The Hdac Inhibitor Panobinostat Promotes Expression Of Delta 133p53 Variants In Tp53 Wild-Type Bladder Cancer Cells

Histone deacetylase inhibitors (HDACi) are a broad group of promising anticancer therapeutics, that show radiosensitizing effects in a broad panel of cancers. The mechanisms of radiosensitization are not fully elucidated. We have shown that panobinostat (PAN), a hydroxamic acid HDACi, significantly radiosensitize RT112 bladder carcinoma cells at low nanomolar concentrations. TP53 point mutations are common in bladder cancer, but little is known about expression of p53 variants in bladder tumors. ∆133p53 variants (N-terminal truncation of the first 132 amino acids and exon 9 splice variants α, β and γ) have reportedly dominant-negative effects on full length p53 in other tumor types. We are investigating the differences in Δ133p53 expression after PAN treatment in RT112 (TP53 wild type) cells. Upregulation of Δ133p53 variants may lead to downregulation of full length p53 expression. This could result in abrogation of cell cycle check points, and thus lethal unrepaired double-strand breaks (DSB) following IR. First, RT112 bladder cells were plated and incubated with PAN for 24h at 10-200 nM to establish toxicity. And also plated and incubated with PAN at 10-50 nM and subsequently irradiated at 2, 4, 6 and 8 Gy. Cells were then trypsinized and seeded at 700 cells per 10 cm plate for clonogenic assays, and plates stained and counted after 14 days. RT112 cells were also plated and treated with 50 nM PAN for 24h (or with DMSO as control). Cells were collected, RNA extracted and DNase I treated, and cDNAs prepared according to standard protocols. A semi-quantitative RT-PCR method was used to detect α, β and γ Δ133p53 variants and transcript identity confirmed using Sanger sequencing. IC50 dose for 24h PAN incubation is 27 nM (SER at this dose is 1.36). Panobinostat radiosensitizes RT112 bladder cancer cells at both10 nM (SER = 1.25) and 50 nM (SER = 1.54). By semi-quantitative RT-PCR we observed a three-fold upregulation of the Δ133p53 α variant and 1.5-fold upregulation of the β variant, compared to DMSO treated control cells. Validation of the γ variant is ongoing. We observed radiosensitising effects of panobinostat, which may be explained, at least partially, by upregulation of p53 dominant-negative Δ133p53 α, β and γ variants with PAN treatment. We shall further investigate this phenomenon, using lower nanomolar concentrations of PAN and combined PAN-radiation treatment, and correlate effects on both Δ133p53 expression and cell-cycle/DSB repair in more bladder cancer cell lines.