Abstract
Hypoxia, a major phenomenon in solid tumors, can promote the metastatic potential of tumor cells which is associated with chemoresistance and poor prognosis. It was reported that various angiogenesis factors including VEGF and HIF, were associated in cancer development and progression by hypoxia. In addition, both epithelial-mesenchymal transition (EMT) and cancer stem cells play an important role in malignant progression in many human tumors. We investigated the effect of hypoxic stress on the angiogenesis, EMT and stemness acquisition in lung cancer. Normal lung cell (BEAS-2B) and lung cancer cell lines (A549, H292, H226 and H460) were incubated in either normoxic or hypoxic (below 1% O2) conditions. For transcriptome analysis, mRNA of BEAS-2B and A549 cell lines were analyzed using next-generation sequencing (HiSeq 2500 system). For further validation, angiogenesis markers were analyzed by western blotting. EMT was assessed with western blotting, wound healing assay and Matrigel invasion assay, and stem cell characteristics were assessed with RT-PCR, immunostaining, soft agar colony formation assay, sphere formation assay and in vivo mice tumor model. Next-generation sequencing revealed significant changes in the expression of angiogenesis, EMT and stem cell markers after hypoxic stress. Among the angiogenesis markers, VEGF and HIF-2α were increased. EMT markers related in hypoxia showed decrease in E-cadherin and increase in fibronectin, vimentin, N-cadherin, α-SMA, Snail, Slug, ZEB1 and ZEB2. Stem cell markers such as CXCR4, Oct4 and Nanog were increased at least one lung cancer cell line in hypoxic condition compared with in normoxic condition. Functional assays for EMT and stemness acquisition indicated that hypoxic stress increased wound healing, Matrigel invasion, sphere formation and in vivo mice tumor formation. These results suggest that hypoxia induces angiogenesis markers expression which is associated with EMT and stemness acquisition in lung cancer.
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