Inhibition of Class I Histone Deacetylases 1 and 2 Promotes Urothelial Carcinoma Cell Death by Various Mechanisms

Abstract

Class I histone deacetylases HDAC1 and HDAC2 contribute to cell proliferation and are commonly upregulated in urothelial carcinoma. To evaluate whether specific inhibition of these enzymes might serve as an appropriate therapy for urothelial carcinoma, siRNA-mediated knockdown and specific pharmacologic inhibition of HDAC1 and HDAC2 were applied in urothelial carcinoma cell lines (UCC) with distinct HDAC1 and HDAC2 expression profiles. HDACs and response marker proteins were followed by Western blotting and qRT-PCR. Effects of class I HDAC suppression on UCCs were analyzed by viability, colony forming, and caspase-3/7 assays; flow cytometry, senescence and lactate dehydrogenase cytotoxicity assays; and immunofluorescence staining. Whereas single knockdowns of HDAC1 or HDAC2 were impeded by compensatory upregulation of the other isoenzyme, efficient double knockdown of HDAC1 and HDAC2 reduced proliferation by up to 80% and induced apoptosis-like cell death in all UCCs. Clonogenic growth was cell line-and HDAC-dependently reduced, with double knockdown of HDAC1 and HDAC2 being usually most efficient. Class I HDAC-specific inhibitors, especially the more specific HDAC1/2 inhibitors romidepsin and givinostat, significantly reduced proliferation of all UCCs (IC 50 , 3.36 nmol/L-4.59 mmol/L). Romidepsin and givinostat also significantly inhibited clonogenic growth of UCCs, with minor effects on nontumorigenic controls. Intriguingly, these compounds induced primarily S-phase disturbances and nonapoptotic cell death in UCCs. Thus, although both ways of inhibiting HDAC1/2 share mechanisms and efficaciously inhibit cell proliferation, their modes of action differ substantially. Regardless, combined inhibition of HDAC1/2 appears to represent a promising strategy for urothelial carcinoma therapy.Mol Cancer Ther; 15(2); 299-312. Ó2016 AACR.

Evaluation of the Therapeutic Potential of the Novel Isotype Specific HDAC Inhibitor 4SC-202 in Urothelial Carcinoma Cell Lines

“…Experiments were performed in the urothelial cancer cell lines (UCCs) VM-CUB1, RT-112, SW-1710, 639-V, UM-UC-3, 5637 and T-24 (provided by Dr. M. A. Knowles, Leeds, UK; Dr. J. Fogh, New York, USA; Dr. B. Grossmann, Houston, USA; DSMZ, Braunschweig, Germany) representing the heterogeneous phenotypes of urothelial carcinoma and different HDAC expression patterns [20, 24]. To investigate the tumor specificity of 4SC-202, experiments were further performed in two different urothelial control cell lines, HBLAK (spontaneously immortalized from primary human bladder epithelial cells, provided by CELLnTEC, Bern, Switzerland) and TERT-NHUC (TERT-immortalized normal human urothelial cells, provided by Dr. M. A. Knowles, Leeds, UK), and in the non-malignant, non-urothelial cell line HEK-293 (provided by Dr. V. Kolb-Bachofen, Duesseldorf, Germany) as previously described [24].…”

“…To investigate the tumor specificity of 4SC-202, experiments were further performed in two different urothelial control cell lines, HBLAK (spontaneously immortalized from primary human bladder epithelial cells, provided by CELLnTEC, Bern, Switzerland) and TERT-NHUC (TERT-immortalized normal human urothelial cells, provided by Dr. M. A. Knowles, Leeds, UK), and in the non-malignant, non-urothelial cell line HEK-293 (provided by Dr. V. Kolb-Bachofen, Duesseldorf, Germany) as previously described [24]. UCCs and HEK-293 cells were cultured in DMEM GlutaMAX-I (Gibco, Life Technologies, Darmstadt, Germany) supplemented with 10 % fetal calf serum (Biochrom, Berlin, Germany) at 37 °C and 5 % CO 2 .…”

“…Throughout, the previous characterized pan-HDACi SAHA served as control HDACi [20, 24]. The previously characterized class I HDACi romidepsin (histone acetylation, morphology, class I HDAC expression after HDACi treatment) and givinostat (morphology) were used as additional controls or for comparison with 4SC-202 [24]. As a positive control in LDH (lactate dehydrogenase) measurements, cells were treated with bortezomib (50 nM, S1013, Selleck Chemicals) or actinomycin D (4 μg/ml, A1410, Sigma Aldrich).…”

“…For example, selective inhibition of HDAC8 inhibited cell proliferation and clonogenic growth in a preclinical neuroblastoma cell culture model and, albeit less efficiently, in urothelial cancer cell lines [22, 23]. In a recent analysis on selective inhibition of class I HDACs, simultaneous and selective inhibition of the class I HDACs HDAC1 and HDAC2 resulted in significant decreases of cell viability, proliferation and clonogenicity associated with accumulation of cells in the G2/M cell cycle phase [24]. …”

Histone deacetylase inhibitors as cancer therapeutics

“…To this I would add that isoform-specific inhibition may also affect unknown molecular functions of other HDACs isoforms unwittingly, which are not related to the Zn-dependent active site, and there are indications that this may be the case from studies comparing HDAC siRNA knockdowns with selective HDACis (18). Thus, "isoform-specific" inhibition may be specific for the particular enzymatic activity attributed to the isoform, but may well have nonisoform-specific effects on different (and perhaps unknown) functions of the other HDAC isoforms.…”