Abstract 1190: Pretreatment with 1,25 dihydroxyvitamin D3 modulates p73 levels and activity to increase pro-apoptotic effects of cisplatin in bladder cancer
Authors: Bunch Brittany L. , Johnson Candace S. , Trump Donald L. 
Published: July 2016

Abstract
Cisplatin-based combination chemotherapy is the standard treatment for advanced bladder cancer. While there is a substantial initial response rate and occasional complete response, long term control is uncommon. There is a substantial need to develop new therapeutic approaches to bladder cancer. 1,25 dihydroxyvitamin D3 (1,25D3), the active metabolite of vitamin D, enhances the anti-tumor effects of cisplatin in preclinical bladder cancer models. We sought to evaluate the mechanism of 1,25D3 potentiation of cisplatin cytotoxicity through in vitro studies in bladder cancer cell lines. Our studies suggest that enhanced cytotoxicity is mediated through modulation of p73. In a clonogenic assay, the surviving fraction after treatment with 1,25D3 (0.1 uM) for 24 hrs followed by cisplatin (0.1 ug/ml) for 48 hrs increases from 64% to 97% in T24 cells transfected with shRNA targeting p73. The ratio of the pro-apoptotic TAp73 isoform to the anti-apoptotic ΔNp73 isoform is important in determining cellular response to cisplatin. Our studies demonstrate that pretreatment with 1,25D3 followed by cisplatin significantly increases the ratio of TA/ΔNp73 mRNA transcripts 2-fold and increases BAX, the transcriptional target of TAp73, approximately 3-fold. Protein levels of TAp73 and BAX are also increased 2- and 3-fold, respectively, as determined by western blot. Protein stability of TAp73 was determined following cycloheximide treatment for 1, 2, and 4 hrs. After 4 hrs of cycloheximide treatment, protein levels of TAp73 decrease by approximately 50% in all treatment groups except the 1,25D3 and cisplatin group, in which the level of TAp73 protein was maintained. This suggests an increase in TAp73 protein stability after treatment. Using a TransAm p53 binding assay after p53 immunodepletion, 1,25D3 and cisplatin treatment increases DNA binding of TAp73 approximately 2-fold. Protein levels of TAp73 upstream activators, c-Abl and p38, also increase approximately 2-fold after 1,25D3 treatment, suggesting a mechanism of action for increased stability and activation of TAp73. Lastly, pharmacologic inhibition of p38 activation using SB203580 prevents the synergistic effects of 1,25D3 and cisplatin. Taken together, these data suggest that the mechanism of synergy for 1,25D3 and cisplatin combination therapy is through an increase in TAp73 stability and pro-apoptotic transcriptional activity. These findings suggest that 1,25D3 may have potential to be used in combination with cisplatin to increase the apoptotic response. Further studies are being performed to confirm the effects of treatment in an in vivo model, looking both at short term molecular response and long term therapeutic response to treatment. Supported by NCI grants CA067267 and CA016056.
Citation Format: Brittany L. Bunch, Candace S. Johnson, Donald L. Trump. Pretreatment with 1,25 dihydroxyvitamin D3 modulates p73 levels and activity to increase pro-apoptotic effects of cisplatin in bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1190.