Hypoxia increases gefitinib-resistant lung cancer stem cells through the activation of insulin-like growth factor 1 receptor

Akiko Murakami; Fumiyuki Takahashi; Fariz Nurwidya; Isao Kobayashi; Kunihiko Minakata; Muneaki Hashimoto; Takeshi Nara; Motoyasu Kato; Ken Tajima; Naoko Shimada; Shin-ichiro Iwakami; Mariko Moriyama; Hiroyuki Moriyama; Fumiaki Koizumi; Kazuhisa Takahashi

doi:10.1371/journal.pone.0086459

Hypoxia increases gefitinib-resistant lung cancer stem cells through the activation of insulin-like growth factor 1 receptor

PLoS One. 2014 Jan 28;9(1):e86459. doi: 10.1371/journal.pone.0086459. eCollection 2014.

Authors

Affiliations

¹ Department of Respiratory Medicine, Juntendo University, Graduate School of Medicine, Tokyo, Japan ; Research Institute for Diseases of Old Ages, Juntendo University, Graduate School of Medicine, Tokyo, Japan.
² Department of Molecular and Cellular Parasitology, Juntendo University, Graduate School of Medicine, Tokyo, Japan.
³ Juntendo University Shizuoka Hospital, Shizuoka, Japan.
⁴ Pharmaceutical Research and technology institute, Kinki University, School of Medicine, Osaka, Japan.
⁵ Shien-Lab, National Cancer Center Hospital, Tokyo, Japan.

Abstract

Accumulating evidence indicates that a small population of cancer stem cells (CSCs) is involved in intrinsic resistance to cancer treatment. The hypoxic microenvironment is an important stem cell niche that promotes the persistence of CSCs in tumors. Our aim here was to elucidate the role of hypoxia and CSCs in the resistance to gefitinib in non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9 and HCC827, which express the EGFR exon 19 deletion mutations, were exposed to high concentration of gefitinib under normoxic or hypoxic conditions. Seven days after gefitinib exposure, a small fraction of viable cells were detected, and these were referred to as "gefitinib-resistant persisters" (GRPs). CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1-all genes involved in stemness-were highly expressed in GRPs in PC9 and HCC827 cells, and PC9 GRPs exhibited a high potential for tumorigenicity in vivo. The expression of insulin-like growth factor 1 (IGF1) was also upregulated and IGF1 receptor (IGF1R) was activated on GRPs. Importantly, hypoxic exposure significantly increased sphere formation, reflecting the self-renewal capability, and the population of CD133- and Oct4-positive GRPs. Additionally, hypoxia upregulated IGF1 expression through hypoxia-inducible factor 1α (HIF1α), and markedly promoted the activation of IGF1R on GRPs. Knockdown of IGF1 expression significantly reduced phosphorylated IGF1R-expressing GRPs under hypoxic conditions. Finally, inhibition of HIF1α or IGF1R by specific inhibitors significantly decreased the population of CD133- and Oct4-positive GRPs, which were increased by hypoxia in PC9 and HCC827 cells. Collectively, these findings suggest that hypoxia increased the population of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC by activating IGF1R. Targeting the IGF1R pathway may be a promising strategy for overcoming gefitinib resistance in EGFR mutation-positive NSCLC induced by lung CSCs and microenvironment factors such as tumor hypoxia.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

AC133 Antigen
Animals
Antigens, CD / metabolism
Carcinogenesis / drug effects
Carcinogenesis / genetics
Carcinogenesis / pathology
Carcinoma, Non-Small-Cell Lung / drug therapy
Carcinoma, Non-Small-Cell Lung / genetics
Carcinoma, Non-Small-Cell Lung / metabolism*
Carcinoma, Non-Small-Cell Lung / pathology*
Cell Hypoxia / drug effects
Cell Hypoxia / genetics
Cell Line, Tumor
Cell Separation
Drug Resistance, Neoplasm / drug effects*
Drug Resistance, Neoplasm / genetics
ErbB Receptors / genetics
Gefitinib
Gene Expression Regulation, Neoplastic / drug effects
Gene Knockdown Techniques
Glycoproteins / metabolism
Humans
Hypoxia-Inducible Factor 1, alpha Subunit / antagonists & inhibitors
Hypoxia-Inducible Factor 1, alpha Subunit / metabolism
Insulin-Like Growth Factor I / genetics
Insulin-Like Growth Factor I / metabolism
Lung Neoplasms / drug therapy
Lung Neoplasms / genetics
Lung Neoplasms / metabolism
Lung Neoplasms / pathology*
Mice, Inbred NOD
Mutation / genetics
Neoplasm Transplantation
Neoplastic Stem Cells / metabolism
Neoplastic Stem Cells / pathology*
Octamer Transcription Factor-3 / metabolism
Peptides / metabolism
Quinazolines / pharmacology*
Quinazolines / therapeutic use
Receptor, IGF Type 1 / antagonists & inhibitors
Receptor, IGF Type 1 / genetics
Receptor, IGF Type 1 / metabolism*
Spheroids, Cellular / drug effects
Spheroids, Cellular / pathology
Up-Regulation / drug effects

Substances

AC133 Antigen
Antigens, CD
Glycoproteins
Hypoxia-Inducible Factor 1, alpha Subunit
Octamer Transcription Factor-3
POU5F1 protein, human
PROM1 protein, human
Peptides
Prom1 protein, mouse
Quinazolines
Insulin-Like Growth Factor I
ErbB Receptors
Receptor, IGF Type 1
Gefitinib

Grants and funding

This study was supported by Grant-in-Aids for Scientific Research No. 23591906 (Fumiyuki Takahashi) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.