Epidermal growth factor modulates prostate cancer cell invasiveness regulating urokinase-type plasminogen activator activity

Summary

Urokinase-type plasminogen activator receptor (uPAR) and Epidermal Growth Factor Receptor (EGFR) are ubiquitous receptors involved in the control of a variety of cellular processes frequently found altered in cancer cells. The EGFR has been recently described to play a transduction role of uPAR stimuli, mediating uPA-induced proliferation in highly malignant cells that overexpress uPAR. We compared the uPA production, the presence of uPAR, AR, EGFR and Her2 with the chemotaxis and the Matrigel invasion in ten human PCa cell lines and observed that: (1) the levels of Her2, but not of EGFR, as well as the uPA secretion, cell motility and Matrigel invasion were statistically higher in AR negative than in AR positive PCa cells; (2) the uPA secretion and uPAR expression were positively related to Matri-gel invasion; (3) the EGF was able to stimulate chemotaxis and Matrigel invasion in a dose-dependent manner; (4) the EGF-induced cell migration was statistically higher in AR negative than in AR positive cells with a similar increase with respect to basal value (about 2.6 fold); (5) the Matrigel invasion was statistically higher in AR negative than in AR positive PCa cells also if the increment of Matrigel invasion after EGF treatment was statis- tically higher in AR positive respect to AR negative cells; (6) the EGF induced uPA secretion and its membrane uptake through the increment of uPAR; and (7) these effects were blocked by EGFR/Her2 tyrosine kinase inhibitors with IC₅₀ lower than those needed to inhibit cell proliferation and required PI3K/Akt, MAPK and PI-PLC activities as verified by inhibition experiments. These enzymatic activities were regulated in different manners in PTEN positive and negative cells. In fact, the inhibition of PI3K blocked the EGF-induced invasiveness in PTEN positive cells but not in PTEN negative cells, in which PI3K activity was not influenced by EGFR/Her2 activation, whereas the inhibition of MAPK was able to block the invasive phenomena in both cell types. Taken together, our data suggest that the blockade of EGFR could attenuate the invasive potential of PCa cells. In addition, considering that the EGFR expression is related to higher Gleason grade of PCa and that EGFR levels are increased after anti androgenic therapy, this therapeutic approach could slow down the metastasis formation which represents the most dramatic event of PCa progression.

Keywords

Prostatic carcinoma - EGFR - uPA - tyrosine kinase inhibitors

• References

• 1 Velutu TJ. Structure, function and transforming potential of the epidermal growth factor receptor. Mol Cell Endocrinol 1990; 70: 205-16.
• 2 Itoh N, Patel U, Skinner MK. Developmental and hormonal regulation of transforming growth factoralpha and epidermal growth factor receptor gene expression in isolated prostatic epithelial and stromal cells. Endocrinology 1998; 139: 1369-77.
• 3 Vizoso F, Villar C, Rodriguez JC. et al. C-erb B-2 oncoprotein content in colorectal cancer and in surrounding mucosa: relationship with clinicopathologic parameters and prognostic significance. Int J Surg Investig 2000; 1: 483-93.
• 4 Tsutsui S, Kataoka A, Ohno S. et al. Prognostic and predictive value of epidermal growth factor receptor in recurrent breast cancer. Clin Cancer Res 2002; 8: 3454-60.
• 5 Bunn PA, Franklin W. Epidermal growth factor receptor expression, signal pathway, and inhibitors in non-small cell lung cancer. Semin Oncol 2002; 29: 38-44.
• 6 Alper O, Bergmann-Leitner ES, Bennett TA. et al. Epidermal growth factor receptor signalling and the invasive phenotype of ovarian carcinoma cells. J Natl Cancer Inst 2001; 93: 1375-84.
• 7 Scher I H, Sarkis A, Reuter V. et al. Changing pattern of expression of the epidermal growth factor receptor and transforming growth factor alpha in the progression of prostatic neoplasms. Clin Cancer Res 1995; 1: 545-50.
• 8 Skacel M, Ormsby AH, Pettay JD. et al. Aneusomy of chromosomes 7, 8, and 17 and amplification of HER-2/neu and epidermal growth factor receptor in Gleason score 7 prostate carcinoma: a differential fluorescent in situ hybridization study of Gleason pattern 3 and 4 using tissue microarray. Hum Pathol 2001; 32: 1392-7.
