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Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death.

Xia HG, Najafov A, Geng J, Galan-Acosta L, Han X, Guo Y, Shan B, Zhang Y, Norberg E, Zhang T, Pan L, Liu J, Coloff JL, Ofengeim D, Zhu H, Wu K, Cai Y, Yates JR, Zhu Z, Yuan J, Vakifahmetoglu-Norberg H - J. Cell Biol. (2015)

Bottom Line: Here we show that metabolic stress triggered by perturbation of receptor tyrosine kinase FLT3 in non-acute myeloid leukemia cells sensitizes cancer cells to autophagy inhibition and leads to excessive activation of chaperone-mediated autophagy (CMA).Importantly, our proteome analysis revealed that HK2 is a CMA substrate and that its degradation by CMA is regulated by glucose availability.Our study delineates a novel pharmacological strategy to promote the degradation of HK2 in cancer cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115.

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Combination treatment of AC220 and spautins induces CMA. (A) WB quantification of mutant p53 levels in ES2 (top) for the indicated time points and in MDA-MB-231, Sum159, and MDA-MB-435 (bottom) cells treated with AC220 and/or C43 for 16 h. (B) Mutant p53 levels in scramble (SCR) or FLT3 siRNA–transfected ES2 cells treated with C43 for 24 h. This experiment used the same blot as that in Fig. 1 E with additional antibodies. (C) p53 levels in ES2 cells treated with C43 and AC220 for 24 h in the absence or presence of proteasome (MG132) or lysosomal inhibitor (ClQ). (D) p53, IκB, and GAPDH levels in ES2 and Sum159 cells treated with AC220 and/or C43 for the indicated time points. (E) WB of p53, Lamp2A, and Hsc70 levels in nontargeting (N.T.), Hsc70, or Lamp2A siRNA–transfected ES2 cells treated with AC220 and/or C43 for 24 h. (F) The cellular ATP levels (fold) in ES2 cells treated with AC220 and/or C43 in the presence or absence of zVAD or 7N-1 for 16 h. (G) Cell death indicated as Annexin V/PI positivity (fold) and WB of PARP-1 and caspase-3 cleavage in ES2 cells treated with AC220 and/or C43 in the presence or absence of zVAD or 7N-1 for 24 h. STS was used as a positive cell death inducer. (H) Cell death (fold) of nontargeting (N.T.), Lamp2A, or Hsc70 siRNA–transfected ES2 cells treated with AC220 and/or C43 for 24 h. Anti–α-tubulin was used as a loading control. Cells were treated with 0.1% DMSO (control: vehicle) or 1 µM AC220 and 10 µM C43, unless otherwise stated. In all the experiments, treatment groups were compared with the control group, unless otherwise indicated. Error bars indicate ±SD. *, P < 0.05; **, P < 0.01.
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fig3: Combination treatment of AC220 and spautins induces CMA. (A) WB quantification of mutant p53 levels in ES2 (top) for the indicated time points and in MDA-MB-231, Sum159, and MDA-MB-435 (bottom) cells treated with AC220 and/or C43 for 16 h. (B) Mutant p53 levels in scramble (SCR) or FLT3 siRNA–transfected ES2 cells treated with C43 for 24 h. This experiment used the same blot as that in Fig. 1 E with additional antibodies. (C) p53 levels in ES2 cells treated with C43 and AC220 for 24 h in the absence or presence of proteasome (MG132) or lysosomal inhibitor (ClQ). (D) p53, IκB, and GAPDH levels in ES2 and Sum159 cells treated with AC220 and/or C43 for the indicated time points. (E) WB of p53, Lamp2A, and Hsc70 levels in nontargeting (N.T.), Hsc70, or Lamp2A siRNA–transfected ES2 cells treated with AC220 and/or C43 for 24 h. (F) The cellular ATP levels (fold) in ES2 cells treated with AC220 and/or C43 in the presence or absence of zVAD or 7N-1 for 16 h. (G) Cell death indicated as Annexin V/PI positivity (fold) and WB of PARP-1 and caspase-3 cleavage in ES2 cells treated with AC220 and/or C43 in the presence or absence of zVAD or 7N-1 for 24 h. STS was used as a positive cell death inducer. (H) Cell death (fold) of nontargeting (N.T.), Lamp2A, or Hsc70 siRNA–transfected ES2 cells treated with AC220 and/or C43 for 24 h. Anti–α-tubulin was used as a loading control. Cells were treated with 0.1% DMSO (control: vehicle) or 1 µM AC220 and 10 µM C43, unless otherwise stated. In all the experiments, treatment groups were compared with the control group, unless otherwise indicated. Error bars indicate ±SD. *, P < 0.05; **, P < 0.01.

