Targeted silencing of elongation factor 2 kinase suppresses growth and sensitizes tumors to doxorubicin in an orthotopic model of breast cancer

Abstract

Eukaryotic elongation factor 2 kinase (eEF-2K), through its phosphorylation of elongation factor 2 (eEF2), provides a mechanism by which cells can control the rate of the elongation phase of protein synthesis. The activity of eEF-2K is increased in rapidly proliferating malignant cells, is inhibited during mitosis, and may contribute to the promotion of autophagy in response to anti-cancer therapies. The purpose of this study was to examine the therapeutic potential of targeting eEF-2K in breast cancer tumors. Through the systemic administration of liposomal eEF-2K siRNA (twice a week, i.v. 150 µg/kg), the expression of eEF-2K was down-regulated in vivo in an orthotopic xenograft mouse model of a highly aggressive triple negative MDA-MB-231 tumor. This targeting resulted in a substantial decrease in eEF2 phosphorylation in the tumors, and led to the inhibition of tumor growth, the induction of apoptosis and the sensitization of tumors to the chemotherapy agent doxorubicin. eEF-2K down-modulation in vitro resulted in a decrease in the expression of c-Myc and cyclin D1 with a concomitant increase in the expression of p27(Kip1). A decrease in the basal activity of c-Src (phospho-Tyr-416), focal adhesion kinase (phospho-Tyr-397), and Akt (phospho-Ser-473) was also detected following eEF-2K down-regulation in MDA-MB-231 cells, as determined by Western blotting. Where tested, similar results were seen in ER-positive MCF-7 cells. These effects were also accompanied by a decrease in the observed invasive phenotype of the MDA-MB-231 cells. These data support the notion that the disruption of eEF-2K expression in breast cancer cells results in the down-regulation of signaling pathways affecting growth, survival and resistance and has potential as a therapeutic approach for the treatment of breast cancer.

Figure 1. Effect of eEF-2K down-regulation on breast cancer cell proliferation and clonogenicity.

(A) Knockdown of eEF-2K by siRNA. siRNA specifically targeting eEF-2K inhibits its expression in MDA-MB-231 cells. Cells were transfected with eEF-2K siRNA (5–75 nM) for 48 h, and cell lysates were subjected to Western blot analysis. (B) Effect of knockdown of eEF-2K on cell proliferation. MDA-MB-231 cells were transfected with eEF-2K siRNA, and after 48 h proliferation was detected by an MTS assay. (C) Overexpression of eEF-2K in MDA-MB-231 cells. Cells were transiently transfected with an expression vector encoding GST-tagged wild type eEF-2K, and cell lysates (48 h) were subjected to Western blot analysis. For the effect of overexpression of eEF-2K on cell proliferation, MDA-MB-231 cells were transiently transfected with eEF-2K or a vector control, and cell proliferation was measured using an MTS assay after 96 h. (D–E) Effect of eEF-2K knockdown on colony formation. Knockdown of eEF-2K by siRNA (50 nM) significantly inhibited the number of colonies formed by MDA-MB-231 (D) and MCF-7 (E) cells (*p<0.05). Cells were transfected every 4 days with control or eEF-2K siRNA.

Figure 2. Downstream molecular effects of knockdown or overexpression of eEF-2K in MDA-MB-231 cells.

Cells were transiently transfected with eEF-2K siRNA or GST-eEF-2K plasmid, and cell lysates (72 h) were subjected to Western blot analysis. (A–B) Knockdown of eEF-2K by siRNA increases expression levels of p27^Kip1 (A), while decreasing cyclin D1 (A) and c-Myc (B) levels. (C–D) eEF-2K knockdown additionally inhibits the activity of Src and FAK as indicated by reduced p-Src (Tyr-416) (C) and p-FAK (Tyr-397) (D). (E) Conversely, overexpression of eEF-2K resulted in increased p-Src (Tyr-416) levels. (F) Knockdown of eEF-2K also inhibits the activity of Akt as indicated by reduced p-Akt (Ser-473).

Figure 3. Systemic in vivo targeting of eEF-2K by liposomal siRNA.

