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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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Both proteasome inhibition and autophagy inhibition induced HIF2α accumulation. (A) Caki-1 cells, (B) RPE cells were treated with 10 µM MG132 or 200 nM bafilomycin A1 for indicated time. (C) HEK293T stable cell line expressing HIF2α-GFP were treated with DMSO, 10 µM MG132 or 200 nM bafilomycin A1 for indicated time. Whole cell lysates were analyzed by immunoblot using antibodies against HIF2α, p62, LC3B, VHL, GFP p53 or β-actin. (D) Quantitation of HIF2α relative amount. HIF2α band intensity was analyzed using ImageJ software. HIF2α levels in non-treated cells or DMSO treated cells were normalized to 1. Data represent mean±S.D., n=3. *, p<0.05, **, p<0.001, compared with control cells. Con, control. MG, MG132. BM, bafilomycin A1. (E) HEK293T stable cell line expressing HIF2α-GFP were treated with 50 µM chloroquine for 8 hrs. CQ, chloroquine. (F) HEK293T stable cell line expressing HIF2α were treated with DMSO, 10 µM MG132 or 200 nM bafilomycin A1 for 8 hrs. Cytoplasmic and nuclear extracts were separated and analyzed by immunoblot using antibodies against GFP, cytoplasm protein LDHA and nuclear membrane protein Lamin A.
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Figure 1: Both proteasome inhibition and autophagy inhibition induced HIF2α accumulation. (A) Caki-1 cells, (B) RPE cells were treated with 10 µM MG132 or 200 nM bafilomycin A1 for indicated time. (C) HEK293T stable cell line expressing HIF2α-GFP were treated with DMSO, 10 µM MG132 or 200 nM bafilomycin A1 for indicated time. Whole cell lysates were analyzed by immunoblot using antibodies against HIF2α, p62, LC3B, VHL, GFP p53 or β-actin. (D) Quantitation of HIF2α relative amount. HIF2α band intensity was analyzed using ImageJ software. HIF2α levels in non-treated cells or DMSO treated cells were normalized to 1. Data represent mean±S.D., n=3. *, p<0.05, **, p<0.001, compared with control cells. Con, control. MG, MG132. BM, bafilomycin A1. (E) HEK293T stable cell line expressing HIF2α-GFP were treated with 50 µM chloroquine for 8 hrs. CQ, chloroquine. (F) HEK293T stable cell line expressing HIF2α were treated with DMSO, 10 µM MG132 or 200 nM bafilomycin A1 for 8 hrs. Cytoplasmic and nuclear extracts were separated and analyzed by immunoblot using antibodies against GFP, cytoplasm protein LDHA and nuclear membrane protein Lamin A.

Mentions: HIF2α is degraded by the proteasome after oxygen-dependent prolyl hydroxylation and VHL-dependent ubiquitination (1, 3). Under normoxia, HIF2α protein levels were very low in the VHL wildtype Caki-I RCC and in the retinal pigment epithelium (RPE) cell lines (Figure 1A, 1B), indicating that HIF2α is constitutively degraded in the presence of intact VHL. When these cell lines were treated with the proteasome inhibitor MG132, there was a gradual accumulation in HIF2α protein levels (Figure 1A, 1B). After 8 hours of treatment, HIF2α levels increased 6–7 fold (Figure 1A, 1B, 1D). These results confirmed that the proteasome was involved in constitutive degradation of HIF2α.


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)

Both proteasome inhibition and autophagy inhibition induced HIF2α accumulation. (A) Caki-1 cells, (B) RPE cells were treated with 10 µM MG132 or 200 nM bafilomycin A1 for indicated time. (C) HEK293T stable cell line expressing HIF2α-GFP were treated with DMSO, 10 µM MG132 or 200 nM bafilomycin A1 for indicated time. Whole cell lysates were analyzed by immunoblot using antibodies against HIF2α, p62, LC3B, VHL, GFP p53 or β-actin. (D) Quantitation of HIF2α relative amount. HIF2α band intensity was analyzed using ImageJ software. HIF2α levels in non-treated cells or DMSO treated cells were normalized to 1. Data represent mean±S.D., n=3. *, p<0.05, **, p<0.001, compared with control cells. Con, control. MG, MG132. BM, bafilomycin A1. (E) HEK293T stable cell line expressing HIF2α-GFP were treated with 50 µM chloroquine for 8 hrs. CQ, chloroquine. (F) HEK293T stable cell line expressing HIF2α were treated with DMSO, 10 µM MG132 or 200 nM bafilomycin A1 for 8 hrs. Cytoplasmic and nuclear extracts were separated and analyzed by immunoblot using antibodies against GFP, cytoplasm protein LDHA and nuclear membrane protein Lamin A.
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Figure 1: Both proteasome inhibition and autophagy inhibition induced HIF2α accumulation. (A) Caki-1 cells, (B) RPE cells were treated with 10 µM MG132 or 200 nM bafilomycin A1 for indicated time. (C) HEK293T stable cell line expressing HIF2α-GFP were treated with DMSO, 10 µM MG132 or 200 nM bafilomycin A1 for indicated time. Whole cell lysates were analyzed by immunoblot using antibodies against HIF2α, p62, LC3B, VHL, GFP p53 or β-actin. (D) Quantitation of HIF2α relative amount. HIF2α band intensity was analyzed using ImageJ software. HIF2α levels in non-treated cells or DMSO treated cells were normalized to 1. Data represent mean±S.D., n=3. *, p<0.05, **, p<0.001, compared with control cells. Con, control. MG, MG132. BM, bafilomycin A1. (E) HEK293T stable cell line expressing HIF2α-GFP were treated with 50 µM chloroquine for 8 hrs. CQ, chloroquine. (F) HEK293T stable cell line expressing HIF2α were treated with DMSO, 10 µM MG132 or 200 nM bafilomycin A1 for 8 hrs. Cytoplasmic and nuclear extracts were separated and analyzed by immunoblot using antibodies against GFP, cytoplasm protein LDHA and nuclear membrane protein Lamin A.
Mentions: HIF2α is degraded by the proteasome after oxygen-dependent prolyl hydroxylation and VHL-dependent ubiquitination (1, 3). Under normoxia, HIF2α protein levels were very low in the VHL wildtype Caki-I RCC and in the retinal pigment epithelium (RPE) cell lines (Figure 1A, 1B), indicating that HIF2α is constitutively degraded in the presence of intact VHL. When these cell lines were treated with the proteasome inhibitor MG132, there was a gradual accumulation in HIF2α protein levels (Figure 1A, 1B). After 8 hours of treatment, HIF2α levels increased 6–7 fold (Figure 1A, 1B, 1D). These results confirmed that the proteasome was involved in constitutive degradation of HIF2α.

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