Abstract 1494: Silencing Notch-1 and Notch-3 promotes epithelial-to-mesenchymal transition (EMT) and self-renewal potential in human bladder cancer.
Authors: Wolfgang Jaeger, Tetsutaro Hayashi, Shannon Awrey, Kilian Gust, Thomas Cordonnier, Na Li, Ralph Buttyan, Estelle Li, Ladan Fazli, Theodorus Van Der Kwast, Bas W. G. Van Rhijn, Peter C. Black
Journal: Cancer Research (2013)

Abstract Background: Beside its regulatory functions during development, the Notch pathway plays an eminent role in the tumorigenesis of malignancies. There are four different Notch receptors, which demonstrate context-dependent activity and can work antagonistically to each other. We have previously described a role for Notch-2 in promoting EMT and invasion in bladder cancer (BCA). Our objective here was to elucidate the role of Notch-1 and -3 in EMT and invasion, hypothesizing that both promote the epithelial, less invasive phenotype. Methods: Immunohistochemistry was performed on a tissue microarray (TMA) of 104 tumors from patients with muscle invasive BCA. Notch-1 and -3 expression was assessed in a panel of bladder cancer cell lines by Western blot (WB) and correlated to previously established EMT status (based on E-cadherin and ZEB1) and invasive ability. Notch-1 and -3 were silenced by lentiviral shRNA vector in 3 cell lines with high constitutive Notch-1 and -3 expression (UM-UC1, UM-UC6 and UM-UC15). Protein and mRNA expression of genes relevant to EMT, invasion and stemness were profiled by WB, real-time PCR, immunofluorescence and FACS. Proliferation, migration and invasion were measured by crystal violet, scratch, and Boyden chamber assays, respectively. Self-renewal potential was assessed by clonogenic assay and spheroid formation in MammoCult™ medium. Results: High expression of Notch-3 receptor was associated with lower pathological stage (p&amp;lt;0.05) and absence of lymphovascular invasion (p&amp;lt;0.05) in the cystectomy specimens. Notch-1 expression did not correlate with pathologic variables. WB revealed expression of Notch-1 and -3 in epithelial bladder cancer cell lines only. Silencing of Notch-1 or Notch-3 in the selected epithelial cell lines promoted EMT at the mRNA and protein levels. Accordingly, phenotypic changes were seen in the form of enhanced migration and invasion but unaltered proliferation. In addition, colony and spheroid formation was increased after specific Notch-3 silencing. The simultaneous knock down of Notch-1 and -3 showed no additive effect. Conclusion: Our results provide evidence that Notch-1 and Notch-3 are involved in invasion and progression of BCA by maintaining an epithelial phenotype and inhibiting EMT and self-renewal potential. This is the opposite of what we have seen previously with Notch-2. Beside verification of our observations in metastatic xenograft models, further investigation is needed to elucidate the molecular mechanisms of these alterations. Citation Format: Wolfgang Jaeger, Tetsutaro Hayashi, Shannon Awrey, Kilian Gust, Thomas Cordonnier, Na Li, Ralph Buttyan, Estelle Li, Ladan Fazli, Theodorus Van Der Kwast, Bas W. G. Van Rhijn, Peter C. Black. Silencing Notch-1 and Notch-3 promotes epithelial-to-mesenchymal transition (EMT) and self-renewal potential in human bladder cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1494. doi:10.1158/1538-7445.AM2013-1494