Targeting SHP2 for EGFR inhibitor resistant non-small cell lung carcinoma

Targeted therapy with inhibitors of epidermal growth factor receptor (EGFR) has produced a noticeable benefit to non-small cell lung cancer (NSCLC) patients whose tumors carry activating mutations (e.g. L858R) in EGFR. Unfortunately, these patients develop drug resistance after treatment, due to acquired secondary gatekeeper mutations in EGFR (e.g. T790M). Given the critical role of SHP2 in growth factor receptor signaling, we sought to determine whether targeting SHP2 could have therapeutic value for EGFR inhibitor resistant NSCLC. We show that SHP2 is required for EGF-stimulated ERK1/2 phosphorylation and proliferation in EGFR inhibitor resistant NSCLC cell line H1975, which harbors the EGFR T790M/L858R double-mutant. We demonstrate that treatment of H1975 cells with II-B08, a specific SHP2 inhibitor, phenocopies the observed growth inhibition and reduced ERK1/2 activation seen in cells treated with SHP2 siRNA. Importantly, we also find that II-B08 exhibits marked anti-tumor activity in H1975 xenograft mice. Finally, we observe that combined inhibition of SHP2 and PI3K impairs both the ERK1/2 and PI3K/AKT signaling axes and produces significantly greater effects on repressing H1975 cell growth than inhibition of either protein individually. Collectively, these results suggest that targeting SHP2 may represent an effective strategy for treatment of EGFR inhibitor resistant NSCLCs.     

Gefitinib Inhibits Invasive Phenotype and Epithelial-Mesenchymal Transition in Drug-Resistant NSCLC Cells with MET Amplification

Despite the initial response, all patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). The EGFR-T790M secondary mutation is responsible for half of acquired resistance cases, while MET amplification has been associated with acquired resistance in about 5-15% of NSCLCs. Clinical findings indicate the retained addiction of resistant tumors on EGFR signaling. Therefore, we evaluated the molecular mechanisms supporting the therapeutic potential of gefitinib maintenance in the HCC827 GR5 NSCLC cell line harbouring MET amplification as acquired resistance mechanism. We demonstrated that resistant cells can proliferate and survive regardless of the presence of gefitinib, whereas the absence of the drug significantly enhanced cell migration and invasion. Moreover, the continuous exposure to gefitinib prevented the epithelial-mesenchymal transition (EMT) with increased E-cadherin expression and down-regulation of vimentin and N-cadherin. Importantly, the inhibition of cellular migration was correlated with the suppression of EGFR-dependent Src, STAT5 and p38 signaling as assessed by a specific kinase array, western blot analysis and silencing functional studies. On the contrary, the lack of effect of gefitinib on EGFR phosphorylation in the H1975 cells (EGFR-T790M) correlated with the absence of effects on cell migration and invasion. In conclusion, our findings suggest that certain EGFR-mutated patients may still benefit from a second-line therapy including gefitinib based on the specific mechanism underlying tumor cell resistance.     

mTOR inhibitors radiosensitize PTEN‐deficient non‐small‐cell lung cancer cells harboring an EGFR activating mutation by inducing autophagy

Clinical resistance to gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), in patients with lung cancer has been linked to acquisition of the T790M resistance mutation in activated EGFR or amplification of MET. Phosphatase and tensin homolog (PTEN) loss has been recently reported as a gefitinib resistance mechanism in lung cancer. The aim of this study was to evaluate the efficacy of radiotherapy in non‐small‐cell lung cancer (NSCLC) with acquired gefitinib resistance caused by PTEN deficiency to suggest radiotherapy as an alternative to EGFR TKIs. PTEN deficient‐mediated gefitinib resistance was generated in HCC827 cells, an EGFR TKI sensitive NSCLC cell line, by PTEN knockdown with a lentiviral vector expressing short hairpin RNA‐targeting PTEN. The impact of PTEN knockdown on sensitivity to radiation in the presence or absence of PTEN downstream signaling inhibitors was investigated. PTEN knockdown conferred acquired resistance not only to gefitinib but also to radiation on HCC827 cells. mTOR inhibitors alone failed to reduce HCC827 cell viability, regardless of PTEN expression, but ameliorated PTEN knockdown‐induced radioresistance. PTEN knockdown‐mediated radioresistance was accompanied by repression of radiation‐induced cytotoxic autophagy, and treatment with mTOR inhibitors released the repression of cytotoxic autophagy to overcome PTEN knockdown‐induced radioresistance in HCC827 cells. These results suggest that inhibiting mTOR signaling could be an effective strategy to radiosensitize NSCLC harboring the EGFR activating mutation that acquires resistance to both TKIs and radiotherapy due to PTEN loss or inactivation mutations. J. Cell. Biochem. 114: 1248–1256, 2013. © 2012 Wiley Periodicals, Inc.     