• 9 Di Loreto G, Tortora D’Armiento G. et al. Expression of epidermal growth factor receptor correlates with disease relapse and progression to androgen-independence in human prostate cancer. Clin Cancer Res 2002; 8: 3438-44.
• 10 Bieche I, Onody P, Tozlu S. et al. Prognostic value of ERBB family mRNA expression in breast carcinomas. Int J Cancer 2003; 106: 758-65.
• 11 Tovey SM, Witton CJ, Bartlett JM. et al. Outcome and human epidermal growth factor receptor (HER) 1–4 status in invasive breast carcinomas with proliferation indices evaluated by bromodeoxyuridine labelling. Breast Cancer Res 2004; 6: 346-51.
• 12 Lotti LV, Lanfrancone L, Migliaccio E. et al. Sch proteins are localized on endoplasmic reticulummembranes and are redistributed after tyrosine kinase receptor activation. Mol Cell Biol 1996; 16: 1946-54.
• 13 Wheeler M, Domin J. Recruitment of the class II phosphoinositide 3-kinase C2beta to the epidermal growth factor receptor: role of Grb2. Mol Cell Biol 2001; 21: 6660-7.
• 14 Dreher T, Zentgraf H, Abel U. et al. Reduction of PTEN and p27kip1 expression correlates with tumor grade in prostate cancer. Analysis in radical prostatectomy specimens and needle biopsies. Virchows Arch 2004; 444: 509-17.
• 15 Bonomi P. Clinical studies with non-iressa EGFR tyrosine kinase inhibitors. Lung Cancer 2003; (Suppl. 01) Suppl S43-8.
• 16 Magne N, Fischel JL, Dubreuil A. et al. Influence of epidermal growth factor receptor (EGFR), p53 and intrinsic MAP kinase pathway status of tumour cells on the antiproliferative effect of ZD1839 (“Iressa“). Br J Cancer 2002; 86: 1518-23.
• 17 Vicentini C, Festuccia C, Gravina GL. et al. Prostate cancer cell proliferation is strongly reduced by the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 in vitro on human cell lines and primary cultures. J Cancer Res Clin Oncol 2003; 29: 165-74.
• 18 Ciardiello F, Caputo R, Bianco R. et al. Inhibition of growth factor production and angiogenesis in human cancer cells by ZD1839 (Iressa), a selective epidermal growth factor receptor tyrosine kinase inhibitor. Clin Cancer Res 2001; 7: 1459-65.
• 19 Ciardiello F, Caputo R, Bianco R. et al. Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptor-selective tyrosine kinase inhibitor. Clin Cancer Res 2000; 6: 2053-63.
• 20 Festuccia C, Gravina GL, Angelucci A. et al. Additive antitumour effects of the epidermal growth factor tyrosine kinase inhibitor, (Iressa), and the non-steroidal antiandrogen, bicalutamide (Casodex), in prostate cancer cells in vitro. Int J Cancer. 2005 in press.
• 21 Gravina GL, Festuccia C, Angelucci A. et al. Long termpresence of androgens and anti-androgens modulate EGF-receptor expression and MAP-Kinase phosphorylation in androgen receptor-prostate positive cancer cells. Int J Oncol 2004; 25: 97-104.
• 22 DeClerck YA, Mercurio AM, Stack MS. et al. Proteases, extracellular matrix, and cancer: a workshop of the path B study section. Am J Pathol 2004; 164: 1131-9.
• 23 Zolfaghari A, Djakiew D. Inhibition of chemomigration of a human prostatic carcinoma cell (TSU-pr1) line by inhibition of epidermal growth factor receptor function. Prostate 1996; 28: 232-8.
• 24 Rajan R, Vanderslice R, Kapur S. et al. Epidermal growth factor (EGF) promotes chemomigration of a human prostate tumor cell line, and EGF immunoreactive proteins are present at sites of metastasis in the stroma of lymph nodes and medullary bone. Prostate 1996; 28: 1-9.
• 25 Mignatti P, Rifkin DB. Nonenzymatic interactions between proteinases and the cell surface: novel roles in normal andmalignant cell physiology. Adv Cancer Res 2000; 78: 103-5.
• 26 Andreasen PA, Egelund R, Petersen HH. The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci 2000; 57: 25-40.
