Abstract 3354: Histone deacetylase-1 promotes urothelial cell migration and invasion by modulating p63-pMLC2 signaling

Abstract

Abstract Background: Altered expression of histone deacetylases (HDAC) and p63 transcription factor are associated with poor prognosis in invasive urothelial tumors. The current study focused on HDAC-evoked epigenetic modification of p63 and its downstream effects. Methods: Primary human urothelial cells (HUC) and urothelial cancer cell lines (HT1376, T24, TCCSUP) were cultured either on human bladder fibroblast-embedded collagen-I to establish 3D-organotypic rafts or as 2D-monolayers. Scratch wound assay was used to study the rate of cell migration before studying the differential mRNA and protein expressions of HDAC1,2,3, p63, p21, p27, Rho-kinase1 (ROCK1), 14-3-3 eta and myosin light chain-2 (MLC2). Results: T24 and TCCSUP cells exhibited invasive incidents on 3D-organotypic rafts, which coincided with their enhanced cell migration capabilities. qPCR and Western blotting analysis demonstrated enhanced expression of HDAC1, HDAC3, p27, p-p27-Thr157 and 14-3-3 eta alongside depleted levels of p63, ROCK1, pMLC2-Ser19, pMLC2-Thr18-Ser19 and p21 in invasive cells compared to normal (N=5, p<0.05). These results coincided with the loss of stress fiber formations visualized by immunofluorescence detection of F-actin in invasive cells indicating that cytoskeletal reorganization may be pivotal for cell migration. Treatment with HDAC inhibitors (vorinostat (pan) and entinostat (HDAC1, 3)) attenuated the number of invasive incidents in T24 and TCCSUP cells, restored expression of p63, p21, pMLC2-Ser19, pMLC2-Thr18-Ser19 (N=5, p<0.05), and normalized expression of 14-3-3 eta and p-p27-Thr157. Reappearance of stress fiber formations traversing across the cell after HDAC inhibitor treatments was supportive of these results, while nuclear enrichment of p27 indicated its enhanced cytoplasm-to-nuclear trafficking. Transient knockdown of HDAC1 in the invasive population mimicked the effect of HDAC inhibitors (N=5, p<0.05), while successive depletion of p63 appeared to abolish the effects of HDAC1 knockdown by upregulating 14-3-3 eta and depleting ROCK1 expression, respectively. Conclusions: Inhibition of HDAC1 activity attenuates urothelial cell migration and invasion by restoring the expression of p63 which, (a) upregulates the expression and activity of ROCK1, and (b) suppresses the expression of 14-3-3 eta, thereby alleviating the cytoplasmic p27 levels and facilitating the pMLC2-mediated cytoskeletal remodeling. These findings provide further support and mechanistic evidence for the potential use of HDAC inhibitors to treat invasive/poor-prognosis bladder cancer. Citation Format: Kirtiman Srivastava, Conor Breen, Karen McCloskey. Histone deacetylase-1 promotes urothelial cell migration and invasion by modulating p63-pMLC2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3354. doi:10.1158/1538-7445.AM2017-3354