Original Article | Published:

SPROUTY-2 represses the epithelial phenotype of colon carcinoma cells via upregulation of ZEB1 mediated by ETS1 and miR-200/miR-150

Oncogene volume 35, pages 29913003 (09 June 2016) | Download Citation

Subjects

Abstract

SPROUTY-2 (SPRY2) is a modulator of tyrosine kinase receptor signaling with receptor- and cell type-dependent inhibitory or enhancing effects. Studies on the action of SPRY2 in major cancers are conflicting and its role remains unclear. Here we have dissected SPRY2 action in human colon cancer. Global transcriptomic analyses show that SPRY2 downregulates genes encoding tight junction proteins such as claudin-7 and occludin and other cell-to-cell and cell-to-matrix adhesion molecules in human SW480-ADH colon carcinoma cells. Moreover, SPRY2 represses LLGL2/HUGL2, PATJ1/INADL and ST14, main regulators of the polarized epithelial phenotype, and ESRP1, an epithelial-to-mesenchymal transition (EMT) inhibitor. A key action of SPRY2 is the upregulation of the major EMT inducer ZEB1, as these effects are reversed by ZEB1 knock-down by means of RNA interference. Consistently, we found an inverse correlation between the expression level of claudin-7 and those of SPRY2 and ZEB1 in human colon tumors. Mechanistically, ZEB1 upregulation by SPRY2 results from the combined induction of ETS1 transcription factor and the repression of microRNAs (miR-200 family, miR-150) that target ZEB1 RNA. Moreover, SPRY2 increased AKT activation by epidermal growth factor, whereas AKT and also Src inhibition reduced the induction of ZEB1. Altogether, these data suggest that AKT and Src are implicated in SPRY2 action. Collectively, these results show a tumorigenic role of SPRY2 in colon cancer that is based on the dysregulation of tight junction and epithelial polarity master genes via upregulation of ZEB1. The dissection of the mechanism of action of SPRY2 in colon cancer cells is important to understand the upregulation of this gene in a subset of patients with this neoplasia that have poor prognosis.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Gene Expression Omnibus

References

  1. 1.

    , . Sprouty proteins, masterminds of receptor tyrosine kinase signaling. Angiogenesis 2008; 11: 53–62.

  2. 2.

    , , . The developing story of Sprouty and cancer. Cancer Metastasis Rev 2014; 33: 695–720.

  3. 3.

    , , , . The bimodal regulation of epidermal growth factor signaling by human Sprouty proteins. Proc Natl Acad Sci USA 2002; 99: 6041–6046.

  4. 4.

    , , , , , . Sprouty fine-tunes EGF signaling through interlinked positive and negative feedback loops. Curr Biol 2003; 13: 297–307.

  5. 5.

    , , , , , et al. Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signalling. EMBO J 2002; 21: 4796–4808.

  6. 6.

    , , , , . Sprouty2 acts at the Cbl/CIN85 interface to inhibit epidermal growth factor receptor downregulation. EMBO Rep 2005; 6: 635–641.

  7. 7.

    , , , , , et al. SPROUTY-2 and E-cadherin regulate reciprocally and dictate colon cancer cell tumourigenicity. Oncogene 2010; 29: 4800–4813.

  8. 8.

    , , , , , et al. Sprouty-2 controls c-Met expression and metastatic potential of colon cancer cells: SPROUTY/c-Met upregulation in human colonic adenocarcinomas. Oncogene 2010; 29: 5241–5253.

  9. 9.

    , , , , , et al. Differential gene expression signatures between colorectal cancers with and without KRAS mutations: crosstalk between the KRAS pathway and other signalling pathways. Eur J Cancer 2011; 47: 1946–1954.

  10. 10.

    , , , , , et al. SPRY2 is an inhibitor of the ras/extracellular signal-regulated kinase pathway in melanocytes and melanoma cells with wild-type BRAF but not with the V599E mutant. Cancer Res 2004; 64: 5556–5559.

  11. 11.

