Epithelial–mesenchymal transition (EMT) is a pivotal mechanism for cancer dissemination. However, EMT-regulated individual cancer cell invasion is difficult to detect in clinical samples. Emerging evidence implies that EMT is correlated to collective cell migration and invasion with unknown mechanisms. We show that the EMT transcription factor Snail elicits collective migration in squamous cell carcinoma by inducing the expression of a tight junctional protein, claudin-11. Mechanistically, tyrosine-phosphorylated claudin-11 activates Src, which suppresses RhoA activity at intercellular junctions through p190RhoGAP, maintaining stable cell–cell contacts. In head and neck cancer patients, the Snail–claudin-11 axis prompts the formation of circulating tumour cell clusters, which correlate with tumour progression. Overexpression of snail correlates with increased claudin-11, and both are associated with a worse outcome. This finding extends the current understanding of EMT-mediated cellular migration via a non-individual type of movement to prompt cancer progression.
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cDNA microarray data that support the findings of this study have been deposited in the GEO under the accession code GSE87841 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE87841). The array-CGH data were deposited in the GEO database under the accession code GSE114122 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE114122). The whole-genome sequencing data were deposited in the Sequence Read Archive (SRA) database under the accession code SRP157020 (https://www.ncbi.nlm.nih.gov/sra/SRP157020). Snail-regulated cell-movement and cell-adhesion genes were analysed using the trailblazer gene signature under the accession code GSE58643 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58643). The differentially expressed genes regulated by Snail were analysed using the lymph node metastasis gene signature of HNSCC under the accession code GSE36942 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE36942). Mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD010908 (https://www.ebi.ac.uk/pride/archive/projects/PXD010908). The hyperlink to the PrognoScan dataset derived from this resource for human colorectal cancer is http://dna00.bio.kyutech.ac.jp/PrognoScan-cgi/PrognoScan.cgi?TITLE=Prognostic+value%20of%20CLDN11%20mRNA%20expression%20in%20Colorectal%20cancer&DATA_POSTPROCESSING=None&TEST_NUM=80&MODE=SHOW_GRAPH&PROBE_ID=4037590 and for ovarian cancer is http://dna00.bio.kyutech.ac.jp/PrognoScan-cgi/PrognoScan.cgi?TITLE=Prognostic+value%20of%20CLDN11%20mRNA%20expression%20in%20Ovarian%20cancer&DATA_POSTPROCESSING=None&TEST_NUM=53&MODE=SHOW_GRAPH&PROBE_ID=2006434. Statistical source data for Figs. 1b,c,f, 2a,d,g, 3c–f, 4a,f, 6c–f and 7b–f and Supplementary Figs. 1e,g,h,o, 2b,c,f–i, 3a, 4l,o, 5c, 6f–h and 7a–d have been provided as Supplementary Table 11. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.
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We thank C.-H. Lin and Y.-L. Huang (National Yang-Ming University, Taiwan) for help with mass spectrometry data analysis; Z.-F. Chang (National Taiwan University, Taiwan) for providing the pTriEx–RhoA FLARE.sc Biosensor WT plasmid; M.-C. Hung (The University of Texas M.D. Anderson Cancer Center, TX) for the pHA–CBP, pHA–EECBP and pHA–AACBP plasmids; Y.-J. Lee (National Yang-Ming University, Taiwan) for the LT-3R plasmid; T.-C. Lee (Academia Sinica, Taiwan) for providing the HFW cell line for organotypic culture; and M.-Y. Liao and H.-C. Wu (Academia Sinica, Taiwan) for providing the mouse anti-human EpCAM antibody. We thank H.-Y. Chen (Instrument Center of National Chung-Hsing University, Taiwan) for technical support of the mass spectrometry analysis; S.-R. Chiang (Taipei Veterans General Hospital, Taiwan) for technical support of the IHC assay; J.-I Lai, J.-R. Huang, T.-H. Jeng, Y.-A. Hsieh, Y.-J. Lin, Y. Ho, H.-Y. Yu, C.-Y. Chen, K.-C. Wu, S.-Y. Ho, K.-C. Kao and Y.-J. Chen (Taipei Veterans General Hospital, Taiwan) for collecting the blood from HNSCC patients for the CTC assay; and Y.-J. Zheng for technical support of the CTC assay (Academia Sinica, Taiwan). We thank Welgene Biotech Co., Ltd (Taipei, Taiwan) for technical support of array-CGH, and T.-T. Liu (National Yang-Ming University, Taiwan) for technical support and analysing the WGS data. This work was supported in part by the Division of Experimental Surgery of the Department of Surgery, Taipei Veterans General Hospital. This work was supported by grants from the Ministry of Science and Technology (103-2633-H-010-001, 104-2321-B-010-005, 104-0210-01-09-02, 105-0210-01-13-01, 106-0210-01-15-02 and 107-0210-01-19-01); National Health Research Institutes (NHRI-EX107-10622BI); Taipei Veterans General Hospital (V107C-071, V107D32-001-MY2-1, VTA105-V1-3-2, VTA106-V1-3-3 and 107-V1-3-2); Veterans General Hospital-University System of Taiwan Joint Research Program (VGHUST107-G4-1-3); the Cancer Progression Research Center of National Yang-Ming University granted by the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education; and the Ministry of Health and Welfare, Center of Excellence for Cancer Research (MOHW107-TDU-B-211-114019).
