Genomic Aberrations Associated with Erlotinib Resistance in Non-Small Cell Lung Cancer Cells

ANTICANCER RESEARCH 33: 5223-5234 (2013)

Genomic Aberrations Associated with Erlotinib Resistance in Non-small Cell Lung Cancer Cells

MASAKUNI SERIZAWA1, TOSHIAKI TAKAHASHI2, NOBUYUKI YAMAMOTO2,3 and YASUHIRO KOH1

1Drug Discovery and Development Division, Shizuoka Cancer Center Research Institute, Sunto-gun, Shizuoka, Japan; 2Division of Thoracic Oncology, Shizuoka Cancer Center Hospital, Sunto-gun, Shizuoka, Japan; 3Third Department of Internal Medicine, Wakayama Medical University, Kimiidera, Wakayama, Japan

Abstract. Background/Aim: Mechanisms of resistance to mutations develop resistance, usually within one year of epidermal growth factor receptor (EGFR)-tyrosine kinase treatment. Therefore, there is an urgent need to elucidate the inhibitors (TKIs) in non-small cell lung cancer (NSCLC) underlying mechanisms of resistance in such tumors to are not fully-understood. In this study we aimed to overcome this obstacle (11-14, 17, 24). Recent studies elucidate remaining unknown mechanisms using erlotinib- suggest that mechanisms of acquired resistance to EGFR- resistant NSCLC cells. Materials and Methods: We TKIs can be categorized into three groups: occurrence of performed array comparative genomic hybridization genetic alterations, activation of downstream pathways via (aCGH) to identify genomic aberrations associated with bypass signaling, and phenotypic transformation (15, 16, 21); EGFR-TKI resistance in erlotinib-resistant PC-9ER cells. therapeutic strategies to overcome these resistance Real-time polymerase chain reaction (PCR) and mechanisms are under development. However, although the immunoblot analyses were performed to confirm the results causes of acquired resistance to EGFR-TKIs have been of aCGH. Results: Among the five regions with copy investigated, in more than 30% of patients with acquired number gain detected in PC-9ER cells, we focused on resistance to EGFR-TKI treatment, the mechanisms remain 22q11.2-q12.1 including v-crk avian sarcoma virus CT10 unknown (15). We previously reported that erlotinib-resistant oncogene homolog-like (CRKL), the overexpression of PC-9ER cells, established from PC-9 NSCLC cells with an which seemed to be associated with EGFR-TKI resistance. EGFR-activating mutation, manifest enhanced cell motility Blockade of downstream phosphatidylinositol 3-kinase which can be abolished by the blockade of transforming (PI3K)/v-akt murine thymoma viral oncogene homolog growth factor-β (TGF-β)/SMAD family member-2 (SMAD2) (AKT) signaling using NVP-BEZ235 suppressed the pathway (18). At the same time, constitutive activation of proliferation of PC-9ER cells, implying the involvement of phosphatidylinositol 3–kinase (PI3K)/v-akt murine thymoma acquired CRKL amplification in EGFR-TKI resistance. viral oncogene homolog (AKT) signaling in PC-9ER cells Conclusion: Acquired CRKL amplification was identified as was observed and this might confer resistance to EGFR- contributing to EGFR-TKI resistance; this cell line model TKIs. However, the mechanism responsible for this activated can be utilized to study this resistance mechanism. signaling on the genetic level remains unclear. Array comparative genomic hybridization (aCGH) Despite a dramatic initial response to the epidermal growth analysis is a high-resolution, genome-wide screening factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) technology that allows for rapid survey of genomic gefitinib and erlotinib, the majority of patients with non- instability, especially of copy number alterations, which are small cell lung cancer (NSCLC) with EGFR-activating key genetic aberrations relevant to the development and progression of human cancers (20). Moreover, aCGH analysis is useful as a means to explore mechanisms of resistance to EGFR-TKIs. For example, met proto-oncogene Correspondence to: Yasuhiro Koh, MD, Drug Discovery and (MET) amplification, a major cause of resistance to EGFR- Development Division, Shizuoka Cancer Center Research Institute, TKIs was detected in gefitinib-resistant NSCLC by aCGH 1007 Shimonagakubo Nagaizumi-cho Sunto-gun, Shizuoka, 411- analysis (4). 8777, Japan. Tel: +81 559895222, Fax: +81 559896085, e-mail: [email protected] In the present study, we aimed to clarify genomic aberrations, especially copy number alterations, relevant to Key Words: EGFR-TKI resistance, erlotinib, amplification, copy- EGFR-TKI resistance mechanisms in PC-9ER cells using number alteration, CRKL, non-small cell lung cancer. aCGH analysis.