    , , , , , et al. Effect of common B-RAF and N-RAS mutations on global gene expression in melanoma cell lines. Carcinogenesis 2005; 26: 1224–1232.

  12. 12.

    , , , , , et al. ONCOMINE: a cancer microarray database and integrated data-mining platform. Neoplasia 2004; 6: 1–6.

  13. 13.

    , , , , , et al. SPROUTY2 is a beta-catenin and FOXO3a target gene indicative of poor prognosis in colon cancer. Oncogene 2014; 33: 1975–1985.

  14. 14.

    , , , , , et al. Deregulated expression of sprouty2 and microRNA-21 in human colon cancer: correlation with the clinical stage of the disease. Cancer Biol Ther 2011; 11: 111–121.

  15. 15.

    , , , , , et al. Epigenetic inactivation of the human sprouty2 (hSPRY2) homologue in prostate cancer. Oncogene 2005; 24: 2166–2174.

  16. 16.

    , , , , , et al. Sprouty 2, an inhibitor of mitogen-activated protein kinase signaling, is down-regulated in hepatocellular carcinoma. Cancer Res 2006; 66: 2048–2058.

  17. 17.

    , , , , , et al. Epigenetic inactivation of the ERK inhibitor Spry2 in B-cell diffuse lymphomas. Oncogene 2008; 27: 4969–4972.

  18. 18.

    , , , , , et al. Expression of sprouty2 inhibits B-cell proliferation and is epigenetically silenced in mouse and human B-cell lymphomas. Blood 2009; 113: 2478–2487.

  19. 19.

    , , , , , et al. Sprouty 2 is an independent prognostic factor in breast cancer and may be useful in stratifying patients for trastuzumab therapy. PLoS One 2011; 6: e23772.

  20. 20.

    , , , , , et al. Membrane-anchored serine protease matriptase regulates epithelial barrier formation and permeability in the intestine. Proc Natl Acad Sci USA 2010; 107: 4200–4205.

  21. 21.

    , , , , , et al. Sprouty: how does the branch manager work? J Cell Sci 2003; 116: 3061–3068.

  22. 22.

    , , , , , . activation is required for fibroblast growth factor receptor-induced phosphorylation of Sprouty and suppression of ERK activity. J Cell Sci 2004; 117: 6007–6017.

  23. 23.

    , , , , . Regulation of Sprouty stability by Mnk1-dependent phosphorylation. Mol Cell Biol 2006; 26: 1898–1907.

  24. 24.

    , , . HECT domain-containing E3 ubiquitin ligase Nedd4 interacts with and ubiquitinates Sprouty2. J Biol Chem 2010; 285: 255–264.

  25. 25.

    , , , , , et al. Claudin-1 regulates cellular transformation and metastatic behavior in colon cancer. J Clin Invest 2005; 115: 1765–1776.

  26. 26.

    , . Cell polarity and cancer—cell and tissue polarity as a non-canonical tumor suppressor. J Cell Sci 2008; 121: 1141–1150.

  27. 27.

    , , , , , et al. The transcriptional repressor ZEB1 promotes metastasis and loss of cell polarity in cancer. Cancer Res 2008; 68: 537–544.

  28. 28.

    , , , , , et al. Epithelial integrity is maintained by a matriptase-dependent proteolytic pathway. Am J Pathol 2009; 175: 1453–1463.

  29. 29.

    , , , , , et al. Bidirectional modulation of endogenous EpCAM expression to unravel its function in ovarian cancer. Br J Cancer 2013; 108: 881–886.

  30. 30.

    , , , , , et al. EpCAM contributes to formation of functional tight junction in the intestinal epithelium by recruiting claudin proteins. Dev Biol 2012; 371: 136–145.

  31. 31.

    , , , . Epithelial cell adhesion molecule (EpCAM) regulates claudin dynamics and tight junctions. J Biol Chem 2013; 288: 12253–12268.

  32. 32.

    , , , , , et al. CD44 splice isoform switching in human and mouse epithelium is essential for epithelial-mesenchymal transition and breast cancer progression. J Clin Invest 2011; 121: 1064–1074.

