Skip to main content
Log in

In vitro invasiveness of DU-145 human prostate carcinoma cells is modulated by EGF receptor-mediated signals

  • Research Papers
  • Published:
Clinical & Experimental Metastasis Aims and scope Submit manuscript

Abstract

Prostate carcinomas often present an autocrine stimulatory loop in which the transformed cells both express the EGF receptor (EGFR) and produce activating ligands (TGFα and EGF forms). Up-regulated EGFR signalling has been correlated with tumor progression in other human neoplasia; however, the cell behaviour which is promoted remains undefined. To determine whether an EGFR-induced response contributes to cell invasiveness, we transduced DU-145 human prostate carcinoma cells with either a full-length (WT) or a mitogenically-active but motility-deficient truncated (c'973) EGFR. The DU-145 Parental and two transgene sublines all produced EGFR and TGFα, but the transduced WT and c'973 EGFR underwent autocrine downregulation to a lesser degree, with more receptor remaining intact. DU-145 cells transduced with WT EGFR transmigrated a human amniotic basement membrane matrix (Amgel) to a greater extent than did Parental DU-145 cells (175±22%). Cells expressing the c'973 EGFR invaded through the Amgel only to about two thirds the extent of the Parental cells (62±23%). A monoclonal antibody which prevents ligand-induced activation of EGFR decreased the invasiveness of WT-expressing cells by half and Parental cells by a fifth, but had little effect on the invasiveness of c′973-expressing cells; with the result that in the presence of antibody, all three cell lines transmigrated the Amgel to the same extent. The different levels of invasiveness between the three sublines were independent of cell proliferation. These findings demonstrated that EGFR-mediated signals increase tumor cell invasiveness and suggested that domains in the carboxy-terminus are required to signal invasiveness. As an initial investigation into the mechanisms underlying the EGFR-mediated enhanced invasiveness, we determined whether these cells presented different collagenolytic activity, as the major constituents of Amgel are collagen types I and IV. All three sublines secreted easily detectable levels of gelatin-directed proteases and TIMP-1, with WT cells secreting equivalent or lower levels of proteases. The proteolytic balance in these cells did not correlate with invasiveness. These data suggest that the TGFα-EGFR autocrine loop promotes invasiveness and that this is accomplished by signalling cell properties other than differential secretion of collagenolytic activity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Stracke, ML, Murata, J, Aznavoorian, S and Liotta, LA, 1994, The role of the extracellular matrix in tumor cell metastasis.In vivo,8, 49–58

    Google Scholar 

  2. Price, JE, 1994, Analyzing the metastatic phenotype.J Cell Biochem,56, 16–22.

    Google Scholar 

  3. Jarrard, DF, Blitz, BF, Smith, RC, Patai, BL and Rukstalis, DB, 1994, Effects of epidermal growth factor on prostate cancer cell line PC3 growth and invasion.Prostate,24, 46–53.

    Google Scholar 

  4. Crowley, CW, Cohen, RL, Lucas, BK, Lui, G, Shuman, MA and Levinson, AD, 1993, Prevention of metastasis by inhibition of the urokinase receptor.Proc Natl A cad Sci (USA),90, 5021–5.

    Google Scholar 

  5. Monsky, WL, Kelly, T, Lin, CYet al. 1993, Binding and localization of Mr 72,000 matrix metalloproteinase at cell surface invadopodia.Cancer Res,53, 3159–64.

    Google Scholar 

  6. Aznavoorian, S, Murphy, AN, Stetler-Stevenson, WG and Liotta, LA, 1993, Molecular aspects of tumor cell invasion and metastasis.Cancer,71,1368–83.

    Google Scholar 

  7. Sato, H, Takino, T, Okada, Yet al. 1994, A matrix metalloproteinase expressed on the surface of invasive tumour cells.Nature,370, 61–5.

    Google Scholar 

  8. Strongin, AY, Mariner, BL, Grant, GA and Goldberg, GI, 1993, Plasma membrane-dependent activation of the 72 kDa type IV collagenase is prevented by complex formation with TIMP-2.J Biol Chem,268,14033–9.

    Google Scholar 

  9. Ponton, A, Coulombe, B and Skup, D, 1991, Decreased expression of tissue inhibitor of metalloproteinases in metastatic tumor cells leading to increased levels of collagenase activity.Cancer Res,51, 2138–43.

