Presentation Authors: Feng He, Fenglin Zhang, Yan Liu*, Ellen Shapiro, Herbert Lepor, Moon-shong Tang, Chuanshu Huang, Xue-Ru Wu, New York, NY
Introduction: There is presently a major discrepancy between the critical role of PTEN knockout in the genesis of muscle-invasive bladder cancer (MIBC) in mice and the very low frequency (6-8%) of PTEN mutation or deletion in the human counterpart. We undertook this study to examine the alternative mechanism(s) of PTEN inactivation, and found hyper-phosphorylation of PTEN, which functionally disables PTEN, to be extremely prevalent in human MIBC. This has several major theranostic implications.
Methods: The status of PTEN and its phosphorylation were first assessed in a panel of human bladder cancer (BC) cell lines, and then in a collection of fresh human BC specimens by Western blotting, and then in a cohort of archived low-grade non-invasive and high-grade invasive BC, both with matching adjacent normal urothelial controls, by immunohistochemical (IHC) staining. In the case of PTEN phosphorylation, phosphorylation site-specific antibodies were used. The effects of PTEN phosphorylation in functionally inactivating PTEN were established by transfecting PTEN-lacking BC cell lines (UMUC3 and J82) with wild-type PTEN or PTEN bearing phosphorylation-incompatible mutations or deletion. Their effects on cell growth, migration, AKT phosphorylation, cell-cycle progression and apoptosis were assessed in vitro and in xenograft mouse models.
Results: All but two BC cell lines (UMUC3 and J82) expressed PTEN and whenever PTEN was expressed, it was hyper-phosphorylated at the C-terminus. By Western blotting of fresh human BC specimens, 10/12 (83%) of the low-grade non-muscle-invasive BC (NMIBC) and high-grade muscle-invasive MIBC contained PTEN, and in all the positive tumors PTEN was hyper-phosphorylated at the C-terminus. By IHC, phosphorylated PTEN was significantly higher in BC, particularly MIBC than matching normal urothelia. Functional studies showed that PTEN phosphorylation drove PTEN from the membrane into the cytoplasm where it was much less active, and that wild-type PTEN was much less effective than PTEN bearing phosphorylation-incompatible amino acids or C-terminal deletion in growth inhibition in vitro and in nude mice.
Conclusions: PTEN phosphorylation is a major mechanism of inactivation in human bladder cancer, and it should be used as biomarker, instead of PTEN itself, for diagnosing, subtyping and predicting the progression of bladder cancer.
Source of Funding: NIH