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Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells.

Xie CM, Liu XY, Sham KW, Lai JM, Cheng CH - Autophagy (2014)

Bottom Line: However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood.Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206.In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences; The Chinese University of Hong Kong; Hong Kong, China.

ABSTRACT
EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca (2+)/calmodulin-dependent protein kinase III, functions in downregulating peptide chain elongation through inactivation of EEF2 (eukaryotic translation elongation factor 2). Currently, there is a limited amount of information on the promotion of autophagic survival by EEF2K in breast and glioblastoma cell lines. However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood. In this study, contrary to the reported autophagy-promoting activity of EEF2K in certain cancer cells, EEF2K is shown to negatively regulate autophagy in human colon cancer cells as indicated by the increase of LC3-II levels, the accumulation of LC3 dots per cell, and the promotion of autophagic flux in EEF2K knockdown cells. EEF2K negatively regulates cell viability, clonogenicity, cell proliferation, and cell size in colon cancer cells. Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206. In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation. Knockdown of AMPK or ULK1 significantly abrogates EEF2K silencing-induced increase of LC3-II levels, accumulation of LC3 dots per cell as well as cell proliferation in colon cancer cells. In conclusion, silencing of EEF2K promotes autophagic survival via activation of the AMPK-ULK1 pathway in colon cancer cells. This finding suggests that upregulation of EEF2K activity may constitute a novel approach for the treatment of human colon cancer.

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Figure 3.EEF2K silencing promotes cell survival in human colon cancer cells. (A) Representative western blot demonstrating the knockdown efficiency of EEF2K siRNA and the overexpression efficiency of EEF2K in both HT-29 and HCT-116 cells. Cells were transfected with control siRNA (siCTL), EEF2K siRNA (siEEF2K), empty vector (Vector), or EEF2K plasmids (EEF2K) for 48 h. (B and C) The effects of EEF2K knockdown and EEF2K overexpression on cell viability. HT-29 or HCT-116 cells were transfected as in (A), and then assessed by the MTT assay. (D and E) The effects of EEF2K knockdown and EEF2K overexpression on colony formation. HT-29 or HCT-116 cells were transfected as in (A). After 48 h transfection, cells were seeded into 6-well plates at the density of 150 cells per well for control siRNA and EEF2K siRNA groups (D) and 200 cells per well for the empty vector and EEF2K overexpression groups (E), incubated at 37 °C for 12 to 14 d, stained with crystal violet (0.5% w/v) and imaged. Colonies with 50 or more cells were counted. (F and G) The effects of EEF2K knockdown and EEF2K overexpression on cell size. HT-29 or HCT-116 cells were transfected as in (A). After 48 h transfection, cells were imaged using a Nikon fluorescence microscope. Scale bar: 20 μm. Cell size was analyzed using the MetaMorph software. The amounts of cell size in more than 50 cells for each group were quantified. (H and I) The effects of EEF2K knockdown and EEF2K overexpression on cell number. HT-29 or HCT-116 cells were transfected as in (A). Cell number was quantified after 48 h transfection. (J) The effect of EEF2K siRNA on oxaliplatin induced apoptosis. HCT-116 cells were transfected with control siRNA or EEF2K siRNA for 24 h, and then treated with vehicle (0.1% DMSO) or oxaliplatin (25 μM) for 48 h. Cells were stained with ANXA5-PI. The percentage of apoptotic cells (ANXA5+) was analyzed by flow cytometry. (K) The effect of EEF2K overexpression on oxaliplatin-induced apoptosis. HT-29 cells were transfected with empty vector (Vector), or EEF2K plasmids (EEF2K) for 24 h, and then treated with vehicle (0.1% DMSO) or oxaliplatin (25 μM) for 48 h. Cells were stained with ANXA5-PI and analyzed by flow cytometry as in (J). All quantitative data shown represent the means ± SEM of at least 3 independent experiments. *P < 0.05, $P < 0.01 and #P < 0.001, vs. the siCTL group (B, D, F, and H), the vector group (C, E, G, and I), or the oxaliplatin treatment only (J and K).
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Figure 3: Figure 3.EEF2K silencing promotes cell survival in human colon cancer cells. (A) Representative western blot demonstrating the knockdown efficiency of EEF2K siRNA and the overexpression efficiency of EEF2K in both HT-29 and HCT-116 cells. Cells were transfected with control siRNA (siCTL), EEF2K siRNA (siEEF2K), empty vector (Vector), or EEF2K plasmids (EEF2K) for 48 h. (B and C) The effects of EEF2K knockdown and EEF2K overexpression on cell viability. HT-29 or HCT-116 cells were transfected as in (A), and then assessed by the MTT assay. (D and E) The effects of EEF2K knockdown and EEF2K overexpression on colony formation. HT-29 or HCT-116 cells were transfected as in (A). After 48 h transfection, cells were seeded into 6-well plates at the density of 150 cells per well for control siRNA and EEF2K siRNA groups (D) and 200 cells per well for the empty vector and EEF2K overexpression groups (E), incubated at 37 °C for 12 to 14 d, stained with crystal violet (0.5% w/v) and imaged. Colonies with 50 or more cells were counted. (F and G) The effects of EEF2K knockdown and EEF2K overexpression on cell size. HT-29 or HCT-116 cells were transfected as in (A). After 48 h transfection, cells were imaged using a Nikon fluorescence microscope. Scale bar: 20 μm. Cell size was analyzed using the MetaMorph software. The amounts of cell size in more than 50 cells for each group were quantified. (H and I) The effects of EEF2K knockdown and EEF2K overexpression on cell number. HT-29 or HCT-116 cells were transfected as in (A). Cell number was quantified after 48 h transfection. (J) The effect of EEF2K siRNA on oxaliplatin induced apoptosis. HCT-116 cells were transfected with control siRNA or EEF2K siRNA for 24 h, and then treated with vehicle (0.1% DMSO) or oxaliplatin (25 μM) for 48 h. Cells were stained with ANXA5-PI. The percentage of apoptotic cells (ANXA5+) was analyzed by flow cytometry. (K) The effect of EEF2K overexpression on oxaliplatin-induced apoptosis. HT-29 cells were transfected with empty vector (Vector), or EEF2K plasmids (EEF2K) for 24 h, and then treated with vehicle (0.1% DMSO) or oxaliplatin (25 μM) for 48 h. Cells were stained with ANXA5-PI and analyzed by flow cytometry as in (J). All quantitative data shown represent the means ± SEM of at least 3 independent experiments. *P < 0.05, $P < 0.01 and #P < 0.001, vs. the siCTL group (B, D, F, and H), the vector group (C, E, G, and I), or the oxaliplatin treatment only (J and K).