Mentions: C43 treatment under nutritional limiting or confluent conditions significantly accelerates the activation of CMA, which leads to the degradation of mutant p53 (Vakifahmetoglu-Norberg et al., 2013). We found that the combination of AC220 with either C43 or A70 treatment can similarly enhance the degradation of mutant p53 proteins in nonconfluent conditions in ES2 cells (Figs. 3 A and S3 A), whereas no significant change of p53 mRNA levels were observed (Fig. S3 B). The level of degradation of mutant p53 upon AC220 and C43 was comparable with the effect of C43 treatment observed in confluent conditions (Fig. S3 C). Significant reduction of mutant p53 in MDA-MB-231, Sum159, and MDA-MB-435 cell lines was also observed upon combination treatment (Fig. 3 A, bottom). In contrast, single treatment of C43, A70, or AC220 had no effect on the mutant p53 protein levels under nonconfluent conditions (Figs. 3 A and S3, A and C). The knockdown of FLT3 levels did not affect the levels of mutant p53 protein, but subsequent treatment with C43 induced degradation of mutant p53 (Fig. 3 B). Furthermore, while the proteasome inhibitor MG132 did not show an effect on the levels of mutant p53 protein, lysosomal inhibitor chloroquine (ClQ) attenuated AC220 and C43 combination-induced mutant p53 degradation in ES2 cells (Fig. 3 C), suggesting that mutant p53 is degraded through the lysosomal pathway. The reduction in mutant p53 levels upon AC220 and C43 treatment was accompanied by increased degradation of GAPDH and IκB, two known substrates of CMA (Aniento et al., 1993; Cuervo et al., 1998; Fig. 3 D). Additionally, siRNA-mediated knockdown of either Hsc70 or Lamp2A, the key mediators of CMA, in ES2 cells attenuated mutant p53 depletion induced by the combination treatment (Fig. 3 E), demonstrating that inhibition of FLT3 by AC220 in combination with C43 activates CMA in proliferating cells under nutritionally rich conditions.


Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death.

Xia HG, Najafov A, Geng J, Galan-Acosta L, Han X, Guo Y, Shan B, Zhang Y, Norberg E, Zhang T, Pan L, Liu J, Coloff JL, Ofengeim D, Zhu H, Wu K, Cai Y, Yates JR, Zhu Z, Yuan J, Vakifahmetoglu-Norberg H - J. Cell Biol. (2015)