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Table I. Chromosome aberrations in PC-9ER1 cells.

Chr Cytoband Start End Overlap Number of Log2 Log2 Number of Genes (hg19) (hg19) detected in aCGH probes ratio ratio genes included in PC-9ER1 included (amplifi- (deletion) included in chromosome and in genomic cation) chromosome aberration PC-9ER4 aberration aberration

1 p35.2- 31,964,879 32,473,479 l 204 –1.07 8 LOC284551, TINAGL1, HCRTR1, PEF1, p35.1 COL16A1, BAI2, SPOCD1, PTP4A2 1 p31.1 72,341,387 72,544,038 104 –1.10 1 NEGR1 2 q14.2- 120,609,630 122,530,342 l 724 1.09 12 PTPN4, EPB41L5, TMEM185B, RALB, q14.3 INHBB, LOC84931, GLI2, TFCP2L1, CLASP1, RNU4ATAC, MKI67IP, TSN 2 q22.2 142,338,612 142,570,218 115 –1.47 1 LRP1B 3 p14.2 60,070,343 60,113,818 l 23 –2.73 1 FHIT 3 p14.2 60,196,167 60,329,532 l 63 –2.83 1 FHIT 3 p14.2 60,344,382 60,375,603 l 18 –3.33 1 FHIT 3 p14.2 60,536,419 60,603,993 l 33 2.46 1 FHIT 3 p14.2 62,479,806 62,541,800 l 33 –1.55 1 CADPS 3 p13 71,345,692 71,395,229 l 27 –1.50 1 FOXP1 8 q24.12- 119,739,051 130,277,369 l 3,329 1.56 45 TNFRSF11B, COLEC10, MAL2, NOV, q24.21 ENPP2, TAF2, DSCC1, DEPDC6, COL14A1, MRPL13, MTBP, SNTB1, HAS2, HAS2AS, ZHX2, DERL1, WDR67, FAM83A, LOC100131726, C8orf76, ZHX1, ATAD2, WDYHV1, FBXO32, KLHL38, ANXA13, FAM91A1, FER1L6, TMEM65, TRMT12, RNF139, TATDN1, NDUFB9, MTSS1, LOC157381, ZNF572, SQLE, KIAA0196, NSMCE2, TRIB1, FAM84B, POU5F1B, LOC727677, MYC, PVT1 8 q24.23 136,467,502 136,771,745 l 120 1.24 1 KHDRBS3 10 p13-p11.1 16,013,438 38,629,589 l 7,704 –1.01 90 PTER, C1QL3, RSU1, CUBN, TRDMT1, VIM, ST8SIA6, PTPLA, STAM, FAM23A, MRC1, MRC1L1, SLC39A12, CACNB2, NSUN6, ARL5B, PLXDC2, NEBL, C10orf113, C10orf114, C10orf140, MLLT10, DNAJC1, COMMD3, BMI1, SPAG6, PIP4K2A, ARMC3, MSRB2, PTF1A, C10orf67, OTUD1, KIAA1217, PRINS, ARHGAP21, PRTFDC1, ENKUR, THNSL1, LOC100128811, GPR158, MYO3A, GAD2, APBB1IP, C10orf50, LOC731789, PDSS1, ABI1, NCRNA00202, ANKRD26, YME1L1, MASTL, ACBD5, LOC387646, PTCHD3, RAB18, MKX, ARMC4, MPP7, WAC, BAMBI, LYZL1, LOC387647, SVIL, KIAA1462, MTPAP, LOC729668, MAP3K8, LYZL2, ZNF438, LOC220930, ZEB1, ARHGAP12, KIF5B, EPC1, CCDC7, C10orf68, ITGB1, NRP1, PARD3, CUL2, CREM, CCNY, GJD4, FZD8, ANKRD30A, ZNF248, ZNF25, ZNF33A, ZNF37A, LOC100129055 13 q31.3 94,063,273 94,377,155 l 163 –2.82 1 GPC6 15 q22.2 60,661,598 60,713,059 l 32 –1.43 2 ANXA2, NARG2 19 q13.11 34,965,663 34,992,122 16 –1.01 1 WTIP 22 q11.21- 20,889,601 29,555,904 l 4,243 1.16 108 MED15, POM121L4P, TMEM191A, q12.1 PI4KA, SERPIND1, SNAP29, CRKL, AIFM3, LZTR1, THAP7, FLJ39582, MGC16703, P2RX6, SLC7A4, P2RX6P,

