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.     

Alternative Signaling Pathways as Potential Therapeutic Targets for Overcoming EGFR and c-Met Inhibitor Resistance in Non-Small Cell Lung Cancer

The use of tyrosine kinase inhibitors (TKIs) against EGFR/c-Met in non-small cell lung cancer (NSCLC) has been shown to be effective in increasing patient progression free survival (PFS), but their efficacy is limited due to the development of resistance and tumor recurrence. Therefore, understanding the molecular mechanisms underlying development of drug resistance in NSCLC is necessary for developing novel and effective therapeutic approaches to improve patient outcome. This study aims to understand the mechanism of EGFR/c-Met tyrosine kinase inhibitor (TKI) resistance in NSCLC. H2170 and H358 cell lines were made resistant to SU11274, a c-Met inhibitor, and erlotinib, an EGFR inhibitor, through step-wise increases in TKI exposure. The IC₅₀ concentrations of resistant lines exhibited a 4–5 and 11–22-fold increase for SU11274 and erlotinib, respectively, when compared to parental lines. Furthermore, mTOR and Wnt signaling was studied in both cell lines to determine their roles in mediating TKI resistance. We observed a 2–4-fold upregulation of mTOR signaling proteins and a 2- to 8-fold upregulation of Wnt signaling proteins in H2170 erlotinib and SU11274 resistant cells. H2170 and H358 cells were further treated with the mTOR inhibitor everolimus and the Wnt inhibitor XAV939. H358 resistant cells were inhibited by 95% by a triple combination of everolimus, erlotinib and SU11274 in comparison to 34% by a double combination of these drugs. Parental H2170 cells displayed no sensitivity to XAV939, while resistant cells were significantly inhibited (39%) by XAV939 as a single agent, as well as in combination with SU11274 and erlotinib. Similar results were obtained with H358 resistant cells. This study suggests a novel molecular mechanism of drug resistance in lung cancer.     

Mapping inhibitor response to the in-frame deletions,insertions and duplications of epidermal growth factor receptor (EGFR) in non-small cell lung cancer

Human epidermal growth factor receptor (EGFR) has become a well-established target for the treatment of non-small cell lung cancer (NSCLC). However, a large number of in-frame deletion, insertion and duplication mutations in the EGFR tyrosine kinase (TK) domain have been observed to alter drug response to such a kinase target. Thus, a systematic investigation of the intermolecular interactions between the clinical small-molecule agents and various EGFR in-frame mutants would help to establish a complete picture of drug response to kinase mutations in lung cancer, and to design new EGFR inhibitors with high potency and selectivity to target drug-resistant mutants. Here, we describe a combined pipeline to explore the drug response of five representative EGFR inhibitors, including three FDA-approved agents (gefitinib, erlotinib and lapatinib) and two compounds under clinical development (AEE788 and TAK-285) to a number of clinically relevant EGFR in-frame mutations, aiming at a comprehensive understanding of molecular mechanism and biological implication underlying drug resistance and sensitivity to EGFR in-frame mutations. It was found that the insertion and duplication mutations in exon 20 can generally cause drug resistance to EGFR due to the reduced size of kinase’s active pocket, while deletion mutations in exon 19 associate closely with increased inhibitor sensitivity to EGFR by establishing additional non-bonded interactions across complex interface, including hydrogen bonds, cation–π interactions and hydrophobic contacts.     

Aberrant trafficking of NSCLC-associated EGFR mutants through the endocytic recycling pathway promotes interaction with Src@

Background  

Epidermal growth factor receptor (EGFR) controls a wide range of cellular processes, and altered EGFR signaling contributes to human cancer. EGFR kinase domain mutants found in non-small cell lung cancer (NSCLC) are constitutively active, a trait critical for cell transformation through activation of downstream pathways. Endocytic trafficking of EGFR is a major regulatory mechanism as ligand-induced lysosomal degradation results in termination of signaling. While numerous studies have examined mutant EGFR signaling, the endocytic traffic of mutant EGFR within the NSCLC milieu remains less clear.     

Sulforaphane attenuates EGFR signaling in NSCLC cells

Background

EGFR, a receptor tyrosine kinase (RTK), is frequently overexpressed and mutated in non-small cell lung cancer (NSCLC). Tyrosine kinase inhibitors (TKIs) have been widely used in the treatment of many cancers, including NSCLC. However, intrinsic and acquired resistance to TKI remains a common obstacle. One strategy that may help overcome EGFR-TKI resistance is to target EGFR for degradation. As EGFR is a client protein of heat-shock protein 90 (HSP90) and sulforaphane is known to functionally regulate HSP90, we hypothesized that sulforaphane could attenuate EGFR-related signaling and potentially be used to treat NSCLC.