Recent Development of the Second and Third Generation Irreversible Epidermal Growth Factor Receptor Inhibitors

Recent reports suggested that essential directions for new lung cancer, breast carcinoma therapies, as well as the roomier realm of targeted cancer therapies were provided through targeting the epidermal growth factor receptor (EGFR). Patients who carrying non‐small cell lung carcinoma (NSCLC) with activating mutations in EGFR initially respond well to the EGFR inhibitors erlotinib and gefitinib, which were located the active site of the EGFR kinase and designed to act as competitive inhibitors of combining with the ATP. However, patients who were treated with the erlotinib and gefitinib will relapse because of the emergence of drug‐resistant mutations, with T790M mutations accounting for approximately 60% of all resistance. In order to overcome drug resistance, Pharmaceutical chemistry experts recently devoted great endeavors to the development of second‐generation irreversible selective inhibitors which covalently modify Cys797 or Cys773 at the ATP binding cleft. Nevertheless, these inhibitors have not reached ideal effect of experts in patients with T790M positive mutation and apparently because of the dose‐limiting toxicities associated with inhibition of wild type EGFR. A novel class of ‘third generation’ EGFR TKIs have been developed that is sensitising and T790M mutant‐specific whilst sparing WT EGFR, representing a significant breakthrough in the treatment in NSCLC patients with acquired resistance harboring these genotypes. Herein, we provides an overview of the second and third generation inhibitors currently approved, in clinical trial and also encompasses novel structures of discovery. This review mainly focuses on drug resistance, their mechanisms of action, development of structure–activity relationships and binding modes.     

Integrin beta1 over-expression associates with resistance to tyrosine kinase inhibitor gefitinib in non-small cell lung cancer

The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) such as gefitinib and erlotinib have been widely used in treating patients with advanced non-small cell lung cancer (NSCLC). However, acquired resistance to EGFR TKI almost occurs in every patient eventually. To identify its potential mechanism, we established a human NSCLC cell line PC9/AB2 which was 576-fold decrease in gefitinib sensitivity compared with its parental PC9 cell lines. No EGFR-T790M mutation or abnormal expression of c-Met protein was found in PC9/AB2 cells. Over-expression of integrin β1 was found, accompanied with increase of the cells' adhesion and migration. To further confirm the role of integrin β1 in gefitinib acquired resistance, we transferred its siRNA-expressing plasmid and its whole cDNA expressing plasmid into PC9/AB2 and into PC9 cells, respectively. The sensitivity of NSCLC cells to gefitinib was negatively correlated with integrin β1 expression levels. All these data suggest that up-regulation of integrin β1 might be an important factor for gefitinib resistance in NSCLC cell line PC9/AB2.     

Pemetrexed induces ROS generation and cellular senescence by attenuating TS-mediated thymidylate metabolism to reverse gefitinib resistance in NSCLC

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) are strongly recommended for non-small-cell lung cancer (NSCLC) patients harbouring active EGFR mutations, while drug resistance makes exploring resistance mechanisms and seeking effective therapeutic strategies urgent endeavours. Thymidylate synthetase (TYMS or TS) is a dominant enzyme in thymidylate nucleotide metabolism. In this study, we found a positive correlation between TS expression and overall survival (OS) and disease-free survival (DFS) in lung adenocarcinoma. The examination of gene sets from 140 NSCLC patients received EGFR-TKI therapy demonstrated a negative correlation between high TS expression and the efficacy of EGFR-TKI therapy. 24 tissue specimens from NSCLC patients exhibited upregulated TS mRNA expression in NSCLC patients resistant to gefitinib. The NSCLC cell PC9 and HCC827 sensitive to gefitinib and relatively resistant PC9/GR and HCC827/GR cells were used to demonstrate the knockdown of TS restored the sensitivity of resistant cells to gefitinib. Furthermore, pemetrexed effectively suppressed TS-mediated thymidylate metabolism and induced ROS generation, DNA damage and cellular senescence, thereby hampering cancer progression and restoring sensitivity to gefitinib. Our findings illuminate the potential mechanism of TS-triggered gefitinib resistance and indicate inhibition of TS by pemetrexed can potentiate the effect of gefitinib in NSCLC. Pemetrexed combined with gefitinib has potent anti-progression potential in gefitinib-resistant NSCLC. This study suggests that NSCLC patients with both high TS expression and EGFR-driving mutations might benefit more from a combination strategy of EGFR-TKI and pemetrexed-based chemotherapy than EGFR-TKI monotherapy, which has profound clinical implications and therapeutic value.     

