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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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VHL-dependent HIF2α accumulation during autophagy inhibition. (A) 786-O parental cells or 786-O stable cell line expressing VHL-wt-Venus were treated with 10 µM MG132 for indicated time. (B) 786-O parental cells or 786-O stable cell line expressing VHL-wt-Venus were treated with 200 nM bafilomycin A1 for indicated time. (C) Quantitation of HIF2α relative amount. HIF2α band intensity was analyzed using ImageJ software. HIF2α levels in control 786-O stable cell line expressing VHL-wt-Venus cells were normalized to 1. Data represent mean±S.D., n=3. **, p<0.001, compared with control cells. (D) HIF2α target gene expression. 786-O stable cell line expressing VHL-wt-Venus were treated with 200 nM bafilomycin A1 for 16 hrs. Total RNA were analyzed by Real-Time PCR using primers specific for HIF2α, VEGFA, TGFA and CCND1. mRNA level in control cells are normalized to 1. Data represent mean±S.D., n=3. **, p<0.001, compared with control cells. (E) 786-O cells were treated with 200 nM bafilomycin A1 for indicated time with or without 8-hr co-treatment with 100 µM CoCl2. (F) 786-O stable cell lines expressing HA-wt-VHL or HA-W117R-VHL were treated with 200 nM bafilomycin A1 for indicated time. (G) 786-O stable cell lines expressing GFP, VHL-wt-Venus, VHL-R167Q-Venus or VHL-F148A-Venus were treated with 200 nM bafilomycin A1 for indicated time. (H) 786-O stable cell lines expressing VHL-wt-Venus were treated with 200 nM bafilomycin A1 for 8 hrs. Cytoplasmic and nuclear extracts were separated. Cell lysates were analyzed by immunoblot using antibodies against HIF2α, p62, LC3B, VHL, GFP, LDHA, LaminA or β-actin. Con, control. MG, MG132. BM, bafilomycin A1.
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Figure 2: VHL-dependent HIF2α accumulation during autophagy inhibition. (A) 786-O parental cells or 786-O stable cell line expressing VHL-wt-Venus were treated with 10 µM MG132 for indicated time. (B) 786-O parental cells or 786-O stable cell line expressing VHL-wt-Venus were treated with 200 nM bafilomycin A1 for indicated time. (C) Quantitation of HIF2α relative amount. HIF2α band intensity was analyzed using ImageJ software. HIF2α levels in control 786-O stable cell line expressing VHL-wt-Venus cells were normalized to 1. Data represent mean±S.D., n=3. **, p<0.001, compared with control cells. (D) HIF2α target gene expression. 786-O stable cell line expressing VHL-wt-Venus were treated with 200 nM bafilomycin A1 for 16 hrs. Total RNA were analyzed by Real-Time PCR using primers specific for HIF2α, VEGFA, TGFA and CCND1. mRNA level in control cells are normalized to 1. Data represent mean±S.D., n=3. **, p<0.001, compared with control cells. (E) 786-O cells were treated with 200 nM bafilomycin A1 for indicated time with or without 8-hr co-treatment with 100 µM CoCl2. (F) 786-O stable cell lines expressing HA-wt-VHL or HA-W117R-VHL were treated with 200 nM bafilomycin A1 for indicated time. (G) 786-O stable cell lines expressing GFP, VHL-wt-Venus, VHL-R167Q-Venus or VHL-F148A-Venus were treated with 200 nM bafilomycin A1 for indicated time. (H) 786-O stable cell lines expressing VHL-wt-Venus were treated with 200 nM bafilomycin A1 for 8 hrs. Cytoplasmic and nuclear extracts were separated. Cell lysates were analyzed by immunoblot using antibodies against HIF2α, p62, LC3B, VHL, GFP, LDHA, LaminA or β-actin. Con, control. MG, MG132. BM, bafilomycin A1.

Mentions: The basal level of HIF2α in 786-O parental cells was relatively high (Figure 2A–2C), and was dramatically reduced with the stable expression of exogenous VHL (Figure 2A–2C). These results indicate that 786-O cells are incapable of degrading HIF2α due to the absence of functional VHL. In 786-O parental cells, neither MG132 nor bafilomycin A1 could significantly increase the already high HIF2α protein levels (Figure 2A–2C). Both agents led to accumulation of HIF2α protein in VHL-expressing 786-O cells (Figure 2A–2C). These results indicate that VHL is required not only for proteasome-mediated HIF2α degradation, but also for autophagy-mediated degradation. In contrast to HIF2α, VHL protein level was only enhanced by MG132 (Figure 2A) but not bafilomycin A1 (Figure 2B), suggesting that VHL was exclusively degraded by the proteasome, and bafilomycin A1-induced HIF2α accumulation was not due to the side effect of proteasomal inhibition.


