Afatinib-resistant Cell Lines Research Articles

Molecular profiling of afatinib-resistant non-small cell lung cancer cells in vivo derived from mice

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. NSCLC patients with overexpressed or mutated epidermal growth factor receptor (EGFR) related to disease progression are treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). Acquired drug resistance after TKI treatments has been a major focus for development of NSCLC therapies. This study aimed to establish afatinib-resistant cell lines from which afatinib resistance-associated genes are identified and the underlying mechanisms of multiple-TKI resistance in NSCLC can be further investigated. Nude mice bearing subcutaneous NSCLC HCC827 tumors were administered with afatinib at different dose intensities (5–100 mg/kg). We established three HCC827 sublines resistant to afatinib (IC50 > 1 μM) with cross-resistance to gefitinib (IC50 > 5 μM). cDNA microarray revealed several of these sublines shared 27 up- and 13 down-regulated genes. The mRNA expression of selective novel genes — such as transmembrane 4 L six family member 19 (TM4SF19), suppressor of cytokine signaling 2 (SOCS2), and quinolinate phosphoribosyltransferase (QPRT) — are responsive to afatinib treatments only at high concentrations. Furthermore, c-MET amplification and activations of a subset of tyrosine kinase receptors were observed in all three resistant cells. PHA665752, a c-MET inhibitor, remarkably increased the sensitivity of these resistant cells to afatinib (IC50 = 12–123 nM). We established afatinib-resistant lung cancer cell lines and here report genes associated with afatinib resistance in human NSCLC. These cell lines and the identified genes serve as useful investigational tools, prognostic biomarkers of TKI therapies, and promising molecule targets for development of human NSCLC therapeutics.

Inhibition of osteopontin overcomes acquired resistance to afatinib in EGFR-mutant non-small-cell lung cancer.

BackgroundWe aimed to explore a novel therapeutic strategy to conquer acquired resistance to second generation EGFR-TKI afatinib in EGFR-mutant NSCLC.MethodsFirstly, we established afatinib-resistant cell lines using increasing concentrations of afatinib. Secondly, we over-expressed or silenced the expression of osteopontin (OPN) using in-vitro transfection. Further, western blot analysis was used to detect the expressions of OPN and epithelial-mesenchymal transition (EMT) biomarkers. Finally, cell proliferation was evaluated by MTT assay.ResultsAfatinib (≤5.0 µmol/L)-resistant H1650 (H1650-AR) and H1975 (H1975-AR) cells were successfully established, and grew faster compared with both parental cells at the same time interval. Western blot analysis revealed that afatinib significantly promoted the expressions of OPN and EMT biomarkers in H1975-AR and H1650-AR cells. Gain and loss assays validated that OPN over-expression promoted acquired resistance to afatinib, and induced the expressions of EMT biomarkers in H1650-AR and H1975-AR cells. Conversely, silencing of OPN not only significantly sensitized resistant cells to afatinib, but also suppressed EMT progression in H1650-AR and H1975-AR cells.ConclusionsThese results demonstrated that OPN was required for acquired resistance of EGFR-mutant NSCLC cells to afatinib.

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib.

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

Abstract 1832: Acquired resistance mechanisms to afatinib in lung cancer cells harboring HER2 alterations

Abstract Human epidermal growth factor receptor 2 (HER2) plays an important role in the pathogenesis of various cancers. HER2 alterations have been suggested to be a therapeutic target in non-small cell lung cancer (NSCLC), just as in breast and gastric cancers. We previously reported that the pan-HER inhibitor afatinib could be a useful therapeutic agent as HER2-targeted therapy for patients with NSCLC harboring HER2 alterations. However, acquired resistance to afatinib was observed in the clinical setting, similar to the case for other HER inhibitors. Thus, elucidation of the mechanisms underlying the development of acquired drug resistance and exploring means to overcome acquired drug resistance are important issues in the treatment of NSCLC. In this study, we experimentally established afatinib-resistant cell lines from NSCLC cell lines harboring HER2 alterations (Calu3, H2170, and H1781), by stepwise-escalation or high-concentration exposure methods, and investigated the mechanisms underlying the acquisition of drug resistance. The established cell lines showed several unique afatinib-resistance mechanisms, including MET amplification, loss of HER2 amplification and gene expression, epithelial-to-mesenchymal transition (EMT) and acquisition of cancer stem cell (CSC)-like features. The afatinib-resistant cell lines showing MET amplification were sensitive to the combination of afatinib plus crizotinib (a MET inhibitor), both in vitro and in vivo. The resistant cell lines with the loss of HER2 amplification obtained MEK inhibitors sensitivity in vitro and in vivo. Other resistant cell lines that showed EMT or had acquired CSC-like features remained sensitive to docetaxel, like the parental cells. These findings may provide clues to countering the resistance to afatinib in NSCLC patients with HER2 alterations. Citation Format: Hidejiro Torigoe, Kazuhiko Shien, Tatsuaki Takeda, Takahiro Yoshioka, Kei Namba, Hiroki Sato, Ken Suzawa, Hiromasa Yamamoto, Junichi Soh, Masakiyo Sakaguchi, Shuta Tomida, Shinichiro Miyoshi, Shinichi Toyooka. Acquired resistance mechanisms to afatinib in lung cancer cells harboring HER2 alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1832.

