Mp45-05 urothelium-specific and temporally controlled bi-allelic inactivation of both pten and p53 triggers basal-subtype muscle-invasive bladder cancer

INTRODUCTION AND OBJECTIVES: PTEN is a tumor suppressor whose inhibitory activity towards phosphoinositide 3 kinase replies on membrane association, a process that is tightly regulated by electrostatic charges of PTEN tail domain. Precisely how and to what extent this domain affects PTEN’s activity remains unclear and is the subject of the present study. METHODS: Urothelial carcinoma cell lines without PTEN (UMUC3 and J82), or that expressing a mutated PTEN (T24), were transiently transfected with vector only, vector containing a full-length PTEN or vector containing a tailless PTEN, and their effects on cell proliferation, AKT activity, cell cycle and apoptosis were assessed under different culture conditions. To circumvent the extreme deleterious effects of the tailless PTEN, stable transfectants of UMUC3 were created that only expressed the tailless PTEN (or the full-length PTEN as a control) upon doxycycline exposure (e.g., stable transfection of CMV/rtTA and TRE-tailless PTEN (or TRE/full-length PTEN). The effects of their inducible expression were assessed in vitro and in xenograft mouse models. RESULTS: Introduction of full-length PTEN and tailless PTEN into PTEN-deficient UMUC3 and J82 cells inhibited proliferation at comparable levels in serum free and low serum (2%) media. However, in high serum (10%) medium, tailless PTEN was three times more inhibitory than full-length PTEN. This was due primarily to the phosphorylation of the serine/threonine clusters of the full-length PTEN’s tail domain, an effect absent in the tailless PTEN. Similar results were observed with transiently transfected T24 cells which expressed an endogenous mutant PTEN. UMUC3, J82 and T24 transfected with tailless PTEN could not sustain long-term culture because of its extreme deleterious effects, prompting us to establish doxycycline-inducible expression cell lines. UMUC3 inducibly expressing tailless PTEN exhibited significantly greater inhibition of proliferation, AKT phosphorylation, G1/S transition and significantly more apoptosis than UMUC3 inducibly expressing full-length PTEN. Finally, xenografted UMUC3 cells inducibly expressing full-length PTEN established tumors, albeit 30% smaller than the vector only controls. In contrast, those inducibly expressing tailless PTEN were devoid of any tumor in nude mice. CONCLUSIONS: Our in vitro and in vivo data are the first demonstration of the high potency of tailless PTEN as a tumor suppressor. They suggest that delivering tailless PTEN via viral vectors or other vehicles can be an effective approach to tackle advanced urothelial carcinomas with wild-type, mutated or deleted PTEN.