Proteomic Analysis of Human Small Cell Lung ... - ACS Publications

Small cell lung cancer (SCLC) is the leading cause of cancer death, with a high propensity for aggressiveness and metastasis even in an early stage. T...
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Proteomic Analysis of Human Small Cell Lung Cancer Tissues: Up-Regulation of Coactosin-Like Protein-1 Hye-Cheol Jeong,† Gwang-Il Kim,‡ Sang-Ho Cho,‡ Kwang-Hyung Lee,§ Jung-Jae Ko,§ Jeong-Hee Yang,§ and Kwang-Hoe Chung*,§ Division of Respiratory and Critical Medicine, Department of Internal Medicine, Department of Pathology, and Department of Applied Bioscience, College of Life Science, CHA University, Sungnam, Republic of Korea Received July 10, 2010

Small cell lung cancer (SCLC) is the leading cause of cancer death, with a high propensity for aggressiveness and metastasis even in an early stage. Thus, identification of biomarkers as early diagnostics and treatment is needed. In this study, we investigated differentially regulated proteins between human SCLC tissues and normal bronchial epithelium by proteomic analysis using twodimensional electrophoresis (2-DE) and MALDI-TOF mass spectrometry. Seven proteins and protein isoforms, including, γ-actin, tubulin R-1B, laminin B1, coactosin-like protein-1 (COTL-1), ubiquitin carboxyl-terminal esterase L1, ubiquitin-conjugating enzyme E2-25K, and carbonic anhydrase 1, were up-regulated more than 2 fold in SCLC tissues. In particular, up-regulated COTL-1 expression was validated by Western blot analysis, immunohistochemistry, and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Moreover, most SCLC tissues (93%; 28/30) were COTL-1-positive in immunohistochemistry, whereas only 16% (10/64) of nonsmall cell lung cancer (NSLC) tissues were. Taken together, this SCLC proteomic data may help in establishing a human SCLC proteome database. COTL-1 may be a biomarker or a therapeutic target in SCLC patients. Keywords: small cell lung cancer • biomarker • proteomic analysis • immunohistochemical stain • coactosin-like protein-1

Introduction Lung cancer is the leading cause of cancer mortality worldwide among both males and females, with more than 1 million deaths annually. In the United States, there were an estimated 219 440 new lung cancer cases and 159 390 deaths in 2009.1 Small cell lung cancer (SCLC) accounts for about 16% of lung cancer diagnosed each year. Because SCLC is particularly aggressive, and characterized by rapid growth, metastasis, and relapse, most SCLC patients are not cured by surgical resection and have poor prognosis. Although SCLC is sensitive to radiotherapy and chemotherapy, the median survival in patients with limited disease is 12-24 months, and extensive disease is 7-11 monthssless than 5% of SCLC patients can survive over 5 years.2 Despite advances in diagnostic capabilities and treatment strategies, lung cancer mortality has not significantly changed over the past several decades. Proteomic analysis is a powerful tool for identifying lung cancer modules that involves protein separation by twodimensional polyacrylamide gel electrophoresis (2-D PAGE) and protein identification by matrix-associated laser desorp* To whom correspondence should be addressed. Kwang-Hoe Chung, Ph.D., Department of Applied Bioscience, College of Life Science, CHA University, 222 Yatap-dong, Sungnam 463-836, Republic of Korea. Tel: +8231-725-8379. Fax: +82-31-725-8350. E-mail: [email protected]. † Division of Respiratory and Critical Medicine, Department of Internal Medicine. ‡ Department of Pathology. § Department of Applied Bioscience. 10.1021/pr100714b

 2011 American Chemical Society

tion/ionization (MALDI) coupled to time-of-flight (TOF) mass spectrometry (MS).3 Proteome-based approaches can identify post-translational processingsproteolysis, glycosylation, phosphorylation, and acetylation, etc.sand correlate mRNA expression with protein synthesis, two limitations of mRNA microarray studies. Nonsmall cell lung cancer (NSCLC) has been tested with proteomic data from serum or tissue samples.4-6 Yanakisawa et al. reported that the proteomic pattern of NSCLC tissues could be used to predict histology, stage, and patient survival.7 In addition, Taguchi et al. discovered a proteomic pattern from NSCLC serum that could predict the treatment response to gefitinib or erlotinib.8 For SCLC, comparative proteomics in SCLC cell lines identified up-regulated proteins for cellular proliferation and tumor antigens.9 Membrane proteomics with a lung cancer cell line resistant to doxorubicin showed up-regulation of sarco/endoplasmic reticulum Ca2+ ATPase.10 Clinical studies showed that plasma chromogranin A levels increased in SCLC and NSCLC and could be a treatment response marker or monitor for disease recurrence.11 In addition, the haptoglobin R-subunit and hepatocyte growth factor are up-regulated in serum samples from SCLC patients.12 SCLC cell lines and biopsy samples have high mRNA levels of splice variants of actinin-4 that have a high binding affinity for filamentous actin polymer, a possible biomarker.13 A proteomic pattern and common surrogate markers in tumors of neuroendocrine origin, includJournal of Proteome Research 2011, 10, 269–276 269 Published on Web 11/03/2010

