Available online 30 September 2023, 106474

Author links open overlay panel, ABSTRACT

Resistance to radio and chemotherapy in Glioblastoma (GBM) is correlated with its malignancy, invasiveness, and aggressiveness. The Rho GTPase pathway plays important roles in these processes, but its involvement in the GBM response to genotoxic treatments remains unsolved. Inhibition of this signaling pathway has emerged as a promising approach for the treatment of CNS injuries and diseases, proving to be a strong candidate for therapeutic approaches. To this end, Rho-associated kinases (ROCK), classic downstream effectors of small Rho GTPases, were targeted for pharmacological inhibition using Y-27632 in GBM cells, expressing the wild-type or mutated p53 gene, and exposed to genotoxic stress by gamma ionizing radiation (IR) or cisplatin (PT). The use of the ROCK inhibitor (ROCKi) had opposite effects in these cells: in cells expressing wild-type p53, ROCKi reduced survival and DNA repair capacity (reduction of γH2AX foci and accumulation of strand breaks) after stress promoted by IR or PT; in cells expressing the mutant p53 protein, both treatments promoted longer survival and more efficient DNA repair, responses further enhanced by ROCKi. The target DNA repair mechanisms of ROCK inhibition were, respectively, an attenuation of NHEJ and NER pathways in wild-type p53 cells, and a stimulation of HR and NER pathways in mutant p53 cells. These effects were accompanied by the formation of reactive oxygen species (ROS) induced by genotoxic stress only in mutant p53 cells but potentiated by ROCKi and reversed by p53 knockdown. The p53-dependent actions of ROCKi were completely abolished by N-acetyl-L-cysteine (NAC) treatment or Rac1 knockdown, as Rac-GTP levels were specifically elevated by ROCK inhibition only in mutant p53 cells. Combining IR or PT and ROCKi treatments broadens our understanding of the sensitivity and resistance of, respectively, GBM expressing wild-type or mutant p53 to genotoxic agents. Our proposal may be a determining factor to improve the efficiency and assertiveness of CNS antitumor therapies based on ROCK inhibitors.

SIGNIFICANCE

The use of ROCK inhibitors in association with radio or chemotherapy modulates GBM resistance and sensitivity depending on the p53 activity, suggesting the potential value of this protein as therapeutic target for tumor pre-sensitization strategies.

Section snippetsINTRODUCTION

Small GTPases of the Rho-family (e.g., RhoA, Rac1, Cdc42) are key regulators of cytoskeletal and cell adhesion dynamics and a constant crosstalk between their activities is necessary for the regulation of the actin cytoskeleton and various stages of the motility cycle (Haga and Ridley, 2016). The serine/threonine kinases ROCK1 and ROCK2 are main downstream effectors of RhoA, being master regulators of actomyosin and, therefore, of different processes such as force generation, cell invasion,

Bioinformatics analysis

Data of gene mutations and expression of ROCK1/2 in tumor samples and its impact in patient survival was obtained in the database PanCancer Atlas for Glioblastoma Multiforme studies, a large-scale collaboration initiated and supported by the National Cancer Institute (NCI) and National Human Genome Research Institute (NHGRI) from The Cancer Genome Atlas (TCGA) consortium. The analyses were performed in the cBioPortal platform, freely available in: https://www.cbioportal.org (Cerami et al., 2012

ROCK as a potential target to the response to radio and chemotherapy in glioblastoma patients

Rho kinase (ROCK) controls a variety of biological responses and plays important regulatory roles in the cytoskeleton (Fig. 1A) of gliomas becoming a potential drug candidate (Mulherkar and Tolias, 2020). Both ROCK isoforms (ROCK1 and ROCK2) present high incidence of mutations in the Rho-binding domain (RBD) and in the Pleckstrin homology (PH) domain of GBM patients (Fig. 1B). Approximately 7% of GBM patients with primary tumors harbor ROCK 1/2 mutations while this rate is approximately 60% in

DISCUSSION

The ROCK1/2 inhibitor Y-27632 has been widely used for research in vitro and in vivo to investigate biological and pharmacological functions of ROCK in several diseases and cancer scenarios. However, the progress of this inhibitor and similar compounds to the advance of clinical trials for antitumor therapy is still quite limited mainly due to their opposing effects in different tumor models and cellular contexts (Barcelo et al., 2023). A recent work from our group showed that inhibition of Rho

Funding

This work was supported by grants from the Sao Paulo Foundation - FAPESP (Grant Nos. 2008/58264-5, 2015/03983-0, 2018/01753-6, and 2022/04243-1), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES (Grant No. 88887.136364/2017-00) and the National Council for Scientific and Technological Development - CNPq (Grant No. 230420/2016/7) to FLF. YTM was the recipient of a FAPESP PhD fellowship (Grant No. 2017/01451-7).

CRediT authorship contribution statement

Fabio Luis Forti: Conceptualization, supervision, writing, reviewing and editing. Yuli Thamires Magalhaes: Methodology, data curation, investigation, validation, original draft preparation and visualization

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Authors thank: i) Prof. Elza T. S. Hojo, Prof. Carlos F. M. Menck and Dr. Veridiana Munford for the donation and validation of GBM cell lines; ii) Prof. Alexandre B. Cardoso and Prof. Nicolas C. Hoch for the use of microscopes Leica DMi8 widefield and Zeiss LSM 780 confocal; iii) Prof. Bianca S. Zingales and the technician Marcelo N. Silva for equipment usage and reagents exchange; iv) Prof. Pio Colepicolo for use of plate reader Tecan equipment Infinite®; v) Izaura Nobuko Toma and Benedita de

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