Acquisition of cancer stem cell-like properties in non-small cell lung cancer with acquired resistance to afatinib.

Afatinib is an irreversible epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) that is known to be effective against the EGFR T790M variant, which accounts for half of the mechanisms of acquired resistance to reversible EGFR-TKIs. However, acquired resistance to afatinib was also observed in clinical use. Thus, elucidating and overcoming the mechanisms of resistance are important issues in the treatment of non-small cell lung cancer. In this study, we established various afatinib-resistant cell lines and investigated the resistance mechanisms. EGFR T790M mutations were not detected using direct sequencing in established resistant cells. Several afatinib-resistant cell lines displayed MET amplification, and these cells were sensitive to the combination of afatinib plus crizotinib. As a further investigation, a cell line that acquired resistance to afatinib plus crizotinib, HCC827-ACR, was established from one of the MET amplified-cell lines. Several afatinib-resistant cell lines including HCC827-ACR displayed epithelial-to-mesenchymal transition (EMT) features and epigenetic silencing of miR-200c, which is a suppresser of EMT. In addition, these cell lines also exhibited overexpression of ALDH1A1 and ABCB1, which are putative stem cell markers, and resistance to docetaxel. In conclusion, we established afatinib-resistant cells and found that MET amplification, EMT, and stem cell-like features are observed in cells with acquired resistance to EGFR-TKIs. This finding may provide clues to overcoming resistance to EGFR-TKIs.

Inhibition of IGF1R signaling abrogates resistance to afatinib (BIBW2992) in EGFR T790M mutant lung cancer cells.

Non-small cell lung cancer (NSCLC) patients with an epidermal growth factor receptor (EGFR) mutation have benefited from treatment of reversible EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib. Acquisition of a secondary mutation in EGFR T790M is the most common mechanism of resistance to first generation EGFR TKIs, resulting in therapeutic failure. Afatinib is a second generation of EGFR TKI that showed great efficacy against tumors bearing the EGFR T790M mutation, but it failed to show the improvement on overall survival of lung cancer patients with EGFR mutations possibly because of novel acquired resistance mechanisms. Currently, there are no therapeutic options available for lung cancer patients who develop acquired resistance to afatinib. To identify novel resistance mechanism(s) to afatinib, we developed afatinib resistant cell lines from a parental human-derived NSCLC cell line, H1975, harboring both EGFR L858R and T790M mutations. We found that activation of the insulin-like growth factor 1 receptor (IGF1R) signaling pathway contributes to afatinib resistance in NSCLC cells harboring the T790M mutation. IGF1R knockdown not only significantly sensitizes resistant cells to afatinib, but also induces apoptosis in afatinib resistance cells. In addition, combination treatment with afatinib and linsitinib shows more than additive effects on tumor growth in in vivo H1975 xenograft. Therefore, these finding suggest that IGF1R inhibition or combination of EGFR-IGF1R inhibition strategies would be potential ways to prevent or potentiate the effects of current therapeutic options to lung cancer patients demonstrating resistance to either first or second generation EGFR TKIs.