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ing 2 cases of SCLC, have been reported. Genetic mutations of p53, Rb, and high levels of c-Met expression are also risk factors for SCLC.15 However, the identification of selective biomarkers and efficient therapeutics for SCLC is still a challenge, with relatively few proteomic data with human SCLC tissues available, partly because of difficulties in tissue acquisition due to the metastatic character of SCLC at the time of diagnosis. We therefore measured differentially expressed proteins in human SCLC tissues and normal bronchial epithelium by proteomic analysis to identify diagnostic and therapeutic targets.

Materials and Methods Materials. Urea, thiourea, CHAPS, DTT, benzamidine, benzonase, acrylamide, iodoacetamide, bis-acrylamide, SDS, ammonium bicarbonate, acetonitrile, trifluoroacetic acid, acetic acid, R-cyano-4-hydroxycinnamic acid, and Coomassie Blue G-250 were purchased from Sigma-Aldrich (St. Louis, MO). Pharmalyte (pH 3.5-10) was from GE Healthcare Bio-Sciences Corp. (Uppsala, Sweden) and IPG DryStrips (pH 4-10 NL, 24 cm) were Genomine Inc. (Pohang, Korea). Modified porcine trypsin (sequencing grade) from Promega (Madison, WI), protease inhibitor cocktail from Roche Molecular Biochemicals (Indianapolis, IN), Bradford’s reagent from Bio-Rad (Hercules, CA), and Poros 20 R2 resin from Perspective Biosystems (Framingham, MA) were used. Preparation of Tissue Samples. Six cases of SCLC tissues and six cases of corresponding normal bronchial epithelium from healthy individuals were obtained by fiberoptic bronchoscopic biopsy from the Bundang CHA General Hospital of CHA University, Korea. The patients and healthy individuals signed an informed consent form for the study, which was reviewed by the Institutional Review Board of CHA University Hospital (Seongnam, Korea). The lung cancer tissue samples were obtained from a “core” part of the tumor at the time of diagnosis before any therapy. For each tissue, the surface epithelium was selectively procured by dissection with special care for minimal contamination and avoiding adjacent noncancerous tissue, and was then immediately snap-frozen in liquid nitrogen. They were classified histologically according to Lauren’s classification after H&E staining. Preparation of Protein Extracts. Two hundred micrograms of the proteins from the collected tissue samples were suspended in 0.5 mL of 50 mM Tris buffer containing 7 M urea, 2 M thiourea, 4% (w/v) 3-[(3-cholamidopropy)dimethyammonio]-1-propanesulfonate (CHAPS), 1% (w/v) dithiothreitol (DTT), 2% (v/v) Pharmalyte, 1 mM benzamidine, and 16 µL protease inhibitor cocktail. The tissue samples were homogenized and centrifuged at 12 000× g for 1 h at 15 °C to harvest supernatant. The protein concentration was measured by the Bradford protein quantitation method (Bio-Rad, Hercules, CA). 2-D PAGE. For 2-D PAGE analysis, the IPG DryStrips pH 4-10 nonlinear, 24 cm (Genomine Inc., Pohang, Korea) were rehydrated in swelling buffer containing 7 M urea, 2 M thiourea, 1% (w/v) DTT, 1% Pharmalyte, and 2% (w/v) CHAPS. Protein lysates (200 µg) were cup-loaded into the rehydrated IPG strip, and isoelectric focusing (IEF) was performed at 20 °C using a Multiphor II apparatus and EPS 3500 XL power supply (GE Healthcare Bio-Sciences Corp., Uppsala, Sweden) according to the manufacturer’s instruction. For IEF, the voltage was linearly increased from 150 to 3500 V for 3 h for sample entry followed by constant 3500 V, with focusing complete after 96 kV/h. Prior to running the second dimension, the strips were incubated 270

