Jin Kyung Rho

Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer

Human non–small cell lung cancers (NSCLCs) with activating mutations in EGFR frequently respond to treatment with EGFR-targeted tyrosine kinase inhibitors (TKIs), such as erlotinib, but responses are not durable, as tumors acquire resistance. Secondary mutations in EGFR (such as T790M) or upregulation of the MET kinase are found in over 50% of resistant tumors. Here, we report increased activation of AXL and evidence for epithelial-to-mesenchymal transition (EMT) in multiple in vitro and in vivo EGFR-mutant lung cancer models with acquired resistance to erlotinib in the absence of the EGFR p.Thr790Met alteration or MET activation. Genetic or pharmacological inhibition of AXL restored sensitivity to erlotinib in these tumor models. Increased expression of AXL and, in some cases, of its ligand GAS6 was found in EGFR-mutant lung cancers obtained from individuals with acquired resistance to TKIs. These data identify AXL as a promising therapeutic target whose inhibition could prevent or overcome acquired resistance to EGFR TKIs in individuals with EGFR-mutant lung cancer.

EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers

The involvement of the MET oncogene in de novo and acquired resistance of non-small cell lung cancers (NSCLC) to tyrosine kinase inhibitors (TKIs) has been reported, but the precise mechanism by which MET overexpression contributes to TKI-resistant NSCLC remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression and their dysregulation has been implicated in tumorigenesis. To understand the role of microRNAs in TKI-resistant NSCLC, we examined TK receptor-mediated microRNA changes. Here we report that miR-30b/c and miR-221/222, modulated by both EGF and MET receptors, and miR-103, -203, controlled only by MET, play important roles in gefitinib-induced apoptosis and epithelial-mesenchymal transition (EMT) of NSCLC cells, in vitro and in vivo, by inhibiting the expression of Bim, APAF-1, PKC-ε and SRC genes. The finding suggests that modulation of specific microRNAs may provide a therapeutic approach for future treatment of NSCLC.

Epithelial to mesenchymal transition derived from repeated exposure to gefitinib determines the sensitivity to EGFR inhibitors in A549, a non-small cell lung cancer cell line

Epithelial to mesenchymal transition (EMT) has been reported to be related with reduced sensitivity to EGFR tyrosine kinase (EGFR-TK) inhibitors. We performed this study to investigate whether this phenomenon would play a role in acquired resistance to gefitinib. In this study, we established a gefitinib-resistant subline (A549/GR), which was derived from the parental A549 cell line by chronic, repeated exposure to gefitinib. Compared with the A549 cells, the A549/GR cells were approximately 7.7-fold more resistant to gefitinib and they showed the cross-resistance against other EGFR-TK inhibitors, including CL-387,758, erlotinib and ZD6478. Phenotypic changes such as a spindle-cell shape and increased pseudopodia formation suggesting EMT was present in the A549/GR cells. These changes were accompanied by a decrease of E-cadherin and an increase of vimentin, which is a mesenchymal marker. In addition, the ability of invasion and migration was increased in the A549/GR cells. TGF-beta1 treatment for 72 h also induced EMT in the A549 cells and this transition led to resistance to gefitinib. Conversely, this was reversed through the removal of TGF-beta1. In conclusion, induction of EMT may contribute to the decreased efficacy of therapy in primary and acquired resistance to gefitinib.

Erratum: EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers (Nat. Med. (2012) 18 (74-82))

The involvement of the MET oncogene in de novo and acquired resistance of non-small cell lung cancers (NSCLC) to tyrosine kinase inhibitors (TKIs) has been reported, but the precise mechanism by which MET overexpression contributes to TKI-resistant NSCLC remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression and their dysregulation has been implicated in tumorigenesis. To understand the role of microRNAs in TKI-resistant NSCLC, we examined TK receptor-mediated microRNA changes. Here we report that miR-30b/c and miR-221/222, modulated by both EGF and MET receptors, and miR-103, -203, controlled only by MET, play important roles in gefitinib-induced apoptosis and epithelial-mesenchymal transition (EMT) of NSCLC cells, in vitro and in vivo, by inhibiting the expression of Bim, APAF-1, PKC-ε and SRC genes. The finding suggests that modulation of specific microRNAs may provide a therapeutic approach for future treatment of NSCLC.

Upregulation of CXCR4 is functionally crucial for maintenance of stemness in drug-resistant non-small cell lung cancer cells.

