOs as described above and incubated overnight. After 24 h When breast carcinoma cancer cell lines were treated with 4Ei1 of incubation with 4EASO or its control, gemcitabine (Eli Lilly) was fl combined with the drug 5- uorouracil there was enhanced added to the appropriate wells at a final concentration of 8 nM cytostatic and/or cytotoxic effects compared with each compound 25 and incubated for an additional 48 h. Cells were then counted using alone. One tactic that was employed to decrease eIF4E a hemacytometer with trypan blue exclusion. Each experiment was abundance in breast carcinoma subtypes was with treatments performed in triplicate, with data normalized to that of untreated 26 that used short-interfering RNA against eIF4E. eIF4E knockdown cells. Group comparisons were performed using two-sided Student’s led to growth inhibition of breast cancer cell lines and also t-test and a P value of o0.05 was taken as significant. decreased the expression of the oncogenic proteins, Cyclin D1, Bcl-2 and Bcl-xL.