Mentions: It has been reported that targeting EEF2K by siRNA reduces cancer growth in glioma and breast cancer cells.17,21 Contrary to this, silencing of EEF2K significantly promoted colon cancer cell viability and colony formation, suggesting that EEF2K negatively regulates cell proliferation in human colon cancer cells (Fig. 3A, B, and D). These findings were confirmed by the decrease of cell viability and colony formation in EEF2K-overexpressing cells as compared with control (Fig. 3A, C, and E). In order to further validate the observation on cell survival in EEF2K-silenced cells, we analyzed cell size and cell number in EEF2K knockdown cells as well as in EEF2K overexpressed cells. Both cell size and cell number in EEF2K-depleted cells were significantly increased compared with the control group (Fig. 3F and H), while the cell size and cell number were decreased in EEF2K-overexpressing cells (Fig. 3G and I). Taken together, EEF2K silencing promotes cell growth and cell proliferation in human colon cancer cells. This finding is further substantiated by the result that EEF2K silencing significantly attenuated the antitumor efficacy of oxaliplatin against colon cancer cells (Fig. 3J). Our findings in colon cancer cells are in accordance with other reports that knockdown of EEF2K by siRNA as well as reduction of EEF2 phosphorylation at effective concentrations by the EEF2K inhibitor A-484954 has little inhibitory effect on cancer cell growth in certain cancer cells including lung cancer and prostate cancer under both serum and serum-free conditions.23 In addition, overexpression of EEF2K could significantly enhance the antitumor efficacy of oxaliplatin against colon cancer cells, indicating that increase of EEF2K activity can be used to treat colon cancer (Fig. 3K).


Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells.

