Proteomics Identified Overexpression of SET Oncogene Product and

Article pubs.acs.org/jpr

Proteomics Identified Overexpression of SET Oncogene Product and Possible Therapeutic Utility of Protein Phosphatase 2A in Alveolar Soft Part Sarcoma Daisuke Kubota,†,‡ Akihiko Yoshida,§ Akira Kawai,‡ and Tadashi Kondo*,† †

Division of Pharmacoproteomics, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan Division of Musculoskeletal Oncology and §Pathology and Clinical Laboratory Division, National Cancer Center Hospital, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan

‡

S Supporting Information *

ABSTRACT: Alveolar soft part sarcoma (ASPS) is an exceedingly rare sarcoma refractory to standard chemotherapy. Although several molecular targeting drugs have been applied for ASPS, their clinical significance has not yet been established, and novel therapeutic strategies have long been required. The aim of this study was to identify proteins aberrantly regulated in ASPS and to clarify their clinical significance. Protein expression profiling of tumor and nontumor tissues from 12 ASPS patients was performed by 2-D difference gel electrophoresis and mass spectrometry. We found that the expression of 145 proteins differed significantly. Among them, further investigation was focused on the SET protein, which has multifunctional roles in cancers. Immunohistochemistry confirmed overexpression of SET in all 15 ASPS cases examined. Gene silencing of SET significantly decreased cell proliferation, invasion, and migration against a background of induced apoptosis. SET is known to be an inhibitor of phosphatase 2A (PP2A), which functions as a tumor suppressor by inhibiting the signal transduction pathway and inducing apoptosis. We found that a PP2A activator, FYN720, decreased cell proliferation through apoptosis. Together, our findings may suggest the possible contribution of SET to the tumor progression and the utility of FYN720 for treatment of ASPS. KEYWORDS: alveolar soft part sarcoma, SET, PP2A, 2D-DIGE

1. INTRODUCTION Alveolar soft part sarcoma (ASPS) is an exceedingly rare sarcoma that accounts for fewer than 1% of all soft tissue sarcomas.1 While patients can often achieve prolonged survival after surgery, even if metastases are present, most eventually succumb to the disease as a result of late metastasis. Both standard cytotoxic chemotherapy and radiation therapy have no significant survival advantage, and an effective molecular targeting drug has long been sought. ASPS is characterized by the presence of chromosomal rearrangement at 17q25 and Xp11.2, engendering a fusion gene, ASPSCR1-TFE3, which is a superactivated chimeric transcription factor.2 Recent global gene expression profiling has identified an array of genes that are possibly regulated by the ASPSCR1-TFE3 fusion gene, and these genes include potentially therapeutic targets.3−5 One of the critical genes induced by ASPSCR1-TFE3 and playing a role in disease progression is the angiogenesis-promoting oncogene, MET.3 This molecular background has led to the introduction of antiangiogenic agents such as INF-alpha,6−8 bevacizumab,9,10 sunitinib,11−13 and cediranib for ASPS. The clinical efficacies of © 2014 American Chemical Society

these novel molecular targeting agents will be validated in largescale clinical studies. In the present investigation, in order to clarify the molecular pathogenesis of ASPS and identify proteins that might have clinical utility, we performed a proteomic study of ASPS. While proteomics has been applied to a variety of cancers to identify biomarkers and therapeutic targets,14 no proteomics study of ASPS has been reported, except for one in which only one formalin-fixed and paraffin-embedded ASPS tissue was examined using a shotgun approach.15 We identified proteins that were uniquely expressed in the tumor tissues, and further focused on one oncogene product, suppressor of variegation, enhancer of zeste, and trithorax (SET), which was originally reported as a chimeric gene in acute myelocytic leukemia.16 We confirmed the overexpression of SET in ASPS by Western blotting and immunohistochemistry and verified the functional significance of SET by a gene-silencing assay. SET is a specific inhibitor of a Received: September 12, 2013 Published: March 13, 2014 2250

dx.doi.org/10.1021/pr400929h | J. Proteome Res. 2014, 13, 2250−2261

Journal of Proteome Research

Article

tumor suppressor, phosphatase 2A (PP2A),17 which is deregulated in several malignancies.18 We revealed that a PP2A activator, FYN720, exerted antitumor effects on ASPS cells. Our study reveals novel aspects of SET in ASPS and suggests possible therapeutic application of a PP2A inhibitor for this malignancy.

The experiment design for the 2D-DIGE experiments is overviewed in Figure 1A. In brief, the internal standard sample was created by mixing equal portions of all individual samples. Five micrograms each of the internal standard sample and individual sample were labeled with Cy3 and Cy5, respectively (CyDye DIGE Fluor saturation dye, GE, Uppsala, Sweden). These differently labeled protein samples were mixed and separated according to isoelectric point and molecular weight. The first-dimension separation was achieved using Immobiline pH gradient DryStrip gels (24 cm long, pI range 4−7, GE) and Multiphor II (GE). The second-dimension separation was achieved by SDS-PAGE using our original large-format electrophoresis apparatus (33 cm separation distance, Biocraft, Tokyo, Japan).19 The gels were scanned using laser scanners (Typhoon Trio, GE) at appropriate wavelengths for Cy3 or Cy5 (Figure 1B). For all protein spots, the Cy5 intensity was normalized with the Cy3 intensity in the same gel using the ProgenesisSameSpots software package version 3 (Nonlinear Dynamics, Newcastleupon-Tyne, U.K.). All samples were examined in triplicate gels, and the mean normalized intensity value was calculated. The experimental reproducibility of 2D-DIGE was examined by running the identical sample three times and evaluating the correlation of the protein spot intensities on a scatter gram (Figure 1C). Mass spectrometric protein identification was performed according to our previous report.19 In brief, 100 μg of the protein sample was labeled with Cy5 and separated by 2DPAGE as previously mentioned. Protein spots were recovered from the gels by an automated spot recovery machine (Molecular Hunter, AsOne, Osaka, Japan) and subjected to manual in-gel digestion using trypsin.19 The tryptic digests were subjected to liquid chromatography coupled with nanoelectrospray tandem mass spectrometry (Finnigan LTQ Orbitrap XL mass spectrometer, Thermo Electron, San Jose, CA). The Mascot software package (version 2.2; Matrix Science, London, U.K.) was used to search for the mass of each peptide ion peak against the SWISS-PROT database (Homo sapiens, 471 472 sequences in the Sprot-57.5.fasta file). Proteins with a Mascot score of 34 or more were considered to be positively identified.