Reciprocal regulation of miR‐206 and IL‐6/STAT3 pathway mediates IL6‐induced gefitinib resistance in EGFR‐mutant lung cancer cells

Persistently activated IL‐6/STAT3 pathway promotes acquired resistance to targeted therapy with epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs) in non–small‐cell lung cancer (NSCLC) treatment. miR‐206 has been verified to be dysregulated and plays as a negative regulator in lung cancer. However, whether miR‐206 may overcome IL6‐induced gefitinib resistance in EGFR‐mutant lung cancer remains elusive. In this study, we investigated the role of miR‐206 in IL6‐induced gefitinib‐resistant EGFR‐mutated lung cancer cell lines. We showed that forced miR‐206 expression restored gefitinib sensitivity in IL6‐induced gefitinib‐resistant EGFR‐mutant lung cancer cells by inhibiting IL6/JAK1/STAT3 pathway. Specifically, mechanistic investigations revealed that miR‐206 blocked IL‐6/STAT3 signalling via directly targeting the 3'‐UTR of intracellular IL‐6 messenger RNA. Moreover, IL‐6 induced miR‐206 down‐regulation by reducing the cropping process of primary miR‐206 (pri‐miR‐206) into the Drosha/DGCR8 complex. Taken together, our findings reveal a direct role of miR‐206 in regulating IL‐6/STAT3 pathway and contrarily activated IL‐6/STAT3 signalling mediates the miR‐206 maturation process in gefitinib‐resistant EGFR‐mutant lung cancer cells.     

PAR2 blockade reverses osimertinib resistance in non-small-cell lung cancer cells via attenuating ERK-mediated EMT and PD-L1 expression

Osimertinib, as the third-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs), is a first-line molecularly targeted drug for non-small cell lung cancer (NSCLC). However, the emergence of therapeutic resistance to osimertinib markedly impairs its efficiency and efficacy, leading to the failure of clinical applications. Novel molecular targets and drugs are urgently needed for reversing osimertinib resistance in NSCLC. Protease-activated receptor 2 (PAR2) that belongs to a subfamily of G protein-coupled receptors can stimulate the transactivation of EGFR to regulate multiple cellular signalling, actively participating in tumour progression. This study firstly discovered that PAR2 expression was notably enhanced when NSCLC cells became resistant to osimertinib. A PAR2 inhibitor facilitated osimertinib to attenuate EGFR transactivation, ERK phosphorylation, EMT and PD-L1 expression which were associated to osimertinib resistance. The combination of the PAR2 inhibitor and osimertinib also notably blocked cell viability, migration, 3D sphere formation and in vivo tumour growth whereas osimertinib itself lost such inhibitory effects in osimertinib-resistant NSCLC cells. Importantly, this reversal effect of PAR2 blockade was uncovered to depend on ERK-mediated EMT and PD-L1, since inhibition of β-arrestin or ERK, which could be modulated by PAR2, sensitized osimertinib to prevent EMT, PD-L1 expression and consequently overcame osimertinib resistance. Thus, this study demonstrated that PAR2 antagonism could limit ERK-mediated EMT and immune checkpoints, consequently attenuating EGFR transactivation and reactivate osimertinib. It suggested that PAR2 may be a novel drug target for osimertinib resistance, and PAR2 inhibition may be a promising strategy candidate for reversing EGFR-TKI resistance in NSCLC.     

MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells

Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30–40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI.     

Effect of simvastatin on the resistance to EGFR tyrosine kinase inhibitors in a non-small cell lung cancer with the T790M mutation of EGFR

