Extracellular Acidosis Elevates Carbonic Anhydrase Ix In Human Glioblastoma Cells Via Transcriptional Modulation That Does Not Depend On Hypoxia

Most solid tumors display extracellular acidosis, which only partially overlaps with hypoxia and induces distinct adaptive changes leading to aggressive phenotype. Although acidosis is mainly attributable to excessive production of lactic acid, it also involves carbonic anhydrase (CA) IX-mediated conversion of CO2 to an extracellular proton and a bicarbonate ion transported to cytoplasm. CA IX is predominantly expressed in tumors with poor prognosis and its transcription and activity are induced by hypoxia. Here we investigated whether low extracellular pH in absence of hypoxia can influence CA IX expression in cell lines derived from glioblastorna, a tumor type particularly linked with acidosis. Our data show that extracellular acidosis increased the level of CA IX protein, mRNA and the activity of minimal CA9 promoter that contains binding sites for HIF-1 and SP-1 transcription factors. Mutation within each of these two biding sites reduced the promoter activity, but did not eliminate the increase by acidosis. Transfection of HIF-1 alpha cDNA produced additive inducing effect with acidosis. Normoxic acidosis was accompanied by HIF-1 alpha protein accumulation and transiently increased phosphorylation of ERK1/2. Expression of a dominant-negative mutant of ERK2 reduced the CA9 promoter activity in both standard and acidic conditions. Similar result was obtained by inhibitors of MAPK and PI3K pathways, whose combination completely suppressed CA IX expression and abolished induction by acidosis. Altogether, our results suggest that acidosis increases the CA IX expression via a hypoxia-independent mechanism that operates through modulation of the basic CA9 transcriptional machinery.