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A20 promotes metastasis of aggressive basal-like breast cancers through multi-monoubiquitylation of Snail1

Nature Cell Biology volume 19, pages 1260–1273 (2017)Cite this article

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Abstract

Although the ubiquitin-editing enzyme A20 is a key player in inflammation and autoimmunity, its role in cancer metastasis remains unknown. Here we show that A20 monoubiquitylates Snail1 at three lysine residues and thereby promotes metastasis of aggressive basal-like breast cancers. A20 is significantly upregulated in human basal-like breast cancers and its expression level is inversely correlated with metastasis-free patient survival. A20 facilitates TGF-β1-induced epithelial–mesenchymal transition (EMT) of breast cancer cells through multi-monoubiquitylation of Snail1. Monoubiquitylated Snail1 has reduced affinity for glycogen synthase kinase 3β (GSK3β), and is thus stabilized in the nucleus through decreased phosphorylation. Knockdown of A20 or overexpression of Snail1 with mutation of the monoubiquitylated lysine residues into arginine abolishes lung metastasis in mouse xenograft and orthotopic breast cancer models, indicating that A20 and monoubiquitylated Snail1 are required for metastasis. Our findings uncover an essential role of the A20–Snail1 axis in TGF-β1-induced EMT and metastasis of basal-like breast cancers.

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Figure 1: Overexpression of the A20 gene in human malignant breast cancers.
Figure 2: A20 is involved in TGF-β-induced EMT.
Figure 3: Stabilization of the Snail1 protein by A20.
Figure 4: A20 promotes the metastasis of aggressive breast cancer cells.
Figure 5: The ZnF7 domain of A20 induces the monoubiquitylation of Snail1.
Figure 6: Three lysine residues of Snail1 monoubiquitylated by A20 are critical for metastasis.
Figure 7: A20 retains Snail1 protein in the nucleus through inhibition of GSK3β-mediated Snail1 phosphorylation.
Figure 8: A20 is required for cancer stemness, chemoresistance, and TNF-α-induced Snail1 stabilization.

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Acknowledgements

This work was supported by a grant from the National R&D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea (1520120) and in part by National Research Foundation grant of Korea (2015R1A2A2A05001344 and SRC 2017R1A5A1014560) funded by the Ministry of Science, ICR & Future Planning.

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Author notes

  1. Ji-Hyung Lee and Su Myung Jung: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Sungkyunkwan University, Suwon, 16419, Korea

    Ji-Hyung Lee, Su Myung Jung, Jin Seok Park, Dongyeob Seo, Minbeom Kim, Jihoon Ha, Jaewon Lee, Jun-Hyeong Kim, Jun Hwan Kim, Donghyuk Shin, Youn Sook Lee, Sangho Lee & Seok Hee Park

  2. Precision Medicine Research Center, Advanced Institutes of Convergence Technology, Seoul National University, Suwon, 16229, Korea

    Kyung-Min Yang, Eunjin Bae, Akira Ooshima, Jinah Park & Seong-Jin Kim

  3. Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, 06273, Korea

    Sung Gwe Ahn & Joon Jeong

  4. Inflammation Research Center, Unit of Cellular and Molecular (Patho)physiology, VIB, B-9052, Ghent, Belgium

    Geert van Loo

  5. Department of Biomedical Molecular Biology, Ghent University, B-9052, Ghent, Belgium

    Geert van Loo

  6. Department of Transdisciplinary Studies, Graduate School of Convergence Science and Technology, Seoul National University, Suwon, 16229, Korea

    Seong-Jin Kim

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Contributions

J.-H.L. and S.M.J. designed the research, carried out the experimental work, analysed data and wrote the manuscript; E.B., J.S.P. and J.-H.K. performed the animal experiments and immunohistochemical analysis; D.S., M.K., J.H., J.L. and J.H.K. carried out the experimental work and analysed data; K.-M.Y., S.G.A., A.O. and J.J. statistically analysed the public data sets and clinical data of breast cancer patients; J.P., D.S., Y.S.L. and S.L. carried out in vitro ubiquitylation and provided technical assistance; G.L. and S.-J.K. participated in the study design and coordinated the study; S.H.P. designed and conceptualized the research, supervised the experimental work, analysed data and wrote the manuscript.

