Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Identification of genes that regulate epithelial cell migration using an siRNA screening approach

Abstract

To provide a systematic analysis of genes that regulate epithelial cell migration, we performed a high throughput wound healing screen with MCF-10A breast epithelial cells, using siRNAs targeting 1,081 human genes encoding phosphatases, kinases and proteins predicted to influence cell migration and adhesion. The primary screen identified three categories of hits: those that accelerate, those that inhibit and those that impair migration with associated effects on cell proliferation or metabolism. Extensive validation of all the hits yielded 66 high confidence genes that, when downregulated, either accelerated or impaired migration; 42 of these high confidence genes have not been previously associated with motility or adhesion. Time-lapse video microscopy revealed a broad spectrum of phenotypic changes involving alterations in the extent and nature of disruption of cell–cell adhesion, directionality of motility, cell polarity and shape, and protrusion dynamics. Informatics analysis highlighted three major signalling nodes, β-catenin, β1-integrin and actin, and a large proportion of the genes that accelerated migration impaired cell–cell adhesion.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Summary of the wound healing phenotypes and schematic representation of the results from the SMARTpool screen and validation studies.
Figure 2: Quantification and correlation of knockdown with phenotype for individual Accelerated bin siRNAs.
Figure 3: Analysis of cell morphology after transfection with HC Accelerated and Impaired migration siRNAs and quantification of the migration pattern determined by time-lapse imaging.
Figure 4: Analysis of the migration pattern of individual cells from representative HC Accelerated migration subgroups determined by time-lapse imaging.
Figure 5: Time-lapse imaging series of cells transfected with representative HC Accelerated migration siRNAs.
Figure 6: Time-lapse imaging series of cells transfected with representative HC Impaired migration siRNAs.
Figure 7: Network relationships for the major signalling nodes β-catenin, β1-integrin and actin.

Similar content being viewed by others

References

  1. Cram, E. J., Shang, H. & Schwarzbauer, J. E. A systematic RNA interference screen reveals a cell migration gene network in C. elegans. J. Cell Sci. 119, 4811–4818 (2006).

    Article  CAS  Google Scholar 

  2. Wang, X. et al. Analysis of cell migration using whole-genome expression profiling of migratory cells in the Drosophila ovary. Dev. Cell 10, 483–495 (2006).

    Article  CAS  Google Scholar 

  3. MacKeigan, J. P., Murphy, L. O. & Blenis, J. Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance. Nature Cell Biol. 7, 591–600 (2005).

    Article  CAS  Google Scholar 

  4. Michl, P. et al. CUTL1 is a target of TGF-β signaling that enhances cancer cell motility and invasiveness. Cancer Cell 7, 521–532 (2005).

    Article  CAS  Google Scholar 

  5. Collins, C. S. et al. A small interfering RNA screen for modulators of tumor cell motility identifies MAP4K4 as a promigratory kinase. Proc. Natl Acad. Sci. USA 103, 3775–3780 (2006).

    Article  CAS  Google Scholar 

  6. Zhao, Y. & Ding, S. A high-throughput siRNA library screen identifies osteogenic suppressors in human mesenchymal stem cells. Proc. Natl Acad. Sci. USA 104, 9673–9678 (2007).

    Article  CAS  Google Scholar 

  7. Liang, C. C., Park, A. Y. & Guan, J. L. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nature Protoc. 2, 329–333 (2007).

    Article  CAS  Google Scholar 

  8. Simpson, K. J., Dugan, A. S. & Mercurio, A. M. Functional analysis of the contribution of RhoA and RhoC GTPases to invasive breast carcinoma. Cancer Res. 64, 8694–8701 (2004).

    Article  CAS  Google Scholar 

  9. Chan, A. Y. et al. Roles of the Rac1 and Rac3 GTPases in human tumor cell invasion. Oncogene 24, 7821–7829 (2005).

    Article  CAS  Google Scholar 

  10. Huang, F., Khvorova, A., Marshall, W. & Sorkin, A. Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference. J. Biol. Chem. 279, 16657–16661 (2004).