The authors declare no competing interests.
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Integrated supplementary information
(a) Snapshots from phase-contrast videos of cells in 2.5D. Scale bars, 100 μm. The representative data were from two independent experiments with similar results. (b) Western blots of EMT-TFs. Red arrows indicate Slug/Snail. (c) Images of knockdown of EMT-TFs in SAS cells in 2.5D. Scale bars, 100 μm. (d) Upper: snapshots from phase-contrast videos of TE1-pLKO/TE1-shSNAI1 in 2.5D. Scale bars, 100 μm. Lower: directionality of migration (n = 15 cells). The data is representative of three independent experiments with similar results. (e) Upper: schema of the animal experiment. Middle: bioluminescence images of mice at the end of week 3. Lower: quantification of IVIS images (n = 10 mice). The mean values are shown (two-sided Mann-Whitney test). (f) GSEA for Snail-regulated gene signature and lymph node metastasis of HNSCC patients. P-values were calculated based on 1000 permutations by the GSEA algorithm and no adjustments were made for multiple comparisons. NES, normalized enrichment score; FDR, false discovery rate; FWER, family-wise error rate. (g) Left: representative western blots of E-cadherin and Snail in 2D/2.5D. Right: quantification of E-cadherin (two-sided Student’s t test). (h) Left: representative immunofluorescent images of E-cadherin in SAS-pLKO/SAS-shSNAI1 in 2D/2.5D. Scale bars, 10 μm. Right: quantification of E-cadherin mislocalization. (i) Immunoprecipitation-western blots and (j) western blots showing lysine-acetylated Snail in cells cultured in 2D/2.5D. (k) Western blots of indicated proteins in cells treated with DMSO or FAK inhibitor 14 (1 μM) for 24 h. (l), (m) and (n) Immunoprecipitation-western blots/western blots of indicated proteins in SAS cells treated with indicated chemicals for 24 h (FAK inhibitor 14, 1 μM; C646, 10 μM; PD98059, 10 μM). (o) RT-qPCR of the indicated genes in TE1 cells. The P value is estimated by ANOVA. b, c, f, and i– n was from one experiment. g, h and o were analysed from three independent experiments (n = 3). Data represent mean ± s.d. shown in g, h and o. NS, not significant. Uncropped images of all blots are shown in Supplementary Figure 8. Source data are provided in Supplementary Table 11.
