Abstract 2018: Rescue of ΔNp63α inhibits urothelial cell invasion by attenuating epithelial-to-mesenchymal transition (EMT)

Abstract

Abstract Background: Loss of the transcription factor, p63 is associated with poor prognosis in muscle-invasive bladder cancers. The current study aimed to investigate the tumor-suppressive role of p63 in urothelial cancers. Methods: A 3-dimensional (3D) organotypic raft model of urothelial cancer was established by culturing human urothelial cancer cell lines (HT1376, T24) on human bladder fibroblast-embedded collagen-I. Control rafts contained normal primary human urothelial cells (HUC). Cells were also cultured as monolayers on collagen-I coated dishes before harvesting total RNA and protein for qPCR and Western blot analyses respectively. Results: Non-invasive HUC and HT1376 had an epithelial phenotype, characterized by compact and cuboidal morphology whereas invasive T24 cells were elongated with a spindle morphology, typical of mesenchymal cells and indicative of epithelial-to-mesenchymal transition (EMT), which is necessary for invasion. The number of invasive incidents in 3D rafts established with T24 cells was higher compared to those with HUC or HT1376 cells (N=3, p<0.05). Consistent with this, the invasive cell line had reduced mRNA and protein expression of total p63 (N=3), a differentiation marker, and the epithelial marker E-cadherin (N=3), in addition to upregulated expression of the mesenchymal markers N-cadherin (N=3) and fibronectin (N=3) and pERK-Y204 (N=2) compared to the non-invasive cells. Treatment of invasive T24 cells with histone deacetylase (HDAC) inhibitors (vorinostat (pan) or entinostat (HDAC1,3)) attenuated the number of invasive incidents (N=3, p<0.05), and restored protein expression of total p63 (N=3) and E-cadherin (N=2) while normalizing pERK-Y204 (N=2 for T24). The tumor-suppressive properties of the ΔNp63α isoform were studied by adenoviral-mediated overexpression of ΔNp63α in T24 cells, which mimicked the effects of HDAC inhibitors and diminished EMT by rescuing E-cadherin expression while depleting fibronectin (N=2). Overexpression of ΔNp63α also reduced the number of invasive incidents in 3D models established with T24 cells (N=2). Conclusion: Invasive T24 cells exhibited an EMT phenotype, which coincided with depleted p63 expression. Rescue of ΔNp63α expression by treatment with HDAC inhibitors or through experimental overexpression attenuated the EMT phenotype and cell invasion. Citation Format: Kirtiman Srivastava, Victoria Smith, Eric A. Fernandez, Conor Breen, Karen D. McCloskey. Rescue of ΔNp63α inhibits urothelial cell invasion by attenuating epithelial-to-mesenchymal transition (EMT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2018.