Although non-small cell lung cancer (NSCLC) tumors with activating mutations in the epidermal growth factor receptor (EGFR) are highly responsive to EGFR tyrosine kinase inhibitors (TKIs) including gefitinib and erlotinib, development of acquired resistance is almost inevitable. Statins show antitumor activity, but it is unknown whether they can reverse EGFR-TKIs resistance in NSCLC with the T790M mutation of EGFR. This study investigated overcoming resistance to EGFR-TKI using simvastatin. We demonstrated that addition of simvastatin to gefitinib enhanced caspase-dependent apoptosis in T790M mutant NSCLC cells. Simvastatin also strongly inhibited AKT activation, leading to suppression of β-catenin activity and the expression of its targets, survivin and cyclin D1. Both insulin treatment and AKT overexpression markedly increased p-β-catenin and survivin levels, even in the presence of gefitinib and simvastatin. However, inhibition of AKT by siRNA or LY294002 treatment decreased p-β-catenin and survivin levels. To determine the role of survivin in simvastatin-induced apoptosis of gefitinib-resistant NSCLC, we showed that the proportion of apoptotic cells following treatment with survivin siRNA and the gefitinib–simvastatin combination was greater than the theoretical additive effects, whereas survivin up-regulation could confer protection against gefitinib and simvastatin-induced apoptosis. Similar results were obtained in erlotinib and simvastatin-treated HCC827/ER cells. These findings suggest that survivin is a key molecule that renders T790M mutant NSCLC cells resistant to apoptosis induced by EGFR-TKIs and simvastatin. Overall, these data indicate that simvastatin may overcome EGFR-TKI resistance in T790M mutant NSCLCs via an AKT/β-catenin signaling-dependent down-regulation of survivin and apoptosis induction.     

Reduced expression of CYLD promotes cell survival and inflammation in gefitinib‐treated NSCLC PC‐9 cells: Targeting CYLD may be beneficial for acquired resistance to gefitinib therapy

The application of tyrosine kinase inhibitors (TKIs) to the epidermal growth factor receptor (EGFR) has been proven to be highly effective for non‐small‐cell lung cancer (NSCLC). However, patients often evolve into acquired resistance. The secondary mutations in EGFR account for nearly half of the acquired resistance. While the remaining 50% of patients exhibit tolerance to EGFR‐TKIs with unclear mechanism(s). Cylindromatosis (CYLD), a deubiquitinase, functions as a tumor suppressor to regulate cell apoptosis, proliferation, and immune response, and so on. The role of CYLD in NSCLC EGFR‐TKI resistance remains elusive. Here, we found CYLD was upregulated in PC‐9 cells, whereas downregulated in PC‐9 acquired gefitinib‐resistant (PC‐9/GR) cells in response to the treatment of gefitinib, which is consistent with the results in the Gene Expression Omnibus database. Overexpression of CYLD promoted a more apoptotic death ratio in PC‐9/GR cells than that in PC‐9 cells. In addition, silencing the expression of CYLD resulted in an increase of the expression level of interleukin‐6, transforming growth factor‐β and tumor necrosis factor‐α, which may contribute to acquired resistance of PC‐9 cells to gefitinib. Taken together, our data in vitro demonstrate that PC‐9/GR cells downregulated CYLD expression, enhanced subsequent CYLD‐dependent antiapoptotic capacity and inflammatory response, which may provide a possible target for acquired gefitinib‐resistant treatment in NSCLC.     

Gefitinib-mediated Reactive Oxygen Specie (ROS) Instigates Mitochondrial Dysfunction and Drug Resistance in Lung Cancer Cells

Therapeutic benefits offered by tyrosine kinase inhibitors (TKIs), such as gefitinib (Iressa) and erlotinib (Tarceva), are limited due to the development of resistance, which contributes to treatment failure and cancer-related mortality. The aim of this study was to elucidate mechanistic insight into cellular perturbations that accompany acquired gefitinib resistance in lung cancer cells. Several lung adenocarcinoma (LAD) cell lines were screened to characterize epidermal growth factor receptor (EGFR) expression and mutation profile. To circumvent intrinsic variations between cell lines with respect to response to drug treatments, we generated gefitinib-resistant H1650 clone by long-term, chronic culture under gefitinib selection of parental cell line. Isogenic cells were analyzed by microarray, Western blot, flow cytometry, and confocal and transmission electron microscope. We observed that although chronic gefitinib treatment provided effective action against its primary target (aberrant EGFR activity), secondary effects resulted in increased cellular reactive oxygen species (ROS). Gefitinib-mediated ROS correlated with epithelial-mesenchymal transition, as well as striking perturbation of mitochondrial morphology and function. However, gefitinib treatment in the presence of ROS scavenger provided a partial rescue of mitochondrial aberrations. Furthermore, withdrawal of gefitinib from previously resistant clones correlated with normalized expression of epithelial-mesenchymal transition genes. These findings demonstrate that chronic gefitinib treatment promotes ROS and mitochondrial dysfunction in lung cancer cells. Antioxidants may alleviate ROS-mediated resistance.     

