Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell diapedesis

  • Linda Sequeira
  • Cara W. Dubyk
  • Tracy A. Riesenberger
  • Carlton R. Cooper
  • Kenneth L. van Golen
Research Paper


Background The Rho GTPases comprise one of the eight subfamilies of the Ras superfamily of monomeric GTP-binding proteins and are involved in cytoskeletal organization. Previously, using a dominant negative construct, we demonstrated a role for RhoC GTPase in conferring invasive capabilities to PC-3 human prostate cancer cells. Further, we demonstrated that inactivation of RhoC led to morphological changes commensurate with epithelial to mesenchymal transition (EMT) and was accompanied by increased random, linear motility and decreased directed migration and invasion. EMT was related positively to sustained expression and activity of Rac GTPase. In the current study we analyze the individual roles of RhoA, RhoC and Rac1 GTPases in PC-3 cell directed migration, invasion and tumor cell diapedesis across a human bone marrow endothelial cell layer in vitro. Results Use of specific shRNA directed against RhoA, RhoC or Rac1 GTPases demonstrated a role for each protein in maintaining cell morphology. Furthermore, we demonstrate that RhoC expression and activation is required for directed migration and invasion, while Rac1 expression and activation is required for tumor cell diapedesis. Inhibition of RhoA expression produced a slight increase in invasion and tumor cell diapedesis. Conclusions Individual Rho GTPases are required for critical aspects of migration, invasion and tumor cell diapedesis. These data suggest that coordinated activation of individual Rho proteins is required for cells to successfully complete the extravasation process; a key step in distant metastasis.


Prostate cancer Invasion Diapedesis RhoA GTPase RhoC GTPase Rac GTPase 



Prostate cancer


Ras homology


Fetal bovine serum


Bone marrow endothelial cell


Phosphate buffered saline


Normal goat serum


Small hairpin RNA


Epithelial to mesenchymal transition



We would like to thank Drs. Kirk Czymmek and Harry Yao for technical assistance, Dr. Robert Gorman for help with statistical analysis and the University of Delaware Prostate Cancer Working Group for thoughtful input. This study was funded in part by the Department of Defense Prostate Cancer Research Program (W81XWH-04-1-0225 and W81XWH-05-1-0005) and the University of Delaware Research Fund (to K.L.v.G.).