Results

Our study revealed that sulforaphane displayed antitumor activity against NSCLC cells both in vitro and in vivo. The sensitivity of NSCLC cells to sulforaphane appeared to positively correlate with the inhibition of EGFR-related signaling, which was attributed to the increased proteasomal degradation of EGFR. Combined treatment of NSCLC cells with sulforaphane plus another HSP90 inhibitor (17-AAG) enhanced the inhibition of EGFR-related signaling both in vitro and in vivo.

Conclusions

We have shown that sulforaphane is a novel inhibitory modulator of EGFR expression and is effective in inhibiting the tumor growth of EGFR-TKI-resistant NSCLC cells. Our findings suggest that sulforaphane should be further explored for its potential clinical applications against NSCLC.     

Modeling of Tumor Progression in NSCLC and Intrinsic Resistance to TKI in Loss of PTEN Expression

EGFR signaling plays a very important role in NSCLC. It activates Ras/ERK, PI3K/Akt and STAT activation pathways. These are the main pathways for cell proliferation and survival. We have developed two mathematical models to relate to the different EGFR signaling in NSCLC and normal cells in the presence or absence of EGFR and PTEN mutations. The dynamics of downstream signaling pathways vary in the disease state and activation of some factors can be indicative of drug resistance. Our simulation denotes the effect of EGFR mutations and increased expression of certain factors in NSCLC EGFR signaling on each of the three pathways where levels of pERK, pSTAT and pAkt are increased. Over activation of ERK, Akt and STAT3 which are the main cell proliferation and survival factors act as promoting factors for tumor progression in NSCLC. In case of loss of PTEN, Akt activity level is considerably increased. Our simulation results show that in the presence of erlotinib, downstream factors i.e. pAkt, pSTAT3 and pERK are inhibited. However, in case of loss of PTEN expression in the presence of erlotinib, pAkt level would not decrease which demonstrates that these cells are resistant to erlotinib.     

Profiling of Phospho-AKT,Phospho-mTOR,Phospho-MAPK and EGFR in Non-small Cell Lung Cancer

Activation of numerous pathways has been documented in non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) has emerged as a common therapeutic target. The mitogen-activated protein kinase (MAPK) and AKT signaling pathways are downstream of EGFR and deregulated via genetic and epigenetic mechanisms in many human cancers. We evaluated selected markers in the EGFR pathway with reference to outcome. Tissues from 220 cases of NSCLC patients presented in a tissue microarray were assayed with immunohistochemistry for phosphorylated AKT, phosphorylated MAPK, phosphorylated mTOR, and EGFR and then quantified by automated image analysis. Individually, the biomarkers did not predict. Combined as ratios, p-mTOR/p-AKT, and p-MAPK/EGFR function as prognostic markers of survival (p=0.008 and p=0.029, respectively), however, no significance was found after adjustment (p=0.221, p=0.103). The sum of these ratios demonstrates a stronger correlation with survival (p<0.001) and remained statistically significant after adjustment (p=0.026). The algebraic combination of biomarkers offer the capacity to understand factors that predict outcome better than current approaches of evaluating biomarkers individually or in pairs. Our results show the sum of p-mTOR/p-AKT and p-MAPK/EGFR is a potential predictive marker of survival in NSCLC patients.     

Contrasting Effects of the Cytotoxic Anticancer Drug Gemcitabine and the EGFR Tyrosine Kinase Inhibitor Gefitinib on NK Cell-Mediated Cytotoxicity via Regulation of NKG2D Ligand in Non-Small-Cell Lung Cancer Cells

IntroductionSeveral cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown.MethodsThis study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis.ResultsWe demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a.ConclusionIn keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells.     

Transformation of cells overexpressing a tyrosine kinase by phospholipase D1 and D2.

Phospholipase D (PLD) activity is elevated in response to most mitogenic signals. Two mammalian PLD genes (PLD1 and PLD2) have been cloned and their gene products have been characterized. PLD1 is a downstream target of the Ras/RalA GTPase cascade implicated in mitogenic and oncogenic signaling. Consistent with a role in mitogenic signaling, elevated expression of PLD1 transforms cells overexpressing the epidermal growth factor (EGF) receptor (EGFR). However, PLD2 colocalizes with the EGFR in caveolin-enriched light membrane microdomains. We therefore investigated whether PLD2 could also contribute to the transformation of cells overexpressing a tyrosine kinase. We report here that elevated expression of PLD2 transforms rat fibroblasts overexpressing either the EGFR or c-Src. Since overexpression of a tyrosine kinase is a common genetic alteration in several human cancers, these data suggest that elevation of either PLD1 or PLD2 may contribute to the progression to a malignant phenotype in cells with elevated tyrosine kinase activity.     

