Anti-tumor activity of the eIF4E-targeted drug ribavirin in breast cancer cellsAnti-tumor activity of the eIF4E-targeted drug ribavirin in breast cancer cells 1 Filippa Pettersson, 1 Monica C. Dobocan, 1 Hélène Retrouvey, 2 Biljana Culjkovic, 2 Lotfi Abdellatif Amri, 2Louis Gaboury, 2 Katherine L.B. Borden, and 1 Wilson H. Miller, Jr.

1 Lady Davis Institute and Segal Cancer Center of the Jewish General Hospital, McGill University, Montreal, QC, Canada. 2 Institute for Research in Immunology and Cancer , Université de Montréal, Montréal, QC, Canada

In this study, we explored the potential of targeting the eukaryotic translation initiation factor (eIF4E) with ribavirin as a novel anti-tumor agent in breast cancer. eIF4E is an oncogene that facilitates nuclear export and translation of specific, growth-stimulatory mRNAs, including cyclins, c-myc, survivin, VEGF and others, thereby promoting cell survival. Overexpression of eIF4E also leads indirectly to activation of Akt, providing a positive feed-back loop for eIF4E activation and Akt signaling effects. Ribavirin is an antiviral drug that has been shown to inhibit oncogenic transformation mediated by eIF4E and reduce the clonogenic potential of cancer cells with high eIF4E levels. Ribavirin specifically inhibits translation and/or nuclear export of eIF4E targets in cells both in vitro and in patients, as shown in a recent phase I/II proof-of-principle trial in patients with AML. In this trial, dramatic clinical improvements were observed and reductions in eIF4E levels and activity correlated with clinical response. Importantly, ribavirin is largely non-toxic even at high doses, possibly due to an eIF4E oncogene addiction specific to tumor cells.

eIF4E is overexpressed in more than 50% of breast cancers, and high levels are associated with increased angiogenesis, clinical progression and poor prognosis. Targeting eIF4E with ribavirin may therefore be an attractive therapeutic strategy for this malignancy. We studied the effects of ribavirin in a panel of breast cancer cells, representing luminal and basal-type tumors with various ER, PR and Her2 status. Western blot analysis showed that eIF4E was overexpressed compared to normal breast tissue and predominantly cytoplasmic in all of the cell lines. In addition, we examined eIF4E levels in metastatic skin lesions of three breast cancer patients and found highly elevated levels compared to normal skin. Ribavirin anti-proliferative activity was assessed using a cell viability assay and clonogenic assays were performed to examine changes in both anchorage dependent and –independent growth. At clinically relevant concentrations, the majority of the cell lines responded to ribavirin, with varying sensitivity. Inhibition of cell growth was associated with decreased protein levels of eIF4E targets such as cyclin D1 and survivin, and a reduction in phosphorylation of Akt as well as eIF4E binding protein 1 (4E-BP1) were observed. Cell cycle analysis showed that ribavirin caused a significant S-phase arrest in sensitive cells, while apoptosis was only observed at elevated concentrations of the drug.

This data encourages further study of ribavirin as a breast cancer therapeutic and identification of potential combination regimens. A clinical trial of single agent ribavirin in patients with advanced metastatic breast cancer is planned in the near future.

ABSTRACT The long-term objective of this project is to develop novel therapeutic strategies for the treatment of poor-prognosis, metastatic breast cancer associated with high levels of the eukaryotic translation initiation factor 4E (eIF4E). Our goal is to create an iterative cycle of discovery where novel experimental therapies are evaluated in the clinic and refined in the laboratory based upon the clinical findings.

eIF4E is an oncogene that stimulates expression of specific, growth promoting mRNAs at the post-transcriptional level. In the nucleus, eIF4E acts to facilitate mRNA export, increasing their availability to the translation machinery. In the cytoplasm, eIF4E recruits mRNAs to the ribosome for initiation of m(7)G cap-dependent translation. Importantly, housekeeping transcripts such as GAPDH, are translated independently of eIF4E. The availability of eIF4E is negatively regulated by 4E binding proteins (4E-BP1 and others). Phosphorylation of 4E-BP1 by mTOR allows eIF4E to be released and perform its functions (see Model 1).

eIF4E overexpression has been reported in more than 50% of breast cancers, and is associated with increased angiogenesis, clinical progression and poor prognosis. Overexpression of eIF4E is associated with oncogenic transformation of cells in culture and with tumor formation and increased tumor invasion in mice. This can be explained by the fact that specific mRNAs targeted by eIF4E include many that are critical to cell division, cell growth and angiogenesis, including cyclins, survivin, c-myc, Mcl-1 and VEGF. Overexpression of eIF4E also leads to activation of Akt.

eIF4E is phosphorylated by Mnk1/2, downstream of the ERK and p38 MAP kinases, but the role of this phosphorylation is not fully defined. Mice lacking both Mnk1 and 2 develop normally, however studies have indicated that eIF4E phosphorylation stimulates its mRNA export function and is required for the anti-apoptotic and oncogenic activity of eIF4E. There is increasing evidence that high levels of phospho-eIF4E in cancer cells correlate with proliferation and survival.