Table I. Continued

5224 Serizawa et al: Genomic Aberrations Associated with EGFR-TKI Resistance in NSCLC

Table I. Continued

LOC400891, POM121L8P, RIMBP3C, RIMBP3B, HIC2, PI4KAP2, UBE2L3, YDJC, CCDC116, SDF2L1, PPIL2, YPEL1, MAPK1, PPM1F, TOP3B, VPREB1, LOC96610, ZNF280B, ZNF280A, PRAME, LOC648691, POM121L1P, GGTLC2, RTDR1, GNAZ, RAB36, BCR, ZDHHC8P, IGLL1, C22orf43, LOC91316, RGL4, ZNF70, VPREB3, C22orf15, CHCHD10, MMP11, SMARCB1, DERL3, SLC2A11, MIF, GSTT2, DDTL, DDT, GSTTP1, LOC391322, GSTT1, GSTTP2, CABIN1, SUSD2, GGT5, POM121L9P, CYTSA, ADORA2A, C22orf45, UPB1, C22orf13, SNRPD3, GGT1, C22orf36, LOC644165, POM121L10P, PIWIL3, TOP1P2, SGSM1, TMEM211, KIAA1671, CRYBB3, CRYBB2, IGLL3, LRP5L, ADRBK2, MYO18B, SEZ6L, ASPHD2, HPS4, SRRD, TFIP11, TPST2, CRYBB1, CRYBA4, MIAT, MN1, PITPNB, LOC284900, TTC28, CHEK2, HSCB, CCDC117, XBP1, ZNRF3, C22orf31, KREMEN1 22 q12.1- 29,560,131 30,165,998 l 312 –1.28 17 KREMEN1, EMID1, RHBDD3, q12.2 EWSR1, GAS2L1, RASL10A, AP1B1, SNORD125, RFPL1S, RFPL1, NEFH, THOC5, NIPSNAP1, NF2, CABP7, ZMAT5, UCRC

Materials and Methods aCGH experiments and data analysis. aCGH analysis was performed with 500 ng genomic DNA from PC-9 cells (reference), and PC-9ER1 Cell culture and reagents. The human lung adenocarcinoma cell or PC-9ER4 cells (experimental sample) using SurePrint G3 Human line PC-9 harboring an EGFR-activating mutation (E746-A750del CGH 1×1M Oligo Microarrays (Agilent Technologies, Palo Alto, CA, at exon 19) was provided by Dr. Fumiaki Koizumi (National USA) according to standard procedures. Genomic Workbench Cancer Center Hospital, Tokyo, Japan). Erlotinib-resistant PC-9ER software ver. 7.0.4.0 (Agilent Technologies) was used to identify cells (PC-9ER1 and PC-9ER4) were established from PC-9 cells consecutive genomic aberrations using the statistical algorithm ADM- as previously reported (18). All the cell lines used in this study 2 with a sensitivity threshold of 10.0, and genomic aberration filters were maintained in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) (absolute average of log2 ratios of 1.0 in more than10 consecutive supplemented with 10% heat-inactivated fetal bovine serum (FBS; probes). Gene annotation enrichment analysis of genes involved in regions of genomic aberration was performed with The Database for Invitrogen) in humidified air containing 5% CO2 at 37˚C. Erlotinib and NVP-BEZ235 were purchased from LC laboratories (Woburn, Annotation, Visualization and Integrated Discovery (DAVID) MA, USA). Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov/) (6, 7). The acceptable statistical significance level of the gene annotation Nucleic acid sample preparation. Genomic DNA of PC-9 and PC- enrichment analysis was set at a p-value of lower than 0.1. 9ER cells was extracted using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. In vitro growth inhibition assay. The growth inhibitory effects of DNA concentrations were measured using a spectrophotometer erlotinib and NVP-BEZ235 were examined using a 3-(4,5- (NanoDrop 2000C; Thermo Scientific, Wilmington, DE, USA). dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay as

5225 ANTICANCER RESEARCH 33: 5223-5234 (2013)

Table II. Chromosome aberrations in PC-9ER4 cells.

