Cancer Res-2013-Shien-0008-5472.CAN-12-4136

Published OnlineFirst March 29, 2013.Cancer Res Kazuhiko Shien, Shinichi Toyooka, Hiromasa Yamamoto, et al. manifestation of stem cell-like properties in cancer cells. Acquired resistance to EGFR inhibitors is associated with a Updated version 10.1158/0008-5472.CAN-12-4136doi: Access the most recent version of this article at: Material Supplementary http://cancerres.aacrjournals.org/content/suppl/2013/03/29/0008-5472.CAN-12-4136.DC1.html Access the most recent supplemental material at: Manuscript Author edited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been E-mail alerts related to this article or journal.Sign up to receive free email-alerts Subscriptions Reprints and .pubs@aacr.orgDepartment at To order reprints of this article or to subscribe to the journal, contact the AACR Publications Permissions .permissions@aacr.orgDepartment at To request permission to re-use all or part of this article, contact the AACR Publications on April 9, 2013. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2013; DOI: 10.1158/0008-5472.CAN-12-4136 1 Acquired resistance to EGFR inhibitors is associated with a manifestation of stem cell-like properties in cancer cells Authors: Kazuhiko Shien1, Shinichi Toyooka1, Hiromasa Yamamoto1, Junichi Soh1, Masaru Jida1, Kelsie L. Thu2, Shinsuke Hashida1, Yuho Maki1, Eiki Ichihara3, Hiroaki Asano1, Kazunori Tsukuda1, Nagio Takigawa4, Katsuyuki Kiura3, Adi F. Gazdar5, Wan L. Lam2, Shinichiro Miyoshi1 Affiliations: 1Department of Thoracic Surgery, Okayama University Hospital, Okayama 700-8558, Japan. 2Department of Integrative Oncology, British Columbia Cancer Research Centre, Vancouver, BC V5Z 1L3, Canada. 3Department of Respiratory Medicine, Okayama University Hospital, Okayama 700-8558, Japan. on April 9, 2013. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2013; DOI: 10.1158/0008-5472.CAN-12-4136 2 4Department of General Internal Medicine 4, Kawasaki Medical School, Okayama 700-8505 Japan. 5Hamon Center for Therapeutic Oncology Research and Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Corresponding Author: Shinichi Toyooka, Department of Thoracic Surgery, Okayama University Hospital, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. Phone: +81-86-235-7265; Fax: +81-86-235-7269; E-mail: toyooka@md.okayama-u.ac.jp Running title: Stem cells and acquired resistance to EGFR inhibitors. Key words: non-small cell lung cancer, EGFR mutation, tyrosine kinase inhibitor, acquired resistance, cancer-stem cell Disclosure of potential conflict of interest: The authors disclose no potential conflicts of interest. Word count: 4,327 words. Total number of figures and tables: 4 figures, 3 tables, 5 supplementary figures, 2 supplementary tables, and 1 supplementary method. on April 9, 2013. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2013; DOI: 10.1158/0008-5472.CAN-12-4136 3 Abstract: Acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is a critical problem in the treatment of lung cancer. While several mechanisms have been demonstrated to be responsible for acquired resistance, all mechanisms have not been uncovered. In this study, we investigated the molecular and cellular profiles of the acquired resistant cells to EGFR-TKI in EGFR mutant lung cancers. Four EGFR-mutant cell lines were exposed to gefitinib by stepwise escalation and high-concentration exposure methods, and resistant sublines to gefitinib were established. The molecular profiles and cellular phenotypes of these resistant sublines were characterized. Although previously reported alterations including secondary EGFR T790M mutation, MET amplification, and appearance of epithelial to mesenchymal transition (EMT) features were observed, these 2 drug-exposure methods revealed different resistance mechanisms. The resistant cells with EMT features exhibited down-regulation of microRNA-200c by DNA methylation. Furthermore, the HCC827-derived subline characterized by the high-concentration exposure method exhibited not only EMT features but also stem cell-like properties, including aldehyde dehydrogenase isoform 1 (ALDH1A1) overexpression, increase of side-population, and self-renewal capability. Resistant sublines with stem cell-like properties were resistance on April 9, 2013. