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, 34 (19), 2450-60

Autophagy Mediates HIF2α Degradation and Suppresses Renal Tumorigenesis

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Autophagy Mediates HIF2α Degradation and Suppresses Renal Tumorigenesis

X-D Liu et al. Oncogene.

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.

Conflict of interest statement

Conflict of interest:

The authors declare no conflict of interest.

Figures

Figure 1
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.
Figure 2
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.
Figure 3
Figure 3
VHL-dependent HIF2α decrease during autophagy induction. (A) 786-O parental cells and (B) 786-O stable cell lines expressing VHL-wt-Venus were cultured in DMEM (0) or EBSS (starvation) for 2 or 3 hrs, or treated with 100 nM rapamycin for 3 or 6 hrs. (C) Quantitation of HIF2α relative amount. HIF2α band intensity was analyzed using ImageJ software. HIF2α levels in control cells were normalized to 1. Data represent mean±S.D., n=3. **, p<0.001, compared with control cells. Con, control. St3, starvation 3hr. Rap6, rapamycin 6 hr. (D) HIF2α target gene expression. 786-O stable cell line expressing VHL-wt-Venus were treated with 100 nM rapamycin for 8 hrs. Total RNA were analyzed by Real-Time PCR using primers specific for 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. Con, control. Rap, rapamycin. (E) 786-O stable cell lines expressing VHL-wt-Venus were treated with 100 nM rapamycin or cultured in EBSS (starvation) in the presence or absence of 200 nM bafilomycin A1. Con, control. St, starvation. Rap, rapamycin. BM, bafilomycin A1. (F) Caki-I cells with or without 6-hr pretreatment with 100 µM CoCl2 were washed with PBS 3 times, and followed by continuous culture in DMEM or EBSS (starvation) for 1 or 2 hrs. Cell lysates were analyzed by immunoblot using antibodies against HIF2α, p62, S6, phosphor-S6, LC3B or β-actin.
Figure 4
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.
Figure 5
Figure 5
Atg5 knockout favors proteasomal degradation of HIF2α. (A) Atg5+/+ and Atg5−/− MEFs stably expressing HIF2α-GFP were treated with 10 µM MG132 or 200 nM bafilomycin A1 for 8 hr. (B) Quantitation of HIF2α relative amount. HIF2α band intensity was analyzed using ImageJ software. HIF2α levels in MG132-treated Atg5−/− MEFs were normalized to 10. Con, control. Data represent mean±S.D., n=3. **, p<0.001, compared with same treatment in Atg5+/+ MEFs. Con, control. BM, bafilomycin A1. MG, MG132. (C) Atg5+/+ and Atg5−/− MEFs stably expressing HIF2α-GFP were treated with 2 µM 17-AAG or 100 µM CoCl2 for 8 hr. (D) HEK293T cells stably expressing control shRNA or BECN1 shRNA were transiently transfected with HIF2α-GFP. After 48 hr, cells were further treated with 200 nM bafilomycin A1 (BM) for 6 hr. Con, control. BM, bafilomycin A1. (E) HEK293T cells stably expressing control shRNA or p62 shRNA were transiently transfected with HIF2α-GFP. After 48 hr, cells were further treated with 200 nM bafilomycin A1 (BM) for 6 hr. Con, control. BM, bafilomycin A1. Con, control. BM, bafilomycin A1. MG, MG132. Whole cell lysates were analyzed by immunoblot using antibodies against GFP, p62, ATG5, LC3B, β-actin or GAPDH.
Figure 6
Figure 6
Genetic alteration of autophagy genes in ccRCC. (A) Defining ccRCCs into autophagy gene expression subgroups (ATG-low, ATG-high-1 and ATG-high-2) using seven genes involved in autophagy nucleation. Top, gene expression heatmap of 7-gene signature. Samples were ordered by the 7-gene composite score and separated into low and high expression groups by Kmeans. Bottom, corresponding gene copy number changes and mutation of ATG7 and VHL were plotted based on SNP6 data. Copy number levels of 1N and 3N were marked. (B) Kaplan Meier analysis of ccRCC patient survival stratified by autophagy gene expression levels as defined in Fig. 6A. (C) Mutations of autophagy related genes. Data were organized from TCGA level 2 mutation data.
Figure 7
Figure 7
Decreased protein expression of ATG7 and Beclin 1 in ccRCC. Tissue microarrays containing normal kidney and ccRCC sections were subjected to H&E staining or Immunohistochemistry staining using ATG7 antibody or beclin 1 antibody. (A) Representative images were shown. (B) Percentage of ATG7 or beclin 1 positive cells in ccRCC tumors or normal kidneys. Mann-Whitney test was used for statistical analysis. (C) Working model. HIF2α is ubiquitinated by its E3 ligase VHL. Around 2/3 of ubiquitinated HIF2α are soluble and can be degraded by proteasome system. Around 1/3 of ubiquitinated HIF2α interacts with UBA domain of p62 and forms aggregates. HIF2α aggregates are recruited to autophagosome via p62-LC3 interaction and are degraded after fusion with the lysosome. There is a compensatory interaction between the proteasome and autophagy in HIF2α degradation. On the one hand, autophagy inactivation redirects HIF2α to proteasomal degradation, and heat shock proteins are probably involved in refolding of HIF2α aggregates. On the other hand, proteasome inhibition induces autophagy and promotes the HIF2α-p62 interaction and aggregation. When the components involved in VHL-elonginB-C (VBC) complex, proteasome degradation pathway and autophagy degradation pathway are genetically deleted, mutated, or their expression is decreased at the same time, HIF2α cannot be degraded by the proteasome or autophagy. Part of the accumulated HIF2α will translocate to nucleus, drive the expression of its target genes and initiate RCC development.

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References

    1. Jonasch E, Futreal PA, Davis IJ, Bailey ST, Kim WY, Brugarolas J, et al. State of the science: an update on renal cell carcinoma. Molecular cancer research : MCR. 2012 Jul;10(7):859–880. PubMed PMID: 22638109. Pubmed Central PMCID:3399969. - PMC - PubMed
    1. Nickerson ML, Jaeger E, Shi Y, Durocher JA, Mahurkar S, Zaridze D, et al. Improved identification of von Hippel-Lindau gene alterations in clear cell renal tumors. Clinical cancer research : an official journal of the American Association for Cancer Research. 2008 Aug 1;14(15):4726–4734. PubMed PMID:18676741. Pubmed Central PMCID: 2629664. - PMC - PubMed
    1. Keith B, Johnson RS, Simon MC. HIF1alpha and HIF2alpha: sibling rivalry in hypoxic tumour growth and progression. Nature reviews Cancer. 2012 Jan;12(1):9–22. PubMed PMID: 22169972. Pubmed Central PMCID:3401912. - PMC - PubMed
    1. Shen C, Beroukhim R, Schumacher SE, Zhou J, Chang M, Signoretti S, et al. Genetic and functional studies implicate HIF1alpha as a 14q kidney cancer suppressor gene. Cancer discovery. 2011 Aug;1(3):222–235. PubMed PMID: 22037472. Pubmed Central PMCID: 3202343. Epub 2011/11/01. eng. - PMC - PubMed
    1. Shinojima T, Oya M, Takayanagi A, Mizuno R, Shimizu N, Murai M. Renal cancer cells lacking hypoxia inducible factor (HIF)-1alpha expression maintain vascular endothelial growth factor expression through HIF-2alpha. Carcinogenesis. 2007 Mar;28(3):529–536. PubMed PMID: 16920734. - PubMed

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