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A hypoxia-responsive TRAF6–ATM–H2AX signalling axis promotes HIF1α activation, tumorigenesis and metastasis

An Author Correction to this article was published on 05 June 2020

Abstract

The understanding of how hypoxia stabilizes and activates HIF1α in the nucleus with related oncogenic signals could revolutionize targeted therapy for cancers. Here, we find that histone H2AX displays oncogenic activity by serving as a crucial regulator of HIF1α signalling. H2AX interacts with HIF1α to prevent its degradation and nuclear export in order to allow successful VHL-independent HIF1α transcriptional activation. We show that mono-ubiquitylation and phosphorylation of H2AX, which are strictly mediated by hypoxia-induced E3 ligase activity of TRAF6 and ATM, critically regulate HIF1α-driven tumorigenesis. Importantly, TRAF6 and γH2AX are overexpressed in human breast cancer, correlate with activation of HIF1α signalling, and predict metastatic outcome. Thus, TRAF6 and H2AX overexpression and γH2AX-mediated HIF1α enrichment in the nucleus of cancer cells lead to overactivation of HIF1α-driven tumorigenesis, glycolysis and metastasis. Our findings suggest that TRAF6-mediated mono-ubiquitylation and subsequent phosphorylation of H2AX may serve as potential means for cancer diagnosis and therapy.

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Figure 1: H2AX is overexpressed in tumour tissues and regulates cancer cell proliferation and senescence.
Figure 2: H2AX regulates HIF1α signalling, glycolysis and tumorigenesis.
Figure 3: H2AX maintains nuclear retention and stability of HIF1α.
Figure 4: TRAF6-mediated mUb-H2AX and γH2AX are essential for HIF1α signalling.
Figure 5: Hypoxia-activated TRAF6 constitutes the ATM-γH2AX-HIF1α pathway.
Figure 6: mUb-H2AX formation and γH2AX formation are oncogenic signals.
Figure 7: TRAF6 and H2AX are potential targets for eradication of metastatic tumours.
Figure 8: γH2AX-mediated HIF1α activation predicts metastatic outcome in breast cancer patients.

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Acknowledgements

We thank F. Alt (Harvard University, USA) for providing H2AX null mice. We also thank S. Piccolo (University of Podova, Italy), X. Lin (The University of Texas MD Anderson Cancer Center, USA), J. Chen (The University of Texas MD Anderson Cancer Center, USA), M. D. Bohmann (University of Rochester, USA), M. Eilers (University of Wurzburg, Germany), J. L. Wrana (Mount Sinai Hospital, Canada), X. Yang (University of Pennsylvania, USA), J. Cheng (Shanghai Jiao Tong University, China), E. Yeh (University of Missouri, USA), Z. Lou (Mayo Clinic, USA) and B. Gan (The University of Texas MD Anderson Cancer Center, USA) for reagents and equipment. This work was supported by NIH R01 grants (R01CA182424-01A1, R01CA193813-01), the MD Anderson Cancer Center SPORE development grant, the R. Clark Fellowship award, MD Anderson Cancer Center Prostate Moonshot Program funds, and Start-up funds from Wake Forest University School of Medicine to H.-K.L. and MOST104-2314-B-384-009-MY3 and MOHW104-TDU-M-212-133004 grants from Taiwan to C.-F.L.

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Authors and Affiliations

Authors

Contributions

A.-H.R. and H.-K.L. co-conceived the project, designed the experiments, analysed the data and wrote the manuscript. A.-H.R. performed the experiments with assistance from C.-Y.W., X.Z., F.H., Z.C., Y.S.J., G.J., L.P. and P.-C.C. C.-F.L. performed the immunohistochemistry experiments with human subjects and analysed the data. J.D. and M.J.Y. helped A.-H.R. with the CT/PET imaging and mouse tumour analysis, respectively. M.-H.L., M.-C.H. and D.S. provided the input and suggestions.