Blockade of the ERK or PI3K-Akt signaling pathway enhances the cytotoxicity of histone deacetylase inhibitors in tumor cells resistant to gefitinib or imatinib

Deregulated activation of protein tyrosine kinases, such as the epidermal growth factor receptor (EGFR) and Abl, is associated with human cancers including non-small cell lung cancer (NSCLC) and chronic myeloid leukemia (CML). Although inhibitors of such activated kinases have proved to be of therapeutic benefit in individuals with NSCLC or CML, some patients manifest intrinsic or acquired resistance to these drugs. We now show that, whereas blockade of either the extracellular signal-regulated kinase (ERK) pathway or the phosphatidylinositol 3-kinase (PI3K)-Akt pathway alone induced only a low level of cell death, it markedly sensitized NSCLC or CML cells to the induction of apoptosis by histone deacetylase (HDAC) inhibitors. Such enhanced cell death induced by the respective drug combinations was apparent even in NSCLC or CML cells exhibiting resistance to EGFR or Abl tyrosine kinase inhibitors, respectively. Co-administration of a cytostatic signaling pathway inhibitor may contribute to the development of safer anticancer strategies by lowering the required dose of cytotoxic HDAC inhibitors for a variety of cancers.     

Challenge and countermeasures for EGFR targeted therapy in non-small cell lung cancer

Lung cancer causes the highest mortality compared to other cancers in the world according to the latest WHO reports. Non-small cell lung cancer (NSCLC) contributes about 85% of total lung cancer cases. An extensive number of risk factors are attributed to the progression of lung cancer. Epidermal growth factor receptor (EGFR), one of the most frequently mutant driver genes, is closely involved in the development of lung cancer through regulation of the PI3K/AKT and MAPK pathways. As a representative of precision medicine, EGFR-tyrosine kinase inhibitors (TKIs) targeted therapy significantly relieves the development of activating mutant EGFR-driven NSCLC. However, treatment with TKIs facilitates the emergence of acquired resistance that continues to pose a significant hurdle with respect to EGFR targeted therapy. In this review, the development of current approved EGFR-TKIs as well as the related supporting clinical trials are summarized and discussed. Mechanisms of action and resistance were addressed respectively, which serve as important guides to understanding acquired resistance. We also explored the corresponding combination treatment options according to different resistance mechanisms. Future challenges include more comprehensive characterization of unclear resistance mechanisms in different populations and the development of more efficient and precision synthetic therapeutic strategies.     

Antitumor activity of sorafenib in human cancer cell lines with acquired resistance to EGFR and VEGFR tyrosine kinase inhibitors

Treatment of non small cell lung cancer (NSCLC) and colorectal cancer (CRC) have substantially changed in the last years with the introduction of epidermal growth factor receptor (EGFR) inhibitors in the clinical practice. The understanding of mechanisms which regulate cells sensitivity to these drugs is necessary for their optimal use.An in vitro model of acquired resistance to two tyrosine kinase inhibitors (TKI) targeting the EGFR, erlotinib and gefitinib, and to a TKI targeting EGFR and VEGFR, vandetanib, was developed by continuously treating the human NSCLC cell line CALU-3 and the human CRC cell line HCT116 with escalating doses of each drug. MTT, western blot analysis, migration, invasion and anchorage-independent colony forming assays were conducted in vitro and experiments with established xenografts in athymic nude mice were performed in vivo in sensitive, wild type (WT) and TKI-resistant CALU-3 and HCT116 cell lines.As compared to WT CALU-3 and HCT116 human cancer cells, TKI-resistant cell lines showed a significant increase in the levels of activated, phosphorylated AKT, MAPK, and of survivin. Considering the role of RAS and RAF as downstream signals of both the EGFR and VEGFR pathways, we treated resistant cells with sorafenib, an inhibitor of C-RAF, B-RAF, c-KIT, FLT-3, RET, VEGFR-2, VEGFR-3, and PDGFR-β. Sorafenib reduced the activation of MEK and MAPK and caused an inhibition of cell proliferation, invasion, migration, anchorage-independent growth in vitro and of tumor growth in vivo of all TKI-resistant CALU-3 and HCT116 cell lines.These data suggest that resistance to EGFR inhibitors is predominantly driven by the RAS/RAF/MAPK pathway and can be overcame by treatment with sorafenib.     