(a) A heatmap show the expression of genes related to cell adhesion in SAS-pLKO vs. SAS-shSNAI1 cells. The data was from one experiment. (b) RT-qPCR indicated the expression of cell adhesion molecules and (c) claudins family genes in SAS-pLKO/SAS-shSNAI1 (n = 2 independent experiments). (d) Western blots of claudin-3, -4 and -7 in SAS-pLKO/SAS-shSNAI1 cells. The data was from one experiment. (e) Upper: snapshots from phase-contrast videos of TE1-pLKO/TE1-shCLDN11 cells moving on collagen gels. Scale bars, 100 μm. Lower: directionality of migration (n = 10 cells). (f) Left: representative images of 3D invasion assays. Yellow arrow indicates collectively invasive cells. The y-axis indicates invasion distance and y-axis heights, 60 μm. Right: the relative invasion index (two-sided Student’s t test). (g) MTT assay. The effect of CLDN11 knockdown on viability in SAS cells. The p-value is estimated by ANOVA. (h) Left: representative images of Transwell migration assay of SAS-pLKO/SAS-shCLDN11 cells. Scale bars, 100 μm. Right: quantification of Transwell migration assay (two-sided Student’s t test). (i) Left: schema of the mice experiment. Middle: three representative bioluminescence images of the mice at the end of week 3. Right: quantification of IVIS images (n = 10 mice; two-sided Mann-Whitney U test). (j) Representative images of H&E staining in lymph node samples from mice orthotopic model. Cropped regions are indicated by the yellow rectangle in the overview panels. Red arrows indicate the regions with tumor infiltration. Scale bars, 100 μm. (k) Western blots of claudin-11 in FaDu-WT vs. FaDu-LN cells. WT, wild-type; LN, lymph node. The representative data were from two independent experiments with similar results. (a), (b), (c), (d) and (e) were performed in 2.5D. (f), (g) and (h) were analyzed from three independent experiments (n = 3). Images of (e), (f), (h), and (j) are representative of three independent experiments with similar results. Data represent mean values shown in (b), (c), (g) and (i). Data represent mean ± S.D. shown in (f), (g) and (h). N.D., no detection; N.S., no significance. Uncropped images of all blots are shown in Supplementary Figure 8. Source data is provided in Supplementary Table 11.
(a) RT-qPCR analysis of CLDN3, CLDN4, CLDN7, and SNAI1 in primary HNSCC cells expressing control (pCDH), wild-type Snail (Snail-WT) or unacetylatable Snail (Snail-2R). Caco2 cells was used as a positive control for the expressions of CLDN3, CLDN4 and CLDN7 (n = 2 independent experiments). Data represent mean values. (b) Western blots for showing claudin-3, -4, -7, and Snail in primary HNSCC cells expressing control (pCDH), wild-type Snail (Snail-WT) or unacetylatable Snail (Snail-2R). Caco2 cells was used as a positive control for the expressions of claudin-3, -4, and -7. β-actin was a loading control. The data was from one experiment. Uncropped images of all blots are shown in Supplementary Figure 8. Source data is provided in Supplementary Table 11.
(a) A pull down assay indicated the level of RhoA-GTP in pLKO/shCLDN11 cells and (b) in SAS-WT vs. SAS-LN cells. (c), (d) and (e) Immunoprecipitation-western blots of indicated proteins in SAS cells. (f) Left: representative snapshots from phase-contrast videos of TE1-pLKO/TE1-shGRLF1 cells moving on collagen gels. Scale bars, 100 μm. Lower: directionality of migration (n = 10 cells). The data is representative of three independent experiments with similar results. (g) Western blots analysis of the levels of indicated proteins in SAS-pLKO/SAS-shSNAI1. (h) A pull down assay for detecting the RhoA-GTP, and western blots for examining the levels of indicated proteins in SAS cells (imatinib, 10 μM; SU6656, 10 μM). (i) Western blots for the indicated proteins in pLKO/shSNAI1 cells. (j) Western blots for the indicated proteins in pLKO/shCLDN11 primary HNSCC cells in 3D for 3 h. (k) Immunoprecipitation-western blots for determining the phosphorylation status of Y418 residue of Src family in SAS-pLKO/SAS-shCLDN11 cells. (l) Left: FRET images of the pTriEx-RhoA biosensor-expressed SAS-pLKO/SAS-shSNAI1 cells. Right: quantification of normalized FRET efficiency ratio signal (n = 3 independent experiments). Data represent mean ± S.D. (two-sided Student’s t test). N.S., no significance. (m) Representative immunofluorescent images for showing the localization MLC2-pT18/S19 in SAS-pLKO/SAS-shSNAI1. Yellow arrows indicate the co-localization of MLC2-pT18/S19 and F-actin. Scale bars, 10 μm. (n) A heatmap showing the expression of genes encoding GEFs and GAPs in SAS-pLKO/SAS-shSNAI1. (o) RT-qPCR for validation of the expression of GEFs and GAPs in SAS-pLKO/SAS-shSNAI1 (n = 2 independent experiment). Data represent mean values. (p) Western blots of ARHGAP42 in SAS-pLKO/SAS-shSNAI1. Red arrows indicate the positions of ARHGAP42. All cells were cultured in 2.5D with the exception of (j) in 3D. (a), (b), (h), (k), (m) and (n) was from one experiment. The representative data (c), (d), (e), (g), (i), (j) and (p) were from two independent experiments with similar results. Uncropped images of all blots are shown in Supplementary Figure 8. Source data is provided in Supplementary Table 11.