Corresponding authors

Correspondence to Abdol-Hossein Rezaeian or Hui-Kuan Lin.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 HIF1α signalling is positively correlated with H2AX expression but not with DNA damage markers.

a, Heat map data were generated from analysis of biopsy specimens of 99 untreated patients with stage 1–3 breast cancer registered at MD Anderson Cancer Center (MDACC). Co-expression analysis was validated by using probes hybridized to H2AX, HIF1α, VEGF and PKM2 genes. b, The co-expression of HIF1α target genes was correlated with that of H2AX by the study of 255 untreated breast cancer patients at MDACC. c, LUC or H2AX knockdown MCF10A breast epithelial cells were cultured in normoxic or hypoxic conditions for 8 h, and lysates were collected for IB. d, Vector- or H2AX-overexpressing MDA-MB-231 cancer cells were cultured under hypoxia at the indicated times and harvested for IB. e, LUC or H2AX knockdown MDA-MB-231 cells were grown under normoxia or hypoxia for 24 h and treated with NBDG (50 mM) for 2 h; glucose uptake was quantified by FACS analysis (n = 3 independent experiments, upper). LUC or H2AX knockdown MDA-MB-231 cells were cultured under normoxia or hypoxia for 24 h and lactate production was measured (n = 3 biological replicate samples, lower). (fj) H2ax+/+ and H2ax−/− MEF (f) or Mdc1+/+ and Mdc1−/− MEF or LUC and MDC1 knockdown MDA-MB-231 (g, h) or Rnf8+/+ and Rnf8−/− MEF or LUC and RNF8 knockdown MDA-MB-231 (i,j) cells were cultured in hypoxic conditions at the indicated times and harvested for IB of hypoxia-inducible protein expression. k, LUC or H2AX knockdown MDA-MB-231 cells silenced by three different shRNAs were cultured in normoxic (N) or hypoxic (H = 8 h) conditions and harvested for detection of HIF1α expression level. lH2ax+/+ and H2ax−/− MEF cells were cultured in hypoxic or normoxic conditions for 12 h, and the localization of HIF1α and H2AX proteins was visualized by immunofluorescence microscopy. Image represents 1 out of 3 independent experiments. Scale bar, 20 μm. Statistical significance of triplicated results was assessed by two tailed t-test. Mean ± s.d. P < 0.01 was considered significant. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 2 SHARP1 may exhibit a competition with H2AX.

a, Mock or H2AX or SHARP1 or H2AX along with SHARP1 were transiently overexpressed in MDA-MB-231 cells were cultured under hypoxic conditions for 8 h and harvested for IB analysis. b, Lysates of 293T cells transfected with (Flag)-HIF1α along with HA-H2AX or (HA)-SHARP1 were subjected to IB analysis. c,dH2ax+/+ and H2ax−/− MEF cells were cultured in normoxic or hypoxic conditions for 12 h, and the localization of MRE11 (c) or RNF8 (d) and γH2AX proteins was visualized by immunofluorescence microscopy. Scale bar, 20 μm. γH2AX is induced upon hypoxia without colocalization with DNA damage factors such as MRE11 and RNF8. The experiments a–d performed once. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 3 TRAF6 is an E3 ligase which monoubiquitinates H2AX and interacts with HIF1α.

a, Lysates of 293T cells transfected with hemagglutinin-tagged H2AX (HA-H2AX) and His-Ub, along with various E3 ligases, were subjected to in vivo ubiquitination assay and IB analysis. b, Vector (pMX)- or TRAF6-overexpressing MDA-MB-231 cells were cultured in normoxic or indicated hypoxic time points and subjected to lysis followed by IB analysis. c, 293T cells co-transfected with His-Ub, TRAF6 and HA-H2AX or various H2AX mutants were subjected to lysis for in vivo monoubiquitination assay. H2AX ubiquitination sites were found at Lys (K) 119 and K120. d, Lysates of MDA-231 cells cultured in normoxic or hypoxic conditions for 4 h were immunoprecipitated with control or TRAF6 antibody followed by IB. e, Immunofluorescence assay of the localization of HIF1α and γH2AX proteins from H2ax+/+ and H2ax−/− MEF cells cultured in hypoxic or normoxic conditions for 12 h. Scale bar, 20 μm. Image represents 1 out of 3 independent experiments. f, Multiple alignments of H2AX amino acids are shown; ubiquitination sites K119/120 (highlighted in red letters) and the phosphorylation site S139 (in the SQEY motif, highlighted in blue) are highly conserved among different species. Numbers show the positions of K (lysine) and/or S (serine). Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 4 TRAF6, a hypoxia responsive gene, regulates HIF1α signalling.

