HAF drives the switch of HIF-1α to HIF-2α by activating the NF-κB pathway, leading to malignant behavior of T24 bladder cancer cells

Hypoxia is a characteristic feature of solid tumors, leading to malignant behavior. During this process, HIF family members (HIFs) and the NF-kappa B pathway are activated. In addition, the hypoxia-associated factor (HAF) is reported to participate in the regulation of HIFs. However, the precise relationship among HIFs, HAF and the NF-kappa B pathway in bladder cancer (BC) remains unknown. In the current investigation, T24 BC cells were exposed to hypoxia, or by plasmid transfection to overexpress HAF or RelA (P65) to demonstrate their roles. The results indicate that hypoxia leads to the elevation of HAF plus activation of the NF-kappa B pathway, accompanied by the switch of HIF-1 alpha to HIF-2 alpha, resulting in the enhanced ability of malignancy in T24 cells. In order to further demonstrate the significance of this switch, HIF-1 alpha and HIF-2 alpha were co-transfected into T24 cells with HIF-beta, respectively. The following results indicate that the T24(hif-2 alpha/beta) cells show enhanced ability of malignancy, accompanied by the maintenance of stem-cell markers, but the T24(hif-1 alpha/beta) cells show higher expression of metabolism-related genes. Boyden assays and wound-healing assays indicate the enhanced ability of malignancy for T24(hif-2 alpha/beta). Thus, we conclude that on the hypoxic microenvironment, the switching of HIF-1 alpha to HIF-2 alpha, which is driven by HAF through activating the NF-kappa B pathway, contributes to the malignancy of T24 cells, accompanied by the maintenance of stem-cell markers. This provides us an avenue for understanding the progression of bladder cancer.