Dados do Trabalho

Título

Effect of the CA12 knockdown and TNF-α pathway in glioblastoma stem cell

Introdução

In glioblastoma (GBM), the most aggressive form of brain cancer, 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 environments and potential biomarkers of tumor cells. The two CA12 isoforms have been associated with the aggressive behavior of cancer cells and patient prognosis, and NF-kB may regulate the expression of CA12 on GSCs.

Objetivo

Investigate the correlation of CA12 expression with molecular subtypes of GBM and analyze the effects of its knockdown (CA12KD) using RNA interference. To analyze the regulation of CA12 by NF-KB, we used the TCGA database to correlate CA12 expression with molecular features and patient survival.

Métodos

The gene and protein expression of CA12 was evaluated on mesenchymal, pro-neural cells, and U87 cell lines 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 GSC cells were exposed to TNF-α (10ng/mL) for 6-24h. The CA12 level were measured by qRT-PCR. The chemo-sensitizing effect of CA12KD was assessed on U87 cells treated with temozolomide (100-300mM). GBM TCGA database was used for analyzing in silico the relation of CA12 isoforms and molecular subtype.

Resultados

CA12kD was validated by decreased CA12 gene and protein levels. Gene and protein expression detected two CA12 isoforms (354aa and 343aa). TNF-α increased CA12 and NFκB 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 between CA12 expression and aggressive features and poor patient survival.

Conclusões

The results demonstrated that NF-kB may regulate CA12 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 in patient prognosis and survival. The chemo-sensitizing effect of CA12KD may represent an attractive strategy for adjuvant therapy for GBM.

Palavras-chave

Carbonic Anhydrase 12, Glioblastoma, Hypoxia

Financiador do resumo

FAPESP processo no. 2014/08899-5