a, MDA-MB-231 cancer cells in which LUC or HIF1α was silenced by shRNA were cultured in normoxic or hypoxic conditions for 8 h, and lysates were collected for IB analysis. b, Primary MEF cells cultured in hypoxic conditions for 2 and 4 h were subjected to qPCR of Traf6 mRNA (n = 3, biologically independent extracts). c, Lysates of HEK-293 cells were treated with biotin-labeled TRAF6-DNA or along with biotin-labeled TRAF6-DNA and cold DNA (not Labeled) compared to labeled-DNA alone were subjected to electrophoretic mobility shift assay (EMSA). d, Hela cells were cultured in normoxic or hypoxic conditions and harvested for chromatin immunoprecipitation (ChIP) with HIF1α antibody followed by qPCR for the expression of TRAF6 genes and were compared to GAPDH control. e, Scatter plots were generated for the correlated expression of HIF1α target genes with TRAF6. Tumour tissues were used from 255 untreated patients with stage 1–3 breast cancer. The biopsies were obtained through fine-needle aspiration. Expression analysis was carried out by Nexus Expression 0.3 software (Biodiscovery). f,g, LUC or TRAF6 knockdown MDA-MB-231 cells were subjected to cell cycle analysis (n = 3, independent experiments). Statistical significance of three biological replications was assessed by two tailed t test. Mean ± s.d.,P < 0.01 was considered significant. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 5 mUb-H2AX and γH2AX formation are critical for HIF1α signalling.

aH2ax−/− MEF cells in which H2AX was restored with vector, H2AX-WT or H2AX-KR mutant construct were cultured under hypoxic conditions for the indicated time points and subjected to lysis and IB. b, TRAF6-silenced MDA-MB-231 cells were treated with vehicle or MG132 in hypoxic conditions for 4 h and subjected to lysis and IB. c, H2ax−/− MEF cells reconstituted with vector, H2AX-WT or H2AX-KR or-SA mutant were incubated under hypoxic conditions for 12 h, and the localization of HIF1α was visualized by confocal microscopy. Scale bar, 20 μm. Experiments were performed twice. d, MEF cells cultured in normoxic and hypoxic conditions were treated with or without ATM inhibitor KU55933 for the indicated times, harvested and subjected to lysis for IB. e, MEF cells generated from Atm+/+ or Atm−/− mice cultured in hypoxic conditions for 8 h and subjected to lysis and then to IB analysis. f, 293T cells treated with non-modified (NM) and modified (M, ub-conjugated) peptides (2 ng ml−1) were cultured in normoxic or hypoxic conditions for 4 h, and lysates were collected for IP with ATM antibody, followed by IB. Interaction of ATM to mUb-H2AX was neutralized followed by reduction in γH2AX expression. g, Traf6−/− MEF cells restored with vector, TRAF6-WT or TRAF6-C70A mutant were cultured in hypoxic conditions for various time points and harvested for chromatin fractionation and IB. γH2AX formation was shown in the chromatin fraction of the cells rescued with TRAF6-WT. h, 293T cells stably overexpressing vector, H2AX-WT or H2AX-KR or-SA mutant were cultured in normoxic or hypoxic conditions and harvested for ChIP-qPCR of VEGF using HIF1α antibody (n = 3, biologically independent extracts). Results are representative from three biological replications. Statistical significance was assessed by ANOVA. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 6 H2AX-targeting reduces cancer cell survival, migration and tumourigenesis.