  1. 1.
    American Cancer Society (2008) Cancer facts & figures 2008Google Scholar
  2. 2.
    Alcover J, Fernandez-Conde M, Carrere W, Serrallach M, Carretero P (1994) Treatment of tumor osteopathy in cancer of the prostate. Actas Urol Esp 18(Suppl):409–416PubMedGoogle Scholar
  3. 3.
    Abate-Shen C, Shen MM (2000) Molecular genetics of prostate cancer. Genes Dev 14:2410–2434. doi: 10.1101/gad.819500 PubMedCrossRefGoogle Scholar
  4. 4.
    Aukerman SL, Price JE, Fidler IJ (1986) Different deficiencies in the prevention of tumorigenic-low-metastatic murine K-1735b melanoma cells from producing metastases. J Natl Cancer Inst 77:915–924PubMedGoogle Scholar
  5. 5.
    Price JE, Aukerman SL, Fidler IJ (1986) Evidence that the process of murine melanoma metastasis is sequential and selective and contains stochastic elements. Cancer Res 46:5172–5178PubMedGoogle Scholar
  6. 6.
    Cooper CR, Pienta KJ (2000) Cell adhesion and chemotaxis in prostate cancer metastasis to bone: a mini review. Prostate Cancer Prostatic Dis 3:6–12. doi: 10.1038/sj.pcan.4500387 PubMedCrossRefGoogle Scholar
  7. 7.
    Cooper CR, McLean L, Walsh M et al (2000) Preferential adhesion of prostate cancer cells to bone is mediated by binding to bone marrow endothelial cells as compared to extracellular matrix components in vitro. Clin Cancer Res 6:4839–4847PubMedGoogle Scholar
  8. 8.
    Lehr JE, Pienta KJ (1998) Preferential adhesion of prostate cancer cells to a human bone marrow endothelial cell line. J Natl Cancer Inst 90:118–123. doi: 10.1093/jnci/90.2.118 PubMedCrossRefGoogle Scholar
  9. 9.
    Ridley AJ (1994) Membrane ruffling and signal transduction. Bioessays 16:321–327. doi: 10.1002/bies.950160506 PubMedCrossRefGoogle Scholar
  10. 10.
    Evers EE, van der Kammen RA, ten Klooster JP, Collard JG (2000) Rho-like GTPases in tumor cell invasion. Meth Enzymol 325:403–415. doi: 10.1016/S0076-6879(00)25461-X PubMedCrossRefGoogle Scholar
  11. 11.
    Hall A (1998) Rho GTPases and the actin cytoskeleton. Science 279:509–514. doi: 10.1126/science.279.5350.509 PubMedCrossRefGoogle Scholar
  12. 12.
    Esteve P, Embade N, Perona R et al (1998) Rho-regulated signals induce apoptosis in vitro and in vivo by a p53- independent, but Bcl2 dependent pathway. Oncogene 17:1855–1869. doi: 10.1038/sj.onc.1202082 PubMedCrossRefGoogle Scholar
  13. 13.
    Gampel A, Parker PJ, Mellor H (1999) Regulation of epidermal growth factor receptor traffic by the small GTPase rhoB. Curr Biol 9:955–958. doi: 10.1016/S0960-9822(99)80422-9 PubMedCrossRefGoogle Scholar
  14. 14.
    Yao H, Dashner E, van Golen CM, van Golen KL (2006) RhoC GTPase is required for PC-3 prostate cancer cell invasion but not motility. Oncogene 25:2285–2296. doi: 10.1038/sj.onc.1209260 PubMedCrossRefGoogle Scholar
  15. 15.
    Hakem A, Sanchez-Sweatman O, You-Ten A et al (2005) RhoC is dispensable for embryogenesis and tumor initiation but essential for metastasis. Genes Dev 19:1974–1979. doi: 10.1101/gad.1310805 PubMedCrossRefGoogle Scholar
  16. 16.
    Simpson KJ, Dugan AS, Mercurio AM (2004) Functional analysis of the contribution of RhoA and RhoC GTPases to invasive breast carcinoma. Cancer Res 64:8694–8701. doi: 10.1158/0008-5472.CAN-04-2247 PubMedCrossRefGoogle Scholar
  17. 17.
    West K, Zhang H, Brown M et al (2004) The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL). J Cell Biol 154:161–176. doi: 10.1083/jcb.200101039 CrossRefGoogle Scholar
  18. 18.
    van Golen KL, Wu ZF, Qiao XT, Bao L, Merajver SD (2000) RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells. Neoplasia 2:418–425. doi: 10.1038/sj.neo.7900115 PubMedCrossRefGoogle Scholar
  19. 19.
