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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 2. BECN1 and ATG7 are required for autophagy in response to EEF2K silencing. (A) Silencing of EEF2K upregulates the protein levels of BECN1 and ATG7, but not ATG5. HT-29 or HCT-116 cells were transfected with nontargeting control siRNA (siCTL) or a single EEF2K siRNA duplex (siEEF2K) for 48 h. ATG5, BECN1, ATG7, and ACTB were analyzed by western blot. Data shown are representative of more than 3 independent experiments. (B) Silencing of EEF2K does not change the mRNA levels of ATG5, BECN1, and ATG7. Cells were transfected as in (A). The mRNA levels of ATG5, BECN1, and ATG7 were analyzed by RT-PCR. (C) Effect of MG132 on the protein levels of ATG5, BECN1, and ATG7 in EEF2K knockdown cells. HCT-116 cells were transfected with nontargeting control siRNA (siCTL) or a single EEF2K siRNA duplex (siEEF2K) for 48. Before harvested for western blot, cells were treated with MG132 (10 μM) for 12 h. (D and E) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effects of BECN1 siRNA (D) and ATG7 siRNA (E) on LC3-II levels induced by EEF2K silencing. HT-29 cells were transfected with nontargeting siRNA, siEEF2K, BECN1 siRNA (siBECN1), ATG7 siRNA (siATG7), siEEF2K plus siBECN1, or siEEF2K plus siATG7 for 48 h. All quantitative data shown represent the means ± SEM of at least 3 independent experiments. *P < 0.05 and $P < 0.01, vs. the siEEF2K group. (F) The effects of BECN1 siRNA and ATG7 siRNA on LC3 dots accumulation induced by EEF2K silencing. HT-29 cells were treated with siRNAs against EEF2K, BECN1, or ATG7 as in (D and E). Cells were fixed, stained for LC3, and imaged. The average number of LC3 dots per cell was counted in more than 5 fields with at least 100 cells for each group and expressed as the means ± SEM of 3 independent experiments. #P < 0.001, vs. the EEF2K siRNA group (siEEF2K).
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Figure 2: Figure 2. BECN1 and ATG7 are required for autophagy in response to EEF2K silencing. (A) Silencing of EEF2K upregulates the protein levels of BECN1 and ATG7, but not ATG5. HT-29 or HCT-116 cells were transfected with nontargeting control siRNA (siCTL) or a single EEF2K siRNA duplex (siEEF2K) for 48 h. ATG5, BECN1, ATG7, and ACTB were analyzed by western blot. Data shown are representative of more than 3 independent experiments. (B) Silencing of EEF2K does not change the mRNA levels of ATG5, BECN1, and ATG7. Cells were transfected as in (A). The mRNA levels of ATG5, BECN1, and ATG7 were analyzed by RT-PCR. (C) Effect of MG132 on the protein levels of ATG5, BECN1, and ATG7 in EEF2K knockdown cells. HCT-116 cells were transfected with nontargeting control siRNA (siCTL) or a single EEF2K siRNA duplex (siEEF2K) for 48. Before harvested for western blot, cells were treated with MG132 (10 μM) for 12 h. (D and E) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effects of BECN1 siRNA (D) and ATG7 siRNA (E) on LC3-II levels induced by EEF2K silencing. HT-29 cells were transfected with nontargeting siRNA, siEEF2K, BECN1 siRNA (siBECN1), ATG7 siRNA (siATG7), siEEF2K plus siBECN1, or siEEF2K plus siATG7 for 48 h. All quantitative data shown represent the means ± SEM of at least 3 independent experiments. *P < 0.05 and $P < 0.01, vs. the siEEF2K group. (F) The effects of BECN1 siRNA and ATG7 siRNA on LC3 dots accumulation induced by EEF2K silencing. HT-29 cells were treated with siRNAs against EEF2K, BECN1, or ATG7 as in (D and E). Cells were fixed, stained for LC3, and imaged. The average number of LC3 dots per cell was counted in more than 5 fields with at least 100 cells for each group and expressed as the means ± SEM of 3 independent experiments. #P < 0.001, vs. the EEF2K siRNA group (siEEF2K).

