Hypoxia increases gefitinib-resistant lung cancer stem cells through the activation of insulin-like growth factor 1 receptor.
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.
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.
Akiko
A
Murakami
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.
Fumiyuki
F
Takahashi
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.
Fariz
F
Nurwidya
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.
Isao
I
Kobayashi
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.
Kunihiko
K
Minakata
Department of Molecular and Cellular Parasitology, Juntendo University, Graduate School of Medicine, Tokyo, Japan.
Muneaki
M
Hashimoto
Department of Molecular and Cellular Parasitology, Juntendo University, Graduate School of Medicine, Tokyo, Japan.
Takeshi
T
Nara
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.
Motoyasu
M
Kato
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.
Ken
K
Tajima
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.
Naoko
N
Shimada
Juntendo University Shizuoka Hospital, Shizuoka, Japan.
Shin-ichiro
S
Iwakami
Pharmaceutical Research and technology institute, Kinki University, School of Medicine, Osaka, Japan.
Mariko
M
Moriyama
Pharmaceutical Research and technology institute, Kinki University, School of Medicine, Osaka, Japan.
Hiroyuki
H
Moriyama
Shien-Lab, National Cancer Center Hospital, Tokyo, Japan.
Fumiaki
F
Koizumi
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.
Kazuhisa
K
Takahashi
D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D063646:Carcinogenesis / Q000187:drug effects / Q000235:genetics / Q000473:pathology; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D015687:Cell Hypoxia / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D002469:Cell Separation; D019008:Drug Resistance, Neoplasm / Q000187:drug effects* / Q000235:genetics; D066246:ErbB Receptors / Q000235:genetics; D000077156:Gefitinib; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D016688:Mice, Inbred NOD; D009154:Mutation / Q000235:genetics; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology*; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology* / Q000627:therapeutic use; D017526:Receptor, IGF Type 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018874:Spheroids, Cellular / Q000187:drug effects / Q000473:pathology; D015854:Up-Regulation / Q000187:drug effects
D000071916:AC133 Antigen; D015703:Antigens, CD; D006023:Glycoproteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse; D011799:Quinazolines; D007334:Insulin-Like Growth Factor I; D066246:ErbB Receptors; D017526:Receptor, IGF Type 1; D000077156:Gefitinib