    Google Scholar 

  10. Mohanam, S, Wang, SW, Rayford, Aet al. 1995, Expression of tissue inhibitors of metalloproteinases: negative regulation of human glioblastoma invasionin vivo.Clin Exp Metastasis,13, 57–62.

    Google Scholar 

  11. Schor, SL and Schor, AM, 1990, Characterization of migration-stimulating factor (MSF): evidence for its role in cancer pathogenesis.Cancer Invest,8, 665–7.

    Google Scholar 

  12. Wernert, N, Gilles, F, Fafeur, V,et al. 1994, Stromal expression of c-etsl transcription factor correlates with tumor invasion.Cancer Res,54, 5683–88.

    Google Scholar 

  13. Aaronson, SA, 1991, Growth factors and cancer.Science,254, 1146–53.

    Google Scholar 

  14. Schlegel, J, Merdes, A, Stumm, Get al. 1994, Amplification of the epidermal growth factor receptor gene correlates with different growth behavior in human glioblastoma.Int J Cancer,56, 72–7.

    Google Scholar 

  15. Collins, VP, 1993, Amplified genes in human gliomas.Sem Cancer Biol,4, 27–32.

    Google Scholar 

  16. Neal, DE, Marsh, C, Bennett, MKet al. 1985, Epidermal growth factor receptors in human bladder cancer: comparison of invasive and superficial tumours.Lancet,i 366–8.

    Google Scholar 

  17. Nguyen, PL, Swanson, PE, Jaszcz, W,et al. 1994, Expression of epidermal growth factor receptor in invasive transitional cell carcinoma of the urinary bladder: a multivariate survival analysis.Am J Clin Path,101, 166–76.

    Google Scholar 

  18. Yasui, W, Sumiyoshi, H, Hata, Jet al. 1988, Expression of epidermal growth factor receptor in human gastric and colon carcinomas.Cancer Res,48, 137–41.

    Google Scholar 

  19. Klijn, JG, Berns, PM, Schmitz, PI and Foekens, JA, 1992, The clinical significance of epidermal growth factor receptor (EGF-R) in human breast cancer: a review on 5232 patients.Endocrine Rev,13, 3–17.

    Google Scholar 

  20. Sainsbury, JRC, Farndon, JR, Needham, GK, Malcolm, AJ and Harris, AL, 1987, Epidermal-growth-factor receptor status as predictor of early recurrence of and death from breast cancer.Lancet,i, 1398–402.

    Google Scholar 

  21. Radinsky R, Risin S, Fan Det al. 1995, Level and function of epidermal growth factor receptor predict the metastatic potential of human colon carcinoma cells.Clin Cancer Res, in press,

  22. Yu, D, Wang, SS, Dulski, KM, Tsai, CM, Nicolson, GL and Hung, MC, 1994, c-erbB-2/neu overexpression enhances metastatic potential of human lung cancer cells by induction of metastasis-associated properties.Cancer Res,54, 3260–6.

    Google Scholar 

  23. Holting, T, Siperstein, AE, Clark, OH and Duh, Q-Y, 1995, Epidermal growth factor (EGF)-a and transforming growth factor alpha-stimulated invasion and growth of follicular thyroid cancer cells can be blocked by antagonism to EGF receptor and tyrosine kinasein vitro.Eur J Endocrinol,132, 229–35.

    Google Scholar 

  24. Morris, GL and Dodd, JG, 1990, Epidermal growth factor receptor mRNA levels in human prostatic tumors and cell lines.J Urol 143, 1272–4.

    Google Scholar 

  25. Davies, P and Eaton, CL, 1989, Binding of epidermal growth factor by human normal, hypertrophic, and carcinomatous prostate.Prostate,14, 123–32.

    Google Scholar 

  26. Eaton, CL, Davies, P and Phillips, MEA, 1988, Growth factor involvement and oncogene expression in prostatic tumors.J Steroid Biochem,30, 341–5.

    Google Scholar 

  27. Myers, RB, Kudlow, JE and Grizzle, WE, 1993, Expression of transforming growth factor alpha, epidermal growth factor and the epidermal growth factor receptor in adenocarcinoma of the prostate and benign prostatic hyperplasia.Mod Pathol,6, 733–7.

    Google Scholar 

  28. Ching, KZ, Ramsey, E, Pettigrew, N, D'Cunha, R, Jason, M and Dodd, JG, 1993, Expression of mRNA for epidermal growth factor, transforming growth factor-alpha and their receptor in human prostate tissue and cell lines.Mol Cell Biochem,126, 151–8.

