Elevated BCRP/ABCG2 expression confers acquired resistance to gefitinib in wild-type EGFR-expressing cells

Background

The sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) is strongly associated with activating EGFR mutations. Although not as sensitive as patients harboring these mutations, some patients with wild-type EGFR (wtEGFR) remain responsive to EGFR TKIs, suggesting that the existence of unexplored mechanisms renders most of wtEGFR-expressing cancer cells insensitive.

Methodology/Principal Findings

Here, we show that acquired resistance of wtEGFR-expressing cancer cells to an EGFR TKI, gefitinib, is associated with elevated expression of breast cancer resistance protein (BCRP/ABCG2), which in turn leads to gefitinib efflux from cells. In addition, BCRP/ABCG2 expression correlates with poor response to gefitinib in both cancer cell lines and lung cancer patients with wtEGFR. Co-treatment with BCRP/ABCG2 inhibitors enhanced the anti-tumor activity of gefitinib.

Conclusions/Significance

Thus, BCRP/ABCG2 expression may be a predictor for poor efficacy of gefitinib treatment, and targeting BCRP/ABCG2 may broaden the use of gefitinib in patients with wtEGFR.     

EGF receptor inhibitors increase ErbB3 mRNA and protein levels in breast cancer cells

The potential benefits of drugs directly targeting the ErbB receptors for cancer therapy have led to an extensive development within this field. However, the clinical effects of ErbB receptor-targeting drugs in cancer treatment are limited due to a high frequency of resistance. It has been reported that, when inhibiting the epidermal growth factor receptor (EGFR) with the tyrosine kinase inhibitor gefitinib, increased activation of ErbB3 via MET, or by re-localization of ErbB3 mediates cell survival. Here we show further evidence that members of the ErbB receptor family facilitate resistance to EGFR inhibitor treatment in ErbB2 overexpressing breast cancer cells. We found that gefitinib treatment increased ErbB3 expression, both at protein and mRNA levels. ErbB3 expression was upregulated not only by gefitinib but also by a panel of different EGFR inhibitors, suggesting that inhibition of EGFR in general affects ErbB3 expression. In addition, we found that gefitinib treatment increased ErbB2 expression levels while EGFR inhibitors decreased the activity of ErbB2. Concentrations of gefitinib that decreased phospho-ErbB2 reversely increased ErbB3 levels. We further examined changes induced by gefitinib treatment on mRNA levels of the most common genes known to be involved in breast cancer. As expected, we found that gefitinib downregulated genes whose functions were linked to cellular proliferation, such as Ki-67, topoisomerase II alpha and cyclins, and surprisingly downregulated gene expression of FAS which is involved in apoptotic signaling. Together, our data strongly suggest that resistance to EGFR inhibitors may result from the compensation of other family members and that combinations of anti-cancer drugs are required to increase the sensitivity of these treatments.     

Lipid raft localization of EGFR alters the response of cancer cells to the EGFR tyrosine kinase inhibitor gefitinib

Epidermal growth factor receptor (EGFR) is overexpressed in many cancer types including ~30% of breast cancers. Several small molecule tyrosine kinase inhibitors (TKIs) targeting EGFR have shown clinical efficacy in lung and colon cancers, but no benefit has been noted in breast cancer. Thirteen EGFR expressing breast cancer cell lines were analyzed for response to EGFR TKIs. Seven were found to be EGFR TKI resistant; while shRNA knockdown of EGFR determined that four of these cell lines retained the requirement of EGFR protein expression for growth. Interestingly, EGFR localized to plasma membrane lipid rafts in all four of these EGFR TKI-resistant cell lines, as determined by biochemical raft isolation and immunofluorescence. When lipid rafts were depleted of cholesterol using lovastatin, all four cell lines were sensitized to EGFR TKIs. In fact, the effects of the cholesterol biosynthesis inhibitors and gefitinib were synergistic. While gefitinib effectively abrogated phosphorylation of Akt- and mitogen-activated protein kinase in an EGFR TKI-sensitive cell line, phosphorylation of Akt persisted in two EGFR TKI-resistant cell lines, however, this phosphorylation was abrogated by lovastatin treatment. Thus, we have shown that lipid raft localization of EGFR correlates with resistance to EGFR TKI-induced growth inhibition and pharmacological depletion of cholesterol from lipid rafts decreases this resistance in breast cancer cell lines. Furthermore, we have presented evidence to suggest that when EGFR localizes to lipid rafts, these rafts provide a platform to facilitate activation of Akt signaling in the absence of EGFR kinase activity.     

