YC-1 potentiates the antitumor activity of gefitinib by inhibiting HIF-1α and promoting the endocytic trafficking and degradation of EGFR in gefitinib-resistant non-small-cell lung cancer cells

Abstract

The tyrosine kinase inhibitor (TKI) gefitinib exerts good therapeutic effect on NSCLC patients with sensitive EGFR-activating mutations. However, most patients ultimately relapse due to the development of drug resistance after 6–12 months of treatment. Here, we showed that a HIF-1α inhibitor, YC-1, potentiated the antitumor efficacy of gefitinib by promoting EGFR degradation in a panel of human NSCLC cells with wild-type or mutant EGFRs. YC-1 alone had little effect on NSCLC cell survival but significantly enhanced the antigrowth and proapoptotic effects of gefitinib. In insensitive NSCLC cell lines, gefitinib efficiently inhibited the phosphorylation of EGFR but not the downstream signaling of ERK, AKT and STAT3; however, when combined with YC-1 treatment, these signaling pathways were strongly impaired. Gefitinib treatment induced EGFR arrest in the early endosome, and YC-1 treatment promoted delayed EGFR transport into the late endosome as well as receptor degradation. Moreover, the YC-1-induced reduction of HIF-1α protein was associated with the enhancement of EGFR degradation. HIF-1α knockdown promoted EGFR degradation, showing synergistic antigrowth and proapoptotic effects similar to those of the gefitinib and YC-1 combination treatment in NSCLC cells. Our findings provide a novel combination treatment strategy with gefitinib and YC-1 to extend the usage of gefitinib and overcome gefitinib resistance in NSCLC patients.

Introduction

Gefitinib is a well-known first-generation EGFR tyrosine kinase inhibitor (EGFR-TKI) that exhibits good antitumor activity in molecularly selected NSCLC patients with L858R/del19 mutations of EGFR (Rusch et al., 1997; Brabender et al., 2001; Jemal et al., 2011; Ferlay et al., 2013). However, despite the initial response and excellent disease control with EGFR-TKI therapy, acquired resistance is inevitable and raises an immense challenge in NSCLC therapy (Dancey, 2004; Perez-Soler et al., 2004; Pao and Chmielecki, 2010; Enting and Spicer, 2012; Rosell and Karachaliou, 2016). Among the mechanisms of acquired resistance, the emergence of a second substitution, T790M in exon 20 of EGFR, was responsible for approximately 50–60% of gefitinib-resistant cases (Sequist et al., 2011). The third-generation TKI osimertinib was approved by the FDA for its enhanced ability to bind and inhibit EGFR/T790M; however, new acquired resistance has been developed in a subset of patients with mutations, such as C797S (Thress et al., 2015). Moreover, many NSCLC patients who overexpress wild-type EGFR usually respond poorly to EGFR-TKI treatment (Stinchcom, 2016). This evidence highlights that innovative strategies are urgently needed to overcome both primary and acquired resistance to EGFR-TKIs in NSCLC patients.

EGFR-TKIs can efficiently inhibit the tyrosine kinase activity of EGFR, thus suppressing the unrestrained proliferation of cancer cells. However, accumulating evidence suggests that EGFR has many other functions beyond kinase activity that have been known to play an essential role in cancer pathology (Coker et al., 1994; Ewald et al., 2003). Many EGFR-dependent NSCLC patients display innate or acquired resistance to EGFR-TKI treatment despite the efficient inhibition of tyrosine kinase activity (Zhang et al., 2008; Tan et al., 2016). Studies have shown that EGFR-TKI treatment induced cellular stress and provoked noncanonical pathways of EGFR trafficking and signaling, which provides cancer cells with a survival advantage (Filosto et al., 2011; Orcutt et al., 2011; Filomeni, 2015; Zou et al., 2013). After long-term TKI treatment, the intracellular signaling of EGFR was prone to be shifted from its kinase-dependent to kinase-independent pattern, which was involved in the acquisition of TKI resistance (Engelman and Janne, 2008). Tan et al. reported that EGFR-TKIs elicited endosomal accumulation of inactive EGFR to initiate autophagy in a kinase-independent fashion, providing a survival advantage and facilitating TKI resistance in NSCLCs with wild-type EGFR (Tan et al., 2015). Menard et al. showed that the inhibition of kinase-independent EGFR signaling overcame TKI resistance in NSCLC cells with different EGFR mutations (Ménard et al., 2018). Inspired by these observations, we believe that cotargeting EGFR kinase-dependent and -independent functions may hold new promise for treating EGFR-TKI resistant cancers.

YC-1, 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole, was initially described as an activator of soluble guanylyl cyclase (sGC), prompting antiplatelet aggregation and vascular relaxation (Galle et al., 1999). More recent studies revealed the ability of YC-1 to inhibit tumor growth, suppress angiogenesis and enhance antitumor effects of radiation; these abilities have been associated with its activity to inhibit hypoxia-inducible factor 1α (HIF-1α) (Chun et al., 2004; Chen et al., 2008).

Recently, many studies showed that YC-1 could increase the sensitivity to cisplatin in cisplatin-resistant cancer cells and overcome the radioresistance of hypoxic cancer cells (Moon et al., 2009; Kong et al., 2014; Lee et al., 2017b; Tuttle et al., 2017). However, the detailed mechanism of YC-1 antitumor activity is largely unknown. In this study, we found for the first time that YC-1 exhibited an unexpected ability to sensitize gefitinib-resistant NSCLC cells to gefitinib treatment. YC-1 was capable of regulating the trafficking and degradation of EGFR protein by reducing HIF-1α protein levels and downregulating kinase-independent EGFR signaling in NSCLC cells.