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.
Similar content being viewed by others
Accession codes
References
Brabletz, T. To differentiate or not—routes towards metastasis. Nat. Rev. Cancer 12, 425–436 (2012).
Kalluri, R. & Weinberg, R. A. The basics of epithelial-mesenchymal transition. J. Clin. Invest. 119, 1420–1428 (2009).
Nieto, M. A., Huang, R. Y.-J., Jackson, R. A. & Thiery, J. P. EMT: 2016. Cell 166, 21–45 (2016).
Brabletz, S. & Brabletz, T. The ZEB/miR-200 feedback loop–a motor of cellular plasticity in development and cancer? EMBO Rep. 11, 670–677 (2010).
Vandewalle, C., Van Roy, F. & Berx, G. The role of the ZEB family of transcription factors in development and disease. Cell. Mol. Life Sci. 66, 773–787 (2009).
Zhang, P., Sun, Y. & Ma, L. ZEB1: at the crossroads of epithelial-mesenchymal transition, metastasis and therapy resistance. Cell Cycle 14, 481–487 (2015).
Fischer, K. R. et al. Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature 527, 472–476 (2015).
Zheng, X. et al. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature 527, 525–530 (2015).
Hingorani, S. R. et al. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell 7, 469–483 (2005).
Rhim, A. D. et al. EMT and dissemination precede pancreatic tumor formation. Cell 148, 349–361 (2012).
Higashi, Y. et al. Impairment of T Cell Development in deltaEF1 Mutant Mice. J. Exp. Med. 185, 1467–1480 (1997).
Brabletz, S. et al. Generation and characterization of mice for conditional inactivation of Zeb1. Genesis http://dx.doi.org/10.1002/dvg.23024 (2017).
Martinelli, P. et al. GATA6 regulates EMT and tumour dissemination, and is a marker of response to adjuvant chemotherapy in pancreatic cancer. Gut http://dx.doi.org/10.1136/gutjnl-2015-311256 (2016).
Laklai, H. et al. Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression. Nat. Med. 22, 497–505 (2016).
Erkan, M. et al. The activated stroma index is a novel and independent prognostic marker in pancreatic ductal adenocarcinoma. Clin. Gastroenterol. Hepatol. 6, 1155–1161 (2008).
Özdemir, B. C. et al. Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell 25, 719–734 (2014).
Rhim, A. D. et al. Stromal elements act to restrain, rather than support, pancreatic ductal adenocarcinoma. Cancer Cell 25, 735–747 (2014).
Tsai, J. H., Donaher, J. L., Murphy, D. A., Chau, S. & Yang, J. Spatiotemporal regulation of epithelial-mesenchymal transition is essential for squamous cell carcinoma metastasis. Cancer Cell 22, 725–736 (2012).
Ocaña, O. H. et al. Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer Prrx1. Cancer Cell 22, 709–724 (2012).
Korpal, M. et al. Direct targeting of Sec23a by miR-200s influences cancer cell secretome and promotes metastatic colonization. Nat. Med. 17, 1101–1108 (2011).
Raj, D., Aicher, A. & Heeschen, C. Concise review: stem cells in pancreatic cancer: from concept to translation. Stem Cells 33, 2893–2902 (2015).
Vannier, C., Mock, K., Brabletz, T. & Driever, W. Zeb1 regulates E-cadherin and Epcam (epithelial cell adhesion molecule) expression to control cell behavior in early zebrafish development. J. Biol. Chem. 288, 18643–18659 (2013).
Dosch, J. S., Ziemke, E. K., Shettigar, A., Rehemtulla, A. & Sebolt-Leopold, J. S. Cancer stem cell marker phenotypes are reversible and functionally homogeneous in a preclinical model of pancreatic cancer. Cancer Res. 75, 4582–4592 (2015).
Wellner, U. et al. The EMT-activator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs. Nat. Cell Biol. 11, 1487–1495 (2009).
Herreros-Villanueva, M. et al. SOX2 promotes dedifferentiation and imparts stem cell-like features to pancreatic cancer cells. Oncogenesis 2, e61 (2013).
Sanada, Y. et al. Histopathologic evaluation of stepwise progression of pancreatic carcinoma with immunohistochemical analysis of gastric epithelial transcription factor SOX2: comparison of expression patterns between invasive components and cancerous or nonneoplastic intraductal components. Pancreas 32, 164–170 (2006).