    Google Scholar 

  29. Tillotson, JK and Rose, DP, 1991, Endogenous secretion of epidermal growth factor peptides stimulates growth of DIJ-145 prostate cancer cells.Cancer Lett,60, 109–12.

    Google Scholar 

  30. Stone, K, Mickey, DD, Wunderli, H, Mickey, GH and Paulson, DF, 1978, Isolation of a human prostate carcinoma cell line (DU-145).Int J Cancer,21, 274–81.

    Google Scholar 

  31. Connolly, JM and Rose, DP, 1992, Interactions between epidermal growth factor-mediated autocrine regulation and linoleic acid-stimulated growth of a human prostate cancer cell line.Prostate,20, 151–8.

    Google Scholar 

  32. Yoshida, K, Tsujino, T, Yasui, Wet al. 1990, Induction of growth factor-receptor and metalloproteinase genes by epidermal growth factor and/or transforming growth factor-alpha in human gastric carcinoma cell line MKN-28.Japan J Cancer Res,81, 793–8.

    Google Scholar 

  33. Matrisian, LM and Hogan, BLM, 1990, Growth factor-regulated proteases and extracellular matrix remodeling during mammalian development.Curr Top Dev Biol,24, 219–54.

    Google Scholar 

  34. Thorne, HJ, Jose, DG, Zhang, H, Dempsey, PJ and Whitehead, RH, 1987, Epidermal growth factor stimulates the synthesis of cell-attachment proteins in human breast cancer cell line PMC42.Int J Cancer,40, 207–12.

    Google Scholar 

  35. Lichtner, RB, Wiedemuth, M, Noeske-Jungblut, C and Schirrmacher, V, 1993, Rapid effects of EGF on cytoskeletal structures and adhesive properties of highly metastatic rat mammary adenocarcinoma cells.Clin Exp Metastasis,11, 113–25.

    Google Scholar 

  36. Chen, P, Gupta, K and Wells, A, 1994, Cell movement elicited by epidermal growth factor receptor requires kinase and autophosphorylation but is separable from mitogenesis.J Cell Biol,124, 547–55.

    Google Scholar 

  37. Chen, P, Xie, H, Sekar, MC, Gupta, KB and Wells, A, 1994, Epidermal growth factor receptor-mediated cell motility: phospholipase C activity is required, but MAP kinase activity is not sufficient for induced cell movement.J Cell Biol,127, 847–57.

    Google Scholar 

  38. Siegal, GP, Wang, M-H, Rinehart, CAet al. 1993, Development of a novel human extracellular matrix for quantitation of the invasiveness of human cells.Cancer Lett,69, 123–32.

    Google Scholar 

  39. Wells, A, Welsh, JB, Lazar, CS, Wiley, HS, Gill, GN and Rosenfeld, MG, 1990, Ligand-induced transformation by a non-internalizing EGF receptor.Science,247, 962–4.

    Google Scholar 

  40. Ullrich, A, Coussens, L, Hayflick, JS,et al. 1984, Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells.Nature,307, 418–25.

    Google Scholar 

  41. Welsh, JB, Gill, GN, Rosenfeld, MG and Wells, A, 1991, A negative feedback loop attenuates EGF-induced morphological changes.J Cell Biol,114, 533–43.

    Google Scholar 

  42. Haigler, HT, Maxfield, FR,Willingham, MC and Pastan, I, 1980, Dansylcadaverine inhibits internalization of125-epidermal growth factor in BALB 3T3 cells.J Biol Chem,255, 1239–41.

    Google Scholar 

  43. Kleinman, HK, McGarvey, ML, Liotta, LA, Gehron-Robey, P, Tryggvason, K and Martin GR, 1982, Isolation and characterization of native type-IV collagen from the EHS sarcoma.Biochemistry,24, 6188–93.

    Google Scholar 

  44. Vukicevic, S, Kleinman, HK, Luyten, FP, Roberts, AB, Roche, NS and Reddi, AH, 1992, Identification of multiple active growth factors in basement membrane matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components.Exp Cell Res,202, 1–8.

    Google Scholar 

  45. Sunada, H, Magun, BE, Mendelsohn, J and MacLeod, CL, 1986, Monoclonal antibody against epidermal growth factor receptor is internalized without stimulating receptor phosphorylation.Proc Natl Acad Sci (USA),83, 3825–9.