1 p35.2 31,964,879 32,362,980 l 163 –1.06 7 LOC284551, TINAGL1, HCRTR1, PEF1, COL16A1, BAI2, SPOCD1 2 q14.2- 120,609,630 122,573,259 l 738 1.06 12 PTPN4, EPB41L5, TMEM185B, q14.3 RALB, INHBB, LOC84931, GLI2, TFCP2L1, CLASP1, RNU4ATAC, MKI67IP, TSN 2 q22.1 141,060,716 141,141,730 42 –1.41 1 LRP1B 3 p14.2 60,070,343 60,115,160 l 24 –2.57 1 FHIT 3 p14.2 60,196,167 60,329,532 l 62 –2.72 1 FHIT 3 p14.2 60,344,382 60,375,603 l 18 –3.54 1 FHIT 3 p14.2 60,534,253 60,603,993 l 34 2.32 1 FHIT 3 p14.2 62,483,217 62,544,239 l 32 –1.47 1 CADPS 3 p13 71,345,692 71,396,695 l 28 –1.45 1 FOXP1 6 p12.3 47,018,163 47,188,477 27 1.22 0 7 q33 132,905,968 133,661,629 363 –1.02 1 EXOC4 8 q24.12- 119,739,051 130,277,369 l 3,329 1.56 45 TNFRSF11B, COLEC10, MAL2, q24.21 NOV, ENPP2, TAF2, DSCC1, DEPDC6, COL14A1, MRPL13, MTBP, SNTB1, HAS2, HAS2AS, ZHX2, DERL1, WDR67, FAM83A, LOC100131726, C8orf76, ZHX1, ATAD2, WDYHV1, FBXO32, KLHL38, ANXA13, FAM91A1, FER1L6, TMEM65, TRMT12, RNF139, TATDN1, NDUFB9, MTSS1, LOC157381, ZNF572, SQLE, KIAA0196, NSMCE2, TRIB1, FAM84B, POU5F1B, LOC727677, MYC, PVT1 8 q24.23 136,467,502 136,771,745 l 120 1.26 1 KHDRBS3 10 p13- 16,028,094 19,944,722 l 1,210 –1.41 16 PTER, C1QL3, RSU1, CUBN, p12.31 TRDMT1, VIM, ST8SIA6, PTPLA, STAM, FAM23A, MRC1, MRC1L1, SLC39A12, CACNB2, NSUN6, ARL5B 10 p11.22 32,536,196 33,966,005 l 451 –1.14 5 EPC1, CCDC7, C10orf68, ITGB1, NRP1 10 q21.1 53,927,409 54,292,038 119 –1.16 2 PRKG1, DKK1 13 q31.3 94,077,984 94,369,846 l 150 –3.08 1 GPC6 15 q22.2 60,661,598 60,713,059 l 32 –1.46 2 ANXA2, NARG2 16 p12.3 - p12.220,477,152 23,562,141 974 –1.35 41 ACSM2A, ACSM2B, ACSM1, THUMPD1, ACSM3, ERI2, LOC81691, DCUN1D3, LYRM1, DNAH3, TMEM159, ZP2, ANKS4B, CRYM, NCRNA00169, NPIPL3, LOC100190986, LOC100271836, SLC7A5P2, METTL9, IGSF6, OTOA, RRN3P1, UQCRC2, C16orf65, C16orf52, VWA3A, EEF2K, POLR3E, CDR2, RRN3P3, LOC641298, LOC100132247, LOC653786, HS3ST2, USP31, SCNN1G, SCNN1B, COG7, GGA2, EARS2 16 q23.1 78,824,406 78,954,986 72 –2.49 1 WWOX 22 q11.21- 20,889,601 29,555,904 l 4,245 1.16 108 MED15, POM121L4P, TMEM191A, q12.1 PI4KA, SERPIND1, SNAP29, CRKL, AIFM3, LZTR1, THAP7, FLJ39582, MGC16703, P2RX6, SLC7A4, P2RX6P, LOC400891, POM121L8P,