EMT: A mechanism for escape from EGFR-targeted therapy in lung cancer

Epithelial mesenchymal transition (EMT) is a reversible developmental genetic programme of transdifferentiation of polarised epithelial cells to mesenchymal cells. In cancer, EMT is an important factor of tumour cell plasticity and has received increasing attention for its role in the resistance to conventional and targeted therapies. In this paper we provide an overview of EMT in human malignancies, and discuss contribution of EMT to the development of the resistance to Epidermal Growth Factor Receptor (EGFR)-targeted therapies in non-small cell lung cancer (NSCLC). Patients with the tumours bearing specific mutations in EGFR have a good clinical response to selective EGFR inhibitors, but the resistance inevitably develops. Several mechanisms responsible for the resistance include secondary mutations in the EGFR gene, genetic or non-mutational activation of alternative survival pathways, transdifferentiation of NSCLC to the small cell lung cancer histotype, or formation of resistant tumours with mesenchymal characteristics. Mechanistically, application of an EGFR inhibitor does not kill all cancer cells; some cells survive the exposure to a drug, and undergo genetic evolution towards resistance. Here, we present a theory that these quiescent or slow-proliferating drug-tolerant cell populations, or so-called “persisters”, are generated via EMT pathways. We review the EMT-activated mechanisms of cell survival in NSCLC, which include activation of ABC transporters and EMT-associated receptor tyrosine kinase AXL, immune evasion, and epigenetic reprogramming. We propose that therapeutic inhibition of these pathways would eliminate pools of persister cells and prevent or delay cancer recurrence when applied in combination with the agents targeting EGFR.     

BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations

Background

Epidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in TKI-induced cell death and other secondary mutations involved in resistance remain unclear. The objective of this study was to identify the mechanism of EGFR TKI-induced apoptosis and secondary resistant mutations that affect this process.

Methods and Findings

To study TKI-induced cell death and mechanisms of resistance, we used lung cancer cell lines (with or without EGFR mutations), Ba/F3 cells stably transfected with EGFR mutation constructs, and tumor samples from a gefitinib-resistant patient. Here we show that up-regulation of the BH3-only polypeptide BIM (also known as BCL2-like 11) correlated with gefitinib-induced apoptosis in gefitinib-sensitive EGFR-mutant lung cancer cells. The T790M mutation blocked gefitinib-induced up-regulation of BIM and apoptosis. This blockade was overcome by the irreversible TKI CL-387,785. Knockdown of BIM by small interfering RNA was able to attenuate apoptosis induced by EGFR TKIs. Furthermore, from a gefitinib-resistant patient carrying the activating L858R mutation, we identified a novel secondary resistant mutation, L747S in cis to the activating mutation, which attenuated the up-regulation of BIM and reduced apoptosis.

Conclusions

Our results provide evidence that BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cells and that both attenuation of the up-regulation of BIM and resistance to gefitinib-induced apoptosis are seen in models that contain the common EGFR T790M and the novel L747S secondary resistance mutations. These findings also suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors.     

Targeting mTOR to Overcome Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer Cells

Aims

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells.

Methods

We selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry.

Results

In the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC₅₀ concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels.

Conclusion

Our results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.     

Chemical Genetics Screen Identifies COPB2 Tool Compounds That Alters ER Stress Response and Induces RTK Dysregulation in Lung Cancer Cells

Activating mutations in the epidermal growth factor receptor (EGFR) are common driver mutations in non-small cell lung cancer (NSCLC). First, second and third generation EGFR tyrosine kinase inhibitors (TKIs) are effective at inhibiting mutant EGFR NSCLC, however, acquired resistance is a major issue, leading to disease relapse. Here, we characterize a small molecule, EMI66, an analog of a small molecule which we previously identified to inhibit mutant EGFR signalling via a novel mechanism of action. We show that EMI66 attenuates receptor tyrosine kinase (RTK) expression and signalling and alters the electrophoretic mobility of Coatomer Protein Complex Beta 2 (COPB2) protein in mutant EGFR NSCLC cells. Moreover, we demonstrate that EMI66 can alter the subcellular localization of EGFR and COPB2 within the early secretory pathway. Furthermore, we find that COPB2 knockdown reduces the growth of mutant EGFR lung cancer cells, alters the post-translational processing of RTKs, and alters the endoplasmic reticulum (ER) stress response pathway. Lastly, we show that EMI66 treatment also alters the ER stress response pathway and inhibits the growth of mutant EGFR lung cancer cells and organoids. Our results demonstrate that targeting of COPB2 with EMI66 presents a viable approach to attenuate mutant EGFR signalling and growth in NSCLC.