Singh, S. K. et al. Antithetical NFATc1–Sox2 and p53–miR200 signaling networks govern pancreatic cancer cell plasticity. EMBO J. 34, 517–530 (2015).
Muller, P. A. J. & Vousden, K. H. Mutant p53 in cancer: new functions and therapeutic opportunities. Cancer Cell 25, 304–317 (2014).
Rivlin, N., Koifman, G. & Rotter, V. p53 orchestrates between normal differentiation and cancer. Semin. Cancer Biol. 32, 10–17 (2015).
Morton, J. P. et al. Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer. Proc. Natl Acad. Sci. USA 107, 246–251 (2010).
Lehmann, W. et al. ZEB1 turns into a transcriptional activator by interacting with YAP1 in aggressive cancer types. Nat. Commun. 7, 10498 (2016).
Mock, K. et al. The EMT-activator ZEB1 induces bone metastasis associated genes including BMP-inhibitors. Oncotarget 6, 14399–14412 (2015).
Brabletz, S. et al. The ZEB1/miR-200 feedback loop controls Notch signalling in cancer cells. EMBO J. 30, 770–782 (2011).
Singh, A. et al. A gene expression signature associated with “K-Ras addiction” reveals regulators of EMT and tumor cell survival. Cancer Cell 15, 489–500 (2009).
Nakamura, T., Fidler, I. J. & Coombes, K. R. Gene expression profile of metastatic human pancreatic cancer cells depends on the organ microenvironment. Cancer Res. 67, 139–148 (2007).
Collisson, E. A. et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nat. Med. 17, 500–503 (2011).
Weissmueller, S. et al. Mutant p53 drives pancreatic cancer metastasis through cell-autonomous PDGF receptor β signaling. Cell 157, 382–394 (2014).
Diepenbruck, M. & Christofori, G. Epithelial–mesenchymal transition (EMT) and metastasis: yes, no, maybe? Curr. Opin. Cell Biol. 43, 7–13 (2016).
Ye, X. & Weinberg, R. A. Epithelial–mesenchymal plasticity: a central regulator of cancer progression. Trends Cell Biol. 25, 675–686 (2015).
Bellomo, C., Caja, L. & Moustakas, A. Transforming growth factor [beta] as regulator of cancer stemness and metastasis. Br. J. Cancer 115, 761–769 (2016).
Korpal, M. & Kang, Y. Targeting the transforming growth factor-β signalling pathway in metastatic cancer. Eur. J. Cancer 46, 1232–1240 (2010).
Lehuédé, C., Dupuy, F., Rabinovitch, R., Jones, R. G. & Siegel, P. M. Metabolic plasticity as a determinant of tumor growth and metastasis. Cancer Res. 76, 5201–5208 (2016).
Nieto, M. A. Epithelial plasticity: a common theme in embryonic and cancer cells. Science 342, 1234850 (2013).
Brabletz, T. et al. Variable beta-catenin expression in colorectal cancer indicates a tumor progression driven by the tumor environment. Proc. Natl Acad. Sci. USA 98, 10356–10361 (2001).
Chaffer, C. L. et al. Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state. Proc. Natl Acad. Sci. USA 108, 7950–7955 (2011).
Puisieux, A., Brabletz, T. & Caramel, J. Oncogenic roles of EMT-inducing transcription factors. Nat. Cell Biol. 16, 488–494 (2014).
Chaffer, C. L. et al. Poised chromatin at the ZEB1 promoter enables breast cancer cell plasticity and enhances tumorigenicity. Cell 154, 61–74 (2013).
Gruber, R. et al. YAP1 and TAZ control pancreatic cancer initiation in mice by direct up-regulation of JAK–STAT3 signaling. Gastroenterology 151, 526–539 (2016).
Zhang, W. et al. Downstream of mutant KRAS, the transcription regulator YAP Is essential for neoplastic progression to pancreatic ductal adenocarcinoma. Sci. Signal. 7, ra42-ra42 (2014).
Moffitt, R. A. et al. Virtual microdissection identifies distinct tumor- and stroma-specific subtypes of pancreatic ductal adenocarcinoma. Nat. Genet. 47, 1168–1178 (2015).
