Hypoxia Increases Gefitinib-Resistant Lung Cancer Stem Cells through the Activation of Insulin-Like Growth Factor 1 Receptor

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.

Interplay between receptor tyrosine kinases and hypoxia signaling in cancer

“…Interestingly, YC-1 was reported to downregulate HIF-1α and to suppress IGF1R signaling, thus overcoming gefitinib resistance in NSCLC cells expressing activating EGFR mutations (2.3.) [92]. Moreover, YC-1 was shown to block HIF-1α expression at the posttranscriptional level and consequently to inhibit transcription factor activity of HIF-1α in vitro [181].…”

“…However, due to the development of resistance mechanisms to these drugs, current treatment strategies experience challenging limitations [71]. Following treatment of NSCLC cells that harbor activating EGFR mutations (exon 19 deletion and L858R) by a selective EGFR inhibitor gefitinib, a small fraction of cells that express stem cell markers are reported to survive [92]. Cancer stem cells (CSCs) are maintained in a quiescent state, which is considered to render them more refractory to anticancer drugs, potentially enabling tumor relapse [93,94].…”

“…Cancer stem cells (CSCs) are maintained in a quiescent state, which is considered to render them more refractory to anticancer drugs, potentially enabling tumor relapse [93,94]. Hypoxic microenvironment was reported to be an important stem cell niche that favours CSCs survival [92,95]. For example, hypoxia was shown to increase the population of gefitinib-resistant CSCs by activation of IGF1R [92].…”

“…Hypoxic microenvironment was reported to be an important stem cell niche that favours CSCs survival [92,95]. For example, hypoxia was shown to increase the population of gefitinib-resistant CSCs by activation of IGF1R [92]. Dallas et al suggested that IGF1R plays a critical role in the persistence of CSCs, as expression of the stem cell markers CD133 and CD44 is linked to increased phosphorylated and total IGF1R in chemoresistant colon cancer cells [96].…”

“…Hypoxia-induced activation of IGF1R might be mediated by HIF-1α as the use of the nitric oxide-guanylate cyclase activator YC-1 [97], which downregulates HIF-1α, was shown to suppress receptor signaling. Targeting IGF1R signaling has been suggested to be a promising strategy for overcoming therapy resistance in tumors with activating EGFR mutatios induced by CSCs and hypoxia [92].…”