Table II. Continued

5226 Serizawa et al: Genomic Aberrations Associated with EGFR-TKI Resistance in NSCLC

Table II. Continued

RIMBP3C, RIMBP3B, HIC2, PI4KAP2, UBE2L3, YDJC, CCDC116, SDF2L1, PPIL2, YPEL1, MAPK1, PPM1F, TOP3B, VPREB1, LOC96610, ZNF280B, ZNF280A, PRAME, LOC648691, POM121L1P, GGTLC2, RTDR1, GNAZ, RAB36, BCR, ZDHHC8P, IGLL1, C22orf43, LOC91316, RGL4, ZNF70, VPREB3, C22orf15, CHCHD10, MMP11, SMARCB1, DERL3, SLC2A11, MIF, GSTT2, DDTL, DDT, GSTTP1, LOC391322, GSTT1, GSTTP2, CABIN1, SUSD2, GGT5, POM121L9P, CYTSA, ADORA2A, C22orf45, UPB1, C22orf13, SNRPD3, GGT1, C22orf36, LOC644165, POM121L10P, PIWIL3, TOP1P2, SGSM1, TMEM211, KIAA1671, CRYBB3, CRYBB2, IGLL3, LRP5L, ADRBK2, MYO18B, SEZ6L, ASPHD2, HPS4, SRRD, TFIP11, TPST2, CRYBB1, CRYBA4, MIAT, MN1, PITPNB, LOC284900, TTC28, CHEK2, HSCB, CCDC117, XBP1, ZNRF3, C22orf31, KREMEN1 22 q12.1- 29,560,131 30,165,998 l 313 –1.19 17 KREMEN1, EMID1, RHBDD3, q12.2 EWSR1, GAS2L1, RASL10A, AP1B1, SNORD125, RFPL1S, RFPL1, NEFH, THOC5, NIPSNAP1, NF2, CABP7, ZMAT5, UCRC

Table III. Gene annotation enrichment analysis of the genes detected in five amplification regions.

Pathway KEGG ID p-Value Genes assigned to KEGG pathways

2q14.2-q14.3 8q24.12-q24.21 22q11.21-q12.1

Glutathione metabolism hsa00480 0.0043 GGT5, GSTT1, GSTT2, GGT1 Chronic myeloid leukemia hsa05220 0.0133 MYC MAPK1, CRKL, BCR Cyanoamino acid metabolism hsa00460 0.0459 GGT5, GGT1 Pathways in cancer hsa05200 0.0646 RALB, GLI2 MYC MAPK1, CRKL, BCR Taurine and hypotaurine metabolism hsa00430 0.0649 GGT5, GGT1

KEGG: Kyoto Encyclopedia of Genes and Genomes, http://www.genome.jp/kegg/

described previously (18). Briefly, a 180-μl volume of an solution (5 mg/ml in phosphate-buffered saline) was added to each exponentially growing cell suspension including 1×103 cells was well and the plates were incubated for further 4 h at 37˚C. After seeded into each well of a 96-well plate (Corning Costar, centrifuging the plates, the medium was aspirated from each well, Cambridge, MA, USA) containing medium with 10% FBS, and and 200 μl of Dimethyl sulfoxide (DMSO) was added to each well incubated for 24 h. The cells were exposed to 20 μl of each inhibitor to dissolve the formazan. The growth-inhibitory effect of each at different concentrations and further cultured at 37˚C in a inhibitor was assessed spectrophotometrically (Model 680 humidified atmosphere for 72 h. After the culture period, 20 μl MTT microplate plate reader; Bio-Rad Laboratories, Hercules, CA, USA).

5227 ANTICANCER RESEARCH 33: 5223-5234 (2013)

Figure 1. Continued

5228 Serizawa et al: Genomic Aberrations Associated with EGFR-TKI Resistance in NSCLC

Figure 1. Array comparative genomic hybridization (aCGH) karyograms of three commonly detected significant amplifications, 2q14.2-3 (A), 8q24.12-21 (B), and 22q11.21-q12.1 (C), in PC-9ER1 and PC-9ER4 cells. Regions of copy number gain and loss are denoted by yellow and blue squares, respectively. Black arrows indicate the locations of six genes that were assigned to “Pathways in cancer” (hsa05200) in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database.

Figure 2. Schematic diagram of phosphatidylinositol 3-kinase (PI3K) and extracellular-signal-regulated kinase (ERK) pathways extracted from “Pathways in cancer” (hsa05200) in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. v-crk avian sarcoma virus CT10 oncogene homolog-like (CRKL), extracellular signal-regulated kinase 2 (ERK2); (mitogen-activated protein kinase 1, MAPK1), and v-myc avian myelocytomatosis viral oncogene homolog (MYC) were assigned to these pathways, and are indicated by solid red boxes.

5229 ANTICANCER RESEARCH 33: 5223-5234 (2013)

Figure 4. PC-9 and PC-9ER cells were treated with erlotinib at the indicated concentrations (0.1, 1, and 5 μmol/l) for 12 h. Protein lysates were immunoblotted and probed with the indicated antibodies.

