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The EMT-activator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer

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Abstract

Metastasis is the major cause of cancer-associated death. Partial activation of the epithelial-to-mesenchymal transition program (partial EMT) was considered a major driver of tumour progression from initiation to metastasis. However, the role of EMT in promoting metastasis has recently been challenged, in particular concerning effects of the Snail and Twist EMT transcription factors (EMT-TFs) in pancreatic cancer. In contrast, we show here that in the same pancreatic cancer model, driven by Pdx1-cre-mediated activation of mutant Kras and p53 (KPC model), the EMT-TF Zeb1 is a key factor for the formation of precursor lesions, invasion and notably metastasis. Depletion of Zeb1 suppresses stemness, colonization capacity and in particular phenotypic/metabolic plasticity of tumour cells, probably causing the observed in vivo effects. Accordingly, we conclude that different EMT-TFs have complementary subfunctions in driving pancreatic tumour metastasis. Therapeutic strategies should consider these potential specificities of EMT-TFs to target these factors simultaneously.

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Figure 1: Zeb1 depletion reduces invasion and metastasis in pancreatic cancer.
Figure 2: Depletion of Zeb1 affects phenotypic variability of tumour cells.
Figure 3: Depletion of Zeb1 affects stemness, tumorigenic and colonization capacities.
Figure 4: Depletion of Zeb1 reduces ADM- and PanIN-precursor lesions.
Figure 5: Depletion of Zeb1 reduces phenotypic variability.
Figure 6: Depletion of Zeb1 reduces TGFβ-induced cellular plasticity.
Figure 7: Depletion of Zeb1 reduces metabolic and phenotypic plasticity.

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Acknowledgements

We thank B. Schlund, E. Bauer and J. Pfannstiel, as well as U. Appelt and M. Mroz (Core Unit Cell Sorting and Immunomonitoring, FAU Erlangen, Germany) for technical assistance and R. Eccles for critical reading of the manuscript. We are grateful to D. Saur (Department of Internal Medicine, TU Munich, Germany) for providing the KPCS cell lines. We thank J. C. Wu, from Stanford University, for the MSCV-LUC_EF1-GFP-T2A-Puro plasmid. This work was supported by grants to T.B., S.B., M.B. and M.P.S. from the German Research Foundation (SFB850/A4, B2, Z1 and DFG BR 1399/9-1, DFG 1399/10-1, DFG BR4145/1-1) and from the German Consortium for Translational Cancer Research (DKTK).

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Contributions

A.M.K. planned and carried out experiments and wrote the manuscript. J.M. carried out mouse experiments. M.L.L. carried out drug studies. O.S. generated the floxed Zeb1 allele. M.B. and H.B. carried out bioinformatics analyses. M.B. and D.M. carried out metabolic tests. W.R. carried out MRI analyses. P.B. carried out histological analyses. V.G.B. established mouse models. C.P. generated cell lines. T.H.W. carried out mouse experiments. S.B. generated the floxed Zeb1 allele, and planned and carried out experiments. M.P.S. generated the floxed Zeb1 allele, planned and carried out mouse experiments, was involved in coordination and wrote the manuscript. T.B. planned and coordinated the project, analysed data and wrote the manuscript. M.P.S. and T.B. contributed equally and share senior authorship.

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Correspondence to Marc P. Stemmler or Thomas Brabletz.

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Integrated supplementary information

Supplementary Figure 1 Characterisation of KPC, heterozygously and homozygously Zeb1 depleted KPC tumours.

(a) Representative Zeb1-immunolabeling of a GFP lineage-traced primary tumour showing Zeb1/GFP double-positive tumour cells (arrows). n = 5 independent tumors. Scale bar, 50 μm. (b) Representative consecutive sections of HE and indicated immunohistochemical stainings of four Zeb1 expressing KPC tumours demonstrating the heterogeneity in phenotype, grading and marker expression. A representative differentiated Zeb1-negative KPCZ tumour is shown for comparison. Arrows indicate Zeb1 positive tumour cells in the differentiated KPC tumour. n = 15 KPC, 13 KPCZ independent tumours. Scale bar, 100 μm. (c) Tumour-free survival of KPC mice vs. KPC mice with a heterozygous deletion of Zeb1 (KPCz) (n = 15 KPC, 16 KPCz independent tumours); log-rank (Mantel-Cox) test); tumour volume (0 = start of MRI measurements, n = 12 KPC, 14 KPCz independent tumours); error bars show mean ± S.E.M.; multiple t-tests with correction for multiple comparison using the Holm-Sidak method; grading, local invasion and relative ECM deposition of the respective tumours (n = 31 KPC, 17 KPCz; Mann-Whitney test (two-tailed); percentage of metastasized tumours (n = 35 KPC, 17 KPCz independent tumours; Chi-square test (two-tailed); n.s. = not significant.

Supplementary Figure 2 Characterisation of KPC vs. KPCZ tumours.

Representative images of immunohistochemical and histological stainings of KPC and KPCZ tumours and quantifications of the indicated markers are given. Asterisks label Zeb1-expressing stroma cells in KPCZ tumours. Specific blue MTS staining labels collagen fibres. Scale bars, 100 μm, for lower left image 50 μm. n = 48 KPC, 29 KPCZ independent tumours for Zeb1 and MTS; n = 15 independent tumours for KPC, 13 independent tumours for KPCZ for all other markers, error bars show mean ± S.D.; p < 0.0001, n.s. = not significant, Chi-square test (two-tailed) for Zeb1, E-cadherin and Sox2, unpaired Student’s t-test (two-tailed) for Ki67 and Casp3 (with Welch’s correction), Mann-Whitney test (two-tailed) for ECM and CD31.

