Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors Resistance In Non-small Cell Lung Cancer Research Articles

Tumor-associated macrophages mediate resistance of EGFR-TKIs in non-small cell lung cancer: mechanisms and prospects.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the first-line standard treatment for advanced non-small cell lung cancer (NSCLC) with EGFR mutation. However, resistance to EGFR-TKIs is inevitable. Currently, most studies on the mechanism of EGFR-TKIs resistance mainly focus on the spontaneous resistance phenotype of NSCLC cells. Studies have shown that the tumor microenvironment (TME) also mediates EGFR-TKIs resistance in NSCLC. Tumor-associated macrophages (TAMs), one of the central immune cells in the TME of NSCLC, play an essential role in mediating EGFR-TKIs resistance. This study aims to comprehensively review the current mechanisms underlying TAM-mediated resistance to EGFR-TKIs and discuss the potential efficacy of combining EGFR-TKIs with targeted TAMs therapy. Combining EGFR-TKIs with TAMs targeting may improve the prognosis of NSCLC with EGFR mutation to some extent.

Epithelial-mesenchymal transition is associated with osteopontin-induced EGFR‑TKI resistance in EGFR mutant non-small cell lung cancer.

Resistance restricts the long-term therapeutic efficacy of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in the treatment of non-small cell lung cancer (NSCLC) with positive EGFR mutations. The present study sought to identify the potential protein osteopontin (OPN) involved in EGFR-TKI resistance and examine its therapeutic mechanism in NSCLC. The expression of OPN in NSCLC tissues was evaluated by immunohistochemistry (IHC). Western blot (WB), quantitative real‑time polymerase chain reaction (qRT-PCR), and immunofluorescence staining were used to analyze OPN and epithelial-mesenchymal transition (EMT)-related protein expression in the PC9 and PC9 gefitinib resistance (PC9GR) cells. Enzyme-linked immunosorbent assays (ELISAs) were used to detect the secreted OPN. Cell Counting Kit-8 (CCK-8) assays and flow cytometry were used to examine the effect of OPN on the gefitinib-induced growth and death of PC9 or PC9GR cells. OPN was upregulated in the human NSCLC tissues and cells resistant to EGFR-TKIs. The overexpression of OPN inhibited EGFR-TKI-induced apoptosis and was associated with the formation of EMT. By activating the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT)-EMT pathway, OPN contributed to the development of EGFR-TKI resistance. Reducing OPN expression and inhibiting PI3K/AKT signaling improved EGFR-TKI sensitivity significantly more than the use of either agent alone. This study showed that OPN increased EGFR-TKI resistance in NSCLC through the OPN-PI3K/AKT-EMT pathway. Our findings may provide a possible therapeutic target for overcoming EGFR-TKI resistance in this pathway.

LMNA Reduced Acquired Resistance to Erlotinib in NSCLC by Reversing the Epithelial-Mesenchymal Transition via the FGFR/MAPK/c-fos Signaling Pathway.