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2013; DOI: 10.1158/0008-5472.CAN-12-4136 4 to conventional chemotherapeutic agents but equally sensitive to histone deacetylase and proteasome inhibitors, compared with their parental cells. ALDH1A1 was up-regulated in clinical samples with acquired resistance to gefitinib. In conclusion, our study indicates that the manner of EGFR-TKI exposure influences the mechanism of acquired resistance and the appearance of stem cell-like property with EGFR-TKI treatment. on April 9, 2013. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2013; DOI: 10.1158/0008-5472.CAN-12-4136 5 Introduction Epidermal growth factor receptor (EGFR) mutations are oncogenic alterations in non-small cell lung cancer (NSCLC) (1, 2). First-generation EGFR-tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, have exhibited significant antiproliferative effects against NSCLC with EGFR mutations in preclinical studies (1, 2) and have also resulted in prolonged disease-free survival in randomized phase III studies (3-5). However, patients with EGFR-activating mutations who initially respond to EGFR-TKIs eventually acquire resistance, which is a critical problem in the treatment of patients with advanced NSCLC. Several mechanisms are believed to be responsible for acquired resistance to EGFR-TKI, including secondary EGFR T790M and minor mutations, MET amplification and activation of MET/HGF axis, acquiring an epithelial to mesenchymal transition (EMT) signature, and transformation from NSCLC into small cell lung cancer (SCLC) (6-11). More recently, AXL kinase activation and loss of the EGFR-mutant allele have been reported as possible mechanisms of resistance, but it is likely that additional mechanisms remain to be identified (12, 13). Establishment of resistant sublines is a common experimental method to investigate the mechanism of drug resistance. The properties of resistant cells can vary according to the experimental methods used in the developing process. For example, in on April 9, 2013. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2013; DOI: 10.1158/0008-5472.CAN-12-4136 6 endometrial cancer, cisplatin-resistant cells established by stepwise escalation exposure and high-concentration exposure methods showed different cellular properties that may be either a cause or result of drug resistance (14). This fact suggests that the mechanism of EGFR-TKI resistance may vary according to in vitro culture conditions, resulting in finding of novel features of resistant cells. While the majority of previously reported cells that were resistant to EGFR-TKI were established with stepwise escalation of EGFR-TKI concentration, we successfully established resistant cells with the high-concentration exposure method as well as the stepwise escalation method, and identified novel features of cells resistant to EGFR-TKI. The purposes of this study were to investigate the acquired mechanism of resistance to EGFR-TKI and to explore strategies to overcome resistance to EGFR-TKI. Materials and Methods Cell lines and reagents EGFR-mutant HCC827 (exon19del E746–A750), PC-9 (exon19del E746–A750), HCC4006 (exon19del L747–E749), and HCC4011 (L858R) cells were used. These cell lines except for PC-9 were established by one of the authors (AFG). PC-9 was obtained on April 9, 2013. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2013; DOI: 10.1158/0008-5472.CAN-12-4136 7 from Immuno-Biological Laboratories (Takasaki, Gunma, Japan). All the cell lines were cultured in RPMI 1640 media supplemented with 10 % fetal bovine serum (FBS), and grown in a humidified incubator with 5 % CO2 at 37°C. EGFR-TKI resistant sublines were established by two different methods: parental cells were cultured with stepwise escalation of concentrations of gefitinib from 5 nM to 2 µM over 6 months (stepwise escalation method), or initially high concentration of gefitinib (2 µM) over 6 months (high-concentration method). Finally, gefitinib-resistant sublines named