Abstract
Epithelial to mesenchymal transition (EMT), and the reverse mesenchymal to epithelial transition (MET), are known examples of epithelial plasticity that are important in kidney development and cancer metastasis. Here we identify ASPP2, a haploinsufficient tumour suppressor, p53 activator and PAR3 binding partner, as a molecular switch of MET and EMT. ASPP2 contributes to MET in mouse kidney in vivo. Mechanistically, ASPP2 induces MET through its PAR3-binding amino-terminus, independently of p53 binding. ASPP2 prevents β-catenin from transactivating ZEB1, directly by forming an ASPP2–β-catenin–E-cadherin ternary complex and indirectly by inhibiting β-catenin’s N-terminal phosphorylation to stabilize the β-catenin–E-cadherin complex. ASPP2 limits the pro-invasive property of oncogenic RAS and inhibits tumour metastasis in vivo. Reduced ASPP2 expression results in EMT, and is associated with poor survival in hepatocellular carcinoma and breast cancer patients. Hence, ASPP2 is a key regulator of epithelial plasticity that connects cell polarity to the suppression of WNT signalling, EMT and tumour metastasis.
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Acknowledgements
This work was supported mainly by the Ludwig Institute for Cancer Research Ltd (X.L.), and in part by the International Cooperation and Exchange Project of the National Natural Science Foundation of China (Y.G. and X.L.). Y.G. and J. Zhao are also supported by the National Basic Research Program of China (973 Program: 2010CB945600 and 2010CB833600). Y.W. is supported by the Medical Research Council (MR/J000930/1). S.S., J.R.L., M.S.S. and N.R.S. are funded by Cancer Research UK (grant number C5255/A12678). We thank K. Bryon-Dodd, G. Sutendra, P. Miller, M. White, L. Buti and C. Goding for critical reading of the manuscript. We are grateful to S. Shirasawa for providing HKe3 cells, F. Miller and S. Santner for the 4T1 and MCF10A series of breast cancer cell lines and Y. Fujita for the E-cadherin–Myc construct.
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X.L. and Y.G. coordinated and supervised the project and designed the experiments together with Y.W. and F.B.; Y.W. carried out most of the experiments, analysed data and wrote the paper with X.L.; F.B. carried out experiments and analysed data for Figs 3d, 6d, e, 7a, b, 8a–c and Supplementary Figs 6c, d, 8a; C.R. carried out experiments for Fig. 5f, g and Supplementary Figs 2k, 5b–d, f, g; S.S., J.R.L., M.S.S. and N.R.S. designed, carried out experiments and analysed data with Y.W. for Fig. 7c–e and Supplementary Fig. 7b; V.S. and F.F. carried out immunohistochemistry in breast cancer tissue microarrays designed by H.M. and analysed data for Fig. 8d, e and Supplementary Fig. 8b; C.T. carried out experiments for Fig. 1b and Supplementary Fig. 1a; S.K. carried out experiments for Fig. 1c; J. Zak and S.Z. provided materials for Supplementary Fig. 1b, c; G.W. and A.L. provided the human HCC samples; P.A.O. carried out some experiments related to this project; D.C.H. and J.D. provided HKe3–ER:HRASV12 and MCF10A–ER:HRASV12 cell lines; R.D.G. provided pathological advice; J. Zhao designed and analysed the experiments of HCC with F.B.; X.T. carried out experiments and analysed data with F.B. for Fig. 8a–c and Supplementary Fig. 8a.
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Integrated supplementary information
Supplementary Figure 1 ASPP2 regulates MET in mouse kidney in vivo.
(a) Immunohistochemical staining of ZEB1 in kidney tissues from ASPP2+/+ or ASPP2Δ3/Δ3 mice. Scale bar: 40 μm. (b) Immunofluorescence staining of ZEB1 (green) and E-cadherin (red) in kidney tissues from ASPP2loxP/loxP Cre–ER mice with indicated treatment. TO-PRO-3 (blue) was used to stain nuclei. Scale bar: 40 μm. (c) Protein expression of ZEB1, E-cadherin and β-catenin in kidney tissues from ASPP2loxP/loxP Cre–ER mice with indicated treatment. Scores under the bands are relative levels against control group (1.0). β-Tubulin was used as a loading control. Uncropped images of blots are shown in Supplementary Fig. 9.
Supplementary Figure 2 N-terminal ASPP2 is required and sufficient to induce MET.