For patients exhibiting non-small-cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutations, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are a first-line treatment. However, most patients who initially responded to EGFR-TKIs eventually developed acquired resistance, limiting the effectiveness of therapy. It has long been known that epithelial-mesenchymal transition (EMT) leads to acquired resistance to EGFR-TKIs in NSCLC. However, the mechanisms underlying the resistance dependent on EMT are unknown. This research aimed to reveal the effects of LMNA in the regulation of acquired resistance to erlotinib by EMT in NSCLC. The acquired erlotinib-resistant cells (HCC827/ER) were induced by gradual increase of concentrations of erlotinib in erlotinib-sensitive HCC827 cells. RNA sequencing and bioinformatics analysis were performed to uncover the involvement of LMNA in the EMT process that induced acquired resistance to erlotinib. The effect of LMNA on cell proliferation and migration was measured by clone-formation, wound-healing, and transwell assays, respectively. The EMT-related protein, nuclear shape and volume, and cytoskeleton changes were examined by immunofluorescence. Western blot was used to identify the underlying molecular mechanism of LMNA regulation of EMT. HCC827/ER cells with acquired resistance to erlotinib underwent EMT and exhibited lower LMNA expression compared to parental sensitive cells. LMNA negatively regulated the expression of EMT markers; HCC827/ER cells showed a significant up-regulation of mesenchymal markers, such as CDH2, SNAI2, VIM, ZEB1, and TWIST1. The overexpression of LMNA in HCC827/ER cells significantly inhibited EMT and cell proliferation, and this inhibitory effect of LMNA was enhanced in the presence of 2.5 μM erlotinib. Furthermore, a decrease in LMNA expression resulted in a higher nuclear deformability and cytoskeletal changes. In HCC827/ER cells, AKT, FGFR, ERK1/2, and c-fos phosphorylation levels were higher than those in HCC827 cells; Furthermore, overexpression of LMNA in HCC827/ER cells reduced the phosphorylation of AKT, ERK1/2, c-fos, and FGFR. In conclusion, our findings first demonstrated that downregulation of LMNA promotes acquired EGFR-TKI resistance in NSCLC with EGFR mutations by EMT. LMNA inhibits cell proliferation and migration of erlotinib-resistant cells via inhibition of the FGFR/MAPK/c-fos signaling pathway. These findings indicated LMNA as a driver of acquired resistance to erlotinib and provided important information about the development of resistance to erlotinib treatment in NSCLC patients with EGFR mutations.

Artesunate promoted anti-tumor immunity and overcame EGFR-TKI resistance in non-small-cell lung cancer by enhancing oncogenic TAZ degradation

Lung cancer is the leading cause of cancer-related death worldwide. The development of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and immune checkpoint inhibitors (ICIs) has brought favorable survival benefits to patients with non–small-cell lung cancer (NSCLC); unfortunately, acquired drug resistance remains a major barrier to the treatment of NSCLC. Recent studies have demonstrated that the transcriptional co-activator with a PDZ-binding motif (TAZ, also called WWTR1) induces tumor immune evasion by directly modulating the expression of programmed death ligand 1 (PD-L1), a key therapeutic target for checkpoint immunotherapy. Moreover, aberrant activation of TAZ is also a major mechanism of acquired resistance to EGFR-TKIs in NSCLC. Therefore, TAZ signaling blockade might be an effective strategy to overcome resistance to ICIs and EGFR-TKIs in NSCLC. In this study, we showed for the first time that artesunate effectively reduced TAZ and PD-L1 expression in NSCLC. We further demonstrated that artesunate suppressed TAZ/PD-L1–induced T-cell growth inhibition in vitro and enhanced anti-tumor immunity by recruiting infiltrating CD8 + T-cells in syngeneic mouse models. Artesunate also inhibited the stem cell–like properties of NSCLC cells and suppressed tumor growth in xenografts bearing gefitinib-resistant tumors. In addition, our results of molecular docking and cellular thermal shift assay analysis suggested that artesunate might directly target the TAZ–TEAD complex and induce proteasome-dependent TAZ degradation in NSCLC cells. These results suggest that artesunate enhanced anti-tumor immunity and overcame EGFR-TKI resistance in NSCLC at least in part by suppressing TAZ/PD-L1 signaling.

Long non-coding RNA CASC9 promotes gefitinib resistance in NSCLC by epigenetic repression of DUSP1

Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, has greatly affected clinical outcomes in non-small cell lung cancer (NSCLC) patients. The long noncoding RNAs (lncRNAs) are known to regulate tumorigenesis and cancer progression, but their contributions to NSCLC gefitinib resistance remain poorly understood. In this study, by analyzing the differentially expressed lncRNAs in gefitinib-resistant cells and gefitinib-sensitive cells in the National Institute of Health GEO dataset, we found that lncRNA CASC9 expression was upregulated, and this was also verified in resistant tissues. Gain and loss of function studies showed that CASC9 inhibition restored gefitinib sensitivity both in vitro and in vivo, whereas CASC9 overexpression promoted gefitinib resistance. Mechanistically, CASC9 repressed the tumor suppressor DUSP1 by recruiting histone methyltransferase EZH2, thereby increasing the resistance to gefitinib. Furthermore, ectopic expression of DUSP1 increased gefitinib sensitivity by inactivating the ERK pathway. Our results highlight the essential role of CASC9 in gefitinib resistance, suggesting that the CASC9/EZH2/DUSP1 axis might be a novel target for overcoming EGFR-TKI resistance in NSCLC.