The hypothesis of cancer stem cells has been proposed to explain the therapeutic failure in a variety of cancers including lung cancers. Previously, we demonstrated acquisition of epithelial–mesenchymal transition, a feature highly reminiscent of cancer stem-like cells, in gefitinib-resistant A549 cells (A549/GR). Here, we show that A549/GR cells contain a high proportion of CXCR4+ cells that are responsible for having high potential of self-renewal activity in vitro and tumorigenicity in vivo. A549/GR cells exhibited strong sphere-forming activity and high CXCR4 expression and SDF-1α secretion compared with parent cells. Pharmacological inhibition (AMD3100) and/or siRNA transfection targeting CXCR4 significantly suppressed sphere-forming activity in A549 and A549/GR cells, and in various non-small cell lung cancer (NSCLC) cell lines. A549/GR cells showed enhanced Akt, mTOR and STAT3 (Y705) phosphorylation. Pharmacological inhibition of phosphatidyl inositol 3-kinase or transfection with wild-type PTEN suppressed phosphorylation of Akt, mTOR and STAT3 (Y705), sphere formation, and CXCR4 expression in A549/GR cells, whereas mutant PTEN enhanced these events. Inhibition of STAT3 by WP1066 or siSTAT3 significantly suppressed the sphere formation, but not CXCR4 expression, indicating that STAT3 is a downstream effector of CXCR4-mediated signaling. FACS-sorted CXCR4+ A549/GR cells formed many large spheres, had self-renewal capacity, demonstrated radiation resistance in vitro and exhibited stronger tumorigenic potential in vivo than CXCR4− cells. Lentiviral-transduction of CXCR4 enhanced sphere formation and tumorigenicity in H460 and A549 cells, whereas introduction of siCXCR4 suppressed these activities in A549/GR cells. Our data indicate that CXCR4+ NSCLC cells are strong candidates for tumorigenic stem-like cancer cells that maintain stemness through a CXCR4-medated STAT3 pathway and provide a potential therapeutic target for eliminating these malignant cells in NSCLC.

Clinical and molecular evidences of epithelial to mesenchymal transition in acquired resistance to EGFR-TKIs.

BACKGROUND Epithelial-to-mesenchymal transition (EMT), which was related with an acquired resistance to gefitinib, was found in the A549 lung cancer cell line. However, the clinical feasibility of this finding is still questionable. Here, we investigated whether EMT could be detected in a more clinically suitable situation using patients tumor and cells with deletion mutation on exon 19 of EGFR gene. METHODS HCC827 cell line was used to establish the subline resistant to EGFR-TKIs. The induction of EMT was analyzed by immunostainings and Western blots in resistant cells and biopsied tissue from a patient with acquired resistance to erlotinib. Migration and invasion assay was performed to characterize the resistant cells. EMT-related genes expression was evaluated by cDNA microarray. Phospho-receptor tyrosine kinase array analysis was carried out to find bypass activating signals such as MET. RESULTS We found that EMT developed in a lung cancer patient who had an acquired resistance to erlotinib while there were no known resistant mechanisms such as T790M and MET amplification. CL-387,785-resistant cells (HCC827/CLR) were obtained by long-term exposure to increasing concentrations of CL-387,785 (an irreversible EGFR-TKI). The morphological and molecular maker changes compatible with EMT were also found in HCC827/CLR cells. However, there were also no secondary T790M mutation and MET amplification. Furthermore, the activity of most of tested RTKs including receptor HER family was decreased suggesting that there was no bypass activating signal leading to resistance. These cells showed an enhanced capability for migration (∼1.6-fold) and invasion (∼2.8-fold). CONCLUSION EMT should be considered as one of possible mechanisms for the acquired resistance to EGFR-TKIs in lung cancer cells.

Epithelial-mesenchymal transition leads to crizotinib resistance in H2228 lung cancer cells with EML4-ALK translocation.

Epithelial‐mesenchymal transition (EMT) is associated with reduced sensitivity to many chemotherapeutic drugs, including EGFR tyrosine kinase inhibitors. Here, we investigated if this reduced sensitivity also contributes to resistance to crizotinib, an ALK inhibitor of lung cancer that exhibits the EML4‐ALK translocation. We established a crizotinib‐resistant subline (H2228/CR), which was derived from the parental H2228 cell line by long‐term exposure to increasing concentrations of crizotinib. Characteristics associated with EMT, including morphology, EMT marker proteins, and cellular mobility, were analyzed. Compared with H2228 cells, the growth of H2228/CR cells was independent of EML4‐ALK, and H2228/CR cells showed cross‐resistance to TAE‐684 (a second‐generation ALK inhibitor). Phenotypic changes to the spindle‐cell shape were noted in H2228/CR cells, which were accompanied by a decrease in E‐cadherin and increase in vimentin and AXL. In addition, H2228/CR cells showed increased secretion and expression of TGF‐β1. Invasion and migration capabilities were dramatically increased in H2228/CR cells. Applying TGF‐β1 treatment to parental H2228 cells for 72 h induced reversible EMT, leading to crizotinib resistance, but this was reversed by the removal of TGF‐β1. Suppression of vimentin in H2228/CR cells by siRNA treatment restored sensitivity to crizotinib. Furthermore, these resistant cells remained highly sensitive to the Hsp90 inhibitors, similar to the parental H2228 cells. In conclusion, we suggest EMT is possibly involved in acquired resistance to crizotinib, and that HSP90 inhibitors could be a promising option for the treatment of EMT.