CRKL copy number analysis. The relative copy numbers of CRKL were quantified by quantitative real-time PCR (qPCR) on the Figure 3. v-crk avian sarcoma virus CT10 oncogene homolog-like StepOnePlus™ Real time PCR system (Applied Biosystems, Foster (CRKL) copy numbers in PC-9 and PC-9ER cells. The copy number of City, CA, USA) using 2 ng genomic DNA, PCR primers for each each gene was normalized to that of Long interspersed nuclear element gene and SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Bio, 1 (LINE-1) and human genomic DNA. *p<0.0001 (Student's t-test). The Shiga, Japan). To generate the standard curve for quantification of error bars indicate standard deviations of the mean. target gene copies, serial dilutions of the PCR amplicons of each gene were used (copy numbers from 10 to 107). The copy number of each gene was normalized to Long interspersed nuclear element 1 (LINE- The growth-inhibition curves were fitted using an algorithm 1) and human genome DNA (Clontech, Palo Alto, CA, USA). The log(inhibitor) vs. response-variable slope (four parameters) of primer sequences used for CRKL were according to Cheung et al. (3). GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). Results Immunoblot analysis. The cultured cells were lysed in M-PER mammalian protein extraction reagent containing EDTA-free Halt Genomic aberrations detected in PC-9ER cells with aCGH protease inhibitor cocktail, and Halt phosphatase inhibitor cocktail (Pierce Chemical Co., Rockford, IL, USA). Equal amounts of protein analysis. Tables I and II list the significant genomic aberrations (20 μg) from whole cell lysates [as measured utilizing the BCA detected in PC-9ER1 and PC-9ER4 cells, respectively. protein assay reagent (Pierce Chemical Co.)] were separated on 8% Significant amplifications were observed in five and six sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- genomic regions in PC-9ER1 and PC-9ER4 cells, respectively. PAGE) and transferred to nitrocellulose membranes (Bio-Rad). Five of these amplification regions (2q14.2-3, 3p14.2, 8q24.12- Membranes were blocked with 5% skim milk or 5% bovine serum 21, 8q24.23, and 22q11.21-q12.1), including 167 genes, were albumin (BSA) in Tris-buffered saline-Tween 20 (TBST) for 1 h at common to both PC-9ER1 and PC-9ER4 cells. Significant room temperature, and then incubated overnight with primary antibodies in TBST containing 5% skim milk or 5% BSA at 4˚C. deletions were detected in 13 and 16 genomic regions in PC- After the membranes were washed with TBST, they were incubated 9ER1 and PC-9ER4 cells, respectively. Eight of these deletion with secondary antibodies conjugated to horseradish peroxidase for 1 regions (1p35.2, 3p14.2, 3p13, 10p13-p12.31, 10p11.22, h at room temperature, followed by a TBST wash. Immunoreactive 13q31.3, 15q22.2, 22q12.1-q12.2), including 51 genes, were bands were visualized using Supersignal West Pico chemiluminescent common to both PC-9ER cell lines. substrate (Pierce Chemical Co.). Antibodies against β-Actin (sc- 47778), EGFR (sc-03) and v-crk avian sarcoma virus CT10 oncogene CRKL amplification contributes to EGFR-TKI resistance in homolog-like (CRKL; sc-319) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho- PC-9ER cells. In order to perform functional categorization, EGFR (3777), AKT (9272), phospho-AKT (9271), S6 ribosomal and to assign genes located within the commonly detected protein (2217), and phospho-S6 ribosomal protein (2211) were genomic aberrations in PC-9ER1 and PC-9ER4 cells to purchased from Cell Signaling Technology (Beverly, MA, USA). corresponding signaling and metabolic pathways, gene

5230 Serizawa et al: Genomic Aberrations Associated with EGFR-TKI Resistance in NSCLC

Figure 5. PC-9 and PC-9ER cells were exposed to the indicated concentrations of erlotinib or the NVP-BEZ235. The viability of the cells was measured after 72 h of treatment by the MTT assay.

