Abstract 77: Suppression of ERK1/2 with siRNA restores drug sensitivity in DU145 cells selected for resistance to AG2034
Authors: Obajimi Oluwakemi , Melera Peter 
Published: February 2011

Abstract
We examined the involvement of the extracellular signal regulated kinase (ERK) cascade (Ras/Raf/MEK/ERK) in the promotion of tumor cell growth, survival and the maintenance of ATP pools in DU145 prostate cancer cells selected for resistance to 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4] thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034). The Ras/Raf/MEK/ERK cascade is a subfamily of the MAPK signal transduction pathway and it plays an important role in gene expressions, growth and differentiation of many cell types. The components of this pathway are often altered in tumor cells and conflicting evidence exists regarding its contribution to the development of resistance to several chemotherapeutic drugs. DU145 cells were selected over time for resistance to AG2034 by gradually decreasing hypoxanthine concentrations from 1.7 µM to zero in the continuous presence of 10 nM 5-methyl tetrahydrofolate and 50 nM AG2034. Since the inhibition of glycinamide ribonucleotide formyl-transferase blocks the incorporation of glycine into the purine ring, total cellular ATP was quantified by reverse-phase HPLC and [14C]-Glycine incorporation into ATP was determined by liquid scintillation counting of HPLC-isolated ATP fractions. Total and phosphorylated proteins were determined by western blotting. Quantitative RT-PCR was utilized to characterize mRNA expression levels before and after transient transfection studies with siRNA. The continuous treatment of cells with AG2034 is cytotoxic to parent DU145 cells and inhibits proliferation. However, in the resistance clones, drug presence did not prevent cell growth but rather, markedly reduces the growth rate in comparison to drug-naïve parent cells. While the treatment of parent cells with AG2034 for 24 h depletes total ATP levels, there was no significant difference in total ATP levels between parent cells and resistant clones. Although AG2034 inhibits [14C]-glycine incorporation into ATP in parent cells, this effect is abrogated by drug resistance. Acquired resistance increases ERK expression levels and phosphorylation status. It also increases upstream activators, NRas, BRaf and MEK. In addition, decreasing the expression of ERK by siRNA reduces steady state levels of total and phosphorylated ERK and results in a marked depletion of total ATP levels in resistant clones. In the parent cells however, ERK inhibition resulted in increased ERK phosphorylation but had no effect on total ATP pools. The inhibition of ERK re-sensitizes drug-resistant DU145 cells to AG2034 concomitant with a depletion of cellular ATP pools. These results demonstrate that DU145 cells selected for resistance to AG2034 require the sustenance of ERK1/2 phosphorylation for the maintenance of their ATP pools and suggest that the intrinsic activation of ERK signaling supports growth and survival in these AG2034-resistant clones.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 77.