Combination treatment of AC220 and spautins induces CMA. (A) WB quantification of mutant p53 levels in ES2 (top) for the indicated time points and in MDA-MB-231, Sum159, and MDA-MB-435 (bottom) cells treated with AC220 and/or C43 for 16 h. (B) Mutant p53 levels in scramble (SCR) or FLT3 siRNA–transfected ES2 cells treated with C43 for 24 h. This experiment used the same blot as that in Fig. 1 E with additional antibodies. (C) p53 levels in ES2 cells treated with C43 and AC220 for 24 h in the absence or presence of proteasome (MG132) or lysosomal inhibitor (ClQ). (D) p53, IκB, and GAPDH levels in ES2 and Sum159 cells treated with AC220 and/or C43 for the indicated time points. (E) WB of p53, Lamp2A, and Hsc70 levels in nontargeting (N.T.), Hsc70, or Lamp2A siRNA–transfected ES2 cells treated with AC220 and/or C43 for 24 h. (F) The cellular ATP levels (fold) in ES2 cells treated with AC220 and/or C43 in the presence or absence of zVAD or 7N-1 for 16 h. (G) Cell death indicated as Annexin V/PI positivity (fold) and WB of PARP-1 and caspase-3 cleavage in ES2 cells treated with AC220 and/or C43 in the presence or absence of zVAD or 7N-1 for 24 h. STS was used as a positive cell death inducer. (H) Cell death (fold) of nontargeting (N.T.), Lamp2A, or Hsc70 siRNA–transfected ES2 cells treated with AC220 and/or C43 for 24 h. Anti–α-tubulin was used as a loading control. Cells were treated with 0.1% DMSO (control: vehicle) or 1 µM AC220 and 10 µM C43, unless otherwise stated. In all the experiments, treatment groups were compared with the control group, unless otherwise indicated. Error bars indicate ±SD. *, P < 0.05; **, P < 0.01.
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fig3: Combination treatment of AC220 and spautins induces CMA. (A) WB quantification of mutant p53 levels in ES2 (top) for the indicated time points and in MDA-MB-231, Sum159, and MDA-MB-435 (bottom) cells treated with AC220 and/or C43 for 16 h. (B) Mutant p53 levels in scramble (SCR) or FLT3 siRNA–transfected ES2 cells treated with C43 for 24 h. This experiment used the same blot as that in Fig. 1 E with additional antibodies. (C) p53 levels in ES2 cells treated with C43 and AC220 for 24 h in the absence or presence of proteasome (MG132) or lysosomal inhibitor (ClQ). (D) p53, IκB, and GAPDH levels in ES2 and Sum159 cells treated with AC220 and/or C43 for the indicated time points. (E) WB of p53, Lamp2A, and Hsc70 levels in nontargeting (N.T.), Hsc70, or Lamp2A siRNA–transfected ES2 cells treated with AC220 and/or C43 for 24 h. (F) The cellular ATP levels (fold) in ES2 cells treated with AC220 and/or C43 in the presence or absence of zVAD or 7N-1 for 16 h. (G) Cell death indicated as Annexin V/PI positivity (fold) and WB of PARP-1 and caspase-3 cleavage in ES2 cells treated with AC220 and/or C43 in the presence or absence of zVAD or 7N-1 for 24 h. STS was used as a positive cell death inducer. (H) Cell death (fold) of nontargeting (N.T.), Lamp2A, or Hsc70 siRNA–transfected ES2 cells treated with AC220 and/or C43 for 24 h. Anti–α-tubulin was used as a loading control. Cells were treated with 0.1% DMSO (control: vehicle) or 1 µM AC220 and 10 µM C43, unless otherwise stated. In all the experiments, treatment groups were compared with the control group, unless otherwise indicated. Error bars indicate ±SD. *, P < 0.05; **, P < 0.01.
Mentions: C43 treatment under nutritional limiting or confluent conditions significantly accelerates the activation of CMA, which leads to the degradation of mutant p53 (Vakifahmetoglu-Norberg et al., 2013). We found that the combination of AC220 with either C43 or A70 treatment can similarly enhance the degradation of mutant p53 proteins in nonconfluent conditions in ES2 cells (Figs. 3 A and S3 A), whereas no significant change of p53 mRNA levels were observed (Fig. S3 B). The level of degradation of mutant p53 upon AC220 and C43 was comparable with the effect of C43 treatment observed in confluent conditions (Fig. S3 C). Significant reduction of mutant p53 in MDA-MB-231, Sum159, and MDA-MB-435 cell lines was also observed upon combination treatment (Fig. 3 A, bottom). In contrast, single treatment of C43, A70, or AC220 had no effect on the mutant p53 protein levels under nonconfluent conditions (Figs. 3 A and S3, A and C). The knockdown of FLT3 levels did not affect the levels of mutant p53 protein, but subsequent treatment with C43 induced degradation of mutant p53 (Fig. 3 B). Furthermore, while the proteasome inhibitor MG132 did not show an effect on the levels of mutant p53 protein, lysosomal inhibitor chloroquine (ClQ) attenuated AC220 and C43 combination-induced mutant p53 degradation in ES2 cells (Fig. 3 C), suggesting that mutant p53 is degraded through the lysosomal pathway. The reduction in mutant p53 levels upon AC220 and C43 treatment was accompanied by increased degradation of GAPDH and IκB, two known substrates of CMA (Aniento et al., 1993; Cuervo et al., 1998; Fig. 3 D). Additionally, siRNA-mediated knockdown of either Hsc70 or Lamp2A, the key mediators of CMA, in ES2 cells attenuated mutant p53 depletion induced by the combination treatment (Fig. 3 E), demonstrating that inhibition of FLT3 by AC220 in combination with C43 activates CMA in proliferating cells under nutritionally rich conditions.

Bottom Line: Here we show that metabolic stress triggered by perturbation of receptor tyrosine kinase FLT3 in non-acute myeloid leukemia cells sensitizes cancer cells to autophagy inhibition and leads to excessive activation of chaperone-mediated autophagy (CMA).Importantly, our proteome analysis revealed that HK2 is a CMA substrate and that its degradation by CMA is regulated by glucose availability.Our study delineates a novel pharmacological strategy to promote the degradation of HK2 in cancer cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115.

Show MeSH
Related in: MedlinePlus