Abstract 1834: The molecular characters of acquired resistant non-small cell lung cancer cells to afatinib

Abstract [Background] The non-small cell lung carcinomas (NSCLCs) with activating epidermal growth factor receptor (EGFR) gene mutation show significant response to EGFR-tyrosine kinase inhibitors (TKIs). However, almost all of them acquire resistance to EGFR-TKIs due to various mechanisms, such as T790M mutation or MET amplification. Afatinib is an irreversible TKI for EGFR and HER2 and known to be effective to the EGFR T790M variant. However, a half of the patient responded afatinib acquire resistance by 12 months in clinical use. Thus, to unravel the mechanisms of the resistances and to overcome them are still important issues of EGFR-TKI treatment. In this study, we established various kinds of afatinib resistant cell lines with changing drug exposing condition as we previously reported (Shien K. et al Cancer Res. 2013). We also investigated these cell lines to understand mechanisms of afatinib resistances in NSCLC. [Materials and Methods] Afatinib resistant cell lines were established using four NSCLC cell lines with activating EGFR mutations by exposing afatinib with different procedures, escalation from 1 nM, escalation from 10 nM, intermittent 2 µM and continuous 2 µM procedures. These cell lines were investigated about previously reported resistant related features, T790M mutation, MET amplification, and epithelial to mesenchymal transition (EMT). ALDH1A1 and ABCB1 expression, which were putative stem cell markers, were also investigated. Proliferative inhibitions with various drugs were evaluated using MTS assay. [Results] There were no T790M mutations in all afatinib resistant cell lines. HCC827 sublines exposed afatinib with escalation from 1 nM and intermittent 2 µM, and HCC4011 sublines showed MET amplification. HCC827 sublines exposed afatinib with escalation from 10 nM and continuous 2 µM, and HCC4006 sublines showed EMT features. Furthermore, ALDH1A1 and ABCB1 expression were up-regulated in HCC827 afatinib resistant sublines with EMT features. Next, we examined the effect of afatinib or crizotinib for the afatinib resistant cell line with MET amplification. We found that combination therapy with afatinib and crizotinib was effective to the MET amplified cell. The combination exposure was continued to establish a resistant subline (HCC827-ACR) to combination therapy. The HCC827-ACR was also showed EMT features and up-regulation of stem cell markers. [Conclusions] As in the cases of reversible EGFR-TKI, MET amplification, EMT, and stem cell-like features were emerged in afatinib resistant cells. To develop strategies to resistant cells with stem cell-like property may be critical part to overcome EGFR-TKI resistance. Citation Format: Shinsuke Hashida, Shinichi Toyooka, Tomoaki Ohtsuka, Ken Suzawa, Kazuhiko Shien, Hiromasa Yamamoto, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi. The molecular characters of acquired resistant non-small cell lung cancer cells to afatinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1834. doi:10.1158/1538-7445.AM2014-1834

FGFR1 activation is an escape mechanism in human lung cancer cells resistant to afatinib, a pan-EGFR family kinase inhibitor.

Most NSCLC patients with EGFR mutations benefit from treatment with EGFR-TKIs, but the clinical efficacy of EGFR-TKIs is limited by the appearance of drug resistance. Multiple kinase inhibitors of EGFR family proteins such as afatinib have been newly developed to overcome such drug resistance. We established afatinib-resistant cell lines after chronic exposure of activating EGFR mutation-positive PC9 cells to afatinib. Afatinib-resistant cells showed following specific characteristics as compared to PC9: [1] Expression of EGFR family proteins and their phosphorylated molecules was markedly downregulated by selection of afatinib resistance; [2] Expression of FGFR1 and its ligand FGF2 was alternatively upregulated; [3] Treatment with anti-FGF2 neutralizing antibody blocked enhanced phosphorylation of FGFR in resistant clone; [4] Both resistant clones showed collateral sensitivity to PD173074, a small-molecule FGFR-TKIs, and treatment with either PD173074 or FGFR siRNA exacerbated suppression of both afatinib-resistant Akt and Erk phosphorylation when combined with afatinib; [5] Expression of twist was markedly augmented in resistant sublines, and twist knockdown specifically suppressed FGFR expression and cell survival. Together, enhanced expression of FGFR1 and FGF2 thus plays as an escape mechanism for cell survival of afatinib-resistant cancer cells, that may compensate the loss of EGFR-driven signaling pathway.

Abstract 5649: Investigational study of acquired resistance to the EGFR irreversible inhibitor afatinib (BIBW2992) in wild-type and EGFR-mutant NSCLC cell lines.

