ERK Suppression in DU145 Cells Selected for AG2034 Resistance is Associated with Decreased ATP, GARFT Inactivation and Increased ERK Signaling

Background: Previously, we showed that AG2034, an inhibitor of glycinamide ribonucleotide formyltransferase (GARFT) in the pathway for de novo purine synthesis, is cytotoxic to different prostate cancer cell lines in the absence of inosine and hypoxanthine in the culture media. Therefore, we examined DU145 androgenindependent prostate cancer cells selected for drug resistance over a prolonged period of time, in the absence of media components (inosine and hypoxanthine) required for salvage purine synthesis and in the continuous presence of AG2034. Methods and Results: DU145 cells were selected over time for 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3Hpyrimidino[5,4,6][1,4] thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034) resistance 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 GARFT blocks glycine incorporation into the purine ring, total cellular ATP was quantified by reverse-phase HPLC and [C]-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 small interfering RNA (siRNA). We show that the exposure of parental DU145 cells to AG2034, an inhibitor of GARFT, leads to rapid enzyme inhibition and depletion of ATP. However, resistant DU145 cells are able to re-activate GARFT, restore ATP pools and increase mRNA expression and phosphorylation of ERK1/2 thus, stimulating drug-resistant growth. Moreover, siRNA knockdown of ERK1/2 mRNA in resistant DU145 cells inhibits ERK protein expression and phosphorylation and results in the depletion of ATP and the inhibition of GARFT activity in the absence of changes in GARFT mRNA and protein expression levels. Conclusions: The inhibition of ERK1/2 signaling by siRNA transfection affects AG2034 resistance by inhibiting GARFT activity and depleting ATP pools. Hence, we conclude that ERK plays an important role in the maintenance of the AG2034-resistant DU145 phenotype.