Loss of apical–basal polarity and activation of epithelial–mesenchymal transition (EMT) both contribute to carcinoma progression and metastasis. Here, we report that apical–basal polarity inhibits EMT to suppress metastatic dissemination. Using mouse and human epithelial three-dimensional organoid cultures, we show that the PAR–atypical protein kinase C (aPKC) polarity complex inhibits EMT and invasion by promoting degradation of the SNAIL family protein SNAI1. Under intact apical–basal polarity, aPKC kinases phosphorylate S249 of SNAI1, which leads to protein degradation. Loss of apical–basal polarity prevents aPKC-mediated SNAI1 phosphorylation and stabilizes the SNAI1 protein to promote EMT and invasion. In human breast tumour xenografts, inhibition of the PAR-complex-mediated SNAI1 degradation mechanism promotes tumour invasion and metastasis. Analyses of human breast tissue samples reveal negative correlations between PAR3 and SNAI1 protein levels. Our results demonstrate that apical–basal polarity functions as a critical checkpoint of EMT to precisely control epithelial–mesenchymal plasticity during tumour metastasis.
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We thank the members of the Yang lab for helpful discussions and especially T. Lee for mouse colony maintenance, J. Callender for technical help and K. Yeung for constructive comments. We thank A. G. de Herreros for the SNAI1 EC3 antibody, M. Aumailley for the anti-laminin V antibody, A. Ewald for advice on mouse primary mammary organoid isolation and I. Macara for the pLV-shPAR3-Venus construct used in pilot experiments. We thank the UCSD Shared Microscope Facility and UCSD Cancer Centre Support Grant (grant no. P30 CA23100 from the NCI). This work was supported by grants from the ACS (grant no. RSG-09-282-01-CSM) and NCI (grant nos 1RO1CA168689, 1R01CA174869, 1R01CA206880 and 1R21CA191442) to J.Y., and the NIH (grant nos NIH R35 GM122523 and NIH P01 DK054441) to A.C.N.
Integrated supplementary information
(a) qPCR analysis of relative E-cadherin and Vimentin mRNA levels in MECs and MEOs isolated from TetON-SNAI1 mice before and after doxycycline treatment for 4 days. n=3 independent experiments, paired two-tailed t-test. Error bars represent standard deviation. (b) Bright-field images of TetON-TWIST1 MEOs in 3D and MECs in 2D before and after doxycycline treatment for 4 days. Scale bar, 25 μm. (c) Immunofluorescence images for E-cadherin and Fibronectin in TetON-TWIST1 MEOs and MECs. Scale bar, 25 μm, (d) qPCR analysis of relative E-cadherin and Vimentin mRNA levels in MECs and MEOs isolated from TetON-Twist1 mice before and after doxycycline treatment for 4 days. n=3 independent experiments, paired two-tailed t-test. Error bars represent standard deviation. (e) qPCR analysis of relative Twist1 mRNA levels in TetON-Twist1MECs and MEOs before and after doxycycline induction. n=3 independent experiments, paired two-tailed t-test Error bars represent standard deviation. (f) Immunofluorescence images for TWIST1 in TetON-TWIST1 MEOs and MECs. Scale bar, 25 μm. All immunofluorescence images represent one out of three independent experiments. Source data for graphs can be found in Supplementary Table 3.
(a) Immunofluorescence images for Par3, aPKC, and F-actin. White arrows point to Par3 localization at the apical/basal region and aPKC at the apical membrane. Scale bar in main images, 25 μm and Scale bar in insets, 50 μm. (b) Representative immunofluorescence images of GFP-positive cells in MEOs upon lentiviral infection to determine infection efficiency in primary MEOs. The graph shows the percentage of GFP-positive cells in MEOs. Scale bars, 25μm. n=13 organoids, error bars represent standard deviation. Images show one out of three independent experiments.
(a) Immunoblot for SNAI1 and GAPDH in TetON-SNAI1 MECs and MEOs under various treatment conditions as indicated. CHIR-99021(CHIR) is a selective GSK3 inhibitor. Values indicate relative signal intensities of SNAI1/GAPDH. The graph represents the quantification of relative SNAI1 protein levels. n=3 independent experiments, Error bars represent standard deviation. (b) The predicted phosphorylation sites on human and mouse SNAI1 gene. (c) The list of predicted phospho sites and motifs on human and mouse SNAI1 proteins. SA is the score for surface accessibility. (d) Immunoblot for PKCζ, SNAI1 and GAPDH in 293T cells overexpressing SNAI1 WT and 6SA under various treatment conditions as indicated. The values indicate the relative signal intensity of SNAI1/GAPDH. Data show a representative of three independent experiments. Source data for graphs can be found in Supplementary Table 3 and unprocessed blots in Supplementary Fig. 7.
(a) Endogenous SNAI1 proteins were immunoprecipitated from Caco2 2D vs. 3D cultures and probed for Ub, β-TrCP, phospho-SNAI1(S249) and SNAI1. Immunoblot for aPKC, β-TrCP, SNAI1 and GAPDH in Caco2 2D vs. 3D cultures, Three independent experiments. (b) qPCR analysis of relative SNAI1 mRNA levels normalized to GAPDH in Caco2 organoids expressing shRFP or shPAR3 #3, and #4. n=3 independent experiments, paired two-tailed t-test, Error bars represent standard deviation. (c) Immunoblots for PAR3, SNAI1 and GAPDH in Caco2 organoids expressing the indicated shRNA constructs. Three independent experiments. Source data for graphs can be found in Supplementary Table 3 and unprocessed blots in Supplementary Fig. 7.
(a) FRET imagining of PKC activity in Caco2 organoids co-transfected with the PAR6-scaffolded PKC activity reporter, CKAR-PB1Par6, and mCherry-tagged PKMζ or mCherry-Vec control and treated with 5uM PZ09. The trace for each cell imaged was normalized to its 0-min baseline value and plotted as means. n=3 independent experiments. (b) qPCR analysis of relative SNAI1 mRNA levels in Caco2 organoids in response to PZ09 treatment. n=3 independent experiments, paired two-tailed t-test. Error bars represent standard deviation. (c) Immunofluorescence images for PARζ3, aPKC, SNAI1, F-actin in Caco2 organoids with or without PZ09 treatment. Scale bar, 25 μm. (d) Immunoblot for SNAI1 and GAPDH in Caco2 organoids expressing the indicated shRNA constructs. Three independent experiments. (e) Immunofluorescence images for E-cadherin, Fibronectin, Laminin V, SNAI1 and F-actin in Caco2 organoids expressing shRFP or shPKCζ #3, and shPKCι #4. Scale bar, 25 μm, (f) Immunoblot for aPKC, PKCι, SNAI1 and GAPDH in Caco2 organoids expressing the indicated shRNA constructs. All immunofluorescence images and Western Blots shown represent one out of three independent experiments. Source data for graphs can be found in Supplementary Table 3 and unprocessed blots in Supplementary Fig. 7.
Supplementary Figure 6 Negative regulation of PAR3, aPKC and SNAI1 in human breast cancer progression.
(a) Immunofluorescence images for E-cadherin and Vimentin in Caco2 organoids expressing the indicated shRNA constructs. Scale bars, 25μm. (b) Representative immunofluorescence images for PAR3, aPKC, and SNAI1 in human breast tissue samples. Scale bars in main images, 25μm and scale bars in insets, 50μm, (c) A model describing the feedback mechanism linking the PAR3/aPKC complex and SNAI1 regulation during tumour progression. All images are representative of three independent experiments.
Unprocessed blots corresponding to all the main and supplementary figures.
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Nature Cell Biology (2019)