Alterations in the activation pathways of protein kinase B and Erk in the progression to metastasis of human prostate cancer

Alterations in the activation pathways of Protein Kinase B and Erk in the progression to metastasis of human prostate cancer R. Michael Sharrard and Norman J. Maitland YCR Cancer Research Unit, University of York, York YOIO 5YW, UK Introduction: Progression to invasiveness and metastasis in epithelial tumours is characterised by cell growth and survival independent of growth factors (GFs) and cell-cell and cell-matrix contact. These functions are modulated by Protein Kinase B (PKB/Akt), activated by PIP3 generated by phosphatidylinositol 3-kinase (Pl3K), and by MAP kinases erkl/2 activated through ras, raf and MEK1/2. Enhanced survival of metastatic prostate tumour cells has been linked to deregulation of PKB activation through loss of the tumour suppressor protein PTEN, which normally functions as an antagonist to Pl3K by degrading PIP3. We report here further investigations into alterations of the signalling pathways that activate PKB and erk in prostatic cancer. Materials and methods: PNT2 and PNTI a (prostatic non-tumour epithelial), P4E6 (early prostatic tumour) and LNCaP and PC3 (metastatic prostate tumour) cell lines were grown in medium containing serum, then transferred to serum-free medium overnight. 1 OuM LY294002 (Pl3K inhibitor), 50uM PP2 (src kinase inhibitor) or 50uM PD98059 (MEK inhibitor) were added singly or in combination for 2 hours, followed by treatment with 5 rig/ml EGF or IGFI. Cells were then either fixed and immunostained for PKB/Akt phosphorylated at ser473 and erkl/2 phosphorylated at thr202/tyr204, or harvested and analysed by Western blotting for phosphorylation of these proteins. Results: Using these cell lines, we previously demonstrated that prostate tumour cells show reduced dependence of PKB/Akt activation on GFs or substrate adhesion, concomitant with enhanced sensitivity to the Pl3K inhibitor LY294002. We have further investigated the molecular basis for these changes using specific inhibitors of Pl3K, src, and MEK1/2, monitoring their effects upon PKB and erkl/2 activation by EGF and IGFI. In nontumour cells EGF activates Pl3WPKB via src and MEWerk independently of src, while IGFI activates Pl3WPKB (but not MEWerk) via src-dependent and -independent pathways. The MEK inhibitor PD98059 enhances activation of Pl3WPKB; this effect may operate through interactions of MEK with the Pl3K pathway independent of its erkkinase activity. In P4E6 tumour and PC3 metastatic cells, alterations to pathway effecters between the GF receptors and src lead to GF-independent PKB activation, while in LNCaP further alterations permit constitutive PKB activation independent of src. lmmunocytochemical analysis indicates that the phosphorylation status of both PKB and erk is influenced by the phase of the cell cycle. Thus, in addition to direct stimulation through their respective signalling pathways, GFs may thus affect PKB and erk activation indirectly through effects on cell proliferation. Conclusion: Prostate cancer progression involves complex alterations to signalling pathways regulating proliferation and survival. Elucidation of the molecular lesions and altered patterns of cross-talk between mechanisms activating PKB and erkl/2 in tumours will allow formulation of combinations of specific protein kinase inhibitors to target prostate cancers and their metastases.