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for 10 min in the equilibration buffer (50 mM Tris-Cl, pH 6.8 containing 6 M urea, 2% SDS and 30% glycerol), first with 1% DTT, and second with 2.5% iodoacetamide. The equilibrated strips were then inserted onto 10-16% gradient SDS-PAGE gels (20 × 24 cm). SDS-PAGE was performed using a Hoefer DALT 2D system (Amersham Biosciences) following the manufacturer’s instructions. The 2-D PAGE gels were run at 20 °C for 1700 Vh. Following fixation of the gels for 1 h in a solution of 40% (v/v) methanol containing 5% (v/v) phosphoric acid, the gels were stained with colloidal Coomassie Blue G-250 solution (ProteomeTech, Seoul, Korea) for 5 h and were destained in 1% (v/v) acetic acid for 4 h. 2-D PAGE Gel Image Analysis. Protein spot detection and 2-D pattern matching were performed using PDQuest software (version 7.0, Bio-Rad, CA) according to the protocols provided by the manufacturer. Quantification of each protein spot was normalized by the intensity of total valid spots. For comparison of protein spot densities between SCLC tissues and normal tissues, 64 spots throughout all gels were correspondingly landmarked and normalized. The quantified spots of candidate proteins were compared with the aid of histograms. To ensure the reproducibility of 2-D PAGE experiments, each sample was analyzed in duplicate. In-Gel Digestion with Trypsin and Extraction of Peptides. The in-gel digestion of protein spots was performed as described.16 In brief, the protein spots stained with Coomassie Blue were excised and cut into pieces. The gel pieces were washed for 1 h at room temperature in 25 mM ammonium bicarbonate buffer, pH 7.8, containing 50% (v/v) acetonitrile (ACN). Following dehydration of gel pieces in a SpeedVac for 10 min, the gel pieces were rehydrated in 10 µL (20 ng/µL) of sequencing grade trypsin solution (Promega, Madison, WI). After incubation in 25 mM ammonium bicarbonate buffer, pH 7.8, at 37 °C overnight, the tryptic peptides were extracted with 5 µL of 0.5% TFA containing 50% (v/v) ACN for 40 min with mild sonication. The extracted solution was reduced to 1 µL in a vacuum centrifuge. Prior to mass spectrometric analysis, the resulting peptide solution was subjected to a desalting process using a reversed-phase column.17 A GEloader tip (Eppendorf, Hamburg, Germany) was packed with Poros 20 R2 resin (Perspective Biosystems, MA). After an equilibration step with 10 µL 5% (v/v) formic acid, the peptide solution was loaded on the column and washed with 10 µL of 5% (v/v) formic acid. The bound peptides were eluted with 1 µL of R-cyano-4hydroxycinnamic acid (CHCA) (5 mg/mL in 50% (v/v) ACN/ 5% (v/v) formic acid) and dropped onto a MALDI plate (96 × 2; Applied Biosystems, Foster City, CA). Analysis of Peptides using MALDI-TOF MS and Identification of Proteins. Mass measurement of tryptic peptides was performed with a Voyager-DE STR mass spectrometer (Perspective Biosystems, MA) in reflectron positive ion mode as described.16,18 Close external calibration was performed for every four samples with calibration mixtures of adrenocorticotropic fragment 18-39 (monoisotopic mass, 2465.1989), neurotensin (monoisotopic mass, 1672.9175), and angiotensin I (monoisotopic mass, 1296.6853) as standard calibrants. Mass spectra were acquired for the mass range of 900-3500 Da. The proteins were identified by peptide mass fingerprinting against the Swiss-Prot and NCBI databases using the search program Profound (http://129.85.19.192/profound_bin/WebPro Found.exe, Rockefeller University, Version 4.10.5), MASCOT (http:// www.matrixscience.com), or MS-Fit (http://prospector. ucsf. edu, University of California San Francisco, Version 4.0.5). The