EMT-Mediated Acquired EGFR-TKI Resistance in NSCLC: Mechanisms and Strategies.

Acquired resistance inevitably limits the curative effects of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), which represent the classical paradigm of molecular-targeted therapies in non-small-cell lung cancer (NSCLC). How to break such a bottleneck becomes a pressing problem in cancer treatment. The epithelial-mesenchymal transition (EMT) is a dynamic process that governs biological changes in various aspects of malignancies, notably drug resistance. Progress in delineating the nature of this process offers an opportunity to develop clinical therapeutics to tackle resistance toward anticancer agents. Herein, we seek to provide a framework for the mechanistic underpinnings on the EMT-mediated acquisition of EGFR-TKI resistance, with a focus on NSCLC, and raise the question of what therapeutic strategies along this line should be pursued to optimize the efficacy in clinical practice.

Yiqi Chutan Tang Reduces Gefitinib-Induced Drug Resistance in Non-Small-Cell Lung Cancer by Targeting Apoptosis and Autophagy.

High incidence and mortality rates for non‐small‐cell lung cancer (NSCLC) lead to low survival rates. Epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKI) are commonly first prescribed for NSCLC patients with EGFR mutations. However, most patients with sensitizing EGFR mutations become resistant to EGFR‐TKI after 9–13 months treatment. Yiqi Chutan Tang (YQCT) has been prescribed as a treatment to this issue for over 20 years. In this report, high‐performance liquid chromatography (HPLC) analysis, flow cytometry, western blot analysis, and functional annotation analysis were applied to uncover the molecular mechanisms of YQCT. Our results show the application of YQCT reduces gefitinib‐induced drug resistance, induces slight cell cycle arrest, enhances gefitinib‐induced apoptosis, and activates the autophagy. These results indicate that at the molecular level YQCT can reduce drug resistance and improve anti‐cancer effects when associated with gefitinib, which could be a result of enhancement of apoptosis and autophagy in the EGFR‐TKI resistant cells of NSCLC. This research provides a new treatment strategy for patients with EGFR‐TKI resistance in NSCLC. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Research Progress of the Role of EMT in EGFR-TKIs Resistance of Non-small Cell Lung Cancer

肺癌是全世界范围内发病率和死亡率最高的恶性肿瘤之一，其中大约80%是非小细胞肺癌（non-small cell lung cancer, NSCLC）。目前分子靶向治疗已成为NSCLC的一线治疗方法，其中表皮生长因子受体-酪氨酸激酶抑制剂（epidermal growth factor receptor-tyrosine kinase inhibitors, EGFR-TKIs）越来越多地应用于临床治疗，但EGFR-TKI的获得性耐药成为制约EGFR-TKI继续使用的瓶颈。上皮间质转化（epithelial-mesenchymal transition, EMT）是上皮细胞转化为间质表型细胞的生物学现象，可促进肺癌转移、侵袭以及肿瘤细胞获得干性。此外，EMT也是引起NSCLC对EGFR-TKIs耐药的原因之一。有研究发现，逆转NSCLC细胞的间质表型，耐药的细胞能恢复对吉非替尼的敏感性，提示靶向EMT的治疗，或能阻止甚至是逆转NSCLC细胞对EGFR-TKIs的耐药，本文对EMT在NSCLC EGFR-TKIs耐药中的研究进展作一综述。

Mechanisms of Resistance to EGFR Tyrosine Kinase Inhibitors and Therapeutic Approaches: An Update.

Resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non small cell lung cancer (NSCLC) is mediated by two major mechanisms namely secondary mutation T790M in EGFR and cMET amplification. Other molecular mediators which contribute towards TKI resistance include the activation of compensatory growth signaling, epithelial mesenchymal transition and microRNAs regulating EGFR and cMET levels. In this chapter, we have included the major mechanisms which contribute towards EGFR TKI resistance in NSCLC. Several therapeutic approaches to overcome TKI resistance are also presented which include second and third generation EGFR TKI inhibitors and cMET inhibitors. Further, the rationale to utilize the combination therapies to simultaneously target EGFR and other major oncogene addictive pathway such as ERBB2 and AXL kinase is outlined. Another promising approach to overcome TKI resistance is to potentiate EGFR protein for degradation. These studies will best be utilized when we can identify the oncogene addictions in an individual patient and tailor the therapy/therapies accordingly for the maximum benefits.

Implication of epithelial-mesenchymal transition in IGF1R-induced resistance to EGFR-TKIs in advanced non-small cell lung cancer.

The underlying mechanisms for acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in about 30%-40% of non-small cell lung cancer (NSCLC) patients remain elusive. Recent studies have suggested that activation of epithelial-mesenchymal transition (EMT) and type 1 insulin-like growth factor receptor (IGF1R) is associated with acquired EGFR-TKIs resistance in NSCLC. Our study aims to further explore the mechanism of EMT and IGF1R in acquired EGFR-TKIs resistance in NSCLC cell lines with mutant (PC-9) or wild-type EGFR (H460). Compared to parental cells, EGFR-TKIs-resistant PC-9/GR and H460/ER cells displayed an EMT phenotype and showed overexpression of IGF1R. SiIGF1R in PC-9/GR and H460/ER cells reversed EMT-related morphologies and reversed their resistance to EGFR-TKIs. Exogenous IGF-1 alone induced EMT in EGFR-TKIs-naïve PC-9 and H460 cells and increased their resistance against EGFR-TKIs. Inducing EMT by TGF-β1 in PC-9 and H460 cells decreased their sensitivity to EGFR-TKIs, whereas reversing EMT by E-cadherin overexpression in PC-9/GR and H460/ER cells restored their sensitivity to EGFR-TKIs. These data suggest that IGF1R plays an important role in acquired drug resistance against EGFR-TKIs by inducing EMT. Targeting IGF1R and EMT may be a potential therapeutic strategy for advanced NSCLC with acquired EGFR-TKIs resistance.

Abstract 765: Overcoming acquired EGFRi resistance in NSCLC with targeted beam irradiation in combination with targeted agents

