EGFR Tyrosine Kinase Inhibitors Activate Autophagy as a Cytoprotective Response in Human Lung Cancer Cells

Department of Medical Oncology, Biomedical Research Center, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China.
PLoS ONE (Impact Factor: 3.53). 06/2011; 6(6):e18691. DOI: 10.1371/journal.pone.0018691
Source: PubMed

ABSTRACT Epidermal growth factor receptor tyrosine kinase inhibitors gefitinib and erlotinib have been widely used in patients with non-small-cell lung cancer. Unfortunately, the efficacy of EGFR-TKIs is limited because of natural and acquired resistance. As a novel cytoprotective mechanism for tumor cell to survive under unfavorable conditions, autophagy has been proposed to play a role in drug resistance of tumor cells. Whether autophagy can be activated by gefitinib or erlotinib and thereby impair the sensitivity of targeted therapy to lung cancer cells remains unknown. Here, we first report that gefitinib or erlotinib can induce a high level of autophagy, which was accompanied by the inhibition of the PI3K/Akt/mTOR signaling pathway. Moreover, cytotoxicity induced by gefitinib or erlotinib was greatly enhanced after autophagy inhibition by the pharmacological inhibitor chloroquine (CQ) and siRNAs targeting ATG5 and ATG7, the most important components for the formation of autophagosome. Interestingly, EGFR-TKIs can still induce cell autophagy even after EGFR expression was reduced by EGFR specific siRNAs. In conclusion, we found that autophagy can be activated by EGFR-TKIs in lung cancer cells and inhibition of autophagy augmented the growth inhibitory effect of EGFR-TKIs. Autophagy inhibition thus represents a promising approach to improve the efficacy of EGFR-TKIs in the treatment of patients with advanced non-small-cell lung cancer.