Ribavirin is an inhibitor of eIF4E with potent anti-cancer activity in cells and in patients with leukemia. Ribavirin competes with the m(7)G mRNA cap, inhibiting translation and/or mRNA export of specific growth-promoting transcripts. We conducted a phase I/II proof-of-principle clinical trial examining the efficacy of ribavirin treatment in patients with poor prognosis M4 and M5 AML (ClinicalTrials.gov, NCT00559091, Assouline et al, Blood, 2009, 114(2):257-60). Reduced protein expression of eIF4E targets and decreased Akt activity were observed, together with dramatic clinical improvements. Notably, no treatment-related toxicities were observed, which may be in part due to an eIF4E oncogene addiction specific to tumor cells.

Based on our success in AML and the high prevalence of eIF4E overexpression in breast cancer, we hypothesize that ribavirin, either alone or as part of combination therapy, may effectively target breast tumors overexpressing eIF4E.

The goal of the present study was to assess ribavirin activity in breast cancer cells lines and begin to correlate biological responses with molecular changes and cell line characteristics.

Model 1. Regulation of mRNA export and protein translation by upstream signal transduction pathways and by ribavirin.

Table 1. Characteristics of cell lines used in the study

A.

# Ref: Neve et al. Cancer Cell, 2006, 10(6):515-27* Determined in a cell viability assay, see Figure 2.

AcknowledgementsThis research is funded by a BCRF-AACR Grant for Translational Breast Cancer Research

P-eIF4E

-actin

eIF4E

0 10 20 (M Rib)A. B.

P-eIF4EeIF4E

0 1 2.5 5 7.5 10 (M CGP)

Figure 4. Ribavirin reduction of phospho-eIF4E may mediate growth inhibition. A) MDA-MB-468 cells were treated for three days and protein levels assessed by semi-quantitative Western blot analysis. B) MDA-MB-468 cells were treated with ribavirin in the absence or presence of a selective Mnk inhibitor (CGP57380) which blocks phosphorylation of eIF4E (top). Cell viability was assessed on day 5 using the Cell Titer-Glo™ assay (bottom), and is expressed relative to untreated cells.

phosBP-1

Normal breast FaDu BT MCF-7 231 468 SkBr3 ZR75.1A.

β-actin

eIF4E

pBP-1

BP-1

Figure 1. Expression of eIF4E and BP1 in breast cancer cell lines. A) Western blot of the indicated proteins in a panel of breast cancer cell lines. Normal breast indicates tumor-adjacent tissue removed during surgery. FaDu head and neck carcinoma cells were used as positive control for eIF4E. BT=BT474, 231 and 468 = MDA-MB-231 and -468, respectively. B) Confocal microscopy showing both cytoplasmic and nuclear eIF4E in BT474, MDA-MB-468 and SkBr3.

eIF4E DAPI overlay

B. Cell viability assay (7 days)A.

Ribavirin conc. (M)

Viab

le c

ells

(% o

f con

trol

)

Clonogenic assay (14 days)B.

Figure 2. Ribavirin reduces proliferation and clonogenic survival of breast cancer cell lines. A) The number of viable cells was measured using the Cell Titer-Glo™ assay (Promega) and is expressed relative to untreated cells. B) Anchorage-dependent clonogenic potential was assessed in cells grown in 6-well plates in the presence or absence of ribavirin. The total number of colonies per well was counted on day 14.

MATERIALS & METHODS

BACKGROUND RESULTSRESULTS

SUMMARYSUMMARY

eIF4E protein is present at significant levels in all cell lines tested.

eIF4E is localized to the nucleus and cytoplasm in breast cancer cells.

Ribavirin reduces proliferation and clonogenic potential of these cell lines, to a varying degree.

The cells with the lowest levels of eIF4E appear to be the least sensitive to ribavirin.

The anti-tumor effects of ribavirin are associated with decreased levels of eIF4E targets and reduced AKT activity.

Ribavirin reduces the level of phosphorylated eIF4E, and this may in part mediate growth inhibition.

A.

Patient 1 Patient 2 Patient 3

TumorNormal

Normal Skin (Patient 1) X10 Tumor skin (Patient 1) X10

eIF4

E

eIF4

E

B.

Figure 5. Elevated expression of eIF4E in biopsies from metastatic breast cancer patients. eIF4E was detected by Q-PCR (A) and IHC (B). PCR results were normalized to G6PDH and normalizing to RPL13a provided similar results. For each patient, normal skin was used a calibrator (relative quantity =1). In (B) blue staining represents collagen and brown stain represents eIF4E.

