Molecular Mechanisms Underlying Mutant p53 Gain of Function in Glioblastoma

Glioblastoma is the most invasive and aggressive Central Nervous System tumor. Even with treatment administration, the median survival of patients diagnosed with such neoplasia is approximately 14.6 months. Several molecular mechanisms contribute to the development of this astrocytoma, including mutations in the TP53 gene. Due to mutations, p53 functions are altered in more than 50% of cancers. In addition, the presence of amyloid aggregates formed by p53 mutants is detected in several tumors, including glioblastoma. These amyloid aggregates are related to the gain-of-function phenotype, contributing to chemoresistance. In glioblastoma, resistance to Temozolomide is mediated by the action of the enzyme O6-methylguanine DNA methyl-transferase, whose expression is regulated by aggregates formed by the mutant of p53, M237I. This work aims to investigate the effects of M237I protein deletion in the glioblastoma cell line, T98G, using the CRISPR/CAS9 system. The protein and RNAm levels were analyzed by western blotting and PCR, respectively. The presence of p53 aggregates inside the cells were measured through the immunofluorescence and seprion assays. Cell migration was evaluated through the wound healing and transwell invasion assays. Thus, we were able of generating glioblastoma cell lines lacking the M237I. We demonstrated that M237I knockout cells present a lower pattern of cell migration and invasion compared with controls. Besides, the absence of M237I affects the cell ability of forming spheroids in a three-dimensional culture model. We conclude that p53 mutations present an important role in glioblastoma progression, as M237I knockout is able of reducing cell migration and altering cell morphology.