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Correspondence to Seok Hee Park.

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Integrated supplementary information

Supplementary Figure 1 A20 does not affect the canonical TGF-β/Smad signaling, but stabilizes the Snail1 protein.

(a) A20-knockdown and shGFP-expressing NMuMG cells were transfected with a Smad- specific CAGA-Luc reporter. Cells were treated with TGF-β1 (5 ng ml−1) for 24 h, and luciferase activities were measured and normalized. n.s., not significant. (b) NMuMG cells were reverse-transfected with 20 nM of control siRNA (siCON) or four different siRNAs targeting mouse A20 mRNA. Knockdown efficiency was confirmed by immunoblot analysis with anti-A20 antibody. (c–e) Quantitative real-time RT-PCR analysis of indicated target genes, induced by the TGF-β/Smad-dependent signaling pathway, in A20-knockdown NMuMG cells treated with TGF-β1 for 24 h. The data in a,c,d, and e were statistically analyzed by a t-test and show the mean ± s.d. of n = 3 independent experiments. (f) Stability of the Snail1 protein was measured in A20-knockdown and shGFP-expressing control NMuMG cells in the presence of TGF-β1, followed by treatment of protein translation inhibitor, cycloheximide (CHX, 50 μg ml−1) for the indicated times. Cell lysates were immunoblotted by the indicated antibodies (upper). Data were quantified using ImageJ software (lower). For normalization, expression of β-actin was used as a control. (g) A20-knockdown NMuMG cells were treated with TGF-β1 for 24 h, followed by exposure to proteasomal inhibitor MG132 (10 μM) for 6 h. Cell lysates were immunoblotted with the indicated antibodies. (h) A plasmid encoding Flag-Snail1 was co-transfected with increasing amounts of HA-A20 plasmid into HEK293 cells. Cell lysates were immunoblotted. (i) Panc-1 cells were reverse-transfected with 20 nM of control siRNA or two independent A20 siRNAs (siA20 #1 and siA20 #3) and treated with TGF-β1 for the indicated times. Cell lysates were immunoblotted. (j) A20-knockdown and shGFP-expressing control NMuMG cells were transfected with Flag-Snail1 and then treated with TGF-β1 for 24 h. Cell lysates were immunoblotted. Expression of β-actin was used as a loading control for all immunoblot analysis shown in this figure. Immunoblot images are representative of n = 3 independent experiments. Statistics source data for a and c–e are available in Supplementary Table 3. Unprocessed original scans of blots in b and f–j are in Supplementary Fig. 9.

Supplementary Figure 2 A20 depletion does not affect tumor growth.

(a,e) MCF10CA1a (M4) (a) and 4T1-Luc (e) cells were infected with the indicated lentiviruses expressing shRNAs targeting A20 mRNA. Cell lysates were immunoblotted with the indicated antibodies. Expression of β-actin was used as a loading control. The data are representative of n = 3 independent experiments. (b,f) A20-knockdown MCF10CA1a (M4) (b) or A20-knockdown 4T1-Luc (f) cells were cultured in 6-well plates and harvested at the indicated time points. Cell proliferation was analyzed by counting cell numbers in each well, compared to shGFP-expressing control cells. The data were statistically analyzed by a t-test and show the mean ± s.d. of n = 3 independent experiments. ∗P < 0.05 compared to the shGFP control cells. n.s., not significant. (c,d) 5 × 105 of A20-knockdown and shGFP-expressing MCF10CA1a (M4) cells were orthotopically injected into NOD/SCID mice (n = 6 mice per group). After the mice were sacrificed 5 weeks later, representative primary tumor images were shown in c and tumor volumes were measured (d). (g,h) 5 × 104 of A20-knockdown and shGFP-expressing control 4T1-Luc cells were orthotopically injected into Balb/c mice (n = 6 mice per group) and the mice were sacrificed 5 weeks later. Representative images of primary tumors were shown in g and tumor volumes were measured (h). The data in d and h were statistically analyzed by a t-test and show the mean ± s.d. n = 6 mice per group per experiment. ∗P < 0.05 compared to the shGFP control cells. n.s., not significant. Statistics source data for b,d,f and h are available in Supplementary Table 3. Unprocessed original scans of blots in a and e are in Supplementary Fig. 9.