Abstract Introduction: Non-small cell lung cancer (NSCLC) represents about 85% of all lung cancers. Almost 20% of NSCLC tumors harbor somatic activating mutations of the tyrosine kinase (TK) encoding domain in EGFR gene (exons 18-21). Patients with EGFR mutations treated with specific inhibitors of EGFR-TKI such as gefitinib and erlotinib demonstrated a significantly longer survival. Nevertheless, the majority of patients acquire resistance to these drugs within one year due to a second-site mutation in EGFR (T790M). The irreversible EGFR-TKI, afatinib, has been found to be effective in inhibiting the growth of NSCLC cells with the T790M mutation of EGFR. In this study, we generated different NSCLC cell lines resistant to afatinib to investigate the biological and molecular mechanisms of acquired resistance. Methods: A dose-escalation study to establish afatinib-resistant cell lines was performed in 3 NSCLC cell lines with a different EGFR mutational status: A549 (EGFR-wild type); H-1650 (exon19 delE746-A750); H-1975 (exon21 L858R/exon20 T790M). EGFR exons (18-21) from parental and resistant cells were sequenced by Sanger method while the EGF pathway was studied by qPCR using TaqMan® Array EGF Pathway and relative expression values of 92 genes, compared to parental cells, were obtained by 2−ddCT. Concomitantly, protein expression of some relevant genes such as EGFR and AKT, both phosphorylated and unphosphorylated, were investigated by western blot (WB). Results: All 3 cell lines exhibited different degree of drug resistance and actively proliferated under persistent afatinib pressure (A549: 8.5μM; H-1650: 1.0μM; H-1975: 5.0μM) as compared to parental cultures. WB reported active phosphorylation of EGFR and AKT in resistant cells regardless of EGFR mutational status. This behavior also persisted in absence of EGF stimulation and in cultures maintained in afatinib-free medium for over 2 months. However none of the resistant cell lines harbored new EGFR mutation in the 4 exons (18-21). Gene expression analysis showed an increase of some members of RAS (MRAS, KRAS) and Rho families and also PI3K regulatory subunit 1 (PIK3R1) in A549 and H1975 cells. Notably, SHC3 (Src homology 2 domain containing; transforming protein 3) was up-regulated about 60-fold in H1975 whereas proto-oncogene JUN showed a 6-fold increase in H1650. In addition EGF was down-modulated in both H1650 and H1975. Conclusion: Silencing of EGF in resistant cells together with the EGFR and AKT phosphorylation, suggest that these cultures acquired a different way to activate the EGF pathway. Furthermore in H-1975 the markedly increase of SHC3, an adaptor immediately downstream of EGFR, leads to hypothesize that it could be implicated in the activation of EGFR. Further investigations of SHC3 involvement in EGF pathway activation as well as sequencing analysis of the whole EGFR gene are ongoing. Citation Format: Angela Alama, Simona Coco, Zita Cavalieri, Anna Truini, Cristina Bruzzo, Mariella Dono, Francesco Grossi. Investigational study of acquired resistance to the EGFR irreversible inhibitor afatinib (BIBW2992) in wild-type and EGFR-mutant NSCLC cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5649. doi:10.1158/1538-7445.AM2013-5649

Malpas joins SpecialChem's board of advisers

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. NSCLC patients with overexpressed or mutated epidermal growth factor receptor (EGFR) related to disease progression are treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). Acquired drug resistance after TKI treatments has been a major focus for development of NSCLC therapies. This study aimed to establish afatinib-resistant cell lines from which afatinib resistance-associated genes are identified and the underlying mechanisms of multiple-TKI resistance in NSCLC can be further investigated. Nude mice bearing subcutaneous NSCLC HCC827 tumors were administered with afatinib at different dose intensities (5–100 mg/kg). We established three HCC827 sublines resistant to afatinib (IC50 > 1 μM) with cross-resistance to gefitinib (IC50 > 5 μM). cDNA microarray revealed several of these sublines shared 27 up- and 13 down-regulated genes. The mRNA expression of selective novel genes — such as transmembrane 4 L six family member 19 (TM4SF19), suppressor of cytokine signaling 2 (SOCS2), and quinolinate phosphoribosyltransferase (QPRT) — are responsive to afatinib treatments only at high concentrations. Furthermore, c-MET amplification and activations of a subset of tyrosine kinase receptors were observed in all three resistant cells. PHA665752, a c-MET inhibitor, remarkably increased the sensitivity of these resistant cells to afatinib (IC50 = 12–123 nM). We established afatinib-resistant lung cancer cell lines and here report genes associated with afatinib resistance in human NSCLC. These cell lines and the identified genes serve as useful investigational tools, prognostic biomarkers of TKI therapies, and promising molecule targets for development of human NSCLC therapeutics.