    Kleer CG, van Golen KL, Zhang Y, Wu ZF, Rubin MA, Merajver SD (2002) Characterization of RhoC expression in benign and malignant breast disease: a potential new marker for small breast carcinomas with metastatic ability. Am J Pathol 160:579–584PubMedGoogle Scholar
  20. 20.
    Hendrix MJ, Seftor EA, Seftor RE, Fidler IJ (1987) A simple quantitative assay for studying the invasive potential of high and low human metastatic variants. Cancer Lett 38:137–147. doi: 10.1016/0304-3835(87)90209-6 PubMedCrossRefGoogle Scholar
  21. 21.
    van Golen KL, Wu ZF, Qiao XT, Bao LW, Merajver SD (2000) RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype. Cancer Res 60:5832–5838PubMedGoogle Scholar
  22. 22.
    Aktories K, Hall A (1989) Botulinum ADP-ribosyltransferase C3: a new tool to study low molecular weight GTP-binding proteins. Trends Pharmacol Sci 10:415–418. doi: 10.1016/0165-6147(89)90191-0 PubMedCrossRefGoogle Scholar
  23. 23.
    Aktories K, Braun U, Rosener S, Just I, Hall A (1989) The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3. Biochem Biophys Res Commun 158:209–213. doi: 10.1016/S0006-291X(89)80199-8 PubMedCrossRefGoogle Scholar
  24. 24.
    Walsh PC (1994) Prostate cancer kills: strategy to reduce deaths. Urology 44:463–466. doi: 10.1016/S0090-4295(94)80039-1 PubMedCrossRefGoogle Scholar
  25. 25.
    Kjoller L, Hall A (1999) Signaling to Rho GTPases. Exp Cell Res 253:166–179. doi: 10.1006/excr.1999.4674 PubMedCrossRefGoogle Scholar
  26. 26.
    Takai Y, Sasaki T, Matozaki T (2001) Small GTP-binding proteins. Physiol Rev 81:153–208PubMedGoogle Scholar
  27. 27.
    Price LS, Collard JG (2001) Regulation of the cytoskeleton by Rho-family GTPases: implications for tumour cell invasion. Semin Cancer Biol 1:167–173. doi: 10.1006/scbi.2000.0367 CrossRefGoogle Scholar
  28. 28.
    Sahai E, Marshall CJ (2002) RHO-GTPases and Cancer. Nat Rev Cancer 2:133–142. doi: 10.1038/nrc725 PubMedCrossRefGoogle Scholar
  29. 29.
    van Golen KL, Davies S, Wu ZF et al (1999) A novel putative low-affinity insulin-like growth factor-binding protein, LIBC (lost in inflammatory breast cancer), and RhoC GTPase correlate with the inflammatory breast cancer phenotype. Clin Cancer Res 5:2511–2519PubMedGoogle Scholar
  30. 30.
    Suwa H, Ohshio G, Imamura T et al (1998) Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of the pancreas. Br J Cancer 77:147–152PubMedGoogle Scholar
  31. 31.
    Imamura F, Mukai M, Ayaki M et al (1999) Involvement of small GTPases Rho and Rac in the invasion of rat ascites hepatoma cells. Clin Exp Metastasis 17:141–148. doi: 10.1023/A:1006598531238 PubMedCrossRefGoogle Scholar
  32. 32.
    Clark EA, Golub TR, Lander ES, Hynes RO (2000) Genomic analysis of metastasis reveals an essential role for RhoC. Nature 406:532–535. doi: 10.1038/35020106 PubMedCrossRefGoogle Scholar
  33. 33.
    Fritz G, Just I, Kaina B (1999) Rho GTPases are over-expressed in human tumors. Int J Cancer 81:682–687. doi: 10.1002/(SICI)1097-0215(19990531)81:5<682::AID-IJC2>3.0.CO;2-B PubMedCrossRefGoogle Scholar
  34. 34.
    Moscow JA, He R, Gnarra JR et al (1994) Examination of human tumors for rhoA mutations. Oncogene 9:189–194PubMedGoogle Scholar
  35. 35.
    del Peso L, Hernandez-Alcoceba R, Embade N et al (1997) Rho proteins induce metastatic properties in vivo. Oncogene 15:3047–3057. doi: 10.1038/sj.onc.1201499 PubMedCrossRefGoogle Scholar
  36. 36.
    Marionnet C, Lalou C, Mollier K et al (2003) Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development. Oncogene 22:3500–3505. doi: 10.1038/sj.onc.1206571 PubMedCrossRefGoogle Scholar
  37. 37.
    Horiuchi A, Imai T, Wang C et al (2003) Up-regulation of small GTPases, RhoA and RhoC, is associated with tumor progression in ovarian carcinoma. Lab Invest 83:861–870PubMedGoogle Scholar