2. MATERIALS AND METHODS 2.1. Patients, Cell Line, and Protein Samples

This study included all 15 cases of ASPS that had been diagnosed and treated at the National Cancer Center Hospital between 1996 and 2010. Metastasis was diagnosed by computed tomography. For proteomics, tumor samples were obtained from 12 cases at the time of surgery, snap frozen in liquid nitrogen, and stored until use. The adjacent nontumor tissues were also obtained from areas distant from the tumor margin in 8 of these 12 cases. Immunohistochemistry was performed for all 15 ASPS cases including the 12 cases used for proteomics. Clinical information on the 15 patients is detailed in Table 1. Protein Table 1. Summary of Clinicopathological Data of the 15 ASPS Patients no.

gender

age

location

metastasis

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

female female female female female male female female male female female male female female female

28 26 23 23 16 35 29 14 33 27 33 31 47 26 23

femur retroperitoneal pelvis buttock femur extremity buttock extremity extremity pelvis pelvis femur primary unknown extremity extremity

none recurrence, spleen, bone neck, brain lung lung bone, lung, brain lung none lung lung lung lung brain brain, lung brain, lung

2.3. Western Blotting

Five microgram portions of the protein samples were separated by SDS-PAGE (ATTO, Tokyo, Japan). The separated proteins were subsequently blotted on a nitrocellulose membrane and incubated with a monoclonal antibody against SET (1:1000 dilution, Abcam, Cambridge, MA), pAKT(1:500 dilution, BD Bioscience, Franklin Lakes, NJ), Bad (1:500 dilution, BD), Bid (1:500 dilution, BD), and actin (1:5000, Abcam). The membrane was then reacted with horseradish-peroxidase-conjugated secondary antibody (1:3000 dilution, GE), processed using enhanced chemiluminescence reagents (ECL Prime, GE), and scanned with a LAS-3000 laser scanner (FujiFilm, Tokyo, Japan).

contents of the primary tumor tissues were examined in three cases each of gastrointestinal stromal tumor, osteosarcoma, rhabdomyosarcoma, and epithelioid sarcoma (Supplementary Table 1 in the Supporting Information). Protein extracts from normal tissues were obtained from BioChain (Newark, CA). The ASPS cell line ASPS-KY was kindly provided by Dr. Y Miyagi (Kanagawa Cancer Center Research Institute, Kanagawa, Japan). ASPS-KY was cultured in DMEM (supplemented with 10% fetal bovine serum, 1 mmol/L sodium pyruvate, 1 × nonessential amino acids, and 2 mmol/L glutamine) and incubated at 37 °C in a humidified 5% CO2 atmosphere. This project was approved by the ethical review board of the National Cancer Center, and signed informed consent was obtained from all of the patients included.

2.4. Immunohistochemistry

The most representative areas of tumor and nontumor tissues were sampled for tissue microarray (TMA). The TMAs were assembled with a tissue array instrument (Azumaya, Tokyo, Japan). To reduce sampling bias due to tumor heterogeneity, we used two replicate 2.0-mm-diameter cores from different areas of individual tumors. The TMA consisted of 15 cases of ASPS, including samples from the 12 cases (ASPS1−12) subjected to 2D-DIGE and from 3 newly enrolled cases (ASPS13−15, Table 1). The expression levels of SET were examined immunohistochemically. In brief, 4-μm-thick formalin-fixed, paraffin-embedded tissue sections and TMAs were autoclaved in 10 mmol/L

2.2. Protein Expression Profiling

Proteins were extracted from frozen tissues in accordance with our previous report.19 In brief, frozen tissues were crushed to powder with a Multibeads shocker (Yasui Kikai, Osaka, Japan) in liquid nitrogen. The frozen powder was then treated with urea lysis buffer (6 M urea, 2 M thiourea, 3% CHAPS, 1% Triton X-100) and centrifuged at 15 000 rpm for 30 min. The supernatant was recovered and stored at −80 °C until use. Protein expression profiling was performed by 2D-DIGE with our original large-format electrophoresis apparatus.19 2251



Article

Figure 1. Overview of experiment workflow using 2D-DIGE. (A) Individual samples and internal standard sample were labeled with two different fluorescent dyes, mixed together, and separated by 2D-PAGE. (B) Typical gel image of 2D-DIGE. The protein spot numbers correspond to those in Figure 2, Table 2, and Supplementary Tables 2 and 3 in the Supporting Information. The enlarged image is provided in Supplementary Figure 1 in the Supporting Information. (C) Technical reproducibility was examined by running one identical sample three times. Note that the intensity of at least 93.5% of the protein spots was scattered within a two-fold difference, and the overall correlation coefficient was more than 0.9.

citrate buffer (pH 6.0) at 121 °C for 30 min and incubated with anti-SET antibody (1:500 dilution, Abcam) for 1 h. Immunostaining was carried out by the streptavidin−biotin peroxidase method using an ABC complex/horseradish peroxidase kit (DAKO, Glostrup, Denmark). One pathologist (A.Y.) and one clinician (D.K.) reviewed the stained sections.