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Up-Regulation of Coactosin-Like Protein-1 following mass search parameters were set: peptide mass tolerance, 50 ppm; a mass window between 0 and 100 kDa, allowance of missed cleavage, 2; consideration for variable modifications such as oxidation of methionine and propionamides of cysteines. Only significant hits as defined by each program were considered initially with at least 4 matching peptide masses. Western Analysis. Proteins (20 µg) from the tissue extracts were loaded on 12.5% PAGE and run at 100 V for 2 h in SDS running buffer. Western blot transfer to PVDF membranes (Millipore) in 25 mM Tris base (pH 8.5), 0.2 M glycine, and 20% methanol was performed at 300 mA for 1.5 h. After washing with deionized water and TBST buffer (10 mM Tris, pH 7.6, 233 mM NaCl and 0.1% Tween-20), the membrane was incubated with blocking buffer, 5% nonfat dried milk in TBST, for 1 h at room temperature. To quantify protein expression, primary antibodies of laminin beta 1, ubiquitin carboxylterminal hydrolase L1, γ-actin, tubulin-R, and carbonic anhydrase 1 were purchased from Millipore, and COTL-1 from Protein Tech Group. The membrane was incubated with primary antibody overnight at 4 °C and washed with TBST buffer for 25 min (2 × 10 min, 5 min), and then corresponding secondary antibodies coupled with HRP (1:3,000) were applied for 1 h at 37 °C. After washing in TBST buffer for 25 min (2 × 10 min, 1 × 5 min), the membrane was developed by the ECL method (LumiGLO, KPL). Immunohistochemistry (IHC). Immunohistochemical staining was done on formalin-fixed and paraffin-embedded tissue sections using a standard immunohistochemical technique. Four-micrometer-thick tissue sections were deparaffinized in xylene, rehydrated in graded ethanol series, and treated with an antigen retrieval solution (10 mmol/L sodium citrate buffer, pH 6.0). The sections were incubated with mouse monoclonal anticoactosin-like protein antibody (Protein Tech group; dilution 1:400) for 1.5 h at room temperature. Finally, the tissue sections were incubated with 3′,3′-diaminobenzidine (SigmaAldrich) until the brown color developed, and counterstained with Harris’ modified hematoxylin. For negative controls, primary antibodies were omitted. The sections were scored on the extent and intensity of staining from the three selected cores of the tissue according to a 4-tiered scale of 0 to 3 (with 3 as the most intense staining). The extent of positive immunoreactivity was graded according to the percentage of stained cells in the region of interest: 0 point for 0%, 1 point for 1-25%, 2 points for 25-50%, 3 points for 51-75%, and 4 points for g76%. An overall score was obtained by the sum of the intensity and the extent of the positive staining. Cases with a final score of more than four were defined as positive. Samples were investigated by two independent, experienced pathologists on staining patterns without knowledge of the patient’s clinical status. Statistical Analysis. Statistical analysis was performed using SAS version 4.0. The associations between clinicopathologic factors and immunohistochemical markers were assessed using Fisher’s exact test. P < 0.05 was considered significant. RNA Extraction and RT-qPCR Analysis. RNAs from the patient tumor tissues (range: 12-51 mg) were prepared using the easy-spin (DNA free) total RNA extraction kit according the manufacturer’s instruction (iNtRON Biotechnology, Inc., Korea). The ratio of A260/A280 of isolated RNAs was over 1.9. Two micrograms of total RNAs were used for reverse transcription reaction with oligo-dT primer and Superscript II RNase H reverse transcriptase (Invitrogen) in 20 µL volume. Quantitative

Table 1. Patient Characteristics in Proteomic Analysis

Sex Male Female Median age (years, range) Stage Smoking Current smoker Ex-smoker Nonsmoker

normal

SCLC

5 1 72 (58-80) -

5 1 67 (52-84) Extensive (6)

3 2 1

4 2 2

PCR was performed with 2 ul of cDNA with QuantiTect SYBR Green PCR kit (Quiagen) in 20 µL reaction volume in triplicate by C1000 Thermal Cycler (CFX 96 Real-Time System, Bio Rad). The primers for COTL-1 were: forward 5′- AGTTGACGGAGGGAGTATTT-3′ and reverse 5′- CAGACGCAGGACTGAAGC-3′. The primers for GAPDH were: forward 5′- TGCACCACCAACTGCTTAG- 3′ and reverse 5′- GATGCAGGGATGATGTTC - 3′. The conditions for quantitative PCR were as follows: denaturation; 94 °C for 2 min, 1 cycle, PCR amplication; 94 °C for 15 s (denaturation), 55 °C for 30 s (annealing), 72 °C for 1 min (extension), 50 cycles, final extension; 68 °C for 5 min, 1 cycle.