  33. 33.

    , . Complex changes in alternative pre-mRNA splicing play a central role in the epithelial-to-mesenchymal transition (EMT). Semin Cancer Biol 2012; 22: 417–427.

  34. 34.

    , , , , , et al. ZEB1-responsive genes in non-small cell lung cancer. Cancer Lett 2011; 300: 66–78.

  35. 35.

    , , , , , et al. TGF-beta drives epithelial-mesenchymal transition through deltaEF1-mediated downregulation of ESRP. Oncogene 2012; 31: 3190–3201.

  36. 36.

    , , , , , et al. The transcription factor ZEB1 (deltaEF1) promotes tumour cell dedifferentiation by repressing master regulators of epithelial polarity. Oncogene 2007; 26: 6979–6988.

  37. 37.

    , , , , , et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 2008; 10: 593–601.

  38. 38.

    , , , . The miR-200 family inhibits epithelial-mesenchymal transition and cancer cell migration by direct targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2. J Biol Chem 2008; 283: 14910–14914.

  39. 39.

    , , , , , et al. A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells. EMBO Rep 2008; 9: 582–589.

  40. 40.

    , , , , , et al. A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition. Cancer Res 2008; 68: 7846–7854.

  41. 41.

    , , , . Efficient suppression of FGF-2-induced ERK activation by the cooperative interaction among mammalian Sprouty isoforms. J Cell Sci 2005; 118: 5861–5871.

  42. 42.

    , , , , , et al. Functional cooperation between Snail1 and twist in the regulation of ZEB1 expression during epithelial to mesenchymal transition. J Biol Chem 2011; 286: 12024–12032.

  43. 43.

    , . Epithelial cell polarity, stem cells and cancer. Nat Rev Cancer 2012; 12: 23–38.

  44. 44.

    , , , , , et al. The level of claudin-7 is reduced as an early event in colorectal carcinogenesis. BMC Cancer 2011; 11: 65.

  45. 45.

    , , , , , et al. Defective claudin-7 regulation by Tcf-4 and Sox-9 disrupts the polarity and increases the tumorigenicity of colorectal cancer cells. Cancer Res 2008; 68: 4258–4268.

  46. 46.

    , , , , , . Hypermethylation-modulated downregulation of claudin-7 expression promotes the progression of colorectal carcinoma. Pathobiology 2008; 75: 177–185.

  47. 47.

    , , , , , et al. Zeb1 and twist are more commonly expressed in metastatic than primary lung tumours and show inverse associations with claudins. J Clin Pathol 2011; 64: 136–140.

  48. 48.

    , , , . Cell density-dependent inhibition of epidermal growth factor receptor signaling by p38alpha mitogen-activated protein kinase via Sprouty2 downregulation. Mol Cell Biol 2009; 29: 3332–3343.

  49. 49.

    , , , , , et al. SIX1 promotes epithelial-mesenchymal transition in colorectal cancer through ZEB1 activation. Oncogene 2012; 31: 4923–4934.

  50. 50.

    , , , . Sprouty-2 overexpression in C2C12 cells confers myogenic differentiation properties in the presence of FGF2. Mol Biol Cell 2005; 16: 4454–4461.

  51. 51.

    , , , , , et al. Expression of claudin7 is tightly associated with epithelial structures in synovial sarcomas and regulated by an Ets family transcription factor, ELF3. J Biol Chem 2006; 281: 38941–38950.

  52. 52.

    , , , , , et al. The Wnt antagonist DICKKOPF-1 gene is induced by 1alpha,25-dihydroxyvitamin D3 associated to the differentiation of human colon cancer cells. Carcinogenesis 2007; 28: 1877–1884.

  53. 53.

    , , , , . Highly efficient lentiviral-mediated human cytokine transgenesis on the NOD/Scid background. Blood 2004; 103: 580–582.

  54. 54.

    , , , , , et al. Beta-catenin and Hedgehog signal strength can specify number and location of hair follicles in adult epidermis without recruitment of bulge stem cells. Dev Cell 2005; 9: 121–131.

