Shabbir Rekaya, Quiles Conrado G, Lane Brian, Zeef Leo, Hoskin Peter J, Choudhury Ananya, West Catharine M L, Smith Tim A D
Division of Cancer Sciences, University of Manchester, Manchester M20 4GJ, UK.
Bioinformatics Core Facility, University of Manchester, Manchester M13 6PT, UK.
Cancers (Basel). 2025 Aug 11;17(16):2624. doi: 10.3390/cancers17162624.
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.
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% O) 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.
The number of upregulated genes increased with decreasing O 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% O. 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.
The number of genes upregulated by exposure of bladder cancer cells to hypoxia increases as O 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.
缺氧性癌症具有放射抗性，但基于多个基因表达的生物标志物可识别出能从缺氧调节中获益的患者。大多数鉴定相关基因的研究将培养中的细胞暴露于1%的氧气中，这会激活缺氧诱导因子（HIF）。然而，缺氧肿瘤中的氧浓度是异质性的，范围从<0.1%。由于更低的氧水平可能会影响转录反应，我们旨在研究在不同氧水平下进行基因选择如何影响所鉴定的基因及其预后能力。
将四种肌层浸润性膀胱癌（J82、T24、UMUC3、HT1376）和两种非肌层浸润性膀胱癌（RT4、RT112）细胞系暴露于不同氧水平（20%、1%、0.2%和0.1% O₂）24小时，然后收获并冷冻。提取RNA并使用Clariom S微阵列分析转录组。研究基因表达的差异。将已发表的24基因特征的预后和预测意义与从低氧水平鉴定的基因所生成的特征进行比较。
上调基因的数量随着氧水平的降低而增加。所涉及的生物途径的数量也增加。细胞系之间的差异主导了由缺氧引起的差异。一些基因在肌层浸润性膀胱癌和非肌层浸润性膀胱癌细胞中共同上调，而其他基因仅在肌层浸润性膀胱癌或非肌层浸润性膀胱癌细胞中增加。已发表的24基因膀胱癌缺氧相关特征的中位表达随着氧水平的降低而增加。通过暴露于0.1% O₂，至少在三个细胞系中有77个基因上调。这77个基因的中位表达在TCGA（癌症基因组图谱）的膀胱癌队列中具有临界预后意义。缺氧上调的77个基因中有5个存在于24基因膀胱癌缺氧特征中。这5个基因的中位表达显示出与24基因特征相同的预后，但未能预测从缺氧调节中获益。
随着氧水平从1%降至0.2%再降至0.1%，膀胱癌细胞暴露于缺氧时上调的基因数量增加。肌层浸润性膀胱癌和非肌层浸润性膀胱癌细胞对基因表达的差异上调以及相关的生物途径可能有助于理解膀胱癌侵袭性的遗传学。基于文献检索，这是第一项使用三种缺氧浓度水平评估膀胱癌中基因表达以鉴定差异表达的膀胱癌基因中疾病进展和预后生物标志物的研究。
