Gene Expression in Muscle-Invasive and Non-Muscle-Invasive Bladder Cancer Cells Exposed to Hypoxia

Introduction: Hypoxic cancers are radioresistant, but biomarkers based on expression of multiple genes can identify patients who will benefit from hypoxia modification. Most studies identifying relevant genes exposed cells in culture to 1% oxygen, which activates hypoxia-inducible factor (HIF). However, oxygen concentrations in hypoxic tumours are heterogeneous ranging from <0.1%. As lower oxygen levels would likely affect transcriptional responses, we aimed to investigate how gene selection at different oxygen levels affects the genes identified and their prognostic capability. Methods: Four MIBC (J82, T24, UMUC3, HT1376) and two non-MIBC (RT4, RT112) bladder cancer cell lines were exposed to varying oxygen levels (20%, 1%, 0.2% and 0.1% O2) for 24 h and were then harvested and frozen. RNA was extracted and transcriptomes analysed using Clariom S microarrays. Differences in gene expression were investigated. Prognostic and predictive significance of a published 24-gene signature was compared with one generated from genes identified at lower oxygen levels. Results: The number of upregulated genes increased with decreasing O2 level. The number of biological pathways involved also increased. Differences between cell lines dominated those due to hypoxia. Some genes were commonly upregulated in MIBC and NMIBC cells and others increased exclusively in either MIBC or NMIBC cells. The median expression of a published 24-gene bladder cancer hypoxia-associated signature increased with decreasing oxygen levels. Seventy-seven genes were upregulated in at least three cell lines by exposure to 0.1% O2. The median expression of the 77 genes was of borderline prognostic significance in the bladder cancer cohort in the TCGA (The Cancer Genome Atlas). Five of the seventy-seven genes upregulated by hypoxia were present in the twenty-four-gene bladder hypoxia signature. The median expression of the 5 genes demonstrated identical prognostication to the 24-gene signature but failed to predict benefit from hypoxia modification. Conclusions: The number of genes upregulated by exposure of bladder cancer cells to hypoxia increases as O2 level is decreased from 1% to 0.2% to 0.1%. Differential upregulation of gene expression by MIBC and NMIBC cells and the associated biological pathways may be useful in understanding the genetics of bladder cancer invasiveness. Based on a search of the literature, this is the first study that assessed the expression of genes in bladder cancer using three hypoxic concentration levels to identify biomarkers for disease progression and prognosis among differentially expressed bladder cancer genes.