• 27 Festuccia C, Dolo V, Guerra F. et al. Plasminogen activator system modulates invasive capacity and proliferation in prostatic tumor cells. Clin Exp Metastasis 1998; 16: 513-28.
• 28 Del Rosso M, Fibbi G, Pucci M. et al. Multiple pathways of cell invasion are regulated by multiple families of serine proteases. Clin Exp Metastasis 2002; 19: 193-207.
• 29 Festuccia C, Giunciuglio D, Guerra F. et al. Osteoblasts modulate secretion of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase- 9 (MMP-9) in human prostate cancer cells promoting migration and invasion. Oncol Res 1999; 11: 17-31.
• 30 Schnack Nielsen B, Rank F, Engelholm LH. et al. Urokinase receptor-associated protein (uPARAP) is expressed in connection with malignant as well as benign lesions of the human breast and occurs in specific populations of stromal cells. Int J Cancer 2002; 98: 656-64.
• 31 Ohtani H. Stromal reaction in cancer tissue: pathophysiologic significance of the expression of matrixdegrading enzymes in relation to matrix turnover and immune/inflammatory reactions. Pathol Int 1998; 48: 1-9.
• 32 Costantini V, Sidoni AD, eveglia R. et al. Combined overexpression of urokinase, urokinase receptor, and plasminogen activator inhibitor-1 is associated with breast cancer progression: an immunohistochemical comparison of normal, benign, and malignant breast tissues. Cancer 1996; 77: 1079-88.
• 33 Plas E, Carroll VA, Jilch R. et al. Variations of components of the plasminogen activation system with the cell cycle in benign prostate tissue and prostate cancer. Cytometry 2001; 46: 184-9.
• 34 Festuccia C, Bologna M, Vicentini C. et al. Increased matrix metalloproteinase-9 secretion in shortterm tissue cultures of prostatic tumor cells. Int J Cancer 1996; 69: 386-93.
• 35 Festuccia C, Angelucci A, Gravina G. et al. Bombesin- dependent pro-MMP-9 activation in prostatic cancer cells requires beta1 integrin engagement. Exp Cell Res 2002; 280: 1-11.
• 36 Guerrero J, Santibanez JF, Gonzalez A. et al. EGF receptor transactivation by urokinase receptor stimulus through a mechanisminvolving Src and matrix metalloproteinases. Exp Cell Res 2004; 292: 201-8.
• 37 Scaccianoce E, Festuccia C, Dondi D. et al. Characterization of prostate cancer DU145 cells expressing the recombinant androgen receptor. Oncol Res 2003; 14: 101-12.
• 38 Fenton MA, Shuster TD, Fertig AM. et al. Functional characterization of mutant androgen receptors from androgen-independent prostate cancer. Clin Cancer Res 1997; 3: 1383-8.
• 39 Suzuki H, Ueda T, Ichikawa T. et al. Androgen receptor involvement in the progression of prostate cancer. Endocr Relat Cancer 2003; 10: 209-16.
• 40 Yebra M, Goretzki L, Pfeifer M. et al. Urokinasetype plasminogen activator binding to its receptor stimulates tumor cell migration by enhancing integrinmediated signal transduction. Exp Cell Res 1999; 250: 231-40.
• 41 Tepper CG, Boucher DL, Ryan PE. et al. Characterization of a novel androgen receptor mutation in a relapsed CWR22 prostate cancer xenograft and cell line. Cancer Res 2002; 62: 6606-14.
• 42 Narayan P, Dahiya R. Establishment and characterization of a human primary prostatic adenocarcinoma cell line (ND-1). J Urol 1992; 148: 1600-4.
• 43 Iizumi T, Yazaki T, Kanoh S. et al. Establishment of a new prostatic carcinoma cell line (TSU-Pr1). J Urol 1987; 137: 1304-6.
• 44 Loop SM, Rozanski TA, Ostenson RC. Human primary prostate tumor cell line, ALVA-31a new model for studying the hormonal regulation of prostate tumor cell growth. Prostate 1993; 22: 93-108.
• 45 D'Alessio S, Margheri F, Pucci M. et al. Antisense oligodeoxynucleotides for urokinase-plasminogen activator receptor have anti-invasive and anti-proliferative effects in vitroand inhibit spontaneous metastases of human melanoma in mice. Int J Cancer 2004; 110: 125-33.