    Article  CAS  Google Scholar 

  11. Echeverri, C. J. & Perrimon, N. High-throughput RNAi screening in cultured cells: a user's guide. Nature Rev. Genet. 7, 373–384 (2006).

    Article  CAS  Google Scholar 

  12. Fedorov, Y. et al. Off-target effects by siRNA can induce toxic phenotype. RNA 12, 1188–1196 (2006).

    Article  CAS  Google Scholar 

  13. Jackson, A. L. et al. Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA 12, 1197–1205 (2006).

    Article  CAS  Google Scholar 

  14. Brembeck, F. H., Rosario, M. & Birchmeier, W. Balancing cell adhesion and Wnt signaling, the key role of beta-catenin. Curr. Opin. Genet. Dev. 16, 51–59 (2006).

    Article  CAS  Google Scholar 

  15. Charest, P. G. & Firtel, R. A. Big roles for small GTPases in the control of directed cell movement. Biochem. J. 401, 377–390 (2007).

    Article  CAS  Google Scholar 

  16. Gupton, S. L. et al. Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin. J. Cell Biol. 168, 619–631 (2005).

    Article  CAS  Google Scholar 

  17. Hugo, H. et al. Epithelial — mesenchymal and mesenchymal — epithelial transitions in carcinoma progression. J. Cell Physiol. 213, 374–383 (2007).

    Article  CAS  Google Scholar 

  18. Marmor, M. D., Skaria, K. B. & Yarden, Y. Signal transduction and oncogenesis by ErbB/HER receptors. Int. J. Radiat. Oncol. Biol. Phys. 58, 903–913 (2004).

    Article  CAS  Google Scholar 

  19. Muthuswamy, S. K., Li, D., Lelievre, S., Bissell, M. J. & Brugge, J. S. ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini. Nature Cell Biol. 3, 785–792 (2001).

    Article  CAS  Google Scholar 

  20. Whitehurst, A. W. et al. Synthetic lethal screen identification of chemosensitizer loci in cancer cells. Nature 446, 815–819 (2007).

    Article  CAS  Google Scholar 

  21. Chen, X. & Gumbiner, B. M. Crosstalk between different adhesion molecules. Curr. Opin. Cell Biol. 18, 572–578 (2006).

    Article  CAS  Google Scholar 

  22. Vicente-Manzanares, M., Zareno, J., Whitmore, L., Choi, C. K. & Horwitz, A. F. Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells. J. Cell Biol. 176, 573–580 (2007).

    Article  CAS  Google Scholar 

  23. Omelchenko, T., Vasiliev, J. M., Gelfand, I. M., Feder, H. H. & Bonder, E. M. Rho-dependent formation of epithelial “leader” cells during wound healing. Proc. Natl Acad. Sci. USA 100, 10788–10793 (2003).

    Article  CAS  Google Scholar 

  24. Rupp, P. A. & Kulesa, P. M. A role for RhoA in the two-phase migratory pattern of post-otic neural crest cells. Dev. Biol. 311, 159–171 (2007).

    Article  CAS  Google Scholar 

  25. Haga, H., Irahara, C., Kobayashi, R., Nakagaki, T. & Kawabata, K. Collective movement of epithelial cells on a collagen gel substrate. Biophys. J. 88, 2250–2256 (2005).

    Article  CAS  Google Scholar 

  26. Poujade, M. et al. Collective migration of an epithelial monolayer in response to a model wound. Proc. Natl Acad. Sci. USA 104, 15988–15993 (2007).

    Article  CAS  Google Scholar 

  27. Lee, J. S., Chang, M. I., Tseng, Y. & Wirtz, D. Cdc42 mediates nucleus movement and MTOC polarization in Swiss 3T3 fibroblasts under mechanical shear stress. Mol. Biol. Cell 16, 871–880 (2005).

