DNAR-06. RHO GTPASES AS A POTENTIAL CHEMOSENSITIZER OF TMZ AND CISPLATIN-RESISTANT GBM TUMOR CELLS THROUGH A P53-DEPENDENT MECHANISM

Abstract

Abstract Rho GTPases regulate cellular processes related to tumor progression and their expression/activity determines the invasiveness and aggressiveness of GBM and, despite current aggressive therapies, effective treatments remain desired. Here we proposed that Rho GTPase acts on the genomic stability of GBM and its resistance to TMZ and cisplatin drugs in a p53-dependent mechanism. We selected GBM cell lines with different p53 status – U87-MG and A172 (wt-p53), T98G and U138-MG (mut-p53) – and subjected them to Rho inhibition by C3 toxin and subsequent DNA damage by TMZ treatments or cisplatin. Bioinformatic analysis showed that prolonged exposure to cisplatin induces the transcription of regulatory genes of Rho GTPases. Pulldown assays corroborated this analysis showing an increase in Rho activity after exposure to cisplatin or TMZ. IC50 for TMZ and cisplatin, as well as survival and proliferation assays, were all decreased by Rho inhibition in wt-p53 cells, while HCR assays showed that DNA repair capacity for both genotoxic drugs is decreased by Rho inhibition only in wt-p53 cells. To verify the role of p53 in this mechanism, we knocked down p53 in U87-MG cells, thus avoiding Rho inhibition-induced chemosensitivity and impaired DNA repair. Then, U87-MG cells were subjected to various treatment regimens with low doses of TMZ or cisplatin to obtain a subpopulation of cells resistant to these drugs. Further exposure to DNA damage led to increased p53(Ser15) phosphorylation, p53 nuclear translocation, and F-actin-induced polymerization. Two TMZ-resistant cell clones were selected, both showing a significant increase in TMZ IC50 for survival, while Rho inhibition reduced IC50 in both clones, significantly reversing the acquired resistance of these cells. These results suggest that Rho GTPase plays a role in DNA repair and in the acquired GBM wt-p53 resistance to TMZ and show that this pathway may be a fragile-point against current therapies.