Supplementary Figure 3 Characterisation of differentiaton markers in KPC vs. KPCZ tumours.

(a) Representative images of positive and negative immunohistochemical stainings and statistical analysis for the indicated EMT-TFs. Scale bar, 150 μm. n = 14 independent tumours for KPC, 13 independent tumours for KPCZ, Chi-square test (two-tailed); n.s. = not significant. (b) Representative images of immunohistochemical stainings and statistical analysis for expression of Gata6. Scale bar, 150 μm. n = 14 independent tumours for KPC, 13 independent tumours for KPCZ; error bars show mean ± S.D.; Mann-Whitney test (two-tailed), p < 0.001. (c) Representative images of differentiated KPCZ and undifferentiated KPC primary tumours (PT) and corresponding metastases (Met) with the same phenotype. Immunohistochemical labelling of Zeb1 expressing tumour cells in the KPC PT and Met (arrows). L = liver or lung tissue. n = 19 KPC, 4 KPCZ independent tumours and corresponding metastases. Scale bar, 100 μm.

Supplementary Figure 4 Characterisation of KPC vs. KPCZ tumour derived cell lines.

(a) Bright field image of primary cell lines from KPC and KPCZ tumours as well as HE stainings of the respective tumours after grafting in syngeneic mice and of the respective primary tumours are shown. Scale bars, 100 μm for bright field, 75 μm for HE stainings. (b) MTT viability assay for the isolated tumour cell lines after treatment with the indicated doses of gemcitabine and erlotinib. The calculated IC50 values for gemcitabine are shown. n = 3 biologically independent experiments, error bars show mean ± S.E.M. (c) Tumour onset after subcutaneous injection of 1 × 105 KPC and KPCZ cells into syngeneic mice. n = 4 mice per cell line, error bars show mean ± S.E.M. (d) Tumour grading, grading at invasive regions and relative ECM deposition of one representative tumour/cell line analysed in c) (n = 6 tumours for KPC, n = 5 tumours for KPCZ); error bars show mean ± S.D.;p < 0.05, p < 0.01, Mann-Whitney test (two-tailed).

Supplementary Figure 5 Depletion of Zeb1 affects tumour promoting capacities.

(a) Representative images of one visual field (n = 6 fields/cell line) showing GFP + cells/cell clusters in the lungs (green dots) 2 h after i.v. injection of KPC and KPCZ tumour cells and control lungs. Scale bar, 500 μm. (b) No. of tumours after subcutaneous injection of the indicated cell numbers for the KPC and KPCZ tumour cell lines and calculated fraction of tumourigenic cells. inf = infinite, Chi-square test. (c) Representative images showing spheres of KPC and KPCZ tumour cells. Scale bar, 500 μm and 50 μm for higher magnifications. (d) Percentage of cells in KPC and KPCZ lines positive for the indicated markers or marker combinations; n = 2 biologically independent experiments, error bars show ± S.D. Source data see Supplementary Table 5, Statistics Source Data. Relative mRNA expression levels (qRT-PCR) of indicated genes, mRNA levels of KPC661 was set to 1; n = 3 biologically independent experiments, Mann-Whitney test (two-tailed), p < 0.05, p < 0.01, error bars show mean ± S.E.M.

Supplementary Figure 6 Depletion of Zeb1 reduces early PanIN lesions.

(a) Consecutive sections showing representative HE and PAS stainings of precancerous PanIN lesions in the pancreas of two different 6 month old KC and of one KCZ mice. Specific dark blue PAS staining indicates the mucin-rich PanIN lesions. Scale bars, 2.5 mm and 150 μm for higher magnifications. Quantification of the PanIN area (% of pancreas area). n = 12 KC and 7 KCZ independent mice, error bars show mean ± S.D.; p < 0.01, unpaired Student’s t-test (two tailed) with Welch’s correction. (b) Gene set enrichment analyses (GSEA) of transcriptome data from KPCZ vs. KPC cells reveals reduction of gene signatures associated with cancer mesenchymal transition and Zeb1 targets in KPCZ vs. KPC cell lines. NES = normalized enrichment score; FDR = false discovery rate.

Supplementary Figure 7 Depletion of Zeb1 reduces tumour cell plasticity.

(a) Relative mRNA expression levels (qRT-PCR) of indicated genes in KPC and KPCZ cell lines treated for different times with TGFβ (time points: 0, 6 h, 1, 3, 7, 14, 21 days). mRNA levels of cell line 661 at day 0 were set to 1. n = 3 biologically independent experiments, error bars show mean ± S.E.M. Statistical analysis is shown for the comparison of TGFβ treated to untreated samples (grey bars) of each individual cell line p < 0.05, p < 0.01, p < 0.001, p < 0.0001, unpaired Student’s t-test (one-tailed) Source data see Supplementary Table 5, Statistics Source Data. (b) Table showing log2FC in mRNA expression levels (microarray) of genes previously determined as common ZEB1/YAP targets in KPC and KPCZ cell lines upon TGFβ treatment for 14 days. (cut-off: adj. p-value < 0.05 and log2FC > 0.5). (c) Representative images of consecutive sections of immunohistochemistry for Ck19 and Zeb1 comparing the plasticity of Zeb1 expression in central and invasive tumour regions. Tumours derived from one KPC and one KPCZ cell line are shown. Asterisks label Zeb1 expression in stroma cells, arrows indicate Zeb1 expression in tumour cells at the invasive front. Ck19 expression is shown to identify cancer cells. n = 15 KPC, 13 KPCZ independent tumours, Scale bars, 50 μm and 150 μm for higher magnifications.

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Krebs, A., Mitschke, J., Lasierra Losada, M. et al. The EMT-activator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer. Nat Cell Biol 19, 518–529 (2017). https://doi.org/10.1038/ncb3513

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