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Bombesin is a potent inducer of signal trasduction pathways involved in the proliferation and invasion of androgen-insensitive prostatic tumor cells. This study examines the bombesin-mediated modulation of pericellular proteolysis, monitoring cell capability to migrate and invade basement membranes, using a chemo-invasion assay and analyzing protease production. The results suggest that bombesin could modulate the invasive potential of prostatic cell lines regulating secretion and cell-surface uptake of uPA and MMP-9 activation. In fact, in PC3 and DU145 cells but not in LNCaP cells, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) are induced by bombesin treatment. Bombesin also stimulates cell proliferation and this effect can be inhibited blocking uPA by antibodies and/or uPA inhibitor p-aminobenzamidine. Moreover, HMW-uPA induces cell proliferation in LNCaP cells, which do not produce uPA in the basal conditions, while PC3 and DU145 cell growth is supported by autocrine production of uPA. The increment of uPA activity on the external plasma membrane causes an increased pericellular plasmin activation. This effect is inhibited by antibodies against uPA and by p-aminobenzamidine. Similarly to EGF, bombesin stimulates secretion and activation of MMP-9 and TIMP-1 production. MMP-9 activation can be also obtained by HMW-uPA treatment, suggesting that plasma-membrane-bound uPA can start a proteolytic cascade involving MMP-9. Therefore, in in vitro assays, bombesin is able to modulate pericellular proteolysis and cell proliferation, differently distributing and activating proteolytic activities. This effect can be related to the “non-random” degradation of the extracellular matrix in which membrane uPA-uPAreceptor complexes could start bombesin-induced directional protein degradation during metastatic spread. Int. J. Cancer 75:418–431, 1998. © 1998 Wiley-Liss, Inc.

Infection with erbB‐2 (E) of Ha‐roj (H) oncogene‐transfected cells has been previously shown to cooperatively induce anchorage‐independent growth of the MCF10A human mammary epithelial cell line in vitro, but not to induce nude mouse tumorigenicity. Here we show that oncogene‐transformed MCF10A are able to halt in the lungs of nude mice, a sign of organ colonization potential. We have therefore studied the transformants for in vitromigratory and invasive properties known to correlate with the metastatic potential of human mammary carcinoma cells in nude mice. MCF10A transfected with Ha‐ras, infected with a recombinant retroviral vector containing the human c‐erbB‐2 proto‐oncogene (MCF10A‐HE cells), show a higher invasive index than either the single transfectant (MCF10A‐H) or MCF10A‐erbB‐2 (MCF10A‐E) cells in the Boyden chamber chemotaxis and chemoinvasion assays. The MCF10A‐HE cells also adopted an invasive stellate growth pattern when plated or embedded in Matrigel, in contrast to the spherical colonies formed by the single transformants MCF10A‐H, MCF10A‐E, and the parental cells. Dot‐blot analysis of gelatinase A and TIMP‐2 mRNA levels revealed increasing gelatinase A mRNA levels (HE > E > H > MCF10A) and reduced TIMP‐2 expression in both single and double transformants. Furthermore, MCF10A‐HE cells show more MMP‐2 activity than parental MCF 10A cells or the single transformants. CD44 analysis revealed differential isoform banding for the MCF10A‐HE cells compared to parental ceils, MCF10A‐H and MCF10A‐E, accompanied by increased binding of hyaluronan by the double transformants. Our results indicate that erbB‐2 and Ha‐ros co‐expression can induce a more aggressive phenotype in vitro, representative of the malignancy of mammary carcinomas. © 1995 Wiley‐Liss, Inc.