Xie CM, Liu XY, Sham KW, Lai JM, Cheng CH - Autophagy (2014)

Figure 3.EEF2K silencing promotes cell survival in human colon cancer cells. (A) Representative western blot demonstrating the knockdown efficiency of EEF2K siRNA and the overexpression efficiency of EEF2K in both HT-29 and HCT-116 cells. Cells were transfected with control siRNA (siCTL), EEF2K siRNA (siEEF2K), empty vector (Vector), or EEF2K plasmids (EEF2K) for 48 h. (B and C) The effects of EEF2K knockdown and EEF2K overexpression on cell viability. HT-29 or HCT-116 cells were transfected as in (A), and then assessed by the MTT assay. (D and E) The effects of EEF2K knockdown and EEF2K overexpression on colony formation. HT-29 or HCT-116 cells were transfected as in (A). After 48 h transfection, cells were seeded into 6-well plates at the density of 150 cells per well for control siRNA and EEF2K siRNA groups (D) and 200 cells per well for the empty vector and EEF2K overexpression groups (E), incubated at 37 °C for 12 to 14 d, stained with crystal violet (0.5% w/v) and imaged. Colonies with 50 or more cells were counted. (F and G) The effects of EEF2K knockdown and EEF2K overexpression on cell size. HT-29 or HCT-116 cells were transfected as in (A). After 48 h transfection, cells were imaged using a Nikon fluorescence microscope. Scale bar: 20 μm. Cell size was analyzed using the MetaMorph software. The amounts of cell size in more than 50 cells for each group were quantified. (H and I) The effects of EEF2K knockdown and EEF2K overexpression on cell number. HT-29 or HCT-116 cells were transfected as in (A). Cell number was quantified after 48 h transfection. (J) The effect of EEF2K siRNA on oxaliplatin induced apoptosis. HCT-116 cells were transfected with control siRNA or EEF2K siRNA for 24 h, and then treated with vehicle (0.1% DMSO) or oxaliplatin (25 μM) for 48 h. Cells were stained with ANXA5-PI. The percentage of apoptotic cells (ANXA5+) was analyzed by flow cytometry. (K) The effect of EEF2K overexpression on oxaliplatin-induced apoptosis. HT-29 cells were transfected with empty vector (Vector), or EEF2K plasmids (EEF2K) for 24 h, and then treated with vehicle (0.1% DMSO) or oxaliplatin (25 μM) for 48 h. Cells were stained with ANXA5-PI and analyzed by flow cytometry as in (J). All quantitative data shown represent the means ± SEM of at least 3 independent experiments. *P < 0.05, $P < 0.01 and #P < 0.001, vs. the siCTL group (B, D, F, and H), the vector group (C, E, G, and I), or the oxaliplatin treatment only (J and K).
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Figure 3: Figure 3.EEF2K silencing promotes cell survival in human colon cancer cells. (A) Representative western blot demonstrating the knockdown efficiency of EEF2K siRNA and the overexpression efficiency of EEF2K in both HT-29 and HCT-116 cells. Cells were transfected with control siRNA (siCTL), EEF2K siRNA (siEEF2K), empty vector (Vector), or EEF2K plasmids (EEF2K) for 48 h. (B and C) The effects of EEF2K knockdown and EEF2K overexpression on cell viability. HT-29 or HCT-116 cells were transfected as in (A), and then assessed by the MTT assay. (D and E) The effects of EEF2K knockdown and EEF2K overexpression on colony formation. HT-29 or HCT-116 cells were transfected as in (A). After 48 h transfection, cells were seeded into 6-well plates at the density of 150 cells per well for control siRNA and EEF2K siRNA groups (D) and 200 cells per well for the empty vector and EEF2K overexpression groups (E), incubated at 37 °C for 12 to 14 d, stained with crystal violet (0.5% w/v) and imaged. Colonies with 50 or more cells were counted. (F and G) The effects of EEF2K knockdown and EEF2K overexpression on cell size. HT-29 or HCT-116 cells were transfected as in (A). After 48 h transfection, cells were imaged using a Nikon fluorescence microscope. Scale bar: 20 μm. Cell size was analyzed using the MetaMorph software. The amounts of cell size in more than 50 cells for each group were quantified. (H and I) The effects of EEF2K knockdown and EEF2K overexpression on cell number. HT-29 or HCT-116 cells were transfected as in (A). Cell number was quantified after 48 h transfection. (J) The effect of EEF2K siRNA on oxaliplatin induced apoptosis. HCT-116 cells were transfected with control siRNA or EEF2K siRNA for 24 h, and then treated with vehicle (0.1% DMSO) or oxaliplatin (25 μM) for 48 h. Cells were stained with ANXA5-PI. The percentage of apoptotic cells (ANXA5+) was analyzed by flow cytometry. (K) The effect of EEF2K overexpression on oxaliplatin-induced apoptosis. HT-29 cells were transfected with empty vector (Vector), or EEF2K plasmids (EEF2K) for 24 h, and then treated with vehicle (0.1% DMSO) or oxaliplatin (25 μM) for 48 h. Cells were stained with ANXA5-PI and analyzed by flow cytometry as in (J). All quantitative data shown represent the means ± SEM of at least 3 independent experiments. *P < 0.05, $P < 0.01 and #P < 0.001, vs. the siCTL group (B, D, F, and H), the vector group (C, E, G, and I), or the oxaliplatin treatment only (J and K).
Mentions: It has been reported that targeting EEF2K by siRNA reduces cancer growth in glioma and breast cancer cells.17,21 Contrary to this, silencing of EEF2K significantly promoted colon cancer cell viability and colony formation, suggesting that EEF2K negatively regulates cell proliferation in human colon cancer cells (Fig. 3A, B, and D). These findings were confirmed by the decrease of cell viability and colony formation in EEF2K-overexpressing cells as compared with control (Fig. 3A, C, and E). In order to further validate the observation on cell survival in EEF2K-silenced cells, we analyzed cell size and cell number in EEF2K knockdown cells as well as in EEF2K overexpressed cells. Both cell size and cell number in EEF2K-depleted cells were significantly increased compared with the control group (Fig. 3F and H), while the cell size and cell number were decreased in EEF2K-overexpressing cells (Fig. 3G and I). Taken together, EEF2K silencing promotes cell growth and cell proliferation in human colon cancer cells. This finding is further substantiated by the result that EEF2K silencing significantly attenuated the antitumor efficacy of oxaliplatin against colon cancer cells (Fig. 3J). Our findings in colon cancer cells are in accordance with other reports that knockdown of EEF2K by siRNA as well as reduction of EEF2 phosphorylation at effective concentrations by the EEF2K inhibitor A-484954 has little inhibitory effect on cancer cell growth in certain cancer cells including lung cancer and prostate cancer under both serum and serum-free conditions.23 In addition, overexpression of EEF2K could significantly enhance the antitumor efficacy of oxaliplatin against colon cancer cells, indicating that increase of EEF2K activity can be used to treat colon cancer (Fig. 3K).

