Enhanced Rho GTPase pathway activity promotes acquisition of glioblastoma chemoresistance

Abstract Glioblastoma (GBM) is a highly aggressive tumor primarily treated through surgery, radiotherapy, and chemotherapy. GBM radioresistance involves the activation of the Rho GTPase pathway, actin cytoskeleton polymerization, and the cytoplasmic retention of wild-type p53. Activation of DNA damage response (DDR) pathways and double-strand break (DSB) repair depends on the cytoplasmic availability of G-actin and its nuclear translocation, which facilitates p53 nuclear transport. In this study, we investigated whether DNA damage repair pathways induced by cisplatin (CP) and temozolomide (TMZ) are dependent on Rho pathway activity and actin cytoskeleton dynamics by generating chemoresistant GBM sublines. GBM cells expressing wild-type p53 displayed activation of the Rho pathway and actin polymerization when treated with TMZ or CP, but showed reduced activation of DNA repair signaling, as well as lower levels of p-p53 (Ser15), and p21Cip1. TMZ-resistant clones exhibited constitutive Rho pathway activity, elevated p53 levels, and activation of DDR and DSB repair pathways, but displayed reduced levels of mismatch repair (MMR) proteins. Notably, inhibition of Rho GTPases restored the sensitivity of TMZ- and CP-resistant clones, reversing either transient or permanent chemoresistance in a process entirely dependent on wild-type p53. GBM cells harboring mutant p53 treated with PRIMA-1 also regained sensitivity to chemotherapy following Rho pathway inhibition. These findings were corroborated in GBM spheroid tumor models treated with TMZ and CP under actin cytoskeleton polymerization inhibition. In summary, modulating Rho pathway activity and actin cytoskeleton dynamics is crucial for both the development and reversal of chemoresistance in GBM.