MP34-10 CROSSTALK BETWEEN DOWNSTREAM EFFECTORS OF RECEPTOR TYROSINE KINASE/RAS PATHWAY CAN BE DISRUPTED BY COMBINATORIAL INHIBITION: IMPLICATION IN TARGETED THERAPY

Abstract

INTRODUCTION AND OBJECTIVES: Phosphatase and Tensin Homology (PTEN) antagonizes PI3-kinase/AKT and this activity relies on PTEN-membrane interaction. While loss of PTEN, along with p53 defects, triggers muscle-invasive bladder cancer (BC) in genetically engineered mice, PTEN deletion or mutation in human BC is rare, suggesting that PTEN is inactivated via a different mechanism. Hyperphosphorylation of PTEN’s C-terminus (PTEN-Cp) is thought to dissociate PTEN from the membrane, thus functionally inactivating PTEN. This study investigates the prevalence of PTEN-Cp and its effects on tumor suppression in BC cells. METHODS: Ten cell lines representing different grades/stages of human BC was assessed for PTEN-Cp using site-specific antibodies. cDNAs encoding (i) wild-type PTEN, (ii) mutated PTEN devoid of C-terminal phosphorylation sites and (iii) truncated PTEN lacking the C-terminus were transfected into UMUC3 and J82 BC cell lines that lack the PTEN gene. PTEN-Cp status, PI3K/AKT activation and BC cell proliferation were assessed in vitro and in vivo using xenograft models. PTEN-Cp was also assessed using Western blotting of freshly preserved human BC tissues. RESULTS: Prominent PTEN-Cp was observed in all BC cell lines except UMUC3 and J82 which lack the PTEN gene. Transfection of wild-type PTEN into UMUC3 and J82 inhibited cell growth, albeit only at serum-free or low-serum (2%) conditions. High-serum (10%) caused PTEN to undergo extensive PTEN-Cp, increased AKT activation and cell proliferation. Mutated PTEN devoid of C-terminal phosphorylation sites and truncated PTEN lacking the C-terminus exerted much greater inhibitory effects on AKT and cell growth of UMUC3 and J82. These effects were reproducible when the transfected cells were transplanted subcutaneously into xenograft models. Finally, surveying a cohort of fresh human BC tissues revealed that a great majority of lowgrade non-invasive and high-grade invasive BC expressed PTEN and whenever PTEN was expressed it was highly phosphorylated at the C-terminus. CONCLUSIONS: Our studies provide the first evidence demonstrating that hyper-phosphorylation at the C-terminus is a major mechanism of PTEN inactivation in human BC, that PTEN phosphorylation may serve as a useful biomarker for BC progression, and that the tail-less PTEN is a much more potent tumor suppressor than the fulllength PTEN when used in molecular therapy.