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Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis.

Liu XD, Yao J, Tripathi DN, Ding Z, Xu Y, Sun M, Zhang J, Bai S, German P, Hoang A, Zhou L, Jonasch D, Zhang X, Conti CJ, Efstathiou E, Tannir NM, Eissa NT, Mills GB, Walker CL, Jonasch E - Oncogene (2014)

Bottom Line: Inhibition of autophagy increases HIF2α, whereas induction of autophagy decreases HIF2α.Autophagy inactivation redirects HIF2α to proteasomal degradation, whereas proteasome inhibition induces autophagy and increases the HIF2α-p62 interaction.Importantly, clear-cell renal cell carcinoma (ccRCC) is frequently associated with monoallelic loss and/or mutation of autophagy-related gene ATG7, and the low expression level of autophagy genes correlates with ccRCC progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
Autophagy is a conserved process involved in lysosomal degradation of protein aggregates and damaged organelles. The role of autophagy in cancer is a topic of intense debate, and the underlying mechanism is still not clear. The hypoxia-inducible factor 2α (HIF2α), an oncogenic transcription factor implicated in renal tumorigenesis, is known to be degraded by the ubiquitin-proteasome system (UPS). Here, we report that HIF2α is in part constitutively degraded by autophagy. HIF2α interacts with autophagy-lysosome system components. Inhibition of autophagy increases HIF2α, whereas induction of autophagy decreases HIF2α. The E3 ligase von Hippel-Lindau and autophagy receptor protein p62 are required for autophagic degradation of HIF2α. There is a compensatory interaction between the UPS and autophagy in HIF2α degradation. Autophagy inactivation redirects HIF2α to proteasomal degradation, whereas proteasome inhibition induces autophagy and increases the HIF2α-p62 interaction. Importantly, clear-cell renal cell carcinoma (ccRCC) is frequently associated with monoallelic loss and/or mutation of autophagy-related gene ATG7, and the low expression level of autophagy genes correlates with ccRCC progression. The protein levels of ATG7 and beclin 1 are also reduced in ccRCC tumors. This study indicates that autophagy has an anticancer role in ccRCC tumorigenesis, and suggests that constitutive autophagic degradation of HIF2α is a novel tumor suppression mechanism.

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HIF2α interacted with autophagy and lysosome components. (A) Colocalization between HIF2α-GFP and LC3A-RFP. HEK293T stable cell line expressing HIF2α-GFP were transiently transfected with LC3A-RFP. Representative images with HIF2α-GFP aggregates and autophagosomes were shown. Scale bar, 10um. (B) HEK293T cells were transiently transfected with GFP or HIF2α-GFP. (C) HEK293T cells were transiently transfected with GFP or HIF2α-GFP. After 48 hr, cells were further treated with 200 nM bafilomycin A1 (BM) or 10 µM MG132 (MG) for 6 hr. (D) HEK293T cells were transiently transfected with GFP or LC3A-GFP with or without HIF2α-HA. Cells were lysed in GFB buffer containing 1% Triton X-100. The soluble portion was subjected to immunoprecipation (GFP-Trap) using Chromotek-GFP-Trap beads. The insoluble pellets were dissolved in 1% SDS (Insoluble). The soluble portion (Input) or immunoprecipitated proteins (GFP-Trap) were subjected to immunoblot assay using antibodies against HA, GFP, HIF2α, p62 or LAMP1. Con, control. St3, starvation 3hr. Rap6, rapamycin 6 hr.
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Figure 4: HIF2α interacted with autophagy and lysosome components. (A) Colocalization between HIF2α-GFP and LC3A-RFP. HEK293T stable cell line expressing HIF2α-GFP were transiently transfected with LC3A-RFP. Representative images with HIF2α-GFP aggregates and autophagosomes were shown. Scale bar, 10um. (B) HEK293T cells were transiently transfected with GFP or HIF2α-GFP. (C) HEK293T cells were transiently transfected with GFP or HIF2α-GFP. After 48 hr, cells were further treated with 200 nM bafilomycin A1 (BM) or 10 µM MG132 (MG) for 6 hr. (D) HEK293T cells were transiently transfected with GFP or LC3A-GFP with or without HIF2α-HA. Cells were lysed in GFB buffer containing 1% Triton X-100. The soluble portion was subjected to immunoprecipation (GFP-Trap) using Chromotek-GFP-Trap beads. The insoluble pellets were dissolved in 1% SDS (Insoluble). The soluble portion (Input) or immunoprecipitated proteins (GFP-Trap) were subjected to immunoblot assay using antibodies against HA, GFP, HIF2α, p62 or LAMP1. Con, control. St3, starvation 3hr. Rap6, rapamycin 6 hr.

Mentions: In HEK293T cells, stably expressed HIF2α-GFP was mainly localized to the cytoplasm (Figure 4A), which is consistent with previous fractionation results (Figure 1D). Importantly, some cells contain HIF2α-GFP aggregates (Figure 4A), which are supposed to be degraded by autophagy. We then transiently transfected this cell line with LC3A-RFP, and found that HIF2α-GFP aggregates co-localized with autophagosomes, as indicated by the LC3A-RFP punctate cytoplasmic structures (Figure 4A).


Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis.