Supplementary Figure 3 A20 induces monoubiquitination of the Snail1 protein through ZnF7 domain.

(a) Plasmids encoding Flag-Snail1 and wild type His-Ub were co-transfected with HA-A20, HA-GSK3β and HA-βTrCP1 into NMuMG cells in the indicated combinations. Ni-NTA-mediated pull-down assays were performed and ubiquitinated Snail1 was observed by immunoblotting using anti-Flag antibody. Total cell lysates (TCL) were immunoblotted with the indicated antibodies. (b) Dynamics of the interaction between endogenous A20 and Snail1 in NMuMG cells. Cells were treated with TGF-β1 (5 ng ml−1) for the indicated times, immunoprecitated with anti-Snail1 antibody and immunoblotted with the indicated antibodies. (c) Plasmid encoding wild-type HA-A20 or A20 ZnF7 mutant (HA-A20_ZnF7∗) was co-transfected into HEK293 cells together with Flag-Snail1 plasmid. Cell lysates were immunoprecipitated with anti-HA antibody and subsequently immunoblotted with the indicated antibodies. (d) For in vitro ubiquitination assays, Flag-Snail1 proteins were eluted from HEK293 cells transfected with Flag-Snail1 plasmid, and wild-type GST-A20 and mutant GST-A20_ZnF7∗ proteins were purified from E.coli. The reactions were performed in the indicated combinations and samples were immunoblotted with the indicated antibodies. (e) Plasmids encoding Flag-Snail1 and wild type His-Ub were co-transfected into NMuMG cells with HA-A20. After cells were treated with the ubiquitin isopeptidase inhibitor G5 for 6 h, Ni-NTA pull-down assays were performed, followed by immunoblotting with the indicated antibodies. (f) Plasmid encoding HA-A20 or HA-A20(C103A) mutant was co-transfected into NMuMG cells with HA-GSK3β and HA-βTrCP1 in the presence of His-Ub and Flag-Snail1. After cells were pre-treated with MG132, Ni-NTA pull-down and immunoblot assays were performed. Expression of β-actin was used as a loading control in all immunoblot assays except for d. Immunoblot images in this figure are representative of n = 3 independent experiments. Unprocessed original scans of blots in Supplementary Fig. 3 are in Supplementary Fig. 9.

Supplementary Figure 4 A20 monoubiquitinates three Snail1 lysine residues, which are crucial for Snail1 stability and TGF-β1-induced EMT.

(a) Plasmids encoding wild type Snail1(Flag-Snail1-WT) or Snail1 mutants (Flag-Snail1-N-6KR and Flag-Snail1-C-8KR) were co-transfected into NMuMG cells with wild-type His-Ub and HA-A20 plasmids in the indicated combinations. Ni-NTA-mediated pull-down assays were performed and ubiquitinated Snail1 was observed by immunoblot analysis using anti-Flag antibody. Total cell lysates (TCL) were immunoblotted with the indicated antibodies. (b) A plasmid encoding wild-type Snail1 (Flag-Snail1) or single K-to-R mutants of Snail1 was co-transfected into NMuMG cells in the absence or presence of HA-A20. Cell lysates were immunoblotted with the indicated antibodies. (c) To examine whether A20-mediated monoubiquitination of Snail1 is linked to the phosphorylation of Snail1 by ERK, a plasmid encoding a Snail1 mutant [Flag-Snail1(S82A/S104A)] or wild-type Snail1, was co-transfected into NMuMG cells with or without HA-A20. Cell lysates were immunoblotted with the indicated antibodies. (d) Snail1 depletion in NMuMG cells by lentiviruses expressing different shRNAs was confirmed by immunoblot analysis with anti-Snail1 antibody. (e) Snail1-depleted NMuMG cells were infected with retroviruses expressing wild-type Snail1 (Flag-Snail1-WT) or the Snail1(3KR) mutant (Flag-Snail1-3KR). After treatment with TGF-β1 (5 ng ml−1) for 48 h to induce EMT, cell lysates were immunoblotted with the indicated antibodies. (f) The CDH1-Luc reporter plasmid was co-transfected into Snail1-depleted NMuMG cells with an indicated plasmid. After treatment with TGF-β1 for 48 h, luciferase activities were measured and normalized. The data were statistically analyzed by a t-test and show the mean ± s.d. of n = 3 independent experiments. ∗∗P < 0.01 compared to cells not treated with TGF-β1 in the case of shGFP and compared to cells treated with TGF-β1 in others. Immunoblot images in this figure are representative of n = 3 independent experiments and expression of β-actin was used as a loading control. Statistics source data for f are available in Supplementary Table 3. Unprocessed original scans of blots in a–e are in Supplementary Fig. 9.