Galvan, J. A. et al. Expression of E-cadherin repressors SNAIL, ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer. Br. J. Cancer 112, 1944–1950 (2015).
Bronsert, P. et al. Prognostic significance of Zinc finger E-box binding homeobox 1 (ZEB1) expression in cancer cells and cancer-associated fibroblasts in pancreatic head cancer. Surgery 156, 97–108 (2014).
Singh, A. & Settleman, J. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 29, 4741–4751 (2010).
Eser, S., Schnieke, A., Schneider, G. & Saur, D. Oncogenic KRAS signalling in pancreatic cancer. Br. J. Cancer 111, 817–822 (2014).
Ni, T. et al. Snail1-dependent p53 repression regulates expansion and activity of tumour-initiating cells in breast cancer. Nat. Cell Biol. 18, 1221–1232 (2016).
Turajlic, S. & Swanton, C. Metastasis as an evolutionary process. Science 352, 169–175 (2016).
Caramel, J. et al. A switch in the expression of embryonic EMT-inducers drives the development of malignant melanoma. Cancer Cell 24, 466–480 (2013).
Denecker, G. et al. Identification of a ZEB2-MITF-ZEB1 transcriptional network that controls melanogenesis and melanoma progression. Cell Death Differ. 21, 1250–1261 (2014).
Ye, X. et al. Distinct EMT programs control normal mammary stem cells and tumour-initiating cells. Nature 525, 256–260 (2015).
Tiwari, N. et al. Sox4 is a master regulator of epithelial-mesenchymal transition by controlling Ezh2 expression and epigenetic reprogramming. Cancer Cell 23, 768–783 (2013).
Tran, H. D. et al. Transient SNAIL1 expression is necessary for metastatic competence in breast cancer. Cancer Res. 74, 6330–6340 (2014).
Olive, K. P. et al. Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome. Cell 119, 847–860 (2004).
Jackson, E. L. et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev. 15, 3243–3248 (2001).
Hingorani, S. R. et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 4, 437–450 (2003).
Chan, I. T. et al. Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease. J. Clin. Invest. 113, 528–538 (2004).
Novak, A., Guo, C., Yang, W., Nagy, A. & Lobe, C. G. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 28, 147–155 (2000).
Murray, S. A., Carver, E. A. & Gridley, T. Generation of a Snail1 (Snai1) conditional null allele. Genesis 44, 7–11 (2006).
Meidhof, S. et al. ZEB1-associated drug resistance in cancer cells is reversed by the class I HDAC inhibitor mocetinostat. EMBO Mol. Med. 7, 831–847 (2015).
Dunning, M. J., Smith, M. L., Ritchie, M. E. & Tavare, S. beadarray: R classes and methods for Illumina bead-based data. Bioinformatics 23, 2183–2184 (2007).
Johnson, W. E., Li, C. & Rabinovic, A. Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics 8, 118–127 (2007).
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).
Author information
Authors and Affiliations
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.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
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.
Supplementary information
Supplementary Information
Supplementary Information (PDF 9368 kb)
Supplementary Table 1
Supplementary Information (XLSX 14 kb)
Supplementary Table 2
Supplementary Information (XLS 376 kb)
Supplementary Table 3
Supplementary Information (XLSX 11 kb)
Supplementary Table 4
Supplementary Information (XLSX 12 kb)
Supplementary Table 5
Supplementary Information (XLSX 29 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ncb3513
- Springer Nature Limited
This article is cited by
-
The mechanism of PFK-1 in the occurrence and development of bladder cancer by regulating ZEB1 lactylation
BMC Urology (2024)
-
Extracellular matrix marker LAMC2 targets ZEB1 to promote TNBC malignancy via up-regulating CD44/STAT3 signaling pathway
Molecular Medicine (2024)
-
Corneal injury repair and the potential involvement of ZEB1
Eye and Vision (2024)
-
lncRNA FGD5-AS1 is required for gastric cancer proliferation by inhibiting cell senescence and ROS production via stabilizing YBX1
Journal of Experimental & Clinical Cancer Research (2024)
-
A thermo-sensitive hydrogel with prominent hemostatic effect prevents tumor recurrence via anti-anoikis-resistance
Journal of Nanobiotechnology (2024)