AIM:To investigate whether hypoxia induces dedifferentiation of non-small cell lung cancer(NSCLC) cells and whether a hypoxia-inducible factor(HIF) inhibitor is able to suppress the process.METHODS:Human lung adenocarcinoma A549 cells and squamous carcinoma QG56 cells were cultured under normoxic(21%O_2) or hypoxic(4%or 1%O_2) conditions.The expression of the following genes were examined by reverse transcription-polymerase chain reaction,Western blotting and/or immunofluorescence:HIF-1α and HIF-2αsubunits;differentiation marker genes,namely surfactant protein C(SP-C)(type Ⅱ alveolar cell marker),CC10(type I alveolar cell marker) and aquaporin 5(AQP5)(Clara cell marker);and stem cell-associated genes,namely CD133,0CT4,and Musashi-1(MSI1).The tumor sphere-forming ability of the cells was evaluated by culturing them in serum-free growth factor-rich medium containing epidermal growth factor(EGF) and fibroblast growth factor(FGF).CD133 expression in hypoxic regions in A549 tumors was examined by double-immunostaining of tissue cryosections with an anti-2-nitroimidazole EF5 antibody and an anti-CD133 antibody.The metastatic ability of A549 cells was examined macroscopically and histologically after injecting them into the tail vein of immunocompromised mice.RESULTS:A549 cells primarily expressed SP-C,and QG56 cells expressed CC10 and AQP5.Exposure of A549 cells to hypoxia resulted in a marked downregulation of SP-C and upregulation of CD133,OCT4,and MSI1 in a time-dependent manner.Moreover,hypoxia mimetics,namely desferrioxamine and cobalt chloride,elicited similar effects.Ectopic expression of the constitutively active HIF-la subunit also caused the downregulation of SP-C and upregulation of CD133 and MSI1 but not OCT4,which is a direct target of HIF-2.Hypoxia enhanced the sphere-forming activity of A549 cells in serum-free medium containing EGF and FGF.Similarly,hypoxia downregulated the expression of CC10 and AQP5 genes and upregulated CD133,OCT4,and MSI1 genes in QG56 cells.TX-402(3-amino-2-quinoxalinecarbonitrile 1,4-dioxide),which is a small molecule inhibitor of the expression of HIF-1α and HIF-2αsubunits under hypoxic conditions,inhibited the upregulation of SP-C’and hypoxia-induced down-regulation of CD133,OCT4,and MSI1.Notably,TX-402 significantly suppressed the hypoxia-enhanced lung-colonizing ability of A549 cells.CONCLUSION:Hypoxia induces the de-differentiation of NSCLC cells into cancer stem cell-like cells,and HIF inhibitors are promising agents to prevent this process. AIM: To investigate whether hypoxia induces dedifferentiation of non-small cell lung cancer (NSCLC) cells and whether a hypoxia-inducible factor (HIF) inhibitor is able to suppress the process. METHODS: Human lung adenocarcinoma A549 cells and squamous carcinoma QG56 cells were cultured under normoxic (21% O 2) or hypoxic (4% or 1% O 2) conditions.The expression of the following genes were examined by reverse transcription-polymerase chain reaction, Western blotting and / or immunofluorescence: HIF-1α and HIF-2αsubunits ; differentiation marker genes, comprising surfactant type C (type II alveolar cell marker), CC10 (type I alveolar cell marker) and aquaporin 5 (AQP5) (Clara cell marker); and stem cell- associated genes CD133, OCT4, and Musashi-1 (MSI1). The tumor sphere-forming ability of the cells was evaluated by culturing in serum-free growth factor-rich medium containing epidermal growth factor (EGF) and fibroblast growth factor (FGF). CD133 expression in hypoxic regions in A549 tumors was e xamined by double-immunostaining of tissue cryosections with an anti-2-nitroimidazole EF5 antibody and an anti-CD133 antibody. The metastatic ability of A549 cells was examined macroscopically and histologically after injecting them into the tail vein of immunocompromised mice .RESULTS: A549 cells were expressed SP-C, and QG56 cells expressed CC10 and AQP5.Exposure of A549 cells to hypoxia resulted in a marked downregulation of SP-C and upregulation of CD133, OCT4, and MSI1 in a time-dependent manner. Moreover, hypoxia mimetics, both desferrioxamine and cobalt chloride, elicited similar effects. Ectopic expression of the constitutively active HIF-la subunit also caused the downregulation of SP-C and upregulation of CD133 and MSI1 but not OCT4, which is a direct target of HIF-2. Hypoxia enhanced the sphere-forming activity of A549 cells in serum-free medium containing EGF and FGF.Similarly, hypoxia downregulated the expression of CC10 and AQP5 genes and upregulated CD133, OCT4, and MSI1 genes in QG56 is a small molecule inhibitor of the expression of HIF-1α and HIF-2α subunits under hypoxic conditions, inhibited the upregulation of SP-C ’ and hypoxia-induced down-regulation of CD133, OCT4, and MSI1.Notably, TX-402 significantly suppressed the hypoxia-enhanced lung-colonizing ability of A549 cells.CONCLUSION: Hypoxia induces the de- differentiation of NSCLC cells into cancer stem cell- like cells, and HIF inhibitors are promising agents to prevent this process.