Supplementary Figure 5 Three lysine residues of Snail1 are essential for breast cancer metastasis.

(a) 4T1-Luc cells stably expressing wild-type Snail1 (Flag-Snail1-WT) or the Snail1(3KR) mutant (Flag-Snail1-3KR) were generated by infection with recombinant retroviruses. Expression of Flag-Snail1-WT or Flag-Snail1-3KR in 4T1-Luc cells were confirmed by immunoblot analysis with anti-Flag antibody. (b,c) 5 × 104 of 4T1-Luc cells stably expressing Flag-Snail1-WT or Flag-Snail1-3KR were orthotopically injected into Balb/c mice (n = 6 mice per group). As a control, the same amounts of 4T1-Luc cells infected with retroviruses expressing empty vector (Mock) were used. After the mice were sacrificed 5 weeks later, representative images of primary tumors (b) were shown and tumor volumes (c) were measured. The data in c were statistically analyzed by a t-test and show the mean ± s.d., compared to control 4T1-Luc cells (Mock). n = 6 mice per group per experiment. n.s., not significant. (d) Generation of recombinant 4T1-Luc cell lines expressing Flag-Snail1-WT or Flag-Snail1-3KR in A20-depleted and shGFP background by consecutive retroviral and lentiviral infections. A20 depletion and Snail1 expression were confirmed by immunoblot analysis. (e–h) Each recombinant 4T1-Luc cell line (5 × 104 cells) was orthotopically injected into Balb/c mice (n = 6 mice per group). Bioluminescence imaging was monitored at the indicated time points (e). After the mice were sacrificed 35 days later, lungs were removed and stained with India ink. Representative images and the numbers of metastatic nodules (f), images of primary tumors (g) and tumor volumes (h) were shown. The data in f and h were statistically analyzed by a t-test and show the mean ± s.d. n = 6 mice per group per experiment. ∗∗P < 0.01 and ∗∗∗P < 0.001 compared to the indicated groups. n.s.; not significant. Immunoblot images in a and d are representative of n = 3 independent experiments and expression of β-actin was used as a loading control. Statistics source data for c,f, and h are available in Supplementary Table 3. Unprocessed original scans of blots in a, and d are in Supplementary Fig. 9.

Supplementary Figure 6 A20-mediated Snail1 monoubiquitination is required for nuclear retention of Snail1 and interaction with transcriptional co-repressors.

(a) A plasmid encoding HA-GSK3β was transfected into HEK293 cells with or without A20 expression plasmid. Cell lysates were immunoprecipitated with anti-A20 antibody and subsequently immunoblotted. (b) NMuMG cells were infected with retroviruses expressing wild-type Snail1 (Flag-Snail1-WT) or the Snail1(3KR) mutant (Flag-Snail1-3KR). After cells were stained with anti-Flag antibody and DAPI, the localization of Snail1 protein was observed by confocal microscopy. Scale bars, 20 μm. (c) β-TrCP1 depletion in NMuMG cells by different siRNAs targeting β-TrCP1 mRNA or control siRNA (siCON) was confirmed by immunoblot analysis with anti-β-TrCP1 antibody. (d) β-TrCP1-depleted (siβTrCP1#2) NMuMG cells were transfected with a plasmid encoding Flag-Snail1-WT or Flag-Snail1-3KR in the absence or presence of HA-A20. Cell lysates were immunoblotted. (e) A20-depleted and control shGFP-expressing NMuMG cells were treated with TGF-β1 (5 ng ml−1) for 24 h, followed by exposure to MG132 (10 μM) for 4 h and fractionated into cytoplasmic and nuclear extracts. Both extracts were immunoblotted with the indicated antibodies. Expressions of tubulin and lamin were used as cytoplamic and nuclear markers, respectively, and loading controls. (f) A plasmid encoding Flag-Snail1-WT or Flag-Snail1-3KR was co-transfected into NMuMG cells with or without HA-A20 plasmid. Cell lysates were immunoprecipitated with anti-Flag antibody and subsequently immunoblotted. (g) Chromatin immunoprecipitation analysis (ChIP) on NMuMG cells transfected with a plasmid encoding Flag-Snail1-WT or Flag-Snail1-3KR. Chromatin fragments were immunoprecipitated with anti-Flag antibody. PCR primers for E-cadherin promoter region were used to amplify the DNA isolated from the immunoprecipated chromatins and input samples. The data in quantitative real-time PCR (lower panel) were statistically analyzed by a t-test and show the mean ± s.d. of n = 3 independent experiments. ∗∗∗P < 0.01 compared to IgG control. n.s.; not significant. Images shown in this figure are representative of n = 3 independent experiments. Expression of β-actin was used as a loading control for the immunoblot analysis except for e. Statistics source data for g are available in Supplementary Table 3. Unprocessed original scans of blots in a and c–f are in Supplementary Fig. 9.