(siRNA-1), 5′-rCrArGrArGUUrGrArArGUrGrArCrArGrArATT3′ (siRNA-2), and 5′-rGrCrAUUrAUUUrGrArCrCrArGrArGUUTT −3′ (siRNA-3). A total of 5 × 103 ASPS cells, ASPS-KY, were seeded into each well of a 96-well plate (Coaster, Cambridge, MA). The following day, the cell monolayer was washed with prewarmed sterile phosphate-buffered saline. Cells were transfected with the appropriate siRNA using DharmaFECT transfection reagents (Thermo Fisher, Waltham, MA) in accordance with the manufacturer’s protocol. Twenty-four hours later the culture medium for the transfected cells was switched to medium A, whereas the conditioned medium was not changed.

2.5. Gene Silencing Assay

SET-specific siRNAs were purchased from Sigma-Aldrich, and control stealth siRNA was from Life Technologies. The target sequences were 5′-rGrCrAUUrAUUUrGrArCrCrArGrArGUUTT-3′ 2252



Article

2.6. Cell Proliferation and Invasion Assay

To examine the effects of SET and an inhibitor for PP2A on the ASPS cells, cell proliferation was examined after treatment with siRNAs or a PP2A activator, FTY720 (Selleck Chemicals, Houston, TX). FTY720 was dissolved with dimethyl sulfoxide (DMSO, Wako, Osaka, Japan), and added to the culture medium at an appropriate concentration. Cell proliferation was examined by the tetrazolium-based colorimetric MTT assay. In brief, the cells were transfected with the appropriate siRNA using DharmaFECT transfection reagent for 24 h. Alternatively, the cells were incubated with various concentrations of FTY720. Then, 20 μL of the reagent from Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) was added to each well containing ASPS-KY cells. After 2 h of incubation, the optical density was measured at a wavelength of 450 nm using a microplate reader (SAFARI, TECAN, Männedorf, Switzerland). Cell invasion before and after the treatments was evaluated using the BD BioCoat Invasion Chamber (BD Bioscience) in accordance with the manufacturer’s protocol. In brief, the cells were transfected with the appropriate siRNA using DharmaFECT transfection reagent for 24 h. The cells were seeded onto the membrane in the upper chamber of the trans-well at a concentration of 5 × 105 in 500 μL of serum-free medium. The medium in the lower chamber contained 10% fetal calf serum as a source of chemoattractants. Cells that passed through the Matrigel-coated membrane were stained with DiffQuick (Sysmex, Kobe, Japan), and the cells were counted. All experiments were performed at least three times, in duplicate or triplicate, and statistical significance was calculated by t test using the SPSS statistical software package (IBM, Armonk, NY).

3. RESULTS 3.1. Protein Expression Profiling Identifies the Set Oncogene Product in ASPS

Protein expression profiles were created by 2D-DIGE, which is advantageous in that gel-to-gel variations can be compensated for by normalizing the expression data for individual samples with those of the common internal standard sample using two different fluorescent dyes (Figure 1A). Such normalization was performed for 2300 protein spots (Figure 1B, the enlarged image is Supplementary Figure 1 in the Supporting Information, and the intensity of all 2300 protein spots is shown in Supplementary Table 2 in the Supporting Information). The large-format electrophoresis apparatus and the internal standard sample resulted in highly reproducible protein expression profiling, as shown by scatter gram for three independent experiments on one identical sample (Figure 1C). The comparison between tumor and nontumor tissues resulted in the identification of 145 protein spots showing significant differences in intensity (p value less than 0.01 and more than two-fold intensity difference, Figure 2 and Table 2). Mass spectrometry detected the corresponding proteins for these 145 spots. The differential intensities of the protein spots and the results of protein identification are summarized in Figure 2. Detailed data for the identified proteins and supportive data for positive protein identification are provided in Supplementary Table 3 in the Supporting Information.

Figure 2. Protein spots with different intensities and their annotation by mass spectrometry. Differences in the intensity of protein spots between tumor tissues and noncancerous adjacent tissues are exhibited in the form of a heat-map. Any intensity showing more than a two-fold difference with statistical significance (p < 0.05) was considered to be positive. Mass spectrometry annotation of the protein spots is demonstrated on the right side of the heat-map.

considered to be a therapeutic target.20 By Western blotting, we confirmed the overexpression of SET in tumor tissues compared with noncancerous adjacent normal tissues (Figure 3A). SET was observed as a single band with the expected molecular weight (Figure 3A). Immunohistochemistry demonstrated nuclear localization of SET with preferential expression in tumor cells relative to nontumor cells (Figure 3B). We confirmed the overexpression of SET in the 15 cases of ASPS (Supplementary

3.2. Immunological Validation of SET Overexpression in Primary Tumor Tissues of ASPS

We focused in detail on the higher expression of the SET oncogene product. The product of the SET is a multifunctional protein contributing to progression of various cancers and is 2253