EGFR pathway targeting drugs in head and neck cancer in the era of immunotherapy

Receptor tyrosine kinases (RTKs) are cell surface receptors that bind growth factor ligands and initiate cellular signaling. Of the 20 classes of RTKs, 7 classes, I-V, VIII, and X, are linked to head and neck cancers (HNCs). We focus on the first class of RTK, epidermal growth factor receptor (EGFR), as it is the most thoroughly studied class. EGFR overexpression is observed in 20% of tumors, and expression of EGFR variant III is seen in 15% of aggressive chemoradiotherapy resistant HNCs. Currently, the EGFR monoclonal antibody (mAb) cetuximab is the only FDA approved RTK-targeting drug for the treatment of HNCs. Clinical trials have also included EGFR mAbs, with tyrosine kinase inhibitors, and small molecule inhibitors targeting the EGFR, MAPK, and mTOR pathways. Additionally, Immunotherapy has been found to be effective in 15 to 20% of patients with recurrent or metastatic HNC as a monotherapy. Thus, attempts are underway for the combinatorial treatment of immunotherapy and EGFR mAbs to determine if the recruitment of immune cells in the tumor microenvironment can overcome EGFR resistance.     

Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.     

EGFR signaling pathway occupies an important position in cancer‐related downstream signaling pathways of Pyk2

Proline‐rich tyrosine kinase 2 (Pyk2) is a member of focal adhesion kinase (FAK) non‐receptor tyrosine kinase family and has been found to promote cancer cell survival, proliferation, migration, invasion, and metastasis. Pyk2 takes part in different carcinogenic signaling pathways to promote cancer progression, including epidermal growth factor receptor (EGFR) signaling pathway. EGFR signaling pathway is a traditional carcinogenic signaling pathway, which plays a critical role in tumorigenesis and tumor progression. FAK inhibitors have been reported to fail to get the ideal anti‐cancer outcomes because of activation of EGFR signaling pathway. Better understanding of Pyk2 downstream targets and interconnectivity between Pyk2 and carcinogenic EGFR signaling pathway will help finding more effective targets for clinical anti‐cancer combination therapies. Thus, the interconnectivity between Pyk2 and EGFR signaling pathway, which regulates tumor development and metastasis, needs to be elucidated. In this review, we summarized the downstream targets of Pyk2 in cancers, focused on the connection between Pyk2 and EGFR signaling pathway in different cancer types, and provided a new overview of the roles of Pyk2 in EGFR signaling pathway and cancer development.     

Epidermal growth factor receptor mutation in combination with expression of MIG6 alters gefitinib sensitivity

Background  

Epidermal growth factor receptor (EGFR) signaling plays an important role in the regulation of cell proliferation, survival, metastasis, and invasion in various tumors. Earlier studies showed that the EGFR is frequently overexpressed in non-small-cell lung cancer (NSCLC) and EGFR mutations at specific amino acid residues in the kinase domain induce altered responsiveness to gefitinib, a small molecule EGFR tyrosine kinase inhibitor. However, the mechanism underlying the drug response modulated by EGFR mutation is still largely unknown. To elucidate drug response in EGFR signal transduction pathway in which complex dynamics of multiple molecules involved, a systematic approach is necessary. In this paper, we performed experimental and computational analyses to clarify the underlying mechanism of EGFR signaling and cell-specific gefitinib responsiveness in three H1299-derived NSCLC cell lines; H1299 wild type (H1299WT), H1299 with an overexpressed wild type EGFR (H1299EGFR-WT), and H1299 with an overexpressed mutant EGFR L858R (H1299L858R; gefitinib sensitive mutant).     

EGFR and c-Met Cross Talk in Glioblastoma and Its Regulation by Human Cord Blood Stem Cells