(A) Treatment schedule of nude mice bearing MDA-MB-231 tumors with liposomal siRNA (control and eEF-2K) and/or doxorubicin. About 2 weeks after tumor cell injection (MDA-MB-231 cells), targeting of eEF-2K was achieved by systemic (i.v. from tail vein) administration of DOPC-liposomal eEF-2K siRNA (150 µg/kg or about 4 µg/mouse) twice a week and/or doxorubicin once a week (i.p., 4 mg/kg), for 4 weeks. (B) Tumor weight of L-Control and L-eEF-2K siRNA treated mice. Therapeutic targeting of the eEF-2K gene was achieved by systemically administering (i.v. twice a week, 150 µg/kg) DOPC-liposomal siRNA (L-eEF-2K siRNA#1 and siRNA#2) in nude mice bearing MDA-MB-231 tumors. Non-silencing DOPC-liposomal siRNA (L-Control siRNA) was used as a control. After 4 weeks of treatment, tumor weight was measured. (C) The mean weight of mice after four weeks of treatment. No toxic effects were observed in mice exposed to liposomal eEF-2K siRNA ± doxorubicin for four weeks, compared with the control group. Mice appeared healthy and did not lose weight during treatment. (D–E) TUNEL assay indicating apoptosis (D) and quantification (% apoptosis) of TUNEL assay (E) in tumors in mice treated with L-eEF-2K siRNA and/or doxorubicin. Brown staining in the TUNEL assay is a positive indicator of apoptosis. (F) In vivo targeting of eEF-2K enhances the efficacy of chemotherapy. Mice bearing MDA-MB-231 tumors were given L-eEF-2K siRNA or L-Control siRNA (i.v. twice a week, 150 µg/kg) and doxorubicin once a week (i.p., 4 mg/kg) for 4 weeks, after which tumor weights were measured.

Figure 4. Molecular effects of in vivo targeting of eEF-2K by liposomal siRNA.

Following treatment with L-eEF-2K siRNA, tumor tissues were removed from mice and subjected to Western blot analysis. (A–B) Treatment of mice with L-eEF-2K siRNA results in the knockdown of eEF-2K in different tumors (A–B), with the consequent reduction in p-eEF2 (Thr-56) levels (B). (C) L-eEF-2K siRNA treatment induces apoptosis in tumors as indicated by caspase-9 cleavage and a decrease in anti-apoptotic Bcl-2 levels. (D) Depletion of eEF-2K enhances doxorubicin-induced Bcl-2 and HIF1α down-regulation.

Figure 5. Effect of down-regulation of eEF-2K by siRNA on doxorubicin efficacy against breast cancer cells.

(A) Western blot analysis demonstrated that knockdown of eEF-2K in doxorubicin-treated MDA-MB-231 cells further reduced cyclin D1 levels while increasing p27^Kip1 levels. (B) Knockdown of eEF-2K using two different siRNA enhances efficacy of doxorubicin-induced (0.1 and 0.5 ìM) apoptosis of MDA-MB-231 breast cancer cells. Apoptosis was detected by Annexin V staining and the percentage of positive cells was quantified by FACS analysis after 48 h of doxorubicin treatment. (C) Down-regulation of eEF-2K enhances inhibition of Src and FAK activity and increases caspase-9 cleavage in doxorubicin-treated MCF-7/DoxR cells.

Figure 6. Effect of knockdown or overexpression of eEF-2K on invasion of MDA-MB-231 cells.

Depletion of eEF-2K by siRNA inhibits invasion (A), while overexpression of eEF-2K increases invasion (B) of MDA-MB-231 cells in Matrigel (72 h).

Figure 7. Knockdown of eEF-2K enhances the inhibitory effect of paclitaxel on MDA-MB-231 cells.

Down-regulation of eEF-2K by siRNA enhances the efficacy of paclitaxel-induced (2 nM) inhibition of colony formation in highly aggressive and metastatic MDA-MB-231 breast cancer cells. (A) Cell colonies stained with crystal violet and (B) Quantification of colony formation represented graphically.

Figure 8. Targeting eEF-2K disrupts signal transduction pathways that promote tumorigenesis in breast cancer cells.

The phosphorylation of eEF2 by eEF-2K leads to an increase in the phospho-eEF2/eEF2 ratio in transformed breast cancer cells, which is predicted to influence protein translation, possibly facilitating the selective translation of mRNAs possessing structured 5′-UTRs encoding for proteins such as c-Myc, Bcl-2 and cyclin D1. eEF-2K facilitates basal c-Src activation through a mechanism that is currently unknown. c-Src is an important mediator of many downstream effects of receptor tyrosine kinases, including the EGFR family, and mediates basal activation of Akt by eEF-2K. Pathways facilitated by eEF-2K are implicated in cell growth, survival, motility and angiogenesis, supporting the notion that it may be a potential cancer therapeutic target. Known mechanisms are represented as solid arrows. Potential mechanisms are represented as dashed arrows.