Supplementary Figure 7 A20 expression is induced by the Smad-independent noncanonical pathway upon TGF-β1 treatment.

(a) Gating strategy of CD44(+)/CD24(-) cancer cell populations in A20-depleted and control M4 (MCF10CA1a) cells. M4 cells were initially gated by FSC-A versus FSC-H for single cells and these separated cells were further gated by FSC-A versus SSC-A for the exclusion of debris. Live cells were finally gated by using fixable dye, APC-Cy7. (b) After NMuMG cells were pre-treated with the TGF-β type I receptor inhibitor SB431542 (10 μM) for 1 h, they were treated with TGF-β1 (5 ng ml−1) for the indicated times. A20 expression and Smad2 phosphorylation were monitored by immunoblot analysis. (c,d) NMuMG cells expressing Smad4-specific shRNAs or GFP-specific control shRNA were treated with TGF-β1 for the indicated times. A20 expression was analyzed by immunoblot (c) and quantitative real-time RT-PCR (d) analysis. In qRT-PCR analysis, expression of Gapdh mRNA was used for normalization. The data in d were statistically analyzed by a t-test and show the mean ± s.d. of n = 3 independent experiments. All data of immunoblot analysis shown in this figure are representative of n = 3 independent experiments. Expression of β-actin was used as a loading control for the immunoblot analysis. Statistics source data for d are available in Supplementary Table 3. Unprocessed original scans of blots in b and c are in Supplementary Fig. 9.

Supplementary Figure 8 A20 expression is correlated with relapse-free survival of human breast cancer patients.

(a) Using Kaplan-Meier (KM) Plotter Tool (http://kmplot.com/analysis)66, the correlation between A20 expression and the relapse-free survival rates of breast cancer patients was analyzed in two independent public GEO datasets (left; GSE9195, right; GSE2603). P values were calculated using a log-rank test. HR = hazard ratio (b) Proposed model demonstrating Snail1 stabilization by A20-mediated multi-monoubiquitination. In the absence of A20, Snail1 is phosphorylated by GSK3β at one of serine 107, 111, 115 and 119 residues and exported from the nucleus to the cytoplasm. Additional phosphorylation occurs at one of the serine 96 and 100 residues by GSK3β in the cytoplasm. β-TrCP1 subsequently recognizes these Snail1 phosphorylations and builds a K48-linked polyubiquitin chain on Snail1, resulting in proteasomal degradation. In the presence of A20, Snail1 is monoubiquitinated by A20 at multiple sites of lysine 206, 234 and 235 residues in the nucleus. This multi-monoubiquitination inhibits the interaction between Snail1 and GSK3β. Thus, GSK3β-mediated Snail1 phosphorylation is decreased and Snail1 stability in the nucleus is increased, eventually promoting EMT and metastasis.

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Lee, JH., Jung, S., Yang, KM. et al. A20 promotes metastasis of aggressive basal-like breast cancers through multi-monoubiquitylation of Snail1. Nat Cell Biol 19, 1260–1273 (2017). https://doi.org/10.1038/ncb3609

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