4E-BP1

AKT

mTOR

eIF4A4E-BP1

p38

Mnk

eIF4G

ERK

Growth and stress signals

Translation OFF

Translation ON

TSC1/2

PDK1

PIP3

PTENPI3K

Growth factors, hormones, cytokines etc.

GF-R

P

Ribavirin

Ribavirin

Ribavirin (via NBS1)

nucleus

cytoplasm

mRNA export

P P

P

P

P

P

PP P

P

PP

PP

Model 1. Regulation of mRNA export and protein translation by upstream signal transduction pathways and by ribavirin.

P

Cells and Reagents: MCF-7, MDA-MB-468, MDA-MB-321 and ZR75.1 cells were maintained in DMEM media supplemented with 10% fetal bovine serum (FBS) and antibiotics. BT-474 and SkBr3 cells were grown in RPMI 1640 with 10% FBS and antibiotics. FaDu cells were maintained in EMEM media supplemented with 10% FBS, antibiotics and amino acids (1X, Wisent). All cell culture media and FBS were obtained from Wisent. Lyophilized Ribavirin was purchased from Kemprotec Ltd, UK. The Mnk inhibitor CGP57380 was acquired from Sigma.

Cell Viability Assay: Cells were cultured in 96-well plates (Costar) in the presence or absence of various Ribavirin concentrations, and/or 10 μM CGP57380. The media was changed and the cells re-treated every 2 or 3 days. Cell viability was assessed at day 5 or 7 using the Cell Titer-Glo™ assay (Promega) according to the manufacturer’s instructions.

Clonogenic Assay: Cells were seeded in 6-well plates (Falcon) at 300 cells/well and kept in the presence or absence of Ribavirin (10 μM or 20 μM) for 2 weeks, with the media changed and the cells re-treated every 2 or 3 days. SRB (sulforhodamine B) staining was used to determine the number of colonies per well. Cells were fixed in 10% TCA (in PBS) for 30 min at 4 ◦C. After 5 washes with water, the plates were left to air-dry, then stained with 0.4% (w/v) SRB in 1% acetic acid for at least 30 min at room temperature. Plates were washed 4 times with 1% acetic acid. The plates were once again air-dried, after which all visible colonies were counted.

Western blotting: Cells were treated with the indicated doses of Ribavirin for the indicated time periods and subsequently lysed in lysis buffer (1% Triton X-100, 150 mM NaCl and 50 mM Tris-HCL, pH 8.0) supplemented with protease and phosphatase inhibitors. Fifty micrograms of lysates were used for Western blotting to detect phospho-eIF-4E (Ser209), phospho-4E-BP1 (Thr37/46), total 4E-BP1, Survivin, phospho-Akt (Ser473) and total Akt (all from Cell Signaling), total eIF-4E (BD Biosciences), as well as Cyclin D1, Cyclin E and Mcl-1 (all from Santa Cruz). Either β-actin (Sigma) or GAPDH (Cell Signaling) was used to confirm equal protein loading.

Immunofluorescence: Cells were plated onto 18 x 18 mm cover slips (Fisher) in 6-well plates (Falcon). After 24h, they were fixed with methanol, permeabilized with 0.5% Triton-X 100 (in PBS), then blocked with 10% goat serum (in PBS). An eIF-4E-FITC antibody (BD Biosciences) was used to show the cellular distribution of eIF-4E, and the VECTASHIELD® Mounting Medium with DAPI was used to visualize the nuclei. Cells were viewed with a confocal microscope 100X objective, further enhanced by a 2X magnification.

Immunohistochemistry: Slides were prepared and stained at the pathology core facility at the Institute for Research in Immunology and Cancer, Université de Montréal.

Quantitative PCR: cDNA was prepared from 5 μg total RNA, using Superscript II reverse transcriptase (Invitrogen). The forward primer 5’-AGGAGGTTGCTAACCCAGAACACT-3’ and the reverse primer 5’-AAAGTGAGTAGTCACAGCCAGGCA-3’ were used for the quantification of eIF-4E gene expression using the 7500 Fast Real-Time PCR System with SYBR Green based detection (Applied Biosystems).

Figure 3. Ribavirin downregulates eIF4E targets in ribavirin-sensitive breast cancer cells. A) and B) MDA-MB468 cells were treated with ribavirin for 3 days and protein levels assessed by semi-quantitative Western blot analysis. C) ZR75.1 cells were treated as in A.

Cyclin E

GAPDH

Mcl-1

0 10 20 μM Ribavirin

Cyclin D1

Survivin

GAPDH

0 10 20 40

MDA-MB-468

μM Ribavirin

C.

0 10 20 40μM Ribavirin

ZR75.1P-Akt

Akt

P-BP1

BP1

β-actin

MDA-MB-468

B.P-Akt

Akt

GAPDH

0 10 20 40μM Ribavirin

MDA-MB-468

P-BP1

BP1