as HCC827-GR-step, PC-9-GR-step, HCC4006-GR-step, and HCC4011-GR-step were established by stepwise escalation method, and HCC827-GR-high1, HCC827-GR-high2, PC-9-GR-high, HCC4006-GR-high, and HCC4011-GR-high were established by high-concentration method. Regarding HCC827-GR-high1 and high2, these two sublines were independently established from different cultures with high-concentration method. The identities of all the parental and resistant cells were confirmed by analyzing the short tandem repeat profile using the Cell ID System (Promega, Madison, WI) and ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions. Clonal resistant cells were isolated by limiting dilution. on April 9, 2013. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2013; DOI: 10.1158/0008-5472.CAN-12-4136 8 Details about the reagents used in the cell proliferation assay and the antibodies for Western blot analysis, fluorescence immunocytochemistry, and immunohistochemistry are included in Supplementary Methods. Determination of cell proliferation Cell proliferation was determined by a modified MTS assay with CellTiter® 96 Aqueous One Solution Reagent (Promega) as previously reported (15). The antiproliferative effects are shown as IC50, which is the concentration of the drug required to inhibit cell proliferation by 50 %. Western blot analysis The detailed protocol for the Western blotting has been described previously (15). Monoclonal anti-actin antibody, used as an equal loading control, was purchased from Merch KGaA (Darmstadt, Germany). The following secondary antibodies were used: goat anti-rabbit or anti-mouse IgG-conjugated horseradish peroxidase (HRP) (Santa Cruz Biotechnology, Santa Cruz, CA). To detect specific signals, the membranes were examined using ECL Prime Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK). Phospho-receptor tyrosine kinase (RTK) array and phospho-kinase arrays on April 9, 2013. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2013; DOI: 10.1158/0008-5472.CAN-12-4136 9 A Human Phospho-RTK Array Kit and a Human Phospho-kinase Array Kit (R&D Systems, Minneapolis, MN) were used to measure the relative level of tyrosine phosphorylation of 42 distinct receptor tyrosine kinases (RTKs) and the relative level of phosphorylation of 46 distinct intracellular kinases. Both arrays were performed according to the manufacturer’s instructions. Fluorescence immunocytochemistry The cells were cultured and fixed by 4 % formaldehyde on chamber slides. Primary antibodies against EGFR, E-cadherin, vimentin, ALDH1A1, and ABCB1 were used. Further details are provided in Supplementary Methods. DNA and RNA extraction Genomic DNAs were isolated from cell lines, frozen tumors, or paraffin embedded tumor by using DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA), standard phenol-chloroform (1:1) extraction followed by ethanol precipitation, or QIAmp DNA FFPE Tissue Kit (Qiagen), respectively. Total RNAs were extracted from cell lines using RNeasy Plus Mini Kit (Qiagen). The complementary DNA (cDNA) was synthesized from total RNA using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems) according to the manufacturer’s instructions. on April 9, 2013. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2013; DOI: 10.1158/0008-5472.CAN-12-4136 10 Direct sequencing, PCR-based length polymorphism assay, and sub-cloning We determined the mutational status of EGFR, KRAS, NRAS, and BRAF genes by direct sequencing, and PCR conditions are provided in Table S1A. EGFR exon19 deletion was also detected with PCR-based length polymorphism assay which have previously reported (16). For sub-cloning, PCR products were cloned into pCR2.1-TOPO vector using TOPO TA cloning kit (Invitrogen, Carlsbad, CA). One hundred clones were randomly selected for PCR-based length polymorphism assay. Analyses of copy number by qPCR and FISH assays Copy number gains (CNGs) of EGFR and MET genes were determined by real-time quantitative PCR (qPCR) assay using Power SYBR Green PCR Master Mix (Applied Biosystems) as previously reported (17, 18). Primer sequences are provided in Table S1B. In brief, gene dosage of each target and LINE-1 gene, a reference gene, was calculated using the standard curve method. Relative copy number of each sample was determined by comparing the ratio of target gene to LINE-1 in each sample with the ratio of these genes in human genomic DNA (EMD Biosciences, Darmstadt, Germany). Based on our previous study, we defined high-level amplification as values greater than 4 in cell lines and those greater than 5 in clinical samples (17, 18). on April 9, 2013. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2013; DOI: 10.1158/0008-5472.CAN-12-4136 11 A dual-color fluorescence in situ hybridization (FISH) assay was performed using the LSI EGFR spectrumOrange/CEP7 spectrumGreen probe (Vysis, Downers Grove, IL) according to the manufacturer’s instructions. Twenty metaphase spreads and 200 interphase nuclei were analyzed in each slide. Hybridoma production and TKI sensitivity analysis The parental HCC827 cells were fused with HCC827-GR-high2 using Sendai virus (Hemagglutinating Virus of Japan) envelope (HVJ-E) GenomONE™-CF (Ishihara Sangyo Kaisha Ltd., Osaka, Japan) according to the manufacturer’s instructions. In brief, HCC827 cells stained with PKH26 Red fluorescent Cell Linker Kit (Sigma-Aldrich, St Louis, MO) were mixed at a ratio of 1:1 HCC827-GR-high2 cells stained with PKH67 Green fluorescent Cell Linker Kit (Sigma-Aldrich). The fused cells were confirmed as double-fluorescent positive cells in fluorescent microscopy. Cells were treated with 2 µM of gefitinib and the presence of double-fluorescent positive and single-fluorescent positive cells (HCC827 and HCC827-GR-high2) was examined 14 days after. Expression profiling analysis RNA from cells was profiled on Illumina HumanHT-12 V4 Expression BeadChip arrays according to the Illumina protocol. The array measures expression levels for over on April 9, 2013. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2013; DOI: 10.1158/0008-5472.CAN-12-4136 12 47,000 transcripts derived from the NCBI RefSeq Release 38. BRB array tools (version 4.2) were used to perform robust spline normalization on background corrected data to generate log2 transformed normalized data. Fold change in expression for individual probes was calculated and probes with fold changes exceeding 2-fold or below 2-fold were considered over- and under-expressed, respectively (Table S2). mRNA and miRNA expression analysis by qRT-PCR Messenger RNA (mRNA) expression analysis by quantitative real-time RT-PCR (qRT-PCR) was performed on cDNA using TaqMan probes and the TaqMan Universal PCR Master Mix (Applied Biosystems). In miRNA expression analysis, the miRNA was isolated with TaqMan MicroRNA Cells-to-CT Kit (Ambion, Austin, TX), and RT reaction was performed with TaqMan MicroRNA Reverse Transcriptional Kit systems (Applied Biosystems) using TaqMan single RT primers for each miRNA. Primer and probe sets (Table S1C, D) were purchased from Applied Biosystems and used according to manufacturer’s instructions. PCR amplification was conducted on an ABI StepOne Real-Time PCR Instrument (Applied Biosystems) and gene expression was calculated using the comparative CT method. Three replicates per sample were assayed for each gene. To quantify the relative changes in gene expression, the 2(-ΔΔCT) method was used and reactions were normalized to endogenous control gene glyceraldehyde-3-phosphate on April 9, 2013. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2013; DOI: 10.1158/0008-5472.CAN-12-4136 13 dehydrogenase (GAPDH) expression levels in mRNA expression analysis, and miR-374 expression level in miRNA expression analysis, respectively. DNA methylation analysis DNA was subjected to bisulfate treatment using Epitect Bisulfite Kit (Qiagen) according to the manufacturer’s protocol. DNA methylation status was examined by bisulfite genomic sequencing and MSP as previously reported (19). Primers are listed in Table S1E, F. Sphere formation assays in serum-free cultures A total of 5 × 103 of cells were plated in 24-well plates with Ultra-Low Attachment surface (Corning Inc., Lowell, MA), and cultured in serum-free DMEM/F12 (Invitrogen) supplemented with 20 ng/mL epiderma