Requirement of epidermal growth factor receptor tyrosine 845, Src, and Stat5b in the neurotensin-induced proliferation of human prostate cancer cells.

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Neuroendocrine (NE) cells have been shown to contribute to the progression of prostate cancer, and we have previously demonstrated that NE-like cells derived from LNCaP prostate cancer cells secrete greater amounts of neurotensin than undifferentiated LNCaP’s. Since PC3 androgen-independent prostate cancer cells express the neurotensin receptor (a G-protein-coupled receptor), we have examined the molecular mechanisms of growth stimulation by neurotensin in PC3 cells. Following a 48 h treatment of serum-starved PC3 cells with 100 nM neurotensin, up to a 2-fold increase in cell number was observed; corresponding increases in DNA synthesis (BrdU incorporation) were measured. Neurotensin-induced proliferation was also observed in androgen-dependent LNCaP prostate cancer cells, but not in RWPE-1 normal prostate epithelial cells. Similar data were obtained by treatment of PC3 cells with conditioned medium from NE-like cells, and this effect was partially inhibited by pre-incubation of PC3 cells with an EGFR inhibitor. Neurotensin induced a time-dependent increase in both general EGFR tyrosine and Tyr⁸⁴⁵ phosphorylation, and these effects were blocked by specific inhibition of Src (which mediates Tyr⁸⁴⁵ phosphorylation) or EGFR. Phosphorylation of Src and Stat5b (a downstream effector of Tyr⁸⁴⁵) was also induced by neurotensin, and these increases were abrogated by specific inhibition of EGFR or Src. Expression of EGFR (Tyr⁸⁴⁵) or Stat5b (Tyr⁶⁹⁹) mutants in PC3 cells resulted in the loss of neurotensin-induced stimulation of DNA synthesis, relative to wild-type controls. Moreover, neurotensin-induced increases in proliferation and DNA synthesis were abolished by selective inhibition of EGFR, Src, and matrix metalloproteinases (MMP’s). These data indicate that neurotensin can stimulate mitogenic activation of PC3 cells through a mechanism involving MMP’s, EGFR transactivation, EGFR Tyr⁸⁴⁵, Src, and Stat5b.