  55. 55.

    , , , , , et al. beta-catenin confers resistance to PI3K and AKT inhibitors and subverts FOXO3a to promote metastasis in colon cancer. Nat Med 2012; 18: 892–901.

  56. 56.

    , , , , , et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 2003; 4: 249–264.

  57. 57.

    , , , . GATExplorer: genomic and transcriptomic explorer; mapping expression probes to gene loci, transcripts, exons and ncRNAs. BMC Bioinform 2010; 11: 221.

  58. 58.

    R Development Core TeamR: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing: Vienna, Austria, 2010.

  59. 59.

    , , , , , et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004; 5: R80.

  60. 60.

    , , , , . Controlling the false discovery rate in behavior genetics research. Behav Brain Res 2001; 125: 279–284.

  61. 61.

    , , , , , et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 2005; 102: 15545–15550.

Download references

Acknowledgements

We thank Drs G Goodall, T Brabletz and A García de Herreros for kindly providing us reagents, T Martínez and X Langa for technical assistance and Robin Rycroft for help with the English manuscript. This study was supported by the Ministerio de Economía y Competitividad of Spain and Fondo Europeo de Desarrollo Regional (FEDER) (grant SAF2013-43468-R to AM, SAF2011-29530 to FXR); FEDER-Instituto de Salud Carlos III (RD12/0036/0021 to AM and JMR, RD12/0036/0034 to FXR, RD12/0036/0016 to MS, RD12/0036/0012 to HGP, RD06/0020/0003, PS09/00562 and PI13/00703 to JMR); Comunidad de Madrid (S2010/BMD-2344 Colomics2 to AM); Fundación Científica de la Asociación Española contra el Cáncer (to JMR); US Department of Defense (CA093471 and CA110602 to EH); National Institutes of Health/National Cancer Institute (1R01CA155234-01 to EH); National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (1R21AR062239-01 to EH); and the Melanoma Research Alliance (to EH).

Author information

Author notes

    • A Fernández-Barral
    •  & F Pereira

    These authors contributed equally to this work.

    • M F Segura

    Present address: Vall d’Hebron Institut de Recerca (VHIR), E-08035 Barcelona, Spain.

    • P Ordóñez-Morán

    Present address: Cancer Stem Cell Laboratory, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.

Affiliations

  1. Department of Cancer Biology, Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain

    • A Barbáchano
    • , A Fernández-Barral
    • , F Pereira
    • , P Ordóñez-Morán
    • , J M González-Sancho
    • , A Costales-Carrera
    •  & A Muñoz
  2. Department of Pathology, New York University School of Medicine, New York, USA

    • M F Segura
    • , D Hanniford
    •  & E Hernando
  3. Epithelial Carcinogenesis Group, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain

    • E Carrillo-de Santa Pau
    •  & F X Real
  4. Unidad de Biología Celular, Unidad Funcional de Investigación en Enfermedades Crónicas, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain

    • N Martínez
    •  & J M Rojas
  5. Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain

    • F X Real
  6. Stem cells and Cancer Group, Vall d'Hebron Institute of Oncology, Barcelona, Spain

    • H G Pálmer

Authors

  1. Search for A Barbáchano in:

  2. Search for A Fernández-Barral in:

  3. Search for F Pereira in:

  4. Search for M F Segura in:

  5. Search for P Ordóñez-Morán in:

  6. Search for E Carrillo-de Santa Pau in:

  7. Search for J M González-Sancho in:

  8. Search for D Hanniford in:

  9. Search for N Martínez in:

  10. Search for A Costales-Carrera in:

  11. Search for F X Real in:

  12. Search for H G Pálmer in:

  13. Search for J M Rojas in:

  14. Search for E Hernando in:

  15. Search for A Muñoz in:

Competing interests

The authors declare no conflict of interest.

Corresponding author

Correspondence to A Muñoz.

Supplementary information

About this article

Publication history

Received

Revised

Accepted

Published

DOI

https://doi.org/10.1038/onc.2015.366

Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

Further reading