(a) Amino acid sequence of claudin-11 (accession code: O75508). The cytosolic tail is highlighted in red. (b) Western blots of Y418-phosphorylated Src and Y530-phosphorylated Src in SAS-pLKO/SAS-shCLDN11. The representative data were from three independent experiments with similar results. (c) GAP activity assay for analyzing the p190RhoGAP activity in HEK293T cells co-transfected with the p190RhoGAP expression vector (pHA-GRLF1) and a vector expression wild-type (WT) or different tyrosine residue-mutated CLDN11, (n = 3 independent experiments). Data represent mean ± S.D. (two-sided Student’s t test). N.S., no significance. (d) Pull down assay/western blots for analyzing the GTP-bound/total RhoA in SAS cells transfected with plasmids expressing wild-type or different tyrosine residue-mutated FLAG-tagged claudin-11. The data was from one experiment. (e) Confirmation of the efficacy of different inhibitors with the western blots of T202/Y204-phosphorylated ERK1/2, and total ERK1/2 in SAS cells treated with different tyrosine kinase inhibitors. The representative data were from two independent experiments with similar results. (f) Immunoprecipitation-western blots for showing the level of tyrosine-phosphorylated claudin-11 in SAS treated with different tyrosine kinase inhibitors following by seeding on the thick collagen for 30 min (cetuximab, 10 μg/ml for 30 min; all other tyrosine kinases inhibitors, 10 μM for 30 min). The representative data were from two independent experiments with similar results. (g) Left: representative snapshots from phase-contrast videos SAS cells treated with DMSO, FAK inhibitor 14 or cetuximab moving on collagen gels. Solid line indicates the position of cell groups at t = 0 h; shading indicates the position of the same cell groups at t = 7 h. Scale bars, 100 μm. Right: directionality of migration presented in rose plot diagrams for SAS cells treated within DMSO, FAK inhibitor 14 or cetuximab (n = 20 cells). The data is representative of two independent experiments with similar results. (h) Western blots of integrin β1, FAK, and Y397-phosphorylated FAK in SAS-pLKO vs. SAS-shSNAI1. The data was from one experiment. Uncropped images of all blots are shown in Supplementary Figure 8. Source data is provided in Supplementary Table 11.
Supplementary Figure 6 Correlation between the number of CTC clusters and the clinical course of HNSCC patients.
(a) Images of the CTC clusters from one representative HNSCC patient. 208 CTC clusters were captured on the biomimetic supported lipid bilayer (SLB) coated microfluidic chip conjugated with a mouse anti-human EpCAM antibody in 2 ml whole blood. The cells were stained for pan-cytokeratin (Pan-CK, red), CD45 (green), and nuclei (DAPI, blue). Scale bars, 10 μm. The same CTC capturing experiment was performed in samples from 28 HNSCC patients. (b), (c), (d) and (e) Representative images of CTC clusters and WBCs from HNSCC patients. (b), the cells were stained with the antibody against pan-cytokeratin (Pan-CK, red), CD45 (green), and Snail (cyan). The images are representative of two independent CTC clusters with similar results. (c), the cells were stained with the antibodies against pan-cytokeratin (Pan-CK, red), CD45 (green), and Src-pY418 (cyan). The images are representative of five independent CTC clusters with similar results. (d), the cells were stained with the antibody against pan-cytokeratin (Pan-CK, red), CD45 (green), and p190RhoGAP-pY1087 (cyan). The images are representative of five independent CTC clusters with similar results. (e), the cells were stained with the antibody against pan-cytokeratin (Pan-CK, red), CD45 (green), and p190RhoGAP-pY1105 (cyan). The images are representative of six independent CTC clusters with similar results. All nuclei were stained with DAPI (blue). Scale bars, 10 μm. (f), (g) and (h) Quantification of single CTC in HNSCC patients with different (f) T stages, (g) lymph node metastasis status, and (h) recurrence. The center values indicate the mean values. The p-value is shown in each panel (two-sided Student’s t test). P, primary; R, recurrence; M, metastasis. (i), (j) and (k) Correlation between the amount of CTC clusters and clinical courses in three different HNSCC patients [patient 2 (i), 3 (j), 4 (k)] receiving treatment. Quantification of CTC clusters at different time points of treatment is illustrated. The treatment course of each patient is indicated at the bottom of the panel. Chemo, chemotherapy; CCRT, concurrent chemoradiotherapy; PR, partial response. Source data is provided in Supplementary Table 11.