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)

VHL-dependent HIF2α accumulation during autophagy inhibition. (A) 786-O parental cells or 786-O stable cell line expressing VHL-wt-Venus were treated with 10 µM MG132 for indicated time. (B) 786-O parental cells or 786-O stable cell line expressing VHL-wt-Venus were treated with 200 nM bafilomycin A1 for indicated time. (C) Quantitation of HIF2α relative amount. HIF2α band intensity was analyzed using ImageJ software. HIF2α levels in control 786-O stable cell line expressing VHL-wt-Venus cells were normalized to 1. Data represent mean±S.D., n=3. **, p<0.001, compared with control cells. (D) HIF2α target gene expression. 786-O stable cell line expressing VHL-wt-Venus were treated with 200 nM bafilomycin A1 for 16 hrs. Total RNA were analyzed by Real-Time PCR using primers specific for HIF2α, VEGFA, TGFA and CCND1. mRNA level in control cells are normalized to 1. Data represent mean±S.D., n=3. **, p<0.001, compared with control cells. (E) 786-O cells were treated with 200 nM bafilomycin A1 for indicated time with or without 8-hr co-treatment with 100 µM CoCl2. (F) 786-O stable cell lines expressing HA-wt-VHL or HA-W117R-VHL were treated with 200 nM bafilomycin A1 for indicated time. (G) 786-O stable cell lines expressing GFP, VHL-wt-Venus, VHL-R167Q-Venus or VHL-F148A-Venus were treated with 200 nM bafilomycin A1 for indicated time. (H) 786-O stable cell lines expressing VHL-wt-Venus were treated with 200 nM bafilomycin A1 for 8 hrs. Cytoplasmic and nuclear extracts were separated. Cell lysates were analyzed by immunoblot using antibodies against HIF2α, p62, LC3B, VHL, GFP, LDHA, LaminA or β-actin. Con, control. MG, MG132. BM, bafilomycin A1.
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Related In: Results  -  Collection

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Figure 2: VHL-dependent HIF2α accumulation during autophagy inhibition. (A) 786-O parental cells or 786-O stable cell line expressing VHL-wt-Venus were treated with 10 µM MG132 for indicated time. (B) 786-O parental cells or 786-O stable cell line expressing VHL-wt-Venus were treated with 200 nM bafilomycin A1 for indicated time. (C) Quantitation of HIF2α relative amount. HIF2α band intensity was analyzed using ImageJ software. HIF2α levels in control 786-O stable cell line expressing VHL-wt-Venus cells were normalized to 1. Data represent mean±S.D., n=3. **, p<0.001, compared with control cells. (D) HIF2α target gene expression. 786-O stable cell line expressing VHL-wt-Venus were treated with 200 nM bafilomycin A1 for 16 hrs. Total RNA were analyzed by Real-Time PCR using primers specific for HIF2α, VEGFA, TGFA and CCND1. mRNA level in control cells are normalized to 1. Data represent mean±S.D., n=3. **, p<0.001, compared with control cells. (E) 786-O cells were treated with 200 nM bafilomycin A1 for indicated time with or without 8-hr co-treatment with 100 µM CoCl2. (F) 786-O stable cell lines expressing HA-wt-VHL or HA-W117R-VHL were treated with 200 nM bafilomycin A1 for indicated time. (G) 786-O stable cell lines expressing GFP, VHL-wt-Venus, VHL-R167Q-Venus or VHL-F148A-Venus were treated with 200 nM bafilomycin A1 for indicated time. (H) 786-O stable cell lines expressing VHL-wt-Venus were treated with 200 nM bafilomycin A1 for 8 hrs. Cytoplasmic and nuclear extracts were separated. Cell lysates were analyzed by immunoblot using antibodies against HIF2α, p62, LC3B, VHL, GFP, LDHA, LaminA or β-actin. Con, control. MG, MG132. BM, bafilomycin A1.
Mentions: The basal level of HIF2α in 786-O parental cells was relatively high (Figure 2A–2C), and was dramatically reduced with the stable expression of exogenous VHL (Figure 2A–2C). These results indicate that 786-O cells are incapable of degrading HIF2α due to the absence of functional VHL. In 786-O parental cells, neither MG132 nor bafilomycin A1 could significantly increase the already high HIF2α protein levels (Figure 2A–2C). Both agents led to accumulation of HIF2α protein in VHL-expressing 786-O cells (Figure 2A–2C). These results indicate that VHL is required not only for proteasome-mediated HIF2α degradation, but also for autophagy-mediated degradation. In contrast to HIF2α, VHL protein level was only enhanced by MG132 (Figure 2A) but not bafilomycin A1 (Figure 2B), suggesting that VHL was exclusively degraded by the proteasome, and bafilomycin A1-induced HIF2α accumulation was not due to the side effect of proteasomal inhibition.

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