eIF4E knockdown decreases breast cancer cell growth without activating Akt signaling

Abstract

Activation of translation initiation is essential for the malignant phenotype and is emerging as a potential therapeutic target. Translation is regulated by the expression of translation initiation factor 4E (eIF4E) as well as the interaction of eIF4E with eIF4E-binding proteins (e.g., 4E-BP1). Rapamycin inhibits translation initiation by decreasing the phosphorylation of 4E-BP1, increasing eIF4E/4E-BP1 interaction. However, rapamycin also inhibits S6K phosphorylation, leading to feedback loop activation of Akt. We hypothesized that targeting eIF4E directly would inhibit breast cancer cell growth without activating Akt. We showed that eIF4E is ubiquitously expressed in breast cancer cell lines. eIF4E knockdown by small interfering RNA inhibited growth in different breast cancer cell subtypes including triple-negative (estrogen receptor/progesterone receptor/HER-2-negative) cancer cells. eIF4E knockdown inhibited the growth of cells with varying total and phosphorylated 4E-BP1 levels and inhibited rapamycin-insensitive as well as rapamycin-sensitive cell lines. eIF4E knockdown led to a decrease in expression of cyclin D1, Bcl-2, and Bcl-xL. eIF4E knockdown did not lead to Akt phosphorylation but did decrease 4E-BP1 expression. We conclude that eIF4E is a promising target for breast cancer therapy. eIF4E-targeted therapy may be efficacious in a variety of breast cancer subtypes including triple-negative tumors for which currently there are no targeted therapies. Unlike rapamycin and its analogues, eIF4E knockdown is not associated with Akt activation.

Figure 1

Expression of eIF4E and 4E-BP1 in panel of breast cancer cell lines. A. Cell lysate was harvested from a panel of breast cancer cell lines and separated by SDS-PAGE. Western blotting was performed for p-eIF4E (S209), total eIF4E, p-4E-BP1 (T70), total 4E-BP1 and actin. B. Rapamycin sensitivity of a panel of breast cancer cell lines. Cells plated in 96-well plates in triplicate, and treated with increasing doses of rapamycin. Cell growth was assessed at five days by SRB, and growth was compared to vehicle treated controls (% control). Results represent the average of at least three independent experiments. C. and D. MDA-MB-468 cells were transfected with control siRNA or one of two siRNA sequences for eIF4E (4E1 and 4E3). Cells were lysed at 48, 72 or 96 hours. Western blotting was performed for eIF4E and actin expression.

Figure 2

Effect of eIF4E knockdown on cancer cell growth. A. MCF7, SKBR-3 MDA-MB-468, MDA-MB-231, MDA-MB-435, and MDA-MB-435S cells were transfected with control siRNA or one of two eIF4E siRNA sequences (4E1 and 4E3). One day after transfection, the cells were trypsinized, counted and plated in 96 well plates in triplicates. On day 4, cell growth was assayed and cell growth in untreated cells (no tx) was normalized to 100%. Shown are representative experiments (n≥3). Columns, mean; bars, SD. *, P < 0.05, untreated group versus control siRNA group by two tailed Student's t-test. **, P < 0.05, control siRNA group versus 4E1 or 4E3 siRNA by two tailed Student's t-test. 72 hours after siRNA transfection, cells were lysed and western blotting was performed for eIF4E and actin expression. Representative western blots demonstrating eIF4E knockdown in each cell line is shown. B. Effect of eIF4E knockdown on anchorage-dependent growth. Cells were transfected with control siRNA or one of two eIF4E siRNA sequences (4E1 and 4E3). One day after transfection, the cells were trypsinized, counted and plated in 60-mm plates (1000 cells/plate). After 2 weeks, colonies were stained with crystal violet. No tx, no treatment. C. Effect eIF4E knockdown on cell cycle. MCF7 cells were transfected with control siRNA or one of two eIF4E siRNA sequences (4E1 and 4E3). Four days after transfection, the attached cells were harvested, and FACS analysis was performed. No tx, no treatment. **, P < 0.05, % cells in G1 in control siRNA group versus 4E1 or 4E3 siRNA by two tailed Student's t-test.

Figure 3

Effect of eIF4E knockdown on translation. A. MDA-MB468 cells were transfected with control siRNA or eIF4E siRNA and were cultured for three days to allow the efficient eIF4E knockdown. Cell lysates were then separated on a 15-50% sucrose gradient. After centrifugation, gradients were fractionated by using an ISCO Foxy Jr. fractionator (Teledyne Isco). The A254 of gradient was analyzed with an ISCO UA-6 UV/VIS detector. B. Effect eIF4E knockdown on eIF4E's translational targets. MDA-MB-468, MCF7, MDA-MB-231 or MDA-MB-435 cells were transfected with control siRNA or eIF4E siRNA (4E3). Three days after transfection, cell lysate was harvested, and western blotting was performed for eIF4E, cyclin D1, Bcl2, Bcl-xL, and actin.

Figure 4

Effect eIF4E knockdown on Akt phosphorylation. A. MDA-MB-468, MCF7, MDA-MB-231 or MDA-MB-435 cells were transfected with control siRNA or eIF4E siRNA (4E3). Three days after transfection, cell lysate was harvested, and western blotting was performed for eIF4E, p-Akt (T308), p-Akt (S473), Akt, and actin. B. MDA-MB-435 cells were treated with vehicle or with 1 or 10nM rapamycin for 24 hours. Western blotting was performed for p-Akt (S473) and Akt. C. MDA-MB-468, MCF7, MDA-MB-231 or MDA-MB-435 cells were transfected with control siRNA or eIF4E siRNA (4E3). Three days after transfection, cell lysate was harvested, and western blotting was performed for 4E-BP1, p-4E-BP1 (T70). S6K1, p-S6K1 (T389), eIF4E, and actin.