    Article  CAS  Google Scholar 

  28. Lee, J. S. et al. Nuclear lamin A/C deficiency induces defects in cell mechanics, polarization, and migration. Biophys. J. 93, 2542–2552 (2007).

    Article  CAS  Google Scholar 

  29. Fuja, T. J., Lin, F., Osann, K. E. & Bryant, P. J. Somatic mutations and altered expression of the candidate tumor suppressors CSNK1 epsilon, DLG1, and EDD/hHYD in mammary ductal carcinoma. Cancer Res. 64, 942–951 (2004).

    Article  CAS  Google Scholar 

  30. Debnath, J., Muthuswamy, S. K. & Brugge, J. S. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30, 256–268 (2003).

    Article  CAS  Google Scholar 

  31. O'Brien, J., Wilson, I., Orton, T. & Pognan, F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267, 5421–5426 (2000).

    Article  CAS  Google Scholar 

  32. Root, D. E., Hacohen, N., Hahn, W. C., Lander, E. S. & Sabatini, D. M. Genome-scale loss-of-function screening with a lentiviral RNAi library. Nature Methods 3, 715–719 (2006).

    Article  CAS  Google Scholar 

  33. Irie, H. Y. et al. Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition. J. Cell Biol. 171, 1023–1034 (2005).

    Article  CAS  Google Scholar 

  34. Zaidel-Bar, R., Itzkovitz, S., Ma'ayan, A., Iyengar, R. & Geiger, B. Functional atlas of the integrin adhesome. Nature Cell Biol. 9, 858–867 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We wish to thank Stewart Rudnicki and Caroline Shamu (Institute for Chemical and Cell Biology) for screening assistance; Jim Horn (Department of Cell Biology) for manufacturing the wound healing pin; William Pearson for conceptualizing and generating the interactive website hosted by the Cell Migration Consortium; Sabina Winograd-Katz and Benjamin Geiger (Weizmann Institute) for collaborative creation of the MAR custom siRNA library; Rick Horwitz, Alan Hall, Gaudenz Danuser and Ghassan Mouneimne for stimulating and helpful discussions and critical reading of the manuscript and Lara Petrak (Nikon Imaging Centre) for assistance with time-lapse microscopy. We are also grateful to William Hahn, David Root and The RNAi Consortium for providing shRNA vectors. Funding was from the DOD W81XWH-04-1-0360 (K.J.S.) and the Cell Migration Consortium, supported by a grant from the NIH/NIGMS GM064346 (J.S.B.). A.R., D.L. and A.K. are employees of Thermo Fisher Scientific, which supported this work in part by supplying a subset of the siRNA reagents.

Author information

Authors and Affiliations

Authors

Contributions

K.J.S. designed, performed and analysed experiments and wrote the manuscript; L.M.S. created and managed the screening database and performed the informatics analysis; J.B. performed experiments; A.R. performed the knockdown quantification under the supervision of D.L. and A.K.; J.S.B analysed and discussed data and wrote the manuscript.

Corresponding author

Correspondence to Joan S. Brugge.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures S1, S2, S3, S4, S5, S6, S7, S8 and Supplementary Discussion (PDF 2666 kb)

Supplementary Information

Supplementary Table 1 (XLS 239 kb)

Supplementary Information

Supplementary Table 2 (XLS 87 kb)

Supplementary Information

Supplementary Table 3 (XLS 44 kb)

Supplementary Information

Supplementary Table 4 (XLS 44 kb)

Supplementary Information

Supplementary Table 5 (XLS 36 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Simpson, K., Selfors, L., Bui, J. et al. Identification of genes that regulate epithelial cell migration using an siRNA screening approach. Nat Cell Biol 10, 1027–1038 (2008). https://doi.org/10.1038/ncb1762

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb1762

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing