Identifying pH independent hypoxia induced genes in human squamous cell carcinomas in vitro

Genes upregulated by low oxygen have been suggested as endogenous markers for tumor hypoxia. Yet, most of the genes investigated have shown inconsistent results, which have led to concerns about their ability to be true hypoxia makers. Previous studies have demonstrated that expression of hypoxia induced genes can be affected by extracellular pH (pH(e)).

Five different human cell lines (SiHa, FaDu(DD), UTSCC5, UTSCC14 and UTSCC15) were exposed to different oxygen concentrations and pH (7.5 or 6.3), and gene expression analyzed with microarray (Affymetrix - Human Genome U133 Plus 2.0 Array).

An analysis of two of the cell lines using SAM identified 461 probesets that were able to separate the four groups "Normal oxygen, normal pH", "Low oxygen, normal pH", "Normal oxygen, low pH" and "Low oxygen, low pH". From here it was possible to identify a fraction of probesets induced at low oxygen independent of pH in these two cell lines, this fraction included HIG2, NDRG1, PAI1 and RORA. Further verification by qPCR highlighted the necessity of using more cell lines to obtain a robust gene expression profiles. To specifically select pH independent hypoxia regulated genes across more cell lines, data for FaDu(DD), UTSCC5, UTSCC14 and UTSCC15 were analyzed to identify genes that were induced by hypoxia in each cell line, where the induction was not affected by low pH, and where the gene was not significantly induced by low pH alone. Each cell line had 65-122 probesets meeting these criteria. For genes to be considered as target genes (hypoxia inducible pH independent), genes had to be present in three of four cell lines.

The result is a robust hypoxia profile unaffected by pH across cell lines consisting of 27 genes. This study demonstrates a way to identify hypoxia markers by microarray, where other factors in the tumor microenvironment are taken into account.