Bottom Line: However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood.Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206.In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences; The Chinese University of Hong Kong; Hong Kong, China.

ABSTRACT
EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca (2+)/calmodulin-dependent protein kinase III, functions in downregulating peptide chain elongation through inactivation of EEF2 (eukaryotic translation elongation factor 2). Currently, there is a limited amount of information on the promotion of autophagic survival by EEF2K in breast and glioblastoma cell lines. However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood. In this study, contrary to the reported autophagy-promoting activity of EEF2K in certain cancer cells, EEF2K is shown to negatively regulate autophagy in human colon cancer cells as indicated by the increase of LC3-II levels, the accumulation of LC3 dots per cell, and the promotion of autophagic flux in EEF2K knockdown cells. EEF2K negatively regulates cell viability, clonogenicity, cell proliferation, and cell size in colon cancer cells. Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206. In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation. Knockdown of AMPK or ULK1 significantly abrogates EEF2K silencing-induced increase of LC3-II levels, accumulation of LC3 dots per cell as well as cell proliferation in colon cancer cells. In conclusion, silencing of EEF2K promotes autophagic survival via activation of the AMPK-ULK1 pathway in colon cancer cells. This finding suggests that upregulation of EEF2K activity may constitute a novel approach for the treatment of human colon cancer.

Show MeSH
Related in: MedlinePlus