a, MDA-MB-231 cells stably overexpressing vector, H2AX-WT or H2AX-KR or-SA mutant were harvested for cell viability assay (n = 3, biological replicate samples). b, H2AX knockdown MDA-MB-231 cells rescued with H2AX-WT or an H2AX mutant were seeded in triplicate for a wound healing analysis using confocal microscopy. c, LUCiferase expressing MDA-MB-231 cancer cells in which H2AX was silenced by shRNA and then restored with vector, HIF1α-WT or HIF1α-Mut were injected into the mammary fat pad of nude mice and luciferase expression in tumour growth was counted. d, LUC or H2AX knockdown MDA-MB-231 breast cancer cells were cultured in normoxic or hypoxic conditions for 24 h, and the conditioned media were collected for detection of secreted VEGF by ELISA assay (n = 4, biological replicate samples). e,f, MDA-MB-231 cells in which LUC or H2AX was silenced with two different shRNAs and plated in transwell chambers for the cell migration (e) and invasion (f) assay (n = 4, biological replicate samples). Scale bar, 100 μm. g, MDA-MB-231 cells were treated with non-ubiquitinated (left, CVLLPKKTSAT) and ubiquiitnated H2AX (right, CVLLPK(ub-K)TSAT) peptides (10 ng ml−1) overnight, and cell viability was determined by flow cytometry. Statistical significance was measured using two tailed t-test (e,f) and ANOVA (a,d). Mean ± s.d.,P < 0.01 and P < 0.05 were considered significant.

Supplementary Figure 7 Overexpression of TRAF6 is associated with upregulation of major cancer hallmarks.

a, Transcriptomics gene expression profiles of 225 breast cancer patients (cohort GSE20194, Gene Expression Omnibus database, http://www.ncbi.nlm.nih.gov/geo) were analyzed using the Nexus Expression 3.0 software (BioDiscovery). The gene expression profiles of the highest TRAF6 quartile were compared with the lowest TRAF6 quartile, and then matched with related biological processes and corresponding cancer hallmarks. The size of cancer hallmark symbols signified the magnitude of their upregulation when TRAF6 expression was elevated in breast cancer. bd, The bar graph indicates a significant increase in cancer proliferation (b), metabolomics reprogramming (c), tissue invasion and metastasis (d) which associated with enhanced TRAF6 expression. Enrichment scores were calculated using the Nexus Expression 3.0 software (BioDiscovery). This Circos map was built using the Circos software (www.circos.ca).

Supplementary Figure 8 TRAF6 regulates HIF1α-dependent breast cancer development.

ac, TRAF6 knockdown MDA-MB-231 cells (expressing luciferase) rescued with pBabe vector, HIF1α-WT or HIF1α-Mut (constitutively active mutant) were injected into the mammary fat pad of nude mice (n = 5/group) and tumour sizes were monitored weekly (b) and tumour weight was measured at week 6 (c, n = 5 tumour/group, Mean ± s.d.) d, MSCV-vector or MSCV-miR-145 overexpressing MDA-MB-231 breast cancer cells were subcutaneously injected into the right flank of 6-week-old nude mice (n = 4/group) and tumour volume was measured weekly. e, Primary tissues, cell lines in different human disease with different types of mutation which is involved in functional effect, inheritance mode, translation impact, unclassified mutation, zygosity and wild type were analysed using Ingenuity Pathway (http://www.ingenuity.com). The publication data range was from Jan. 1954-Apr. 2016. f, Activation of TRAF6, H2AX and HIF1α network in cancer genomics was proven by cBioPortal web-based data sets (http://www.cBioPortal.org)1,2. Panels e and f indicate that this network is significantly existed including TRAF6, H2AX and HIF1α gene pairs with co-occurring alterations in many cancer types. Statistical significance was assessed by ANOVA. Mean ± s.d. ns, not significant. P < 0.01 was considered significant.

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Rezaeian, AH., Li, CF., Wu, CY. et al. A hypoxia-responsive TRAF6–ATM–H2AX signalling axis promotes HIF1α activation, tumorigenesis and metastasis. Nat Cell Biol 19, 38–51 (2017). https://doi.org/10.1038/ncb3445

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