CA12 KNOCKDOWN INHIBITS CELL MIGRATION AND SENSITIZES GLIOBLASTOMA CELLS TO TEMOZOLOMIDE

Introduction: In glioblastoma (GBM), the most aggressive form of brain cancer with a dismal prognosis, due to the presence of a heterogeneous population of glioblastoma stem cells (GSC), carbonic anhydrases (CA) have been linked to aggressive and invasive behavior of cells and resistance to therapy. Carbonic anhydrases are tumor-associated enzymes, highly expressed in hypoxic environment, and have been considered as potential biomarkers of tumor cells and a promising target. The CA12 two isoforms have been associated to the aggressive behavior of cancer cells and patient prognosis and the expression of CA12 on GSCs may be regulated by NF-kB. Aim: We propose to investigate the correlation of CA12 expression with molecular subtypes of GBM and analyze the effects of its knockdown (CA12KD), using RNA interference, evaluating cell proliferation, migration and chemo-resistance to temozolomide. Also, to analyze the regulation of CA12 by NF-KB and, using TCGA data base, to correlate CA12 expression with molecular features and patient survival. Methodology: The gene and protein expression of CA12 was evaluated on mesenchymal, pro-neural cells and U87 cell line by conventional PCR, qRT-PCR and western blot. The effect of CA12KD was analyzed by in vitro cell viability and ex-vivo cell migration. The chemo-sensitizing effect of CA12KD was assessed on U87 cells treated with temozolomide (100-300DM). GBM TCGA database was used for analyzing in silico the relation of CA12 isoforms and molecular subtype. Results: CA12kD was validated by decreased levels of CA12 gene and protein. Two CA12 isoforms (354aa and 343aa) were detected by gene and protein expression. TNF-D increased CA12 and NFkB expression. CA12kD inhibited cell migration on GSCs and sensitized GBM cells to treatment with temozolomide. In addition, TCGA data indicated that 52.8% of patients harboring mesenchymal subtype expressed both CA12 isoforms, and classic and pro-neural subtypes (29.6%) expressed only the short isoform, suggesting a correlation of CA12 expression and aggressive features and poor patient survival. Conclusion: The results demonstrated that CA12 may be regulated by NF-kB in GSCs and its activation may be one of the mechanisms that facilitate malignant GSC progression. The CA12 isoforms are expressed on different subtypes of GBM and may be implicated with patient prognosis and survival and the chemo-sensitizing effect of CA12KD represent an attractive strategy at the adjuvant therapy for GBM.