Results The median age of the 6 SCLC patients was 67 years old (range: 52-84 years old); five males and one female. The stages of 6 SCLC patients were all extensive. The median age of the normal control group was 72 years old (range: 58-80 years old); five males and one female (Table 1). Proteome Profiles of SCLC Tissues and the Normal Bronchial Epithelium. Two -D PAGE using nonlinear IPG ranging from pH 4-10 was performed to separate protein extracts (Figure 1A and B). More than 1500 spots were detected on the 2-D PAGE gel with a molecular weight ranging from 14 to 150 kDa and a pI value between 4 and 10. Sixty-four protein spots differentially expressed in SCLC tissues compared with control tissues were nominated on the 2-D gel (Figure 1C). Among these protein spots, 15 spots (# 1615, 1618, 2002, 2019, 3002, 3111, 3206, 6012, 7101, 2016, 2720, 5015, 7012, 8018, and 8020) were significantly up-regulated over 2-fold (Figure 2), and 49 spots were down-regulated (data not shown). MALDI-TOF MS Analysis. The 15 up-regulated protein spots were cut out and subjected to in-gel trypsin digestion followed by MALDI-TOF analysis to identify individual proteins. The PMF maps obtained by MALDI-TOF MS were used for protein identification. Fifteen protein spots were identified: laminin β1, ubiquitin-conjugating enzyme E2-25K, albumin, hypothetical protein LOC374986, ubiquitin carboxyl-terminal esterase L1 (UCHL1; also called protein gene product 9.5, PGP 9.5), γ-actin, tubulin R-1B, hemoglobin-β, erythrocyte 2,3-biphosphoglycerate mutase, coactosin-like protein-1 (COTL-1), hemoglobin, carbonic anhydrase I, and carbonic anhydrase II complex (Table 2). We then tested levels of laminin-β1, ubiquitinconjugating enzyme E2-25K, ubiquitin carboxyl-terminal esterase L1, γ-actin, tubulin R-1B, coactosin-like protein-1 (COTL1), and carbonic anhydrase 1 using specific antibodies in the SCLC tissues. Validation of Up-Regulated COTL-1 Expression in SCLC Tissues by Western Blotting. For Western blotting analysis, we tested protein extracts from 6 SCLC tissues and 5 normal tissues using antibodies to laminin-β1, ubiquitin-conjugating enzyme Journal of Proteome Research • Vol. 10, No. 1, 2011 271

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Figure 1. Representative 2-D PAGE gel images of protein spots in SCLC tissues. More than 1500 spots were detected in (A) normal bronchial epithelium and (B) SCLC tissues. (C) We analyzed 64 differentially expressed protein spots with changes over 2-fold between normal bronchial epithelium and SCLC tissues as labeled by PDQuest software.

Figure 2. Magnified 2-D PAGE gel images of the 15 up-regulated protein spots in (P) 6 SCLC tissues compared with (N) 6 normal bronchial epithelium. Protein spots showing more than 2-fold increased expression in SCLC tissues were presented.

E2-25K, ubiquitin carboxyl-terminal esterase L1, γ-actin, tubulin R-1B, coactosin-like protein-1 (COTL-1), and carbonic anhydrase. COTL-1 expression was upregulated in SCLC tissues, but other proteins were not (Figure 3). Validation of Up-Regulated Expression of COTL-1 in SCLC Tissues by Immunohistochemical Staining. We further evaluated COTL-1 expression in lung cancer tissues using IHC in paraffin samples from 30 SCLC cases, 32 adenocarcinomas, and 32 squamous cell carcinomas from the tissue bank of CHA 272

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hospital from 2000 to 2008. Patient characteristics, including carcinoma type, age, sex, stage, and smoking history, are presented in Table 3. COTL-1 staining in the SCLC group was higher than the NSCLC group, including adenocarcinoma and squamous cell carcinoma (p < 0.0001) (Figure 4). The pathological examination of IHC data has determined that COTL-1 expression in SCLC appeared to be located in almost all neoplastic epithelial cells. These cells were apparently highly mitotic and round shaped with scant cytoplasm, typical SCLC

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Up-Regulation of Coactosin-Like Protein-1 a

Table 2. Identification of Proteins Up-Regulated in SCLC Tissues by Proteomic Analysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

spot no.

protein name

1615 1618 2002 2016 2019c 2720 3002 3111 3206 5015 6012c 7012 7101 8018 8020

laminin B1 laminin B1 ubiquitin-conjugating enzyme E2-25K, coactosin-like protein GA module complexed with albumin hypothetical protein LOC374986 ubiquitin carboxyl-terminal esterase L1 gamma actin tubulin alpha hemoglobin hemoglobin beta carbonic anhydrase erythrocyte 2,3-diphosphoglycerate mutase carbonic anhydrase Ii complex hemoglobin

accession no.