annotation enrichment analysis was performed using DAVID (FHIT), hypocretin (orexin) receptor 1 (HCRTR1), integrin, Bioinformatics Resources. Forty genes included in the beta 1 (ITGB1), neurofilament, heavy polypeptide (NEFH), commonly detected amplified regions in both PC-9ER cells neuropilin 1 (NRP1), protein tyrosine phosphatase-like were assigned to 88 Kyoto Encyclopedia of Genes and (proline instead of catalytic arginine), member A (PTPLA), Genomes (KEGG) pathways, and five pathways with signal transducing adaptor molecule (SH3 domain and ITAM statistical significance were selected (Table III). The CRKL, motif) 1 (STAM) and tRNA aspartic acid methyltransferase 1 mitogen-activated protein kinase 1 (MAPK1), breakpoint (TRDMT1) included in the detected deletion regions, common cluster region (BCR), v-ral simian leukemia viral oncogene to both PC-9ER cell lines, were assigned to 23 KEGG homolog B (RALB), GLI family zinc finger 2 (GLI2) and v-myc pathways, but no statistically significant pathways were found. avian myelocytomatosis viral oncogene homolog (MYC) genes are assigned to “Pathways in cancer” (hsa05200) in the KEGG Discussion pathway database (Table III, Figures 1 and 2). Among these six genes, we focused on CRKL, which encodes a member of In the present study, we explored genomic aberrations the CRK adaptor protein that participates in signal relevant to EGFR-TKI resistance in erlotinib-resistant PC- transduction, and is best known as a substrate of the BCR- 9ER cells using aCGH analysis. The cytoband 22q11.21 ABL kinase in chronic myelogenous leukemia (2, 5). The region including the CRKL gene was seen to be one of the copy number of CRKL in both PC-9ER cell lines was about most commonly amplified regions across multiple human six-fold higher than that in PC-9 cells (Figure 3). Enhanced cancer types in genome-wide scanning with a high-resolution expression of CRKL was also detected in PC-9ER cells by single nucleotide polymorphism array (1). Using aCGH immunoblot analysis (Figure 4). Constitutive phosphorylation analysis, high-level amplification and low-level copy number of AKT not suppressed by erlotinib treatment was observed in gain of the 22q11.21 region were observed in 3% and 11% PC-9ER cells (Figure 4), indicating circumvention of the of lung cancer specimens, respectively (8). CRKL effect of erlotinib. Moreover, blockade of activated PI3K/AKT amplification and its resultant overexpression were reported signaling with the dual PI3K/mechanistic target of rapamycin to be associated with cell proliferation, poor prognosis, cell- (mTOR) inhibitor NVP-BEZ235 effectively abolished cell cycle progression, and cell motility in lung cancer (8, 22). growth of PC-9ER cells (Figure 5). These results imply that These previous reports suggest that CRKL amplification CRKL amplification-induced activation of PI3K/AKT affects diverse biological features of tumor cells. The signaling may contribute to resistance against EGFR-TKIs in involvement of CRKL amplification in resistance to EGFR- PC-9ER cells (Figure 2). Ten genes, adaptor-related protein TKIs has been previously reported only under conditions of complex 1, beta 1 subunit (AP1B1), calcium channel, voltage- forced expression of CRKL, and in only one patient with dependent, beta 2 subunit (CACNB2), fragile histidine triad acquired resistance to erlotinib, whose repeat biopsy