MET and AXL Inhibitor NPS-1034 Exerts Efficacy against Lung Cancer Cells Resistant to EGFR Kinase Inhibitors Because of MET or AXL Activation

In non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations, acquired resistance to EGFR-tyrosine kinase inhibitors (EGFR-TKI) can occur through a generation of bypass signals such as MET or AXL activation. In this study, we investigated the antitumor activity of NPS-1034, a newly developed drug that targets both MET and AXL, in NSCLC cells with acquired resistance to gefitinib or erlotinib (HCC827/GR and HCC827/ER, respectively). Characterization of H820 cells and evaluation of NPS-1034 efficacy in these cells were also performed. The resistance of HCC827/GR was mediated by MET activation, whereas AXL activation led to resistance in HCC827/ER. The combination of gefitinib or erlotinib with NPS-1034 synergistically inhibited cell proliferation and induced cell death in both resistant cell lines. Accordingly, suppression of Akt was noted only in the presence of treatment with both drugs. NPS-1034 was also effective in xenograft mouse models of HCC827/GR. Although the H820 cell line was reported previously to have T790M and MET amplification, we discovered that AXL was also activated in this cell line. There were no antitumor effects of siRNA or inhibitors specific for EGFR or MET, whereas combined treatment with AXL siRNA or NPS-1034 and EGFR-TKIs controlled H820 cells, suggesting that AXL is the main signal responsible for resistance. In addition, NPS-1034 inhibited cell proliferation as well as ROS1 activity in HCC78 cells with ROS1 rearrangement. Our results establish the efficacy of NPS-1034 in NSCLC cells rendered resistant to EGFR-TKIs because of MET or AXL activation or ROS1 rearrangement.

p53 Enhances Gefitinib-Induced Growth Inhibition and Apoptosis by Regulation of Fas in Non–Small Cell Lung Cancer

Treatment with gefitinib, a specific inhibitor of epidermal growth factor receptor tyrosine kinase (EGFR-TK), has resulted in dramatic responses in some patients with non-small cell lung cancer (NSCLC). Most patients who respond to gefitinib have EGFR-TK mutations; however, >10% of patients with EGFR-TK mutations do not respond. Similarly, some patients without EGFR-TK mutations respond to this drug, suggesting that other factors determine sensitivity to gefitinib. Aberrations of the tumor suppressor gene p53 are frequently associated with drug resistance. In this study, we investigated the role of p53 in growth-inhibitory and apoptotic effects of gefitinib in the human NSCLC cell lines NCI-H1299 and A549, which have no EGFR-TK mutations. NCI-H1299 cells, which had a p53-null genotype, were more resistant to gefitinib compared with A549 cells, which were wild-type p53 (IC(50), 40 micromol/L in NCI-H1299 and 5 micromol/L in A549). Treatment of A549 with gefitinib resulted in the translocation of p53 from cytosol to nucleus and the up-regulation of Fas, which was localized to the plasma membrane. In the stable H1299 cell line with tetracycline-inducible p53 expression, induced p53 enhanced growth inhibition and apoptosis by gefitinib through the up-regulation of Fas and restoration of caspase activation. A caspase inhibitor, Z-VAD-fmk, reduced these effects. Conversely, inhibition of p53 using antisense oligonucleotide in A549 caused a significant decrease in apoptosis by gefitinib and down-regulation of Fas under the same conditions. In conclusion, p53 may play a role in determining gefitinib sensitivity by regulating Fas expression in NSCLC.

Combined inhibition of IGFR enhances the effects of gefitinib in H1650: a lung cancer cell line with EGFR mutation and primary resistance to EGFR-TK inhibitors

PurposeH1650 non-small cell lung cancer (NSCLC) cells display primary resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) although they have a deletion mutation on exon 19 of the EGFR gene. We investigated the effect of inhibition of both insulin-like growth factor receptor (IGFR) and EGFR signaling considering that IGFR signaling pathway has been implicated in the development and progression with therapeutic resistance of various cancers including lung cancer.MethodsThree human NSCLC cell lines with an EGFR mutation of PC-9, HCC827 and H1650 were used for experiment. Cell viability and proliferative activity were assessed by MTT and three-dimensional culture assay. Combination index was obtained by CalcuSyn software. The change of EGFR- and IGFR-related signals was evaluated by western blots.ResultsH1650 cells were 1,000 times more resistant to gefitinib and erlotinib than HCC827 and PC-9 cells possessing the same EGFR mutation. Phosphatase and tensin homolog loss and sustained phosphorylation of Akt in spite of treatment with gefitinib were evident only in H1650 cells. Interestingly, IGFR phosphorylation was decreased by gefitinib in HCC827 and PC-9 cells while being maintained in H1650 cells. Combined treatment with the IGFR inhibitors α-IR3 and AG1024 enhanced gefitinib-induced growth inhibition and apoptosis, and down-regulated phosphorylation of Akt, EGFR and IGFR.ConclusionCombined inhibition of IGFR signaling enhances the growth inhibitory and apoptosis-inducing effects of gefitinib, suggesting that this approach could be useful to overcome the primary resistance to EGFR-TKIs in lung cancer.