Original Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Med Genet. Nov 27, 2013; 3(4): 41-54
Published online Nov 27, 2013. doi: 10.5496/wjmg.v3.i4.41
An inhibitor of HIF-α subunit expression suppresses hypoxia-induced dedifferentiation of human NSCLC into cancer stem cell-like cells
Miho Akimoto, Hideko Nagasawa, Hitoshi Hori, Yoshihiro Uto, Yoshio Honma, Keizo Takenaga
Miho Akimoto, Yoshio Honma, Keizo Takenaga, Department of Life Science, Shimane University Faculty of Medicine, Izumo 693-8501, Japan
Hideko Nagasawa, Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan
Hitoshi Hori, Yoshihiro Uto, Department of Life System, Institute of Technology and Science, Graduate School, University of Tokushima, Tokushima 770-8506, Japan
Author contributions: Takenaga K conceived and designed the experiments; Akimoto M, Takenaga K performed the experiments; Akimoto M, Takenaga K, Honma Y analyzed the data; Nagasawa H, Hori H, Uto Y contributed reagents and materials; Takenaga K, Akimoto M wrote the paper.
Supported by Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, (No. 18590280); the Foundation for the Promotion of Cancer Research in Japan; Grant-in-Aid for Japan Arteriosclerosis Research Foundation; and Shimane University “S-TAKUMI Medical Nanotechnology” Project
Correspondence to: Keizo Takenaga, PhD, Associated professor, Department of Life Science, Shimane University Faculty of Medicine, 89-1 Enya, Izumo 693-8501, Japan. biokeizo@med.shimane-u.ac.jp
Telephone: +81-853-202352 Fax: +81-853-202340
Received: June 17, 2013
Revised: August 29, 2013
Accepted: September 3, 2013
Published online: November 27, 2013
Processing time: 187 Days and 19.7 Hours
Abstract

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% O2) or hypoxic (4% or 1% O2) conditions. The expression of the following genes were examined by reverse transcription-polymerase chain reaction, Western blotting and/or immunofluorescence: HIF-1α and HIF-2α subunits; 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, OCT4, 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 down-regulation 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-1α 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-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.

Keywords: Non-small cell lung cancer; Tumor microenvironment; Hypoxia; Hypoxia-inducible factor; Differentiation; Cancer stem cells; Hypoxia-inducible factor inhibitor

Core tip: Hypoxia induces the de-differentiation of human non-small cell lung cancer cells into cancer stem cell-like cells, and TX-402, a small-molecule inhibitor of hypoxia-inducible factor (HIF)-1α and HIF-2α expression, suppresses this hypoxia-induced process and, importantly, the metastatic ability of the cells.