gi gi gi gi gi gi gi gi gi gi gi gi gi gi gi

5031877 5031877 4885417 56966036 55669910 38348384 21361091 178045 57013276 1942686 229149 4502517 52695673 9257153 229752

estd Z

no. of matched peptides

sequence coverage

MW (KDa)n

pIn

intensity rate (+)

2.34 2.31 1.60 2.30 0.91 1.14 2.39 2.38 1.78 2.38 1.74 1.89 1.78 1.97 2.04

18/26 20/30 5/5 9/23 6/7 5/5 10/26 8/8 5/15 10/20 11/11 5/5 6/6 5/5 5/5

29% 31% 29% 45% 13% 10% 42% 31% 23% 67% 67% 18% 22% 31% 43%

66.68 66.88 22.50 15.78 67.20 71.96 25.15 26.15 50.82 15.86 15.97 28.91 31.22 29.15 15.96

5.1 5.1 5.3 5.5 5.6 5.4 5.3 5.6 4.9 6.8 5.2 6.6 6.3 6.9 6.8

2.05 3.34 2.04 6.47 3.61 7.26 4.00 4.98 3.29 85.44 2.77 13.17 2.97 12.93 20.29

a Peptide profiles of the protein spots treated with trypsin were analyzed by MALDI-TOF MS. The Profound program (http://prowl.rockefeller.edu/ prowl-cgi/profound.exe) was used to search a protein database for identification using peptide mass fingerprinting (PMF). n Mass and pI values specified are theoretically matched by database search. Plus (+); the rate of increase in intensity of small cell carcinoma tissue spot to intensity of normal lung tissue spot. c Protein spots represent probable fragments.

Figure 3. Result of Western blot assay. Validation of differential expression of COTL-1 between SCLC tissues and normal bronchial epithelium. N; normal, P; patient.

characteristics. Detailed immunohistological analysis of COTL-1 expression in lung carcinoma tissues is summarized in Table 3. COTL-1 staining rates in squamous cell carcinoma and in adenocarcinoma were 13% and 19%, respectively. In addition, two of five bronchial carcinoid tumors with the same degree of neuroendocrine differentiation also showed weak COTL-1 staining (data not shown). These results demonstrate that upregulation of COTL-1 is SCLC specific and may be a biomarker of SCLC. Validation of Up-Regulated Expression of COTL-1 in SCLC Tissues. For further validation study, we performed RTqPCR analysis with tumor tissues which were used for immunohistochemistry for the detection of COTL-1 mRNA expression. Four SCLC samples (sclc1, 2, 3, 4), 4 lung adenocarcinomas (ad1, 2, 3, 4), and 4 squamous cell carcinomas (sq1, 2, 3, 4), were analyzed. As shown in Figure 5, the levels of COTL-1 gene expression normalized by GAPDH was high in SCLC (relative fold expression was 0.18, 0.6, 1.3, and 2.6 for sclc1, sclc2, sclc3, and sclc4, respectively), moderate in adenocarcinoma (relative fold expression was 1, 0.08, 0.54, and 0.6 for ad1, ad2, ad3, ad4), and low in squamous cell carcinoma (relative fold expression was 0.21, 0.04, 0.002, and 0.24, for sq1, sq2, sq3, and sq4), respectively. This result seemed to correspond to the proteomic analysis data and the following validation studies analyzed by Western blotting and IHC. Taken together, these findings suggest that COTL-1 expression was specifically up-regulated in SCLC.

Discussion In this study, we identified differentially regulated proteins in human SCLC tissues compared with the normal bronchial epithelium by proteomic analysis. Many proteins were cytosk-

