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Identification of Modulators of Tumor and Invasive Suppressor Genes by Different Classes of Histone Deacetylase Inhibitors using High-Throughput Reverse Phase Protein Arrays
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Identification of Modulators of Tumor and Invasive Suppressor Genes by Different Classes of Histone Deacetylase Inhibitors using High-Throughput Reverse Phase Protein Arrays
Justin J. Gould, MD1, Patrick A. Kenney, MD1, Matthew F. Wszolek1, Kimberly M. Rieger-Christ, PhD1, Brasil Neto, MD1, Antonia Holway, PhD2, Brett Spurrier, MS2, John Austin, PhD2, John A. Libertino, MD1, Ian C. Summerhayes, PhD1.
1Lahey Clinic, Burlington, MA, USA, 2Aushon Biosystems, Billerica, MA, USA.
Objective: To identify histone deacetylase inhibitors (HDACi) that can modulate the expression of different tumor and invasive suppressor genes in a panel of bladder carcinoma cell lines using high-throughput reverse phase protein arrays (RPPA).
Materials and Methods: Nine bladder carcinoma cell lines (RT112, RT4, HU456, CUBIII, BC16.1, 5637, UM-UC-3, J82, EJ) representative of a discontinuous model of progression were used to establish the IC50 for four different classes of HDACi’s in clonogenic assays. The HDACi’s included Trichostatin A (TSA), Valproic acid, Apicidin and MS275. Lysates were prepared from cells exposed to a range of concentrations of HDACi, selected around the IC50, over intervals of time ranging between 0-36 hours. 225 cell lysates from each HDACi experiment were arrayed (Aushon Biosystems 2470) with 15 different controls in a 10-fold dilution series, in duplicate, resulting in 4,800 arrayed samples on a single nitrocellulose-coated microscope slide. Individual RPPA were probed with antibodies (E-, N-cadherin, α-, β-, γ-catenin, FAK, gelsolin, desmocollin-2/3, desmoglein-2, plakopholin-3, α-tubulin) validated for use in RPPA by the identification of a single band in western blot analysis. A total of 19,200 data points/antibody were collected and analyzed using P-SCAN and a dose interpolation methodology calculated for each sample series following normalization.
Results: Modulation of expression of α-, γ-catenin and gelsolin was identified in subgroups of bladder carcinoma cell lines following exposure to each of the HDACi’s. Cell lines representative of late-stage disease revealed significant up-regulation of γ-catenin where the latter has been shown to act as a tumor suppressor gene in bladder carcinoma cells. Reduced gelsolin expression, reported associated with bladder tumor progression, was shown to be up-regulated by all HDACi’s in cell lines representative of pre-invasive disease. Confirmation of such events was established in western blot analysis. E-cadherin, an invasive suppressor gene, was recorded up-regulated in the presence of apicidin, valproic acid and MS275 in multiple cell lines displaying constitutive expression of this protein. In contrast, desmoglein was found down-regulated in well-differentiated bladder cell lines following exposure to all four HDACi’s. Modulation in FAK expression was associated with exposure to valproic acid and MS275 in a restricted number of cell lines. No significant change in expression was recorded in the remaining protein targets
Conclusions: The different classes of HDACi’s share similar potential to up-regulate the expression of important tumor and invasive suppressor genes involved in bladder tumorigenesis. Modulation of such genes has been shown to suppress the tumor phenotype identifying HDACi’s as potential therapeutic agents in bladder cancer. The RPPA approach demonstrates the ability to rapidly screen multiple compounds and assess expression profiles of specific protein groups targeted for therapy.
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