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    • "NSCLC Response to EGFR TKI Therapy May Involve Activation of Beclin 1 and Autophagy Our results have direct implications for the treatment of patients with active EGFR mutations and NSCLC. Based on previous data that EGFR TKIs induce autophagy (Gorzalczany et al., 2011; Han et al., 2011) and the belief that autophagy induction may lead to chemoresistance (Amaravadi et al., 2011), there are currently several NIH-sponsored clinical trials that combine autophagy inhibitory agents (e.g., chloroquine and hydroxychloroquine ) with EGFR inhibitors in the treatment of NSCLC. Earlier studies have either claimed that autophagy induction can enhance or limit the response to EGFR TKI therapy, but they suffer from experimental limitations, including (1) the use of NSCLC cell lines with WT EGFR or overexpressed EGFR rather than NSCLCs with TKI-sensitive mutations in EGFR (which are the tumors that clinically respond best to TKI therapy), (2) induction of autophagy with the use of high doses of TKIs, which are not relevant to those prescribed to patients, (3) the use of assays based on mitochondrial activity (which reflect both cell proliferation and death) rather than clonogenic survival assays to determine the effect of autophagy manipulation on NSCLC survival, and most importantly (4) the lack of in vivo studies that directly assess the effects of modulating autophagy on the response of NSCLC to TKIs. "
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    ABSTRACT: Cell surface growth factor receptors couple environmental cues to the regulation of cytoplasmic homeostatic processes, including autophagy, and aberrant activation of such receptors is a common feature of human malignancies. Here, we defined the molecular basis by which the epidermal growth factor receptor (EGFR) tyrosine kinase regulates autophagy. Active EGFR binds the autophagy protein Beclin 1, leading to its multisite tyrosine phosphorylation, enhanced binding to inhibitors, and decreased Beclin 1-associated VPS34 kinase activity. EGFR tyrosine kinase inhibitor (TKI) therapy disrupts Beclin 1 tyrosine phosphorylation and binding to its inhibitors and restores autophagy in non-small-cell lung carcinoma (NSCLC) cells with a TKI-sensitive EGFR mutation. In NSCLC tumor xenografts, the expression of a tyrosine phosphomimetic Beclin 1 mutant leads to reduced autophagy, enhanced tumor growth, tumor dedifferentiation, and resistance to TKI therapy. Thus, oncogenic receptor tyrosine kinases directly regulate the core autophagy machinery, which may contribute to tumor progression and chemoresistance.
    Cell 09/2013; 154(6):1269-84. DOI:10.1016/j.cell.2013.08.015 · 33.12 Impact Factor
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    • "Regarding the mechanisms involved in autophagy induction, recent studies have indicated that both inhibition of PI3K/Akt/mTOR signaling and translocation of p53 into the nucleus could trigger autophagy in cancer cell [32]. It has been reported that many drugs could induce autophagy through inhibition of PI3K/Akt/mTOR pathway [19] [20] [33]. Recently, more attention has been paid on the autophagy modulation function of the tumor suppressor p53 protein. "
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    ABSTRACT: PURPOSE: Erlotinib is a commonly used tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC). Autophagy is a catabolic process in response to stress and deprivation of nutrients. This study aims to investigate whether autophagy confers acquired resistance to erlotinib treatment in NSCLC. METHODS: Four NSCLC cell lines (HCC827, HCC4006, H358 and H1975) with different epidermal growth factor receptor (EGFR) mutation status (exon 19 deletion, exon 19 deletion, wild-type and L858R/T790M respectively) were selected. MTT assay, crystal violet staining and Annexin-V assay were performed to determine cell viability and apoptosis. Autophagic proteins were detected by Western blot. Acidic vesicular organelle (AVO) formation was determined by acridine orange staining. Autophagy inhibitor (chloroquine) and RNA interference were used to demonstrate the biological effect of erlotinib-induced autophagy. RESULTS: In line with EGFR mutation status, it was shown that both HCC827 and HCC4006 cells were sensitive to erlotinib, while H358 and H1975 cell lines were resistant. Erlotinib treatment at clinically relevant concentrations induced autophagy (increased LC3II expression, Atg-5/Atg12 conjugation, formation of AVO and p62 degradation) in sensitive NSCLC cell lines, via p53 nuclear translocation, AMPK activation and mTOR suppression. Addition of chloroquine, as an autophagy inhibitor, enhanced erlotinib sensitivity in sensitive cells. Similarly, silencing of Atg5 or Beclin-1 significantly increased sensitivity to erlotinib in both sensitive cell lines. In contrast, there was no induction of autophagy in resistant H358 and H1975 cell lines upon erlotinib exposure. CONCLUSIONS: Erlotinib can induce both apoptosis and autophagy in sensitive NSCLC cell lines with activating EGFR mutation (exon 19 del). Inhibition of autophagy can further enhance sensitivity to erlotinib in EGFR-mutated NSCLC, suggesting that autophagy may serve as a protective mechanism.
    Lung cancer (Amsterdam, Netherlands) 06/2013; 81(3). DOI:10.1016/j.lungcan.2013.05.012 · 3.74 Impact Factor
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    ABSTRACT: Gefitinib, a small molecule inhibitor of the epidermal growth factor receptor tyrosine kinase, has been shown to induce autophagy as well as apoptosis in tumor cells. Yet, how to use autophagy and apoptosis to improve therapeutic efficacy of this drug against cancer remains to be explored. We reported here that MK-2206, a potent allosteric Akt inhibitor currently in phase I trials in patients with solid tumors, could reinforce the cytocidal effect of gefitinib against glioma. We found that cotreatment with gefitinib and MK-2206 increased the cytotoxicity of this growth factor receptor inhibitor in the glioma cells, and the CompuSyn synergism/antagonism analysis showed that MK-2206 acted synergistically with gefitinib. The benefit of the combinatorial treatment was also shown in an intracranial glioma mouse model. In the presence of MK-2206, there was a significant increase in apoptosis in glioma cells treated with gefitinib. MK-2206 also augmented the autophagy-inducing effect of gefitinib, as evidenced by increased levels of the autophagy marker, LC3-II. Inhibition of autophagy by silencing of the key autophagy gene, beclin 1 or 3-MA, further increased the cytotoxicity of this combinatorial treatment, suggesting that autophagy induced by these agents plays a cytoprotective role. Notably, at 48 hours following the combinatorial treatment, the level of LC3-II began to decrease but Bim was significantly elevated, suggesting a switch from autophagy to apoptosis. On the basis of the synergistic effect of MK-2206 on gefitinib observed in this study, the combination of these two drugs may be utilized as a new therapeutic regimen for malignant glioma.
    Molecular Cancer Therapeutics 11/2011; 11(1):154-64. DOI:10.1158/1535-7163.MCT-11-0606 · 6.11 Impact Factor
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