• 46 Edlund M, Sung SY, Chung LW. Modulation of prostate cancer growth in bone microenvironments. J Cell Biochem 2004; 91: 686-705.
• 47 Sirotnak FM, She Y, Lee F. et al. Studies with CWR22 xenografts in nude mice suggest that ZD1839 may have a role in the treatment of both androgen-dependent and androgen-independent human prostate cancer. Clin Cancer Res 2002; 8: 3870-6.
• 48 Yamanaka I, Koizumi M, Baba T. et al. Epidermal growth factor increased the expression of alpha2beta1-integrin and modulated integrin-mediated signaling in human cervical adenocarcinoma cells. Exp Cell Res 2003; 286: 165-74.
• 49 Lee LT, Huang YT, Hwang JJ. et al. Transinactivation of the epidermal growth factor receptor tyrosine kinase and focal adhesion kinase phosphorylation by dietary flavonoids: effect on invasive potential of human carcinoma cells. Biochem Pharmacol 2004; 67: 2103-14.
• 50 Schiffer CA. Signal transduction inhibition: changing paradigms in cancer care. Semin Oncol 2001; 28: 34-9.
• 51 Bruell D, Stocker M, Huhn M. et al. The recombinant anti-EGF receptor immunotoxin 425(scFv)-ETA' suppresses growth of a highly metastatic pancreatic carcinoma cell line. Int J Oncol 2003; 23: 1179-86.
• 52 van Bokhoven A, Varella-Garcia M, Korch C. et al. Widely used prostate carcinoma cell lines share common origins. Prostate 2001; 47: 36-51.
• 53 van Bokhoven A, Varella-Garcia M, Korch C. et al. Molecular characterization of human prostate carcinoma cell lines. Prostate 2003; 57: 205-25.
• 54 van Bokhoven A, Varella-Garcia M, Korch C. et al. TSU-Pr1 and JCA-1 cells are derivatives of T24 bladder carcinoma cells and are not of prostatic origin. Cancer Res 2001; 61: 6340-4.
• 55 Margheri F, D'Alessio S, Serratì S. et al. Effects of blocking urokinase receptor signaling by antisense oligonucleotides in a mouse model of experimental prostate cancer bone metastases. Gene Ther 2005; 12: 702-14.
• 56 Unlu A, Leake RE. The effect of EGFR-related tyrosine kinase activity inhibition on the growth and invasionmechanisms of prostate carcinoma cell lines. Int J Biol Markers 2003; 18: 139-46.
• 57 Azuma T, Witke W, Stossel TP. et al. Gelsolin is a downstream effector of rac for fibroblast motility. EMBO J 1998; 17: 1362-70.
• 58 McShan GD, Zagozdzon R, Park SY. et al. Csk homologous kinase associates with RAFTK/Pyk2 in breast cancer cells and negatively regulates its activation and breast cancer cell migration. Int J Oncol 2002; 21: 197-205.
• 59 She QB, Solit D, Basso A. et al. Resistance to in PTEN-null HER-overexpressing tumor cells can be overcome through restoration of PTEN function or pharmacologic modulation of constitutive phosphatidylinositol 3'-kinase/Akt pathway signaling. Clin Cancer Res 2003; 9: 4340-6.
• 60 Shien T, Doihara H, Hara H. et al. PLC and PI3K Pathways are important in the inhibition of EGF-induced cell migration by ('Iressa', ZD1839). Breast Cancer 2004; 11: 367-73.
• 61 Yang Z, Bagheri-Yarmand R, Wang RA. et al. The epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 (Iressa) suppresses c-Src and Pak1 pathways and invasiveness of human cancer cells. Clin Cancer Res 2004; 10: 658-67.
• 62 Veit C, Genze F, Menke A. et al. Activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase is required for glial cell line-derived neurotrophic factor-induced migration and invasion of pancreatic carcinoma cells. Cancer Res 2004; 64: 5291-300.
• 63 Chen L, He HY, Li HM. et al. 1/2 and p38 pathways are required for P2Yreceptor-mediated prostate cancer invasion. Cancer Lett 2004; 215: 239-47.
• 64 Gioeli D, Mandell JW, Petroni GR. et al. Activation of mitogen-activated protein kinase associated with prostate cancer progression. Cancer Res 1999; 59: 279-84.