5231 ANTICANCER RESEARCH 33: 5223-5234 (2013) specimen showed copy number gain of CRKL relative to the to lead to the activation of TGFβRIII as a tumor suppressor pre-treatment baseline sample (3). These reported results (19). Inhibin A was reported to attenuate TGF-β signaling by shed light on CRKL amplification as a novel mechanism of down-regulating cell surface expression of the TGF-β co- EGFR-TKI resistance. Here we report, to our knowledge for receptor betaglycan (9). However, the involvement of the first time, that spontaneously-acquired CRKL INHBB in the TGF-β pathway remains an open question. amplification contributes to EGFR-TKI resistance in NSCLC Further investigation on the effect of amplified INHBB in cells selected by long-term exposure to EGFR-TKI erlotinib-resistant PC-9ER cells is required and will shed treatment. The dual PI3K/mTOR inhibitor NVP-BEZ235 light on the biology of EGFR-TKI-resistant NSCLC. effectively abolished cell growth of PC-9ER cells, consistent In conclusion, as far as we are aware of, this is the first with the previous report that combined treatment with report showing the contribution of spontaneously acquired gefitinib and the PI3K inhibitor GDC-0941 led to a CRKL amplification to EGFR-TKI resistance in erlotinib- substantial decrease in cell viability in EGFR-mutant cells resistant NSCLC cells established from EGFR-mutant with forced expression of CRKL (3). These results indicate NSCLC cells by sequential exposure to erlotinib. that blockade of PI3K/AKT signaling may serve as a Furthermore, blockade of PI3K/AKT signaling with the dual potential therapeutic strategy in patients with NSCLC who PI3K/mTOR inhibitor NVP-BEZ235 may serve as a develop acquired resistance to EGFR-TKIs caused by CRKL potential therapeutic approach in patients with NSCLC who amplification. In order to confirm that CRKL amplification develop acquired resistance to EGFR-TKIs caused by CRKL is a novel mechanism underlying EGFR-TKI resistance, it is amplification. These cell lines hold potential as a tool for the important to test biopsies of tumors exhibiting acquired development of therapeutic strategies to overcome CRKL EGFR-TKI resistance for CRKL amplification, and to amplification-mediated EGFR-TKI resistance. investigate the detailed molecular mechanisms and biology of CRKL-amplified EGFR-TKI-resistant tumors. Disclosure We previously reported that erlotinib-resistant PC-9ER cells manifest the enhanced cell motility and this can be abolished The Authors declare no conflicts of interest. by blockade of TGF-β/SMAD2 pathway (18). Interestingly, CRKL is also known to be involved in cell migration and Acknowledgements invasion. CRKL-knockdown in CRKL-amplified NSCLC cells This work was supported by the Japanese Society for the Promotion led to a significant inhibition of both cell migration and cell of Science (JSPS) KAKENHI Grant Number 25871225 (M.S.). The invasion, suggesting that CRKL amplification promotes cell Authors thank Junko Suzuki and Akane Naruoka for technical migration and invasion (8). It is of note that interaction between assistance. CRKL and the TGF-β signaling pathway has also been reported. In glioblastoma cells, TGF-β1-induced migration and References invasiveness were enhanced by forced expression of CRKL and were suppressed by knockdown of CRKL, indicating that 1 Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, Donovan J, Barretina J, Boehm JS, Dobson J, Urashima M, Mc CRKL may regulate TGF-β pathway-mediated cell motility Henry KT, Pinchback RM, Ligon AH, Cho YJ, Haery L, (10). On the contrary, activation of TGF-β signaling promoted Greulich H, Reich M, Winckler W, Lawrence MS, Weir BA, phosphorylation of CRKL in neural crest cells, demonstrating Tanaka KE, Chiang DY, Bass AJ, Loo A, Hoffman C, Prensner J, that CRKL is downstream of TGF-β signaling (23). Detailed Liefeld T, Gao Q, Yecies D, Signoretti S, Maher E, Kaye FJ, molecular mechanisms regulating the interaction between Sasaki H, Tepper JE, Fletcher JA, Tabernero J, Baselga J, Tsao CRKL and the TGF-β signaling pathway still remain largely MS, Demichelis F, Rubin MA, Janne PA, Daly MJ, Nucera C, unknown. Moreover, the association of CRKL and the TGF-β Levine RL, Ebert BL, Gabriel S, Rustgi AK, Antonescu CR, Ladanyi M, Letai A, Garraway LA, Loda M, Beer DG, True LD, pathway in EGFR-TKI-resistant tumors has never been Okamoto A, Pomeroy SL, Singer S, Golub TR, Lander ES, Getz investigated to our knowledge, and this should be studied using G, Sellers WR, and Meyerson M: The landscape of somatic this cell line model and clinical specimens. copy-number alteration across human cancers. Nature 463: 899- Although we have previously reported that TGF-β2 was 905, 2010. overexpressed in PC-9ER cells, significant copy number gain 2 Birge RB, Kalodimos C, Inagaki F and Tanaka S: Crk and CrkL was not observed in the genomic region which included the adaptor proteins: Networks for physiological and pathological TGFB2 gene. We then investigated the regions where genes signaling. Cell Commun Signal 7: 13-35, 2009. related to the TGF-β pathway are included, and detected 3 Cheung HW, Du J, Boehm JS, He F, Weir BA, Wang X, Butaney M, Sequist LV, Luo B, Engelman JA, Root DE, Meyerson M, INHBB amplification of encoding inhibin β (2q14.2-3, Tables Golub TR, Janne PA and Hahn WC: Amplification of CRKL I and II). INHBB is known to regulate the production of induces transformation and epidermal growth factor receptor follicle-stimulating hormone. Binding of testicular inhibin B inhibitor resistance in human non-small cell lung cancers. to TGF-β receptor III (TGFβRIII) in prostate was reported Cancer Discov 1: 608-625, 2011.

5232 Serizawa et al: Genomic Aberrations Associated with EGFR-TKI Resistance in NSCLC

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