Article

Table 2. Summary of Proteins with Differential Expression between ASPS Tumor Tissues and Nontumor Tissues spot no.a

accession no.b

3552 3541 3549 3543

P07477 Q9P032 P35030 Q9NYY3

3089

Q99536

3091 3539 2792 3551 3335 3090

O14773 Q9P1Z9 P07237 P07477 P07339 Q99536

3556 3544 3154 2123 3560 3554 3084

P16402 P47974 P40121 Q9H0J4 P07477 P07477 Q99536

3088

Q99536

3158 3504 3548 1604 2015 1488 2791 2681 489 3333 3558 2976 2883

P40121 P23528 P07477 P07339 P01024 Q96DA2 P07237 P27797 P27797 P07339 P35030 Q12765 Q6ZMW3

761 3384 1406 3499 1568

P08670 Q9Y5Z4 Q9NZD8 Q9P1Z9 Q96C19

1611 2974 3500 3302

P27348 P04264 P11940 O14646

3562 3275 1752 2846 1686 3334 323 2558 2794 326 2695 3575 3182

P07477 Q9P1Z9 Q7Z6Z7 Q13885 P40121 P07339 Q8IWE2 P14314 P07237 Q8IWE2 P27797 P07477 Q9UJ70

identified protein trypsin-1 uncharacterized protein C3orf60 trypsin-3 serine/threonine-protein kinase PLK2 synaptic vesicle membrane protein VAT-1 tripeptidyl-peptidase 1 uncharacterized protein KIAA1529 protein disulfide-isomerase trypsin-1 cathepsin D synaptic vesicle membrane protein VAT-1 histone H1.3 butyrate response factor 2 macrophage-capping protein glutamine-rich protein 2 trypsin-1 trypsin-1 synaptic vesicle membrane protein VAT-1 synaptic vesicle membrane protein VAT-1 macrophage-capping protein cofilin-1 trypsin-1 cathepsin D complement C3 Ras-related protein Rab-39B protein disulfide-isomerase calreticulin calreticulin cathepsin D trypsin-3 secernin-1 echinoderm microtubule-associated protein-like 6 vimentin heme-binding protein 2 maspardin uncharacterized protein KIAA1529 EF-hand domain-containing protein D2 14-3-3 protein theta keratin, type II cytoskeletal 1 putative protein PABPC1-like chromodomain-helicase-DNAbinding protein 1 trypsin-1 uncharacterized protein KIAA1529 E3 ubiquitin-protein ligase HUWE1 tubulin beta-2A chain macrophage-capping protein cathepsin D protein NOXP20 glucosidase 2 subunit beta protein disulfide-isomerase protein NOXP20 calreticulin trypsin-1 N-acetyl-D-glucosamine kinase

fold difference (ratio of means)

pI (cal)c

MW (cal) (Da)c

10−5 10−5 10−5 10−5

0.008515009 0.010363941 0.010390875 0.011751306

6.08 8.48 7.46 8.52

27111 20566 33306 79270

1.5877 × 10−5

0.021958413

5.88

Wilcoxon test p value 1.5877 1.5877 1.5877 1.5877

× × × ×

protein scored

peptide matches

sequence coverage (%)

72 67 58 37

1 1 1 1

8.1 6 4.3 1.3

42122

185

2

8.9

1.5877 1.5877 1.5877 1.5877 1.5877 1.5877

× × × × × ×

10−5 10−5 10−5 10−5 10−5 10−5

0.02956955 0.029933118 0.03101972 0.031037268 0.032525296 0.034147549

6.01 5.74 4.76 6.08 6.1 5.88

61723 192404 57480 27111 45037 42122

149 40 1138 43 254 225

3 1 38 1 4 5

8.2 1.2 35.8 8.1 14.3 21.4

1.5877 1.5877 3.1754 1.5877 1.5877 1.5877 1.5877

× × × × × × ×

10−5 10−5 10−5 10−5 10−5 10−5 10−5

0.035355776 0.036972559 0.037782583 0.039776539 0.041918698 0.043031618 0.043691971

11 8.52 5.88 6.25 6.08 6.08 5.88

22336 52000 38779 181228 27111 27111 42122

32 38 429 42 63 58 356

1 1 8 1 1 1 7

4.1 5.1 26.1 0.5 8.1 8.1 19.8

1.5877 × 10−5

0.044704717

5.88

42122

215

5

12.5

0.00011114 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5

0.04492316 0.046486714 0.051111972 0.052422591 0.053827382 0.054075741 0.056152907 0.059656716 0.059974668 0.060978434 0.062680878 0.062850066 0.062965523

5.88 8.22 6.08 6.1 6.02 7.7 4.76 4.29 4.29 6.1 7.46 4.66 7.17

38779 18719 27111 45037 188569 24835 57480 48283 48283 45037 33306 46980 220270

332 53 79 91 58 36 71 504 94 153 60 86 50

9 1 1 2 1 1 2 14 2 4 1 2 2

21.8 6.6 8.1 6.3 1.3 6.1 4.7 26.6 4.3 11.2 4.3 6.3 0.4

0.0004763 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5

0.065528927 0.067161156 0.068166 0.068985159 0.071337515

5.06 4.58 5.85 5.74 5.15

53676 22861 35223 192404 26794

358 143 82 43 165

15 2 1 1 3

23.6 13.2 3.9 1.2 15

10−5 10−5 10−5 10−5

0.074204821 0.074765405 0.075151195 0.075335702

4.68 8.15 8.88 6.72

28032 66170 30256 197538

74 122 49 40

1 2 1 1

5.7 3.7 7.4 0.5

1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 0.00019052 1.5877 × 10−5

0.076824606 0.07718673 0.079027202 0.079394824 0.079825987 0.079953736 0.080187134 0.080715302 0.081241738 0.081614914 0.083690191 0.083697415 0.084723353