Abstract BACKGROUND: Non-small cell lung cancer (NSCLC) patients undergo primary, adjuvant or neoadjuvant radiotherapy treatment with image-guided radiotherapy (IGRT) being widely used to provide more accurate treatment plans and reduced side effects. Patients who have activating mutations in the epidermal growth factor receptor (EGFR) gene are treated with EGFR tyrosine kinase inhibitors (EGFRi). However, resistance to EGFRi occurs in the majority of patients with the main clinical drivers being secondary gatekeeper mutations (T790M) or amplification of genes such as c-MET and Her2. The small animal radiation research platform (SARRP), which allows the treatment of rodent models of cancer more accurately with planned protocols similar to those utilised in the clinic, was used in combination with EGFRi, to assess the efficacy of combination treatment strategies of irradiation (IR) in EGFRi resistant NSCLC xenografts. METHODS: Resistance to EGFRi in the NSCLC cell line HCC827, which harbours an activating EGFR mutation, was generated through repeated exposure to Gefitinib or Erlotinib. Resistant models were characterised for further mutations in the EGFR gene by direct sequencing and for c-MET, Axl and Her2 over-expression/genomic amplification by quantitative PCR. EGFRi resistant or parental HCC827 cells were implanted subcutaneously into nude mice, xenograft growth was monitored by calliper measurements and treatment initiated when the mean tumour volume reached ∼200mm3. An image guided IR dose of 3Gy/mouse/day (30Gy total) was targeted directly to the xenografts whilst sparing the surrounding normal tissue. Mice were treated with IR either alone or in combination with Erlotinib or Crizotinib; response to treatment was evaluated by tumour volume measurement. RESULTS: EGFR-TKI resistant HCC827 cell lines were generated following successive cycles of treatment. Amplification of c-MET was identified in a number of clones, but no Her2 amplification or gatekeeper mutations; therefore Crizotinib, a c-MET inhibitor, was selected for evaluation in these models. When assessed in vivo, treatment of parental HCC827 xenografts with targeted IR or Erlotinib treatment resulted in tumour regression. However, in the c-MET driven resistant model, both the IR and Erlotinib response was significantly attenuated, resulting in tumour re-growth. Combination testing with Crizotinib restored the efficacy of both IR and Erlotinib to naïve treatment levels confirming the role of c-MET in resistance. CONCLUSIONS: Resistance to EGFRi and IR in a HCC827 model is driven by c-MET amplification. Treatment with Crizotinib restored sensitivity to both EGFRi and IR demonstrating that pre-clinical models of resistance are invaluable in assessing novel agents targeting the EGFR pathway and the development of new combination strategies which seek to prevent/overcome resistance to EGFRi. Citation Format: Andrew McKenzie, Nektaria Papadopoulou, Lucy Page, Simon Jiang, Jane Wrigley, Yinfei Yin, Qian Shi, Martin Page, Ian Wilson, Rajendra Kumari. Overcoming acquired EGFRi resistance in NSCLC with targeted beam irradiation in combination with targeted agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 765. doi:10.1158/1538-7445.AM2015-765

Histone Deacetylation, Instead of Promoter Methylation, Results in the Epigenetic Silencing of Bim and Resistance to Egfr Tki in Nsclc

ABSTRACT Aim: With targeted therapy improving the survival of advanced stage epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) patients, resistannce remains a major issue in the application of EGFR tyrosine kinase inhibitors (TKIs). Bim, a Bcl-2 family pro-apoptotic protein, has been intensively studied for its role in various targeted therapies. Hence we investigated the epigenetic regulation of Bim in NSCLC cell lines and patients in the hope of identifying its relationship with EGFR TKI resistance. Methods: Methylation specific PCR, pyrosequencing and nested quantitative MSP (nested q-MSP) were applied to study the methylation status of BIM in NSCLC cell lines and the BIM methylation profile in NSCLC patients was assessed by nested q-MSP. PC9 and its gefitinib-resistant cell lines were treated with methylation inhibitor 5-aza-2′-deoxycytidine (AZA) and histone deacetylation inhibitor trichostatin A (TSA) before gefitinib treatment. Bim RNA level, cell survival and resistance to gefitinib were examined. The distribution of various levels of methylated BIM and their relationship with EGFR TKI efficacy was statistically analyzed. Results: 8 NSCLC cell lines all carried hypo-methylated BIM. AZA did not increase the expression of Bim in PC9 and its gefitinib-resistant cell lines, nor did it reverse their resistance to gefitinib. Instead, cells experienced less apoptosis from gefitinib when pre-treated with AZA. TSA on the other hand, not only increased the expression of Bim RNA significantly in the three cell lines, but also reversed their resistance to gefitinib. 25 (78.1%) patients with hypo-methylated BIM and 7 (21.9%) with partial or hyper-methylated BIM were identified. Hypo-methylated BIM distributed randomly in different clinicalpathological charcteristics. In both the entire study group and EGFR mutant group, hypo-methylated BIM carriers experienced no significant PFS difference compared with patients with partial or hyper-methylated BIM. Conclusions: All cell lines in the study and the majority of patients were with hypo-methylated BIM. AZA didn't reverse the resistance to EGFR TKI in acquired resistant cell lines PC9/R and PC9/G2, while HDACi TSA did. Thus histone deacetylation, rather than promoter methylation, may play a major role in the epigenetic silencing of BIM and leads to EGFR TKI resistance in NSCLC. Disclosure: All authors have declared no conflicts of interest.

