The study of FGFR3 ⁃ TACC3 fusion gene mediating pyruvate kinase M2 nuclear translocation to promote DNA damage repair in glioblastoma

Abstract

Objective To explore the mechanism of FGFR3⁃TACC3 (F3⁃T3) fusion gene mediating DNA damage repair through promoting pyruvate kinase M2 (PKM2)'s nuclear translocation in glioblastoma. Methods The lentiviral transfection technology was used to constructed stable transitional cell lines U87MG cells and U251MG cells that stably expressing F3 ⁃ T3 and empty vector. In vivo glioblastoma model was constructed by intracranial in situ tumor implantation in athymic mice, and the tumorigenic ability of F3⁃T3 transfected cells in athymic mice of each treatment group was observed by small⁃animal in vivo imaging system. Bioinformatics analysis was performed to analyze gene microarray data exploring the possible biological functions of F3 ⁃ T3 mediating chemoresistance and identify the relationship between survival expectations and PKM2 expression levels in glioblastoma patients from The Cancer Genome Atlas (TCGA) database. Transient transfection of small interference RNA (siRNA) was used to knock down the expression of PKM2 in the F3 ⁃ T3 transfected cells. Proliferative activity of U87MG and U251MG cells treated with different concentrations of temozolomide (TMZ), transfected with siRNA, and TMZ in combination with Compound 3k, a PKM2 inhibitor, was observed in CCK ⁃ 8 cell proliferation assays. Nuclear and cytoplasmic proteins were extracted separately and PKM2 protein expression was observed in whole cell extract, cytoplasmic extract and cytosolic extract after TMZ treatment. Relative expression of PKM2, relative expression of cytosolic phosphorylated histone H2AX (p⁃H2AX) and relative expression of siRNA knockdown PKM2 gene in U87MG and U251MG cells stably expressing the F3 ⁃ T3 fusion gene, detected by Western blotting. Results 1) CCK⁃8 cell proliferation assay showed that the survival rate of U87MG cells in the F3 ⁃ T3 transfected group was higher than that in the empty vector transfected group after treatment with TMZ 640, 320, 160, 80, 40 μmol/L (P = 0.000, 0.000, 0.000, 0.000, 0.004, 0.010), and the survival rate of U251MG cells in the F3 ⁃ T3 transfected group was also higher than that in the empty vector transfected group after TMZ 640, 320, 160, 80, 40, 20, 5 μmol/L (P = 0.000, 0.000, 0.000, 0.002, 0.001, 0.002); the survial rate of U87MG cells in the si⁃PKM2⁃1009 transfected group was lower than that of F3 ⁃ T3 transfected group after TMZ 640, 320, 160, 80, 40, 20, 10, 5, 2.50 μmol/L, the survival rate of U251MG cells in the si⁃PKM2⁃1377 transfected group was also lower than that in F3⁃T3 transfected group after TMZ 640, 320, 160, 80, 40, 20, 5 μmol/L (P = 0.000, 0.000, 0.002, 0.000, 0.002, 0.048, 0.042); and the survival rate of U87MG cells in TMZ + Compound 3k group was lower than TMZ group after TMZ 640, 320, 160, 80, 40, 20 μmol/L (P = 0.000, 0.000, 0.000, 0.000, 0.001, 0.002), and the survival rate of U251MG cells in TMZ + Compound 3k group after treatment with high concentrations of TMZ (640, 320, 160, 80 and 40 μmol/L) was also lower than TMZ group (P = 0.000, 0.000, 0.000, 0.000, 0.003), while the survival rate of U251MG cells in TMZ + Compound 3k group after treatment with low concentrations of TMZ (10, 5 and 2.50 μmol/L) was higher than that of TMZ group (P = 0.000, 0.000, 0.006). 2) An animal model of glioblastoma showed the presence of TMZ resistance in homozygous mice. 3) Bioinformatic analysis showed that the biological function of F3⁃T3 was significantly enriched in the DNA repair pathway (P = 0.000). Survival and overall survival of glioblastoma patients in the TCGA database were lower in the PKM2 high expression group than in the low expression group (P < 0.05). 4) Western blotting showed that the relative expression of p⁃H2AX in U87MG (P = 0.000, 0.000, 0.004) and U251MG (P = 0.000, 0.007, 0.005) cells in the F3 ⁃ T3 transfected group were lower than those in the empty vector transfected group after TMZ treatment for 48 h and then medium change for 24, 36 and 48 h. PKM2 protein incorporation into the nucleus was observed in both U87MG and U251MG cells in the F3⁃T3 transfected group after TMZ treatment, whereas it was not observed in cells in the empty vector transfected group; si⁃PKM2⁃1009 and si⁃ PKM2⁃1377 knocked down the relative expression of p⁃H2AX in U87MG (P = 0.000, 0.001, 0.006) and U251MG (P = 0.000, 0.000, 0.000) cells, respectively. Conclusions In the presence of TMZ, F3 ⁃ T3 promotes PKM2's nuclear translocation and activates DNA damage repair pathways, which in the eventually results resistance of glioblastoma cell to TMZ. PKM2 inhibitors can compromise the resistance glioblastoma cells stably expressing F3⁃T3 fusion gene to TMZ.

DOI: 10.3969/j.issn.1672⁃6731.2023.08.015