6.08 5.74 5.1 4.78 5.88 6.1 4.61 4.33 4.76 4.61 4.29 6.08 5.81

27111 192404 485523 50274 38779 45037 61046 60357 57480 61046 48283 27111 37694

63 42 39 975 154 310 267 316 841 82 209 61 388

1 3 1 18 4 6 6 5 25 2 4 1 6

8.1 1.2 0.3 43.8 8.3 16.5 15.1 10.6 32.5 3.7 9.4 8.1 25

1.5877 1.5877 1.5877 1.5877

× × × ×

2254



Article

Table 2. continued spot no.a

accession no.b

2790 1877 2971 1689 2972

P07237 P62820 Q12765 P52565 Q8N7U6

3085

Q99536

2841 2124 2564 2030 2839 1426

Q96FT9 P60660 P27824 Q9H0J4 Q13885 P84157

992 1635 3441

P60709 P67936 Q92630

1128 3328 3468 1681 252 863 3481 3561 1292 3573 2009 1836 3263 1843 2837 758 1492

P40121 P07339 Q8WWM9 P04264 P14625 Q07960 P62820 P07477 Q9P1Z9 P07477 P00441 P51149 P47756 P51149 Q13885 P14625 Q9BPX1

998 3383 964 3264 1609

P60709 Q9Y5Z4 Q9UNH7 P47756 Q9UL46

2914

P21281

3305

O00299

1548 1583 1211

P06753 P67936 P50213

1581

Q96C19

2847 3311 884 2556 1457 2566 1439

Q13885 P62258 Q07960 P14314 Q8N8N7 P27824 P84157

2180 1247

P04264 P06748

identified protein protein disulfide-isomerase Ras-related protein Rab-1A secernin-1 Rho GDP-dissociation inhibitor 1 EF-hand domain-containing family member B synaptic vesicle membrane protein VAT-1 uncharacterized protein C14orf179 myosin light polypeptide 6 calnexin glutamine-rich protein 2 tubulin beta-2A chain matrix-remodeling-associated protein 7 actin, cytoplasmic 1 tropomyosin alpha-4 chain dual specificity tyrosinephosphorylation-regulated kinase 2 macrophage-capping protein cathepsin D cytoglobin keratin, type II cytoskeletal 1 endoplasmin Rho GTPase-activating protein 1 Ras-related protein Rab-1A trypsin-1 uncharacterized protein KIAA1529 trypsin-1 superoxide dismutase [Cu−Zn] Ras-related protein Rab-7a F-actin-capping protein subunit beta Ras-related protein Rab-7a tubulin beta-2A endoplasmin 17-beta-hydroxysteroid dehydrogenase 14 actin, cytoplasmic 1 heme-binding protein 2 sorting nexin-6 F-actin-capping protein subunit beta proteasome activator complex subunit 2 V-type proton ATPase subunit B, brain isoform chloride intracellular channel protein 1 tropomyosin alpha-3 chain tropomyosin alpha-4 chain isocitrate dehydrogenase [NAD] subunit alpha EF-hand domain-containing protein D2 tubulin beta-2A chain 14-3-3 protein epsilon Rho GTPase-activating protein 1 glucosidase 2 subunit beta glucosidase 2 subunit beta calnexin matrix-remodeling-associated protein 7 keratin, type II cytoskeletal 1 nucleophosmin


pI (cal)c

MW (cal) (Da)c

protein scored

peptide matches


10−5 10−5 10−5 10−5 10−5

0.085599608 0.086095703 0.087093501 0.089182867 0.089364253

4.76 5.93 4.66 5.02 7.5

57480 22891 46980 23250 94558

313 216 42 207 48

7 4 1 5 1

15.4 27.3 2.7 22.1 1.8

1.5877 × 10−5

0.090047239

5.88

42122

186

4

17

10−5 10−5 10−5 10−5 10−5 10−5

0.090304869 0.090851666 0.09097873 0.091020777 0.091496662 0.091543619

4.57 4.56 4.47 6.25 4.78 4.24

23572 17090 67982 181228 50274 21509

44 205 224 45 279 48

1 4 4 1 5 1

9.1 28.5 10.1 0.5 11.9 3.9

1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5

0.092202715 0.093892535 0.094160695

5.29 4.67 9.7

42052 28619 67123

359 613 40

21 67 1

21.1 35.1 4

6.3507 × 10−5 1.5877 × 10−5 1.5877 × 10−5 0.00030166 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 6.3507 × 10−5 1.5877 × 10−5 6.3507 × 10−5 1.5877 × 10−5 3.1754 × 10−5 1.5877 × 10−5 6.3507 × 10−5 6.3507 × 10−5

0.094214275 0.094241211 0.094317704 0.094354027 0.095256618 0.095902059 0.097286337 0.097721585 0.098548367 0.098828882 0.09993381 0.101448964 0.101659636 0.101686834 0.101856539 0.101899313 0.101946674

5.88 6.1 6.32 8.15 4.76 5.85 5.93 6.08 5.74 6.08 5.7 6.4 5.36 6.4 4.78 4.76 5.8

38779 45037 21505 66170 92696 50461 22891 27111 192404 27111 16154 23760 31616 23760 50274 92696 28642