    Google Scholar 

  46. Reddy, CC, Wells, A and Lauffenburger, D, 1994, Proliferative response of fibroblasts expressing internalization-deficient EGF receptors is altered via differential EGF depletion effects.Biotech Prog,10, 377–84.

    Google Scholar 

  47. Heussen, C and Dowdle, EB, 1980, Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates.Anal Biochem,102, 196–202.

    Google Scholar 

  48. Kuo, BS, Korner, G and Bjornsson, TD, 1988, Effects of sulfonylureas on the synthesis and secretion of plasminogen activator from bovine aortic endothelial cells.J Clin Invest,81, 730–7.

    Google Scholar 

  49. Wiley, HS, Herbst, JJ, Welsh, BJ, Lauffenburger, DA, Rosenfeld, MG and Gill GN, 1991, Role of tyrosine kinase activity in endocytosis, compartmentation and downregulation of the EGF receptor.J Biol Chem,266, 11083–94.

    Google Scholar 

  50. Reddy C, Wells A and Lauffenburger DA, 1995, Differences in mitogenic potencies of EGF and TGFα are due to differences in ligand processing.J Cell Physiol, in press.

  51. Chen, WS, Lazar, CS, Lund, KA,et al. 1989, Functional independence of the epidermal growth factor receptor from a domain required for ligand-induced internalization and calcium regulation.Cell,59, 33–43.

    Google Scholar 

  52. Gates, RE and King, LE, 1985, Different forms of the epidermal growth factor receptor kinases have different autophosphorylation sites.Biochemistry,24, 5209–15.

    Google Scholar 

  53. Wells, A, Bishop, JM and Helmeste, D, 1988, Amplified gene for EGF receptor in a human glioblastoma cell line presenting an enzymatically inactive protein.Mol Cell Biol,8, 4561–5.

    Google Scholar 

  54. Felder, S, LaVin, J, Ullrich, A and Schlessinger, J, 1992, Kinetics of binding, endocytosis, and recycling of EGF receptor mutants.J Cell Biol,117, 203–12.

    Google Scholar 

  55. Walton, GM, Chen, WS, Rosenfeld, MG and Gill, GN, 1990, Analysis of deletions of the carboxyl terminus of the epidermal growth factor receptor reveals selfphosphorylation at tyrosine 992 and enhanced in vivo tyrosine phosphorylation of cell substrates.J Biol Chem,265, 1750–4.

    Google Scholar 

  56. Collier, IE, Wilhelm, SM, Eisen, AZet al. 1988, H-ras oncogene transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement collagen.J Biol Chem,263, 6579–87.

    Google Scholar 

  57. Wilhelm, SM, Collier, IE, Mariner, BL, Eisen, AZ, Grant, GA and Goldberg, GI, 1989, SV40-transformed human lung fbroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages.J Biol Chem 264, 17213–21.

    Google Scholar 

  58. McGuire, PG and Seeds, NW, 1989, The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells.J Cell Biochem,40, 215–27.

    Google Scholar 

  59. Goodly LJ, Singh RK, Wang M-H and Siegal GP, 1995,In vivo modulation of human tumor cell growth by normal human extracellular matrix.Tumor Biol in press.

  60. Stephenson, RA, Dinny, CPN, Gohji, D, Ordonez, NG, Killion, JJ and Fidler IJ, 1992, Metastatic model for human prostate cancer using orthopic implantations in nude mice.J Natl Cancer Inst 84, 951–7.

    Google Scholar 

  61. Ware, JL, 1993, Growth factors and their receptors as determinants in the proliferation and metastasis of human prostate cancer.Cancer Metastasis Rev,12, 287–301.

    Google Scholar 

  62. Stetler-Stevenson, WG, Krutzsch, HC and Liotta, LA, 1989, Tissue inhibitor of metalloproteinase (TIMP-2). A new member of the metalloproteinase inhibitor family.J Biol Chem,264, 17374–8.

    Google Scholar 

  63. Sreenath, T, Matrisian, LM, Stetler-Stevenson, W, Gattoni-Celli, S and Pozzatti, RO, 1992, Expression of matrix metalloproteinase genes in transformed rat cell lines of high and low metastatic potential.Cancer Res,52, 4942–7.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Alan Wells.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xie, H., Turner, T., Wang, MH. et al. In vitro invasiveness of DU-145 human prostate carcinoma cells is modulated by EGF receptor-mediated signals. Clin Exp Metast 13, 407–419 (1995). https://doi.org/10.1007/BF00118180

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00118180

Keywords

Navigation