Abstract LB-158: P73, a potential marker for chemoresponsiveness to cisplatin therapy and survival in muscle invasive bladder cancer (MIBC)
Authors: Greenspan Rebecca D. , Krishnan Nithya , Morrison Carl , Omilian Angela , Bshara Wiam , Worral Amber , Attwood Kristopher , Trump Donald L. , Woloszynska-Read Anna , Johnson Candace S. 
Published: August 2015

Intrinsic and/or acquired resistance to cisplatin is a major obstacle in the treatment of advanced bladder cancer (BC). It is important to find markers that predict tumor responsiveness to cisplatin-based chemotherapy. P73, a p53 homologue, plays an important role in cisplatin sensitivity. TAp73, a major isoform of p73, utilizes an extrinsic promoter (P1) and is pro-apoptotic. We utilized Illumina 450K methylation arrays to interrogate over 150 BC patient samples and found 9 distinct CpGs in the P1 promoter region that were hypermethylated in tumors compared to adjacent normal tissues (p&lt;.05). To determine the functional link between promoter methylation and transcriptional regulation of TAp73, we treated BC cell lines (253J, HT1376, and T24) with DNA hypomethylating agent decitabine (DAC) at various doses (0,1,5 μM). We found that a 10-20% decrease in methylation across 5 interrogated CpG sites resulted in a two fold increase in TAp73 expression. We hypothesize that demethylation of the P1 promoter increases TAp73 expression, sensitizing BC cells to cisplatin. To test this, T24 cells were pretreated with DAC (2μM), followed by cisplatin treatment (0.5μg/mL). Clonogenicity of T24 was measured, as well as changes in p73 methylation and expression. Results show a decrease in clonogenic potential following combination therapy. T24 treated with DAC alone or in combination with cisplatin showed a 15% decrease in P1 promoter methylation, while the same was not seen in cells treated with cisplatin alone. Treatment with DAC and cisplatin did not have a clear effect on TAp73 mRNA expression. We are investigating whether TAp73 protein expression reflects the effects of DAC. To determine the clinical value of TAp73 protein expression we utilized tissue microarrays (TMAs) of 354 BC patients. Immunohistochemical staining was assigned H-score indicating intensity of TAp73 expression and percentage of positive nuclei. Results showed lower TAp73 expression to be associated with patient samples of higher tumor grade and staging (p&lt;.001), suggesting that TAp73 expression is lost as the disease progresses from superficial to advanced BC. Consequently, low TAp73 protein expression was associated with shorter overall survival (p&lt;.05). To further investigate clinical utility of TAp73 promoter methylation and expression, we analyzed tumor samples from 24 patients treated with cisplatin in a neoadjuvant setting. Results showed hypermethylation in tumor compared with non-tumor samples (p&lt;.05), but no significant difference in mRNA expression was observed. Studies continue to focus on understanding the mechanism of cisplatin resistance in the context of epigenetic dysregulation and utilization of DAC as a potentiating agent of cispatin-based chemotherapy in muscle invasive BC.

Supported by NCI grants CA067267 and CA016056.

Citation Format: Rebecca D. Greenspan, Nithya Krishnan, Carl Morrison, Angela Omilian, Wiam Bshara, Amber Worral, Kristopher Attwood, Donald L. Trump, Anna Woloszynska-Read, Candace S. Johnson. P73, a potential marker for chemoresponsiveness to cisplatin therapy and survival in muscle invasive bladder cancer (MIBC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-158. doi:10.1158/1538-7445.AM2015-LB-158