(a) Immunofluorescence staining of ASPP2 (green), E-cadherin (green), ZEB1 (red) or Vimentin (green) in a panel of MCF10A cells. Arrows indicate junctional ASPP2 or E-cadherin. Scale bar: 10 μm. (b) Protein expression of ASPP2, E-cadherin, ZEB1, Vimentin and β-catenin in a panel of MCF10A cell lines. WT: wild-type β-Tubulin was used as a loading control. Scores under the bands are relative levels against WT (1.0). (c) Fold change in mRNA levels of ASPP2, E-cadherin and ZEB1 in a panel of MCF10A cell lines. (d) Protein expression of ASPP2 in a panel of MCF10A cell lines treated with or without 5-AZ. Scores under the bands are relative levels against untreated WT (1.0). (e) Fold change in mRNA levels of ASPP2, ZEB1 and E-cadherin following 5-AZ treatment in MCF10A-M3 cells. (f) Schematic representation of truncated ASPP2 mutants. (g) Immunofluorescence staining of V5-ASPP2 (green) and ZEB1 (red) in MCF10A-M3 cells transfected with V5-tagged full-length or ASPP2 fragments. DAPI (blue) was used to stain nuclei. Scale bar: 10 μm. Triangles indicate ZEB1 expression in V5 + cells. The percentage of V5 + /ZEB1 down-regulated cells in indicated transfected samples and the percentage of V5 + /with MET morphology change in indicated transfected samples are shown as graphs. Error bars indicate SD. n = 3 samples. 100 V5 positive cells were counted per sample. (h and i) Immunofluorescence staining of V5-ASPP2 (green) and ZEB1 (red) (h) or V5-ASPP2 (green) and E-cadherin (red) (i) in U2OS cells transfected with V5-tagged full-length or ASPP2 fragments. TO-PRO-3 (blue) was used to stain nuclei. Scale bar: 10 μm. Arrows indicate junctional staining of ASPP2 or E-cadherin. Triangles indicate ZEB1 expression in V5 + cells. The percentage of V5 + /E-cadherin + cells or V5 + /ZEB1 down-regulated cells in indicated transfected samples are shown as graphs. Error bars indicate SD. n = 3 samples. 100 V5 positive cells were counted per sample. (j) Immunofluorescence staining of E-cadherin (green) or ZEB1 (red) in MCF10A-M3 cells transfected with E-cadherin-myc plasmid. TO-PRO-3 (blue) was used to stain nuclei. Scale bar: 10 μm. (k) Immunofluorescence staining of E-cadherin (red) or ASPP2 (green) in MDA-MB-231 cells transfected with or without E-cadherin-myc plasmid. Scale bar: 10 μm. (l) Immunofluorescence staining of V5-ASPP2 (green) in MDA-MB-231 cells transfected with full-length V5-tagged ASPP2. TO-PRO-3 (blue) was used to stain nuclei. Scale bar: 10 μm. Data in c and e are the mean of 2 independent experiments performed in duplicate. For source data, see Supplementary Table 1. Uncropped images of blots are shown in Supplementary Fig. 9.
Supplementary Figure 3 ASPP2 depletion promotes EMT and sensitises cells to oncogenic RAS-induced EMT.
(a) Immunofluorescence staining of E-cadherin (red) or ZEB1 (green) in HCT116 cells with indicated RNAi transfections. TO-PRO-3 was used to stain nuclei. Scale bar: 10 μm. (b) Q-RT-PCR analysis of E-cadherin, Snail1, Snail2, ZEB1 and TWIST expression in HCT116 cells transfected with control or ASPP2 RNAi. (c) Immunofluorescence staining of E-cadherin (green) in HCT116 cells with indicated RNAi transfections. TO-PRO-3 (blue) was used to stain nuclei. Scale bar: 10 μm. (d) Immunofluorescence staining of ZEB1 (green) in MCF10A ER:HRAS V12 cells with indicated treatments. TO-PRO-3 (blue) was used to stain nuclei. Scale bar: 10 μm. (e) Q-RT-PCR and western blot of samples from MCF10A-ER:HRASV12 cells with indicated treatments showing effects on ZEB1 and ASPP2. β-Tubulin was used as a loading control. Data in b and e are the mean of 2 independent experiments performed in duplicate. For source data, see Supplementary Table 1. Uncropped images of blots are shown in Supplementary Fig. 9.
Supplementary Figure 4 ASPP2 knockdown induces ZEB1 expression via β-catenin, not p53.
(a) Western blot of lysates from HKe3-ER:HRASV12 cells with indicated treatments showing effects on ZEB1 and p53. GAPDH was used as a loading control. (b) ChIP assays of β-catenin’s ability to bind the ZEB1 promoter in HKe3 ER:HRAS V12 cells with the indicated treatments. qPCR of fragments of the ZEB1 promoter immunoprecipitated in ChIP assays from HKe3 ER:HRAS V12 cells with an antibody against β-catenin and control IgG. Amplified ZEB1 promoter region (−325∼−101) contains β-catenin/TCF binding sites at −161. (c) Protein expression of ZEB1, ASPP2, ASPP1 and β-catenin in HKe3-ER:HRASV12 cells with indicated treatments. β-Tubulin was used as a loading control. (d) Western blot showing the knockdown of β-catenin in MCF10A-WT or M3 cells. GAPDH was used as a loading control. (e) Protein expression of ZEB1, ASPP2 and β-catenin in 4-OHT-treated MCF10A-ER:HRASV12 cells with indicated RNAi transfections. β-Tubulin was used as a loading control. (f) Protein expression of ZEB1, ASPP2 and β-catenin in HepG2 cells with indicated RNAi transfections. β-Tubulin was used as a loading control. Uncropped images of blots are shown in Supplementary Fig. 9.