264 190 174 807 1270 189 68 67 43 79 44 421 102 262 459 245 37

7 4 4 14 75 6 1 1 4 1 1 7 2 6 11 6 1

14.7 11.2 19.5 25.6 33 15.7 7.8 8.1 1.2 8.1 9.1 38.2 8.7 37.7 24 7.3 4.4

10−5 10−5 10−5 10−5 10−5

0.102092468 0.102129229 0.102531665 0.103384254 0.105128204

5.29 4.58 5.81 5.36 5.44

42052 22861 46905 31616 27515

715 156 261 265 94

90 3 6 4 2

34.1 19.5 15.3 13.7 10.9

1.5877 × 10−5

0.105169042

5.57

56807

509

12

20.5

1.5877 × 10−5

0.105232399

5.09

27248

262

4

21.2

1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5

0.105267655 0.106071807 0.106662107

4.68 4.67 6.47

32856 28619 40022

343 426 150

36 9 3

15.1 30.2 8.5

1.5877 × 10−5

0.107781812

5.15

26794

46

1

5

1.5877 × 10−5 1.5877 × 10−5 0.00071446 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5

0.107841883 0.108066218 0.108386461 0.108738059 0.109312543 0.109775315 0.109934952

4.78 4.63 5.85 4.33 5.27 4.47 4.24

50274 29326 50461 60357 38930 67982 21509

319 359 291 194 40 267 172

6 7 7 4 1 6 3

18.9 27.8 10.9 10.2 2.6 11.8 17.6

1.5877 × 10−5 1.5877 × 10−5

0.110359174 0.110641094

8.15 4.64

66170 32726

289 42

6 1

11 3.1

Wilcoxon test p value 1.5877 1.5877 1.5877 1.5877 1.5877

1.5877 1.5877 1.5877 1.5877 1.5877 1.5877

1.5877 1.5877 1.5877 1.5877 1.5877

× × × × ×

× × × × × ×

× × × × ×

2255



Article

Table 2. continued spot no.a

accession no.b

2214 2612 2286 1999 860 1149 3513

O75916 P08670 Q96QU1 P52565 Q9UNH7 P61160 O60423

1345 1814 3180

Q96JI7 P61586 P10809

533

P29083

3181 3522 2952 2254 950 277 1553 1481 1665 2840 498

Q9UJ70 P84077 Q9P1U1 Q14005 Q01105 P27824 P06753 Q49AM3 P63104 Q13885 Q16822

3331

Q5TAX3

3555 2921 2571 3285 1591 1433 3452 1840 746 2560 1546

P16949 P08670 Q9H0J4 P08758 P67936 Q9P1Z9 P09211 P51149 P60709 P14314 Q6ZMW3

3178 3177 2189 3124 3123

P07951 P07951 P35030 P68133 P62736

identified protein regulator of G-protein signaling 9 vimentin protocadherin-15 Rho GDP-dissociation inhibitor 1 sorting nexin-6 actin-related protein 2 probable phospholipid-transporting ATPase IK spatacsin transforming protein RhoA 60 kDa heat shock protein, mitochondrial general transcription factor IIE subunit 1 N-acetyl-D-glucosamine kinase ADP-ribosylation factor 1 actin-related protein 3B pro-interleukin-16 protein SET calnexin tropomyosin alpha-3 chain tetratricopeptide repeat protein 31 14-3-3 protein zeta/delta tubulin beta-2A chain phosphoenolpyruvate carboxykinase [GTP] zinc finger CCHC domaincontaining protein 11 stathmin vimentin glutamine-rich protein 2 annexin A5 tropomyosin alpha-4 chain uncharacterized protein KIAA1529 glutathione S-transferase P Ras-related protein Rab-7a actin, cytoplasmic 1 glucosidase 2 subunit beta echinoderm microtubule-associated protein-like 6 tropomyosin beta chain tropomyosin beta chain trypsin-3 actin, alpha skeletal muscle actin, aortic smooth muscle


pI (cal)c

MW (cal) (Da)c

protein scored

peptide matches


10−5 10−5 10−5 10−5 10−5 10−5 10−5

0.110938705 0.111308534 0.111433545 0.112138698 0.11235212 0.112416534 0.113222024

9.42 5.06 4.94 5.02 5.81 6.3 7.97

77601 53676 217303 23250 46905 45017 149532

36 1077 36 216 79 50 48

1 24 1 5 2 1 1

1.2 41.8 0.6 15.2 5.2 3 1.6

1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5

0.113459904 0.113566968 0.113616827

5.63 5.83 5.7

282621 22096 61187

39 49 133

2 1 3

1 4.1 5.2

1.5877 × 10−5

0.113756065

4.74

49763

41

1

3.2

10−5 10−5 10−5 10−5 10−5 10−5 10−5 10−5 10−5 10−5 10−5

0.114109084 0.114951474 0.115291654 0.115572204 0.116088182 0.117230427 0.117344658 0.117497177 0.119108879 0.119125209 0.119425451

5.81 6.32 5.61 8.34 4.23 1 4.68 8.52 4.73 4.78 7.56

37694 20741 48090 142976 33469 67982 32856 57753 27899 50274 71447

224 274 76 41 278 584 46 42 339 91 134

4 6 1 1 5 13 1 1 13 2 3

14.8 32 2.6 1.3 22.8 5 3.9 2.7 24.9 8.5 6.3

1.5877 × 10−5

0.11952247

8.3

188014

35

1

1

1.5877 × 10−5 0.00215924 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5 3.1754 × 10−5 1.5877 × 10−5 1.5877 × 10−5 1.5877 × 10−5

0.119549402 0.120101433 0.120540368 0.120737507 0.121326925 0.121566905 0.121610737 0.121913756 0.121964836 0.12226935 0.124504715