Receptor tyrosine kinases (RTK) and their ligands control critical biologic processes, such as cell proliferation, migration, and differentiation. Aberrant expression of these receptor kinases in tumor cells alters multiple downstream signaling cascades that ultimately drive the malignant phenotype by enhancing tumor cell proliferation, invasion, metastasis, and angiogenesis. As observed in human glioblastoma (hGBM) and other cancers, this dysregulation of RTK networks correlates with poor patient survival. Epidermal growth factor receptor (EGFR) and c-Met, two well-known receptor kinases, are coexpressed in multiple cancers including hGBM, corroborating that their downstream signaling pathways enhance a malignant phenotype. The integration of c-Met and EGFR signaling in cancer cells indicates that treatment regimens designed to target both receptor pathways simultaneously could prove effective, though resistance to tyrosine kinase inhibitors continues to be a substantial obstacle. In the present study, we analyzed the antitumor efficacy of EGFR inhibitors erlotinib and gefitinib and c-Met inhibitor PHA-665752, along with their respective small hairpin RNAs (shRNAs) alone or in combination with human umbilical cord blood stem cells (hUCBSCs), in glioma cell lines and in animal xenograft models. We also measured the effect of dual inhibition of EGFR/c-Met pathways on invasion and wound healing. Combination treatments of hUCBSC with tyrosine kinase inhibitors significantly inhibited invasion and wound healing in U251 and 5310 cell lines, thereby indicating the role of hUCBSC in inhibition of RTK-driven cell behavior. Further, the EGFR and c-Met localization in glioma cells and hGBM clinical specimens indicated that a possible cross talk exists between EGFR and c-Met signaling pathway.     

Temporal resolution of autophosphorylation for normal and oncogenic forms of EGFR and differential effects of gefitinib

Epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases (RTK). EGFR overexpression or mutation in many different forms of cancers has highlighted its role as an important therapeutic target. Gefitinib, the first small molecule inhibitor of EGFR kinase function to be approved for the treatment of nonsmall cell lung cancer (NSCLC) by the FDA, demonstrates clinical activity primarily in patients with tumors that harbor somatic kinase domain mutations in EGFR. Here, we compare wild-type EGFR autophosphorylation kinetics to the L834R (also called L858R) EGFR form, one of the most common mutations in lung cancer patients. Using rapid chemical quench, time-resolved electrospray mass spectrometry (ESI-MS), and Western blot analyses, we examined the order of autophosphorylation in wild-type (WT) and L834R EGFR and the effect of gefitinib (Iressa) on the phosphorylation of individual tyrosines. These studies establish that there is a temporal order of autophosphorylation of key tyrosines involved in downstream signaling for WT EGFR and a loss of order for the oncogenic L834R mutant. These studies also reveal unique signature patterns of drug sensitivity for inhibition of tyrosine autophosphorylation by gefitinib: distinct for WT and oncogenic L834R mutant forms of EGFR. Fluorescence studies show that for WT EGFR the binding affinity for gefitinib is weaker for the phosphorylated protein while for the oncogenic mutant, L834R EGFR, the binding affinity of gefitinib is substantially enhanced and likely contributes to the efficacy observed clinically. This mechanistic information is important in understanding the molecular details underpinning clinical observations as well as to aid in the design of more potent and selective EGFR inhibitors.     

Laser capture microdissection and protein microarray analysis of human non-small cell lung cancer: differential epidermal growth factor receptor (EGPR) phosphorylation events associated with mutated EGFR compared with wild type

Little is known about lung carcinoma epidermal growth factor (EGF) kinase pathway signaling within the context of the tissue microenvironment. We quantitatively profiled the phosphorylation and abundance of signal pathway proteins relevant to the EGF receptor within laser capture microdissected untreated, human non-small cell lung cancer (NSCLC) (n = 25) of known epidermal growth factor receptor (EGFR) tyrosine kinase domain mutation status. We measured six phosphorylation sites on EGFR to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation of specific multiple phosphorylation sites on the EGFR and downstream proteins. Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Tyr-1148 (p < 0.044) and Tyr-1068 (p < 0.026) and decreased phosphorylation of EGFR Tyr-1045 (p < 0.002), HER2 Tyr-1248 (p < 0.015), IRS-1 Ser-612 (p < 0.001), and SMAD Ser-465/467 (p < 0.011) across all classes of mutated EGFR patient samples compared with wild type. To explore which subset of correlations was influenced by ligand induction versus an intrinsic phenotype of the EGFR mutants, we profiled the time course of 115 cellular signal proteins for EGF ligand-stimulated (three dosages) NSCLC mutant and wild type cultured cell lines. EGFR mutant cell lines (H1975 L858R) displayed a pattern of EGFR Tyr-1045 and HER2 Tyr-1248 phosphorylation similar to that found in tissue. Persistence of phosphorylation for AKT Ser-473 following ligand stimulation was found for the mutant. These data suggest that a higher proportion of the EGFR mutant carcinoma cells may exhibit activation of the phosphatidylinositol 3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (MTOR) pathway through Tyr-1148 and Tyr-1068 and suppression of IRS-1 Ser-612, altered heterodimerization with ERBB2, reduced response to transforming growth factor beta suppression, and reduced ubiquitination/degradation of the EGFR through EGFR Tyr-1045, thus providing a survival advantage. This is the first comparison of multiple, site-specific phosphoproteins with the EGFR tyrosine kinase domain mutation status in vivo.