Supplementary Figure 5 ASPP2 binds β-catenin and negatively regulates the WNT/β-catenin pathway.
(a) Immunofluorescence staining of V5-ASPP2 or β-catenin in MCF10A-M3 cells transfected with control plasmid (Vector) or V5-ASPP2. DAPI (blue) was used to stain nuclei. Scale bar: 10 μm. Arrows indicate junctional staining of ASPP2 or β-catenin. (b) Immunofluorescence staining of ASPP2 (green) and E-cadherin or β-catenin (magenta) in MCF7 cells. Scale bar: 10 μm. (c) Co-immunoprecipitation experiment using in vitro-translated (IVT) β-catenin and V5-tagged ASPP2. A rabbit polyclonal antibody against ASPP2 was used to immunoprecipitate ASPP2. β-catenin and ASPP2 levels were then analysed by western blot. (d) Different amount of total cell lysates from MCF7 and MDA-MB-231 cells, 30 μg or 60 μg used as input respectively, were immunoprecipitated with a rabbit polyclonal anti-ASPP2 antibody or control IgG. β-catenin, E-cadherin and ASPP2 levels are indicated. β-Tubulin was used as a loading control. (e) ASPP2 binds β-catenin and the binding does not require PAR3. Total cell lysates from HKe3 cells transfected with control or PAR3 RNAi were immunoprecipitated with a rabbit polyclonal antibody against ASPP2 or control IgG. β-catenin, PAR3 and ASPP2 levels were then analysed by western blot. IgGL indicates IgG light chain. (f) Immunofluorescence staining showing the effect of WNT3a on endogenous β-catenin in wild-type and ASPP2Δ3/Δ3 MEFs, respectively. Green and red arrows indicate nuclei containing β-catenin in wild-type or ASPP2Δ3/Δ3 MEFs, respectively. Scale bar: 50 μm. (g) Immunofluorescence staining showing the effect of LiCl on endogenous β-catenin in wild-type or ASPP2Δ3/Δ3 MEFs, respectively. Green and red arrows indicate nuclei containing β-catenin in wild-type and ASPP2Δ3/Δ3 MEFs, respectively. Scale bar: 50 μm. Uncropped images of blots are shown in Supplementary Fig. 9.
Supplementary Figure 6 ASPP2 deficiency promotes cell migration and invasion.
(a) Representative phase contrast images of MCF10A-ER:HRASV12 cells cultured in Matrigel with indicated treatments. Scale bar: 40 μm. (b) Protein expression of ASPP2 in MCF10A-ER:HRASV12 cells transfected with control or ASPP2 RNAi for the indicated time. iASPP was used as a loading control. (c) Hep3B or HCC-LM3 cells were infected with ASPP2 shRNA or shNon for 72 h. Cells were then seeded in 96-well plates for MTS assay. (d) Hep3B or HepG2 cells infected with ASPP2 shRNA or shNon were subjected to transwell migration assay or Matrigel invasion assay. ∗P < 0.05. The expressions of ASPP2 were detected by western blot. Data in c and d are the mean from a representative experiment, and error bars indicate SEM. n = 6 wells in c and n = 5 random fields in d. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. The experiments were repeated 5 times. Uncropped images of blots are shown in Supplementary Fig. 9.
Supplementary Figure 7 ASPP2 expression inhibits metastasis in vivo.
(a) Western blot of V5 in 4T1-GFP cells transfected with control plasmids or V5-tagged full-length (1-1128) or N terminal (1-360) ASPP2. (b) Visible tumours were counted in the 4T1-GFP model by virtue of the H&E staining; graph showing total tumour number per mouse brain for each group. Data in b are individual values with mean, and error bars indicate SD. n = 3 mice per group. For source data, see Supplementary Table 1. Uncropped images of blots are shown in Supplementary Fig. 9.
Supplementary Figure 8 ASPP2 expression correlates with EMT status in human HCC, and the ASPP2 expression pattern in breast cancer is also shown.
(a) The relationships between ASPP2 expression and protein expression of β-catenin, ZEB1 and E-cadherin were analysed (Mann–Whitney U-test: P < 0.001). Nuclear β-catenin negative n = 133 and nuclear β-catenin positive n = 40; nuclear ZEB1 negative n = 126 and nuclear ZEB1 positive n = 53; junctional E-cadherin positive n = 69 and junctional E-cadherin negative n = 117. Spread of data denoted by box-and-whisker plot, the central box represents the value from the lower to upper quartile (25th–75th percentile). The middle line represents the median. The whisker ends 1 and 99 percentiles. (b) Representative ASPP2 staining pattern (High or Low ASPP2) in 275 human breast cancer tissue microarray cores. Scale bar: 50 μm. For source data, see Supplementary Table 1.
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Wang, Y., Bu, F., Royer, C. et al. ASPP2 controls epithelial plasticity and inhibits metastasis through β-catenin-dependent regulation of ZEB1. Nat Cell Biol 16, 1092–1104 (2014). https://doi.org/10.1038/ncb3050
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DOI: https://doi.org/10.1038/ncb3050
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