Liu XD, Yao J, Tripathi DN, Ding Z, Xu Y, Sun M, Zhang J, Bai S, German P, Hoang A, Zhou L, Jonasch D, Zhang X, Conti CJ, Efstathiou E, Tannir NM, Eissa NT, Mills GB, Walker CL, Jonasch E - Oncogene (2014)

HIF2α interacted with autophagy and lysosome components. (A) Colocalization between HIF2α-GFP and LC3A-RFP. HEK293T stable cell line expressing HIF2α-GFP were transiently transfected with LC3A-RFP. Representative images with HIF2α-GFP aggregates and autophagosomes were shown. Scale bar, 10um. (B) HEK293T cells were transiently transfected with GFP or HIF2α-GFP. (C) HEK293T cells were transiently transfected with GFP or HIF2α-GFP. After 48 hr, cells were further treated with 200 nM bafilomycin A1 (BM) or 10 µM MG132 (MG) for 6 hr. (D) HEK293T cells were transiently transfected with GFP or LC3A-GFP with or without HIF2α-HA. Cells were lysed in GFB buffer containing 1% Triton X-100. The soluble portion was subjected to immunoprecipation (GFP-Trap) using Chromotek-GFP-Trap beads. The insoluble pellets were dissolved in 1% SDS (Insoluble). The soluble portion (Input) or immunoprecipitated proteins (GFP-Trap) were subjected to immunoblot assay using antibodies against HA, GFP, HIF2α, p62 or LAMP1. Con, control. St3, starvation 3hr. Rap6, rapamycin 6 hr.
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Figure 4: HIF2α interacted with autophagy and lysosome components. (A) Colocalization between HIF2α-GFP and LC3A-RFP. HEK293T stable cell line expressing HIF2α-GFP were transiently transfected with LC3A-RFP. Representative images with HIF2α-GFP aggregates and autophagosomes were shown. Scale bar, 10um. (B) HEK293T cells were transiently transfected with GFP or HIF2α-GFP. (C) HEK293T cells were transiently transfected with GFP or HIF2α-GFP. After 48 hr, cells were further treated with 200 nM bafilomycin A1 (BM) or 10 µM MG132 (MG) for 6 hr. (D) HEK293T cells were transiently transfected with GFP or LC3A-GFP with or without HIF2α-HA. Cells were lysed in GFB buffer containing 1% Triton X-100. The soluble portion was subjected to immunoprecipation (GFP-Trap) using Chromotek-GFP-Trap beads. The insoluble pellets were dissolved in 1% SDS (Insoluble). The soluble portion (Input) or immunoprecipitated proteins (GFP-Trap) were subjected to immunoblot assay using antibodies against HA, GFP, HIF2α, p62 or LAMP1. Con, control. St3, starvation 3hr. Rap6, rapamycin 6 hr.
Mentions: In HEK293T cells, stably expressed HIF2α-GFP was mainly localized to the cytoplasm (Figure 4A), which is consistent with previous fractionation results (Figure 1D). Importantly, some cells contain HIF2α-GFP aggregates (Figure 4A), which are supposed to be degraded by autophagy. We then transiently transfected this cell line with LC3A-RFP, and found that HIF2α-GFP aggregates co-localized with autophagosomes, as indicated by the LC3A-RFP punctate cytoplasmic structures (Figure 4A).

Bottom Line: Inhibition of autophagy increases HIF2α, whereas induction of autophagy decreases HIF2α.Autophagy inactivation redirects HIF2α to proteasomal degradation, whereas proteasome inhibition induces autophagy and increases the HIF2α-p62 interaction.Importantly, clear-cell renal cell carcinoma (ccRCC) is frequently associated with monoallelic loss and/or mutation of autophagy-related gene ATG7, and the low expression level of autophagy genes correlates with ccRCC progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
Autophagy is a conserved process involved in lysosomal degradation of protein aggregates and damaged organelles. The role of autophagy in cancer is a topic of intense debate, and the underlying mechanism is still not clear. The hypoxia-inducible factor 2α (HIF2α), an oncogenic transcription factor implicated in renal tumorigenesis, is known to be degraded by the ubiquitin-proteasome system (UPS). Here, we report that HIF2α is in part constitutively degraded by autophagy. HIF2α interacts with autophagy-lysosome system components. Inhibition of autophagy increases HIF2α, whereas induction of autophagy decreases HIF2α. The E3 ligase von Hippel-Lindau and autophagy receptor protein p62 are required for autophagic degradation of HIF2α. There is a compensatory interaction between the UPS and autophagy in HIF2α degradation. Autophagy inactivation redirects HIF2α to proteasomal degradation, whereas proteasome inhibition induces autophagy and increases the HIF2α-p62 interaction. Importantly, clear-cell renal cell carcinoma (ccRCC) is frequently associated with monoallelic loss and/or mutation of autophagy-related gene ATG7, and the low expression level of autophagy genes correlates with ccRCC progression. The protein levels of ATG7 and beclin 1 are also reduced in ccRCC tumors. This study indicates that autophagy has an anticancer role in ccRCC tumorigenesis, and suggests that constitutive autophagic degradation of HIF2α is a novel tumor suppression mechanism.

Show MeSH
Related in: MedlinePlus