Supplementary Figure 7 Expression of Snail or claudin-11 correlates with an advanced HNSCC and a worse prognosis.
(a) Immunohistochemical expression score of claudin-11 and Snail in adjacent normal tissues vs. HNSCC specimens. Y-axis displays the expression calculated by a modified H-score of claudin-11 (left) or Snail (right). The center values indicate the mean values. The p-value of each panel is shown (two-sided Student’s t test). N, normal; T, tumor. (b) Correlation between Snail expression and CTC counts in HNSCC patients. Left: correlation between Snail expression and clustering CTC counts. Right: correlation between Snail expression and single CTC counts. Each dot represents a HNSCC specimen (n = 21 patients). The Pearson correlation coefficient (r) and p-value are shown. (c) The correlation between the relative expression of SNAI1 and CLDN11 in 43 HNSCC specimens. Each red dot represents a specimen. The Pearson correlation coefficient (r) and p-value are shown. (d) Quantification of the relative CLDN11 (left) and SNAI1 (right) mRNA levels in tumor vs. normal parts of HNSCC patients. The p-value of each panel is shown (two-sided Student’s t test). N, normal; T, tumor. (e) The Kaplan-Meier survival plots to show the prognostic effect of CLDN11 expression in colorectal cancer (GSE17536) and ovarian cancer (DUKE-OC). The data were obtained from the public database PrognoScan. The corresponding p-value is indicated in each panel (two-sided Log-rank test). Source data is provided in Supplementary Table 11.
Uncropped films showing the full blots displayed in the figures.
Supplementary Figures 1–8, and Supplementary Video and Supplementary Table legends.
Upregulated and downregulated genes in cDNA microarray analysis of SAS-pLKO versus SAS-shSNAI1 cells.
List of DNA copy number aberrations in pan-CK+/CD45- CTCs and pan-CK-/CD45+ WBCs from a HNSCC patient.
Genetic alterations of 6 HNCC related genes detected in the pan-CK+/CD45- CTC but not in the matched WBC for patient A (Supplementary Table 3.1) and patient B (Supplementary Table 3.2).
Demographics, CTC counts and IHC results of 28 HNSCC patients.
Demographics and IHC results of 54 HNSCC patients.
Predictors of progression free survival; Cox multivariable analysis.
Demographics and RT-qPCR results of 43 HNSCC patients.
Relationships between CLDN11 expression and prognosis of patients with colorectal cancer and ovarian cancer were investigated using the PrognoScan database.
Information of nucleotide sequences used in the study.
Information of antibodies used in this study.
Statistics source data.
Time-lapse video microscopy of SAS, TE1, TE9 and HSC3 in 2.5D cultured system.
Time-lapse video microscopy of SAS-control and SAS-shSNAI1 in 2.5D cultured system.
Time-lapse video microscopy of TE1-control and TE1-shSNAI1 in 2.5D cultured system.
Movie illustrates a 3D invasion assay of 100% SAS-pLKO-GFP (green), 100% SAS-shSNAI1-RFP (red), or 2% SAS-pLKO-GFP/98% SAS-shSNAI1-RFP cells.
Time-lapse video microscopy of SAS-control and SAS-shCLDN11 in 2.5D cultured system.
Time-lapse video microscopy of TE1-control and TE1-shCLDN11 in 2.5D cultured system.
Time-lapse video microscopy of SAS-control and SAS-shGRLF1 in 2.5D cultured system.
Time-lapse video microscopy of TE1-control and TE1-shGRLF1 in 2.5D cultured system.
Time-lapse video microscopy of SAS treated with DMSO, FAK inhibitor 14 or cetuximab in 2.5D cultured system.
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Li, CF., Chen, JY., Ho, YH. et al. Snail-induced claudin-11 prompts collective migration for tumour progression. Nat Cell Biol 21, 251–262 (2019). https://doi.org/10.1038/s41556-018-0268-z
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