Mentions: A series of autophagy-related (ATG) genes are involved in the process of autophagy. We would like to know whether autophagy induced by silencing of EEF2K contributes to regulation of specific proteins of the ATG family. ATG5 and ATG7 (a ubiquitin-activating enzyme homolog), are required for initiation of autophagy. BECN1 is required for the initiation of autophagosome formation. Previous studies show that autophagy can be induced through ATG5-, BECN1-, or ATG7-dependent or independent signaling pathways.14,25 To determine whether induction of autophagy by EEF2K silencing is related to ATG5, BECN1, or ATG7, we first analyzed the expression levels of ATG5, BECN1, and ATG7 separately by western blot. As shown in Figure 2A, knockdown of EEF2K significantly increased the protein levels of BECN1 and ATG7, but not ATG5. The increase in BECN1 and ATG7 levels in EEF2K-depleted cells is attributed to protein synthesis but not to transcriptional increase (Fig. 2B). In order to further validate the increased BECN1 and ATG7 due to protein synthesis, we blocked protein degradation by MG132. The result showed that protein levels of BECN1 and ATG7 were significantly accumulated in EEF2K-depleted cells after exposure to MG132, suggesting EEF2K silencing does not block protein degradation of BECN1 and ATG7 (Fig. 2C). Taken together, the increase of both BECN1 and ATG7 in EEF2K knockdown cells is not due to blockage of degradation but to protein synthesis. We silenced BECN1 using siRNA in HT-29 cells. The result showed that knockdown of BECN1 could significantly block the accumulation of LC3-II in EEF2K-silenced cells (Fig. 2D). Similar to the effect of BECN1 knockdown, silencing of ATG7 also markedly attenuated the accumulation of LC3-II (Fig. 2E). Moreover, the number of LC3 dots per cell was significantly reduced after silencing of BECN1 or ATG7 in EEF2K knockdown cells (Fig. 2F). Taken together, these results indicate that the upregulation of BECN1 and ATG7 is responsible for autophagy induced by EEF2K silencing.


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 2. BECN1 and ATG7 are required for autophagy in response to EEF2K silencing. (A) Silencing of EEF2K upregulates the protein levels of BECN1 and ATG7, but not ATG5. HT-29 or HCT-116 cells were transfected with nontargeting control siRNA (siCTL) or a single EEF2K siRNA duplex (siEEF2K) for 48 h. ATG5, BECN1, ATG7, and ACTB were analyzed by western blot. Data shown are representative of more than 3 independent experiments. (B) Silencing of EEF2K does not change the mRNA levels of ATG5, BECN1, and ATG7. Cells were transfected as in (A). The mRNA levels of ATG5, BECN1, and ATG7 were analyzed by RT-PCR. (C) Effect of MG132 on the protein levels of ATG5, BECN1, and ATG7 in EEF2K knockdown cells. HCT-116 cells were transfected with nontargeting control siRNA (siCTL) or a single EEF2K siRNA duplex (siEEF2K) for 48. Before harvested for western blot, cells were treated with MG132 (10 μM) for 12 h. (D and E) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effects of BECN1 siRNA (D) and ATG7 siRNA (E) on LC3-II levels induced by EEF2K silencing. HT-29 cells were transfected with nontargeting siRNA, siEEF2K, BECN1 siRNA (siBECN1), ATG7 siRNA (siATG7), siEEF2K plus siBECN1, or siEEF2K plus siATG7 for 48 h. All quantitative data shown represent the means ± SEM of at least 3 independent experiments. *P < 0.05 and $P < 0.01, vs. the siEEF2K group. (F) The effects of BECN1 siRNA and ATG7 siRNA on LC3 dots accumulation induced by EEF2K silencing. HT-29 cells were treated with siRNAs against EEF2K, BECN1, or ATG7 as in (D and E). Cells were fixed, stained for LC3, and imaged. The average number of LC3 dots per cell was counted in more than 5 fields with at least 100 cells for each group and expressed as the means ± SEM of 3 independent experiments. #P < 0.001, vs. the EEF2K siRNA group (siEEF2K).