  38. 38.
    Kamai T, Tsujii T, Arai K et al (2003) Significant association of Rho/ROCK pathway with invasion and metastasis of bladder cancer. Clin Cancer Res 9:2632–2641PubMedGoogle Scholar
  39. 39.
    Shinto E, Tsuda H, Matsubara O, Mochizuki H (2003) Significance of RhoC expression in terms of invasion and metastasis of colorectal cancer. Nippon Rinsho 61(Suppl 7):215–219PubMedGoogle Scholar
  40. 40.
    Wang W, Yang LY, Yang ZL, Huang GW, Lu WQ (2003) Expression and significance of RhoC gene in hepatocellular carcinoma. World J Gastroenterol 9:1950–1953PubMedGoogle Scholar
  41. 41.
    Kondo T, Sentani K, Oue N, Yoshida K, Nakayama H, Yasui W (2004) Expression of RHOC is associated with metastasis of gastric carcinomas. Pathobiology 71:19–25. doi: 10.1159/000072958 PubMedCrossRefGoogle Scholar
  42. 42.
    Sander EE, ten Klooster JP, van Delft S, van der Kammen RA, Collard JG (1999) Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior. J Cell Biol 147:1009–1022. doi: 10.1083/jcb.147.5.1009 PubMedCrossRefGoogle Scholar
  43. 43.
    Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A (1992) The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70:401–410. doi: 10.1016/0092-8674(92)90164-8 PubMedCrossRefGoogle Scholar
  44. 44.
    Ridley AJ, Hall A (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70:389–399. doi: 10.1016/0092-8674(92)90163-7 PubMedCrossRefGoogle Scholar
  45. 45.
    Arthur WT, Burridge K (2001) RhoA inactivation by p190RhoGAP regulates cell spreading and migration by promoting membrane protrusion and polarity. Mol Biol Cell 12:2711–2720PubMedGoogle Scholar
  46. 46.
    Burbelo PD, Miyamoto S, Utani A et al (1995) p190-B, a new member of the Rho GAP family, and Rho are induced to cluster after integrin cross-linking. J Biol Chem 270:30919–30926. doi: 10.1074/jbc.270.52.30919 PubMedCrossRefGoogle Scholar
  47. 47.
    Tikoo A, Czekay S, Viars C et al (2000) p190-A, a human tumor suppressor gene, maps to the chromosomal region 19q13.3 that is reportedly deleted in some gliomas. Gene 257:23–31. doi: 10.1016/S0378-1119(00)00387-5 PubMedCrossRefGoogle Scholar
  48. 48.
    Wennerberg K, Forget M-A, Ellerbroek SM et al (2003) Rnd Proteins function as RhoA antagonists by activationg p190 RhoGAP. Curr Biol 13:1106–1115. doi: 10.1016/S0960-9822(03)00418-4 PubMedCrossRefGoogle Scholar
  49. 49.
    Riento K, Vilalonga P, Garg R, Ridley AJ (2005) Function and Regulation of RhoE. Biochem Soc Trans 33:649–651. doi: 10.1042/BST0330649 PubMedCrossRefGoogle Scholar
  50. 50.
    Wang H-R, Zhan Y, Ozdamar B et al (2003) Regulation of cell polarity and protrusion formation by targeting RhoA for degredation. Science 302:1775–1779. doi: 10.1126/science.1090772 PubMedCrossRefGoogle Scholar
  51. 51.
    Sahai E, Marshall CJ (2003) Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol 5:711–719. doi: 10.1038/ncb1019 PubMedCrossRefGoogle Scholar
  52. 52.
    Wittchen ES, van Buul JD, Burridge K, Worthylake RA (2005) Trading spaces: Rap, Rac, and Rho as architects of transendothelial migration. Curr Opin Hematol 12:14–21. doi: 10.1097/01.moh.0000147892.83713.a7 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Linda Sequeira
    • 1
    • 2
  • Cara W. Dubyk
    • 1
    • 2
  • Tracy A. Riesenberger
    • 1
    • 2
  • Carlton R. Cooper
    • 2
    • 3
  • Kenneth L. van Golen
    • 1
    • 2
  1. 1.The Laboratory of Cytoskeletal PhysiologyThe University of DelawareNewarkUSA
  2. 2.Department of Biological Sciences, The Center for Translational Cancer ResearchThe University of DelawareNewarkUSA
  3. 3.Cancer Biology LaboratoryThe University of DelawareNewarkUSA

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