Antineoplastic effects of specific inhibition of Notch-2 in bladder cancer.

Abstract

e15590 Background: Notch signaling guides cell fate decisions, establishment of cell lineages, stem cell maintenance and differentiation during early development. Notch is re-activated in many malignancies where it has been shown to regulate tumor growth and progression. We report effects of Notch-2 knockdown and treatment with a specific Notch-2 inhibitor, NRR2Mab, in pre-clinical models of bladder cancer. Methods: UM-UC3, UM-UC13 and UM-UC16 cells, representing invasive bladder cancer cells with high Notch-2 expression, were stably transduced with Notch-2 or non-targeting shRNA in a lentiviral vector. A Notch-2-inactivating monoclonal antibody, NRR2Mab, and an isotype-matched, non-targeting antibody were provided by Genentech, Inc. (San Francisco, CA). Cells were grown under adherent (AH) or anchorage-independent conditions (AI) and the effects of Notch-2 silencing or NRR2Mab inactivation were quantitatively assessed for changes in proliferation, migration and invasive activities using in vitro assays, and for expression of Notch-2 or other common stem cell-related genes using quantitative RT-PCR, Western blotting and immunohistochemistry. In vivo effects were evaluated in an orthotopic bladder cancer xenograft model with bioluminescence imaging. Results: Cells transduced with the Notch-2 shRNA or treated with NRR2Mab demonstrated markedly decreased Notch-2 expression. Both treatments diminished cell proliferation under AI but not AH conditions in UM-UC3 and UM-UC13. When grown in AI conditions, UM-UC3 and UM-UC13 were found to be enriched for expression of stem cell genes. Notch-2 silencing or treatment with NRR2Mab diminished the expression of these genes. Both treatments significantly inhibited cell migration and invasion of these cells. In vivo, stable Notch-2 knockdown significantly inhibited xenograft growth for all 3 cell lines. NRR2Mab treatment also inhibited UM-UC13 xenograft growth and metastasis. Conclusions: Our results provide preclinical evidence that Notch-2 may be a useful target to inhibit growth and progression of bladder cancer. Targeting Notch-2 with a specific inhibitory monoclonal antibody warrants further evaluation in this context.