Abstract 1171: Increased expression of PDK4 in bladder cancer cells
Authors: Choudhary Dharamainder , Mikhalyuk Andrew , Pilbeam Carol , Taylor John 
Published: August 2015

Introduction and Objectives. Cancer cells have a high rate of glucose metabolism due to an increased requirement for biomolecules and energy. In some solid tumors, cancer cells divert pyruvate to lactate even in the presence of oxygen (Warburg effect) providing a survival advantage in an acidic and anaerobic environment. Pyruvate dehydrogenase complex (PDC) catalyzes oxidative decarboxylation of pyruvate to acetyl-CoA and links glycolysis to energetic and anabolic functions of the tricarboxylic acid cycle. PDC activity is regulated by pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP). In a laser capture, microarray pilot study we found PDK4 expression to be increased 33-fold in high-grade invasive vs low-grade bladder cancers. The aim of this study was to examine the expression of PDK and PDP genes in bladder cancer cells.

Methods. Bladder cancer (HTB2, HTB4, HTB5, HTB9, HT1376) and benign urothelial (UROtsa) cell lines were cultured in defined media. Total RNA was extracted and quantitative PCR performed. Cell numbers were counted using a Coulter counter. Cell cycle and apoptosis analyses were performed using fluorescence-activated cell sorting. Transient transfections with PDK4 gene specific siRNAs were performed.

Results. In the present study, PDK4 expression was increased (20-100 fold; p&lt;0.01) in bladder tumor vs. benign cell lines. Expression of the other three PDK isoforms was increased 3-5 fold. Investigation of transcription factors known to regulate the expression of PDK4 revealed overexpression of PPARα (8-22 fold; p&lt;0.05-0.001) and HIF1α (3.7 - 13.4 fold; p&lt;0.01 only in HT1376). Expression of PDP1 and PDP2 was also assessed (0.4 - 2.2 fold, not significant). The ratio of PDK/PDP expression was found to be 101 (PDK4/PDP1) and 138 (PDK4/PDP2) for HTB9; 196 and 42 for HT1376; 94 and 50 for HTB5 and 57 and 26.3 for HTB4. To examine the impact of PDK4 inhibition, cells were treated with a known inhibitor, dichloroacetate (DCA), for 0-72 h. A significant decrease in cell number was detected in a dose dependent manner (10-50 mM). HTB2, a low grade cell line, showed a significant decline in cell number (70%) when incubated with 50 mM DCA for 72 h (p&lt; 0.01). HTB5, a high-grade cell line with a higher PDK4 expression, had a greater decline (92%) with same treatment (p&lt; 0.01). Further, we observed growth inhibition by cell count with siRNA knockdown of PDK4. Our preliminary data on cell cycle analysis showed an enhanced number of tumor cells in G1 phase and a decrease in S-phase.

Conclusions. PDK4 gene expression was found to be higher in bladder cancer cell lines. Both inhibition and knock down of PDK4 resulted in impaired cell growth with cell cycle arrest at the G1 phase. Taken together these data suggest that PDK4 may prove to be a novel therapeutic target in the management of bladder cancer.

Source of Funding. The Leo & Anne Albert Charitable Trust

Citation Format: Dharamainder Choudhary, Andrew Mikhalyuk, Carol Pilbeam, John Taylor. Increased expression of PDK4 in bladder cancer cells. [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 1171. doi:10.1158/1538-7445.AM2015-1171