eletal or extracellular matrix (ECM) proteinssactin-γ, tubulinR, and laminin-βsthat are critically involved in cell morphology, locomotion, transport, and division, consistent with the aggressive metastatic propensity of SCLC. Actin exists as a globular monomer called G-actin or a filamentous polymer called F-actin. The dynamics of actin remodeling play an important role in regulating phenotypic changes of premalignant and malignant cells, such as altered morphology, tumor invasion, and altered growth and apoptosis. SCLC cells show scant cytoplasm with round or fusiform shape and a very high mitotic rate,19 and actin is a target of a growing number of anticancer drugs.20 Alterations in the dynamic state of actin could change cell fate or induce cell death caused by actin-binding drugs or by the mutations of actin or/and actinbinding proteins.21,22 The up-regulated expression of gamma actin in SCLC implicates that actin may be responsible for the pathological development and metastasis of SCLC. Together with actin filaments and intermediary filaments, microtubules formed by heterodimers of tubulin-R and tubulin-β subunits are also important cytoskeletal molecules.23 Microtubules are polymeric proteins with distinct roles in maintaining intracellular architecture and trafficking of cellular vesicles. Tubulin-R is a multidomain scaffolding protein that is ubiquitously expressed and consists of 6 SH3 domains. Four N-terminal SH3 domains bind directly to dynamin1 and two C-terminal src-homology 3 (SH3) domains bind multiple actin regulatory proteins. Although tubulin-R is a relatively unknown protein, it may associate with cell motility or cell invasion.24 In previous studies,25,26 tubulin-R contributed to drug resistance in lung cancer. Microtubule targeting drugs, such as taxol, epothilone, and vinca alkaloids have been extensively used to treat many types of cancer. However, the detailed action Journal of Proteome Research • Vol. 10, No. 1, 2011 273

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Table 3. Validation of COTL-1 Expression by IHC in Lung Cancer Patients

cancer type

Median age (range) Sex male female Stage limited extensive I II III IV Smoking current smoker ex-smoker nonsmoker COTL-1 IHCa Intensity non (0) weak (1) moderate (2) intense (3) Extensity 0 1 2 3 4 Positive staining

SCLC (n ) 30)

squamous cell carcinoma (n ) 32)

adenocarcinoma (n ) 32)

70 years (52-86) 63 years (34-81) 68 years (49-82)

27 (87%) 4 (13%)

13 (40%) 19 (60%)

31 (97%) 1 (3%)

3 (9%) 1 (3%) 3 (9%) 25 (78%)

3 (9%) 6 (19%) 13 (41%) 10 (31%)

21 (68%) 9 (29%) 1 (3%)

10 (31%) 3 (9%) 19 (59%)

22 (69%) 9 (28%) 1 (3%)

0 5 2 23

22 5 5 0

26 4 0 2

0 2 1 2 25 28 (93%)

22 3 2 3 2 6 (19%)

26 0 3 2 1 4 (13%)

6 (19%) 25 (81%)

a For IHC, the intensity of staining was graded according to a 4-tiered scale of 0 to 3 (with 3 as the most intense staining). The extent of positive immunoreactivity was graded according to the percentage of stained cells in the region of interest: 0 point for 0%, 1 point for 1-25%, 2 points for 26-50%, 3 points for 51-75%, and 4 points for g76%, respectively. An overall score was obtained by the sum of the intensity and the extent of the positive staining. A case with a final score of more than four was defined as positive. Samples were investigated by two independent experienced pathologists on staining pattern without knowledge of the patient’s clinical status.

mechanism and signaling pathways which lead to cytotoxic effects have not been clearly elucidated. Furthermore, the issues of specificity and sides effects still remain as major concerns in cancer therapy. Intriguingly, recent studies demonstrated that short siRNAs for tubulin betaII, III, and IVb isotypes have enhanced sensitivity to the anticancer drugs in NSCLC, implicating that the specific targeting of cytoskeletal molecules and their isotypes in cancer treatment is increasingly visible.27,28 On this line, the expression and mutational pattern analysis of actin or tubulin isotypes and their involvement to the drug sensitivity in SCLC may provide new therapeutic strategies. In addition, the studies on inter-regulation of tubulin or actin with other cytoskeletal molecules also may help in bring new insights into SCLC therapeutics. Furthermore, in association with cytoskeletal molecules via integrin or transmembrane proteins, ECM clearly functions in cancer biology. For example, laminin-β1 interacts with malignant cancer cells in tumor progression, and is synthesized and secreted in hepatocellular carcinoma29 and expressed in lung cancer cells.30 A growing number of studies targeting cytoskeletal molecules and ECM as anticancer drugs have shown promising outcomes in cancer treatment.31,32 Therefore, our observation of up-regulated expression of actin, tubulin, or laminin in SCLC suggest that these molecules may be future therapeutic targets. 274