Modulation of membrane properties of lung cancer cells by azurin enhances the sensitivity to EGFR-targeted therapy and decreased β1 integrin-mediated adhesion

In lung cancer, the Epidermal Growth Factor Receptor (EGFR) is one of the main targets for clinical management of this disease. The effectiveness of therapies toward this receptor has already been linked to the expression of integrin receptor subunit β₁ in NSCLC A549 cells. In this work we demonstrate that azurin, an anticancer therapeutic protein originated from bacterial cells, controls the levels of integrin β₁ and its appropriate membrane localization, impairing the intracellular signaling cascades downstream these receptors and the invasiveness of cells. We show evidences that azurin when combined with gefitinib and erlotinib, tyrosine kinase inhibitors which targets specifically the EGFR, enhances the sensitivity of these lung cancer cells to these molecules. The broad effect of azurin at the cell surface level was examined by Atomic Force Microscopy. The Young 's module (E) shows that the stiffness of A549 lung cancer cells decreased with exposure to azurin and also gefitinib, suggesting that the alterations in the membrane properties may be the basis of the broad anticancer activity of this protein. Overall, these results show that azurin may be relevant as an adjuvant to improve the effects of other anticancer agents already in clinical use, to which patients often develop resistance hampering its full therapeutic response     

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.     

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.     

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.     

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.     

Can EGFR be a therapeutic target in breast cancer?

Epidermal growth factor receptor (EGFR) is highly expressed in certain cancer types and is involved in regulating the biological characteristics of cancer progression, including proliferation, metastasis, and drug resistance. Various medicines targeting EGFR have been developed and approved for several cancer types, such as lung and colon cancer. To date, however, EGFR inhibitors have not achieved satisfactory clinical results in breast cancer, which continues to be the most serious malignant tumor type in females. Therefore, clarifying the underlying mechanisms related to the ineffectiveness of EGFR inhibitors in breast cancer and developing new EGFR-targeted strategies (e.g., combination therapy) remain critical challenges. Various studies have demonstrated aberrant expression and maintenance of EGFR levels in breast cancer. In this review, we summarize the regulatory mechanisms underlying EGFR protein expression in breast cancer cells, including EGFR mutations, amplification, endocytic dysfunction, recycling acceleration, and degradation disorders. We also discuss potential therapeutic strategies that act directly or indirectly on EGFR, including reducing EGFR protein expression, treating the target protein to mediate precise clearance, and inhibiting non-EGFR signaling pathways. This review should provide new therapeutic perspectives for breast cancer patients with high EGFR expression.     

ANXA2 could act as a moderator of EGFR-directed therapy resistance in triple negative breast cancer

Triple negative breast cancer (TNBC) patients cannot benefit from EGFR-targeted therapy even though the EGFR is highly expressed, because patients exhibit resistance to these drugs. Unfortunately, the molecular mechanisms remain relatively unknown. ANXA2, highly expressed in invasive breast cancer cells, is closely related with poor prognosis, and acts as a molecular switch to EGFR activation. In this study, MDA-MB-231 cells and MCF7 cells were used. Our results showed that ANXA2 expression is inversely correlated with cell sensitivity to gefitinib. Knockdown of ANXA2 expression in MDA-MB-231 cells increased the gefitinib induced cell death. When ANXA2 was overexpressed in MCF7 cells, the gefitinib induced cell death was decreased. Furthermore, we demonstrated that phosphorylation of ANXA2 at Tyr23 is negatively correlated with the sensitivity of TNBC to gefitinib. Altogether, our results suggest a new role of ANXA2 in regulating sensitivity of TNBC MDA-MB-231 cells to the EGFR inhibitor gefitinib.     

EGFR tyrosine kinase inhibitors activate autophagy as a cytoprotective response in human lung cancer cells

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.     

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.     

t-Darpp Promotes Enhanced EGFR Activation and New Drug Synergies in Her2-Positive Breast Cancer Cells