5.76 5.06 6.25 4.94 4.67 5.74 5.43 6.4 5.29 4.33 7.17

17292 53676 181228 35971 28619 192404 23569 23760 42052 60357 220270

130 320 45 930 165 44 205 171 387 230 38

3 7 1 21 4 3 3 4 11 4 1

20.8 14.8 0.5 48.1 17.3 1.2 21.4 24.6 21.3 8.5 0.4

8.436657849 8.672066697 8.755681188 13.86346832 23.794185

4.66 4.66 7.46 5.23 5.23

32945 32945 33306 42366 42381

674 502 66 561 386

13 9 1 16 17

34.9 27.5 4.3 31.8 19.6

Wilcoxon test p value 1.5877 1.5877 1.5877 1.5877 1.5877 1.5877 1.5877

1.5877 1.5877 1.5877 3.1754 1.5877 1.5877 1.5877 1.5877 1.5877 1.5877 1.5877

× × × × × × ×

× × × × × × × × × × ×

0.00030166 0.00071446 0.00730333 0.00409621 0.00730333

a

Spot numbers refer to those in Figure 1B. bAccession numbers of proteins were derived from Swiss-Plot and NBCI nonredundant databases. Theoretical isoelectric point and molecular weight obtained from Swiss-Prot. dMascot score for the identified proteins based on the peptide ions score (p < 0.05) (http://www.matrixscience.com). c

Figure 2 in the Supporting Information). We then examined the expression of SET in normal tissues using Western blotting and found that none of these tissues expressed SET (Figure 3C). We surveyed the expression of SET in different histologic types of sarcomas such as gastrointestinal stromal tumor, osteosarcoma, rhabdomyosarcoma, alveolar soft part sarcoma, and epithelioid sarcoma and found that SET was expressed to various degrees in all of them (Figure 3D).

siRNAs against SET resulted in a marked decrease in SET protein expression (Figure 4A). The cells with reduced SET expression showed significantly diminished cell proliferation relative to cells transfected with control siRNA (Figure 4B). The matrigel invasion assay showed that silencing of SET caused a significant decrease in invasion and migration of ASPS cells (Figure 4C,D). These observations suggested that SET may be involved in tumor progression in ASPS.

3.3. Functional Verification of the Overexpressed SET Oncogene Product in ASPS Cells

3.4. Apoptotic Effects of the PP2A Activator, FTY720, on ASPS Cells

To verify the functional significance of SET overexpression in ASPS, we performed a gene-silencing assay. Transfection of three

SET is known to be an inhibitor of PP2A, which reduces the kinase-dependent signal transduction pathway, causing 2256



Article

Figure 3. Immunological validation of SET overexpression in ASPS tumor cells. (A) Expression of SET was evaluated in tumor tissues and nontumor tissues by Western blotting. SET was preferentially expressed in tumor tissues. (B) Immunohistochemistry demonstrated the expression and localization of SET in tumor and nontumor tissues. Note that SET was absent in nontumor cells that surrounded tumor cells (left panel). Nuclear localization of SET is demonstrated (right panel). The results of immunohistochemistry for 15 cases of ASPS are exhibited in Supplementary Figure 2 in the Supporting Information. (C) Expression of SET in organs and tissues was evaluated by Western blotting. Note that SET expression was observed only in ASPS tumor tissues. (D) Expression of SET in sarcomas of different histologic types was assessed by Western blotting. Note that SET protein was expressed to various degrees in all of the sarcomas examined.

mitochondria-dependent apoptosis.20 We hypothesized that the inhibitory effects of SET on PP2A would be responsible for the tumor progression of SET and that the PP2A pathway might be a novel therapeutic target in ASPS. To address this hypothesis, we examined the effects of a PP2A activator, FTY720, on ASPS cells. FTY720 is used for the treatment of patients with multiple sclerosis, and its application to anticancer therapy has been considered.21 Treatments of ASPS cells with FTY720 markedly suppressed their proliferation in a dose- and time-dependent manner (Figure 5A,B). We found that the PP2A treatment altered the expression of proteins involved in mitochondriamediated apoptosis, including phosphorylated Akt, Bad, and Bid (Figure 5C), consistent with previous reports.22−24

These observations suggested the possible utility of the PP2A activator, FTY720, for treatments of ASPS.

4. DISCUSSION ASPS is refractory to standard chemotherapy and radiation therapy, and novel therapeutic targets have long been sought to improve the clinical outcome.25 Although proteomics is an approach worth trying for ASPS, no such study has been performed previously, probably because of the extreme rarity of ASPS and the difficulty in obtaining adequate clinical samples. In fact, in 2010, only 11 cases of ASPS were registered in Japan,26 and gel-based proteomics such as 2D-DIGE requires frozen tissue samples, which are usually not stored in hospitals. 2257



Article

Figure 4. In vitro functional verification of SET in ASPS cells. (A) SET expression was suppressed by treatment with specific siRNAs against SET relative to treatment with control siRNA. Treatment with siRNAs 1−3 decreased the expression of SET in ASPS cells. (B) Proliferation of cells showing decreased SET expression was significantly inhibited. (C) Appearance of invading cells treated with siRNAs against SET. (D) Number of invasive cells was significantly (p < 0.05) decreased by treatment with siRNAs 1−3.

have a wide dynamic range. Moreover, gel-to-gel variations are compensated for by using a common internal standard sample. We developed our own large-format gel electrophoresis apparatus, which allows wider coverage of the proteome.27 In the present study, we observed 2300 protein spots, and normalized their intensity (Supplementary Table 2 in the Supporting Information). Together with details of the clinical parameters of the cases employed (Supplementary Table 1 in the Supporting Information), our proteome data will be useful for further studies of ASPS. We identified higher expression of SET in ASPS tissues relative to adjacent nontumor tissues (Figure 2). Overexpression of SET was confirmed in all 15 cases of ASPS by Western blotting and immunohistochemistry (Figure 2). We found that SET was generally expressed to various degrees in sarcomas (Figure 2). The clinical significance of differences in the expression level