The role of IGF-1R in EGFR TKI resistance in NSCLC using IHC and AQUA technology.

10556 Background: Epidermal growth factor receptor (EGFR) mutations predict benefit from EGFR tyrosine kinase inhibitors (TKI) in non-small cell lung cancer (NSCLC), however, only 70-80% will respo...

Abstract 626: Therapeutic effect of HGF inhibitors against HGF-induced EGFR-TKI resistance in lung cancer harboring EGFR mutations

Abstract Introduction: Non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR)-activating mutations (EGFRmu) responds favorably to the EGFR tyrosine kinase inhibitors (EGFR-TKIs), gefitinib or erlotinib. However, the responders invariably acquire resistance to EGFR-TKIs. Two mechanisms, second-site point mutation that substitutes methionine for threonine at position 790 (T790M) in EGFR and amplification of MET proto-oncogene, which contribute to acquired resistance to EGFR-TKIs have been reported. As the third mechanism, we recently demonstrated that hepatocyte growth factor (HGF), specific ligand for MET, induced resistance to EGFR-TKIs. The purpose of this study was to examine whether inhibitor of HGF could overcome resistance to EGFR-TKIs induced by HGF in NSCLC cells with EGFRmu. Results: HGF induced the resistance to gefitinib and erlotinib in lung cancer cells (PC-9 and HCC827) with EGFRmu, by restoring Akt phosphorylation through MET but not EGFR or ErbB3. These NSCLC cells also became resistant to EGFR-TKIs when co-cultured in vitro with HGF-producing fibroblasts and co-injected into SCID mice. Importantly, combined use of gefitinib plus anti-HGF antibody or the HGF antagonist, NK4 successfully overcame the fibroblast-induced EGFR-TKI resistance both in vitro and in vivo. Conclusions: These findings indicate that HGF inhibitor, such as anti-HGF antibody and NK4, may be useful for controlling HGF-induced EGFR-TKI resistance in NSCLC harboring EGFRmu in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 626.

Addition of S-1 to the Epidermal Growth Factor Receptor Inhibitor Gefitinib Overcomes Gefitinib Resistance in Non–small cell Lung Cancer Cell Lines withMETAmplification

Most non-small cell lung cancer (NSCLC) tumors with activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to EGFR tyrosine kinase inhibitors (EGFR-TKI) such as gefitinib and erlotinib, but they almost invariably develop resistance to these drugs. A secondary mutation in EGFR (T790M) and amplification of the MET proto-oncogene have been identified as mechanisms of such acquired resistance to EGFR-TKIs. We have now investigated whether addition of the oral fluoropyrimidine derivative S-1 to gefitinib might overcome gefitinib resistance in NSCLC cell lines. The effects of gefitinib on EGFR signaling and on the expression both of thymidylate synthase and of the transcription factor E2F-1 in gefitinib-resistant NSCLC cells were examined by immunoblot analysis. The effects of S-1 (or 5-fluorouracil) and gefitinib on the growth of NSCLC cells were examined in vitro as well as in nude mice. Gefitinib induced down-regulation of thymidylate synthase and E2F-1 in gefitinib-resistant NSCLC cells with MET amplification but not in those harboring the T790M mutation of EGFR. The combination of 5-fluorouracil and gefitinib synergistically inhibited the proliferation of cells with MET amplification, but not that of those with the T790M mutation of EGFR, in vitro. Similarly, the combination of S-1 and gefitinib synergistically inhibited the growth only of NSCLC xenografts with MET amplification. Our results suggest that the addition of S-1 to EGFR-TKIs is a promising strategy to overcome EGFR-TKI resistance in NSCLC with MET amplification.