Trastuzumab has led to improved survival rates of HER2⁺ breast cancer patients. However, acquired resistance remains a problem in the majority of cases. t-Darpp is over-expressed in trastuzumab-resistant cell lines and its over-expression is sufficient for conferring the resistance phenotype. Although its mechanism of action is unknown, t-Darpp has been shown to increase cellular proliferation and inhibit apoptosis. We have reported that trastuzumab-resistant BT.Her^R cells that over-express endogenous t-Darpp are sensitized to EGFR inhibition in the presence (but not the absence) of trastuzumab. The purpose of the current study was to determine if t-Darpp might modulate sensitivity to EGFR inhibitors in trastuzumab-resistant cells. Using EGFR tyrosine kinase inhibitors AG1478, gefitinib and erlotinib, we found that trastuzumab-resistant SK.Her^R cells were sensitized to EGFR inhibition, compared to SK-Br-3 controls, even in the absence of trastuzumab. t-Darpp knock-down in SK.Her^R cells reversed their sensitivity to EGFR inhibition. Increased EGFR sensitivity was also noted in SK.tDp cells that stably over-express t-Darpp. High levels of synergy between trastuzumab and the EGFR inhibitors were observed in all cell lines with high t-Darpp expression. These cells also demonstrated more robust activation of EGFR signaling and showed greater EGFR stability than parental cells. The T75A phosphorylation mutant of t-Darpp did not confer sensitivity to EGFR inhibition nor activation of EGFR signaling. The over-expression of t-Darpp might facilitate enhanced EGFR signaling as part of the trastuzumab resistance phenotype. This study suggests that the presence of t-Darpp in HER2⁺ cancers might predict the enhanced response to dual HER2/EGFR targeting.     

Induction of Autophagy Is an Early Response to Gefitinib and a Potential Therapeutic Target in Breast Cancer

Gefitinib (Iressa^®, ZD1839) is a small molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. We report on an early cellular response to gefitinib that involves induction of functional autophagic flux in phenotypically diverse breast cancer cells that were sensitive (BT474 and SKBR3) or insensitive (MCF7-GFPLC3 and JIMT-1) to gefitinib. Our data show that elevation of autophagy in gefitinib-treated breast cancer cells correlated with downregulation of AKT and ERK1/2 signaling early in the course of treatment. Inhibition of autophagosome formation by BECLIN-1 or ATG7 siRNA in combination with gefitinib reduced the abundance of autophagic organelles and sensitized SKBR3 but not MCF7-GFPLC3 cells to cell death. However, inhibition of the late stage of gefitinib-induced autophagy with hydroxychloroquine (HCQ) or bafilomycin A1 significantly increased (p<0.05) cell death in gefitinib-sensitive SKBR3 and BT474 cells, as well as in gefitinib-insensitive JIMT-1 and MCF7-GFPLC3 cells, relative to the effects observed with the respective single agents. Treatment with the combination of gefitinib and HCQ was more effective (p<0.05) in delaying tumor growth than either monotherapy (p>0.05), when compared to vehicle-treated controls. Our results also show that elevated autophagosome content following short-term treatment with gefitinib is a reversible response that ceases upon removal of the drug. In aggregate, these data demonstrate that elevated autophagic flux is an early response to gefitinib and that targeting EGFR and autophagy should be considered when developing new therapeutic strategies for EGFR expressing breast cancers.     

Gefitinib resistance of cancer cells correlated with TM4SF5-mediated epithelial-mesenchymal transition

Although cancers can be initially treated with the epidermal growth factor receptor (EGFR) inhibitor, gefitinib, continued gefitinib therapy does not benefit the survival of patients due to acquired resistance through EGFR mutations, c-MET amplification, or epithelial-mesenchymal transition (EMT). It is of further interest to determine whether mesenchymal-like, but not epithelial-like, cancer cells can become resistant to gefitinib by bypassing EGFR signaling and acquiring alternative routes of proliferative and survival signaling. Here we examined whether gefitinib resistance of cancer cells can be caused by transmembrane 4 L six family member 5 (TM4SF5), which has been shown to induce EMT via cytosolic p27Kip1 stabilization. Gefitinib-resistant cells exhibited higher and/or sustained TM4SF5 expression, cytosolic p27Kip1 stabilization, and mesenchymal phenotypes, compared with gefitinib-sensitive cells. Conversion of gefitinib-sensitive to -resistant cells by introduction of the T790M EGFR mutation caused enhanced and sustained expression of TM4SF5, phosphorylation of p27Kip1 Ser10 (responsible for cytosolic location), loss of E-cadherin from cell-cell contacts, and gefitinib-resistant EGFR and survival signaling activities. Additionally, TM4SF5 overexpression lessened the sensitivity of NSCLC cells to gefitinib. Suppression of TM4SF5 or p27Kip1 in gefitinib-resistant cells via the T790M EGFR mutation or TM4SF5 expression rendered them gefitinib-sensitive, displaying more epithelial-like and less mesenchymal-like characteristics. Together, these results indicate that TM4SF5-mediated EMT may have an important function in the gefitinib resistance of cancer cells.