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COTL-1 (coactosin like-1) is an F-actin-binding protein predominantly expressed in placenta, lung, and kidney,33 and we found it upregulated in SCLC in proteomic analysis, Western blot (Figure 3), and IHC (Figure 4). COTL-1 is expressed in urinary bladder cancer cells34 as well as in the serum of pancreatic cancer patients, where it elicited both humoral and cellular immunity recognized by HLA-A2 restricted and tumor reactive CTL.35 COTL-1 activates 5-lipoxygenase, an enzyme that converts arachidonic acid to LTA436 and is involved in inflammatory disease, cancer development, and cell survival. In addition, actin binds to 5-lipoxygenase and associates with lipoxygenase metabolites to inhibit lipoxygenase.37,38 Pharmacological inhibitors of lipoxygenase suppress carcinogenesis and tumor metastasis.39 Taken together, COTL-1 expression may interact with actin and 5-lipoxygenase to play a central role in SCLC. To confirm whether the up-regulated expression of COTL-1 was specific to SCLC, we tested human NSCLC tissues, including squamous cell carcinomas and adenocarcinomas. IHC showed strong COTL-1 expression in SCLC (28/30), as for proteomic analysis and Western analysis, but weak COTL-1 expression in squamous cell carcinomas (6/32) and in adenocarcinomas (4/32) (Figure 4, Table 3). Thus, up-regulated COTL-1 expression could be specific for SCLC. As SCLC originates from the neuroendocrine field, and neuroendocrine and neural differentiation would result in the expression of neuroendocrine markers, such as chromogranin A, CD56, gastrin releasing peptides, and IGF-1,40 we also tested for COTL-1 expression in 5 advanced neuroendocrine differentiated carcinoid tumors to study whether it could be useful as a neuroendocrine marker. Two out of five tumors stained weakly for COTL-1, but the others did not, suggesting that COTL-1 expression inversely correlated with the differentiation of neuroendocrine tumors. Indeed, COTL-1 expression was specifically increased in nearly all cases of SCLC with poor neuroendocrine differentiation (data not shown). Meanwhile, Cho et al showed differential expression of p63 and cytoketatin C18 that depended on the developmental stage of SCLC, and suggested them as diagnostic biomarkers.14 Here, we found that the up-regulation of COTL-1 expression was dominant in the extensive stage. Further studies on the SCLC tissue type-for example, primary SCLC, cancer metastasized to the lung from other tissues, or recurrent tissues-and its corelation with COTL-1 regulation could help early detection, prognosis, relapse, and responses to treatment. Protein ubiquitination is an important post-translational modification that functions in tumor growth by regulating protein degradation, the cell cycle, and apoptosis.41,42 SCLC tissues had high levels of ubiquitin carboxyl-terminal esterase L1 (UCHL1) and ubiquitin conjugating enzyme E2. UCHL1 serves as a regulator of protein degradation and apoptosis in breast cancer cells through phosphoinositide 3-kinase (PI3K)/ Akt signaling.43 Most 87.5% (14/16) SCLC tissues showed positive IHC staining for UCHL1, and up-regulated expression of UCHL1 was also found in SCLC cell lines by comparative proteomics.9,44 Intriguingly, an activated form of small ubiquitin like modifier (SUMO-2) was highly up-regulated in lung cancer and could interfere with MDM2 ubiquitination of p53, which, consequently, deregulates the cell cycle.45 Further studies on the mechanism of action of these enzymes may elucidate the dysregulated ubiquitin proteosome pathway in SCLC.

research articles

Up-Regulation of Coactosin-Like Protein-1

Figure 4. Immunohistochemical localization of COTL-1 in (A) normal bronchial epithelium, (B) adenocarcinoma, (C) squamous cell carcinoma, and (D) small lung carcinoma (×200). (E) The magnified view of (D) (×400). Strong COTL-1 staining was observed in SCLC.

Figure 5. RT-qPCR analysis of COTL-1 expression in lung cancer tissues of adenocarcinoma, squamous cell carcinoma, and SCLC.

In conclusion, we have identified differentially expressed proteins, including actin, tubulin, laminin, COTL-1, UCHL-1, and ubiquitin conjugating enzyme E2 in SCLC by proteomic analysis. COTL-1 expression was validated in SCLC by immunohistochemistry and may be a potential biomarker. Further studies are needed on the application of these molecules as diagnostics and treatment for SCLC.

Acknowledgment. This work was supported by a grant (2008-331-E00115) from the Korea Research Grant Foundation and a grant (2009-0093821) from the Priority

Research Centers Program funded by the Ministry of Education, Science and Technology, Republic of Korea.

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