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Figure 2: Figure 2. BECN1 and ATG7 are required for autophagy in response to EEF2K silencing. (A) Silencing of EEF2K upregulates the protein levels of BECN1 and ATG7, but not ATG5. HT-29 or HCT-116 cells were transfected with nontargeting control siRNA (siCTL) or a single EEF2K siRNA duplex (siEEF2K) for 48 h. ATG5, BECN1, ATG7, and ACTB were analyzed by western blot. Data shown are representative of more than 3 independent experiments. (B) Silencing of EEF2K does not change the mRNA levels of ATG5, BECN1, and ATG7. Cells were transfected as in (A). The mRNA levels of ATG5, BECN1, and ATG7 were analyzed by RT-PCR. (C) Effect of MG132 on the protein levels of ATG5, BECN1, and ATG7 in EEF2K knockdown cells. HCT-116 cells were transfected with nontargeting control siRNA (siCTL) or a single EEF2K siRNA duplex (siEEF2K) for 48. Before harvested for western blot, cells were treated with MG132 (10 μM) for 12 h. (D and E) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effects of BECN1 siRNA (D) and ATG7 siRNA (E) on LC3-II levels induced by EEF2K silencing. HT-29 cells were transfected with nontargeting siRNA, siEEF2K, BECN1 siRNA (siBECN1), ATG7 siRNA (siATG7), siEEF2K plus siBECN1, or siEEF2K plus siATG7 for 48 h. All quantitative data shown represent the means ± SEM of at least 3 independent experiments. *P < 0.05 and $P < 0.01, vs. the siEEF2K group. (F) The effects of BECN1 siRNA and ATG7 siRNA on LC3 dots accumulation induced by EEF2K silencing. HT-29 cells were treated with siRNAs against EEF2K, BECN1, or ATG7 as in (D and E). Cells were fixed, stained for LC3, and imaged. The average number of LC3 dots per cell was counted in more than 5 fields with at least 100 cells for each group and expressed as the means ± SEM of 3 independent experiments. #P < 0.001, vs. the EEF2K siRNA group (siEEF2K).
Mentions: A series of autophagy-related (ATG) genes are involved in the process of autophagy. We would like to know whether autophagy induced by silencing of EEF2K contributes to regulation of specific proteins of the ATG family. ATG5 and ATG7 (a ubiquitin-activating enzyme homolog), are required for initiation of autophagy. BECN1 is required for the initiation of autophagosome formation. Previous studies show that autophagy can be induced through ATG5-, BECN1-, or ATG7-dependent or independent signaling pathways.14,25 To determine whether induction of autophagy by EEF2K silencing is related to ATG5, BECN1, or ATG7, we first analyzed the expression levels of ATG5, BECN1, and ATG7 separately by western blot. As shown in Figure 2A, knockdown of EEF2K significantly increased the protein levels of BECN1 and ATG7, but not ATG5. The increase in BECN1 and ATG7 levels in EEF2K-depleted cells is attributed to protein synthesis but not to transcriptional increase (Fig. 2B). In order to further validate the increased BECN1 and ATG7 due to protein synthesis, we blocked protein degradation by MG132. The result showed that protein levels of BECN1 and ATG7 were significantly accumulated in EEF2K-depleted cells after exposure to MG132, suggesting EEF2K silencing does not block protein degradation of BECN1 and ATG7 (Fig. 2C). Taken together, the increase of both BECN1 and ATG7 in EEF2K knockdown cells is not due to blockage of degradation but to protein synthesis. We silenced BECN1 using siRNA in HT-29 cells. The result showed that knockdown of BECN1 could significantly block the accumulation of LC3-II in EEF2K-silenced cells (Fig. 2D). Similar to the effect of BECN1 knockdown, silencing of ATG7 also markedly attenuated the accumulation of LC3-II (Fig. 2E). Moreover, the number of LC3 dots per cell was significantly reduced after silencing of BECN1 or ATG7 in EEF2K knockdown cells (Fig. 2F). Taken together, these results indicate that the upregulation of BECN1 and ATG7 is responsible for autophagy induced by EEF2K silencing.

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