These issues are inherent to rare diseases and conventional proteomics methods and are an obstacle to basic research that requires the use of clinical materials. In the present study, we successfully examined 12 samples of frozen ASPS tissue and validated the results of proteomics in 15 cases, including 3 newly enrolled cases (Table 1). Because all cases of ASPS share a common chromosomal rearrangement and expression of an activated transcription factor, ASPL-TFE3,2 the molecular background of ASPS may be homogeneous. Thus, even from a small number of samples we may be able to obtain results that would shed light on the general background of the disease. We employed 2D-DIGE to create protein expression profiles. In 2D-DIGE, the proteins are extensively separated according to isoelectric point and molecular weight, and the levels of their expression are evaluated in terms of fluorescent signals, which 2258



Article

Figure 5. Effects of the PP2A activator, FTY720, on cell proliferation and apoptosis. (A) Effects of FTY720 on cell viability were examined as a function of FTY720concentration. (B) Time-dependent inhibitory effects of FTY720 (16 μM) on cell viability. (C) Effects of FTY720 at various concentrations on the expression of apoptosis-regulating proteins were examined by Western blotting. Note that treatment of the cells with FTY720 caused a reduction in the expression of pAkt and increased the expression of Bad and Bid. In contrast, Akt expression level was not affected by FTY720 treatments. These proteins were not observed by 2D-DIGE. (D) Activation of PP2A by treatment with FTY720 triggers the apoptosis pathway, resulting in reduced proliferation of ASPS cells.

The molecular mechanisms responsible for SET overexpression are intriguing but still unclear. ASPS is characterized by chromosomal rearrangement and expression of an activated transcription factor, ASPL-TFE3.2 Genes under the control of this fusion gene have been revealed by DNA microarray experiments, and such genes would likely account for the molecular

of SET may be worth investigating in the context of its predictive or prognostic utility. In this study, the SET expression was observed in ASPS and the other sarcomas but not in normal tissues examined, suggesting that SET expression may represent common characters of malignancies including sarcomas. 2259



■

background of hyper-angiogenesis observed in ASPS.3−5 However, SET is not one of the genes that are regulated by ASPL-TFE3.3−5 Cervoni et al. reported overexpression of SET in various cancers such as those of the breast, uterus, colorectum, stomach, and kidney relative to their adjacent normal tissues28 but not in ASPS. Recently, in a study of chronic myeloid leukemia, Piazza et al. found a mutation in SET binding protein 1 (SETBP1), which hindered the degradation of SET and increased the expression level of both SETBPs and SET.29 Elucidation of the functional properties of the protein complex of SET may provide further insight into the etiology of ASPS. Because higher expression of SET is observed in a wide variety of malignancies, it may be attributable to common and important molecular mechanisms and therefore warrants further investigation. It is interesting if SET is a key to reach to common molecular backgrounds among malignancies including ASPS. Because overexpression of SET may be a novel therapeutic target in ASPS, we focused on SET in the present study. SET is a multifunctional protein with an inhibitory effect on the activity of PP2A, which is one of the major cellular serine-threonine phosphatases21 and negatively regulates signaling pathways such as the PI3K/Akt pathway,30 which is aberrantly regulated in various cancers. PP2A is a tumor-suppressor protein, and its functional inactivation has been reported in several cancers.31 Switzer et al. have reported a novel SET interactive peptide, COG112, which inhibits the association between SET and the PP2A catalytic subunit.32 Treatment of cancer cells with CIG112 increases PP2A activity and inhibits Akt signaling and cellular proliferation. In leukemia, activators of PP2A such as forskolin,33 1,9-dideoxy-forskolin,34 and FTY72035 effectively antagonize leukemogenesis. Remarkably, even in imatinib-resistant leukemia cells, restoration of PP2A activity, either by application of a chemical PP2A activator or interference with SET/PP2A interplay, has been reported to inactivate BCR/ABL, trigger growth suppression, enhance apoptosis, and impair leukemogenesis.36 Therefore, PP2A may be a promising drugable therapeutic target for patients who show resistance to treatment. In rare diseases, it may be difficult to develop original drugs because of their low commercial potential; therefore, additional use of existing drugs may be regarded as a more practical approach. Here we focused on FTY720 because it has been used for treatment of multiple sclerosis and malignancies.21 We found that treatment of ASPS cells with FTY720 induced apoptosis and inhibited cell proliferation, invasion, and migration (Figure 3). ASPS exhibits MET autophosphorylation following activation of downstream signaling pathways, and the clinical efficacy of a multikinase inhibitor, sunitinib malate, has been demonstrated in ASPS.12,13,37 Because FTY720 affects the signal transduction pathway through a mechanism differing from than of MET, it would be worth examining the utility of PP2A activator for ASPS patients who show resistance to kinase inhibitors. The effects of FTY720 on ASPS cells should be investigated further using additional cell lines and animal models. In conclusion, we have successfully identified the expression of SET in ASPS using a proteomics approach. The possible application of PP2A activator for treatment of ASPS will be investigated further. Because overexpression of SET was generally observed in sarcomas, the application of the PP2A activators for sarcoma treatments is worth examining. To elucidate the molecular background responsible for overexpression of SET, it is essential to establish ASPS cell lines. We anticipate that our research activity will eventually be of benefit to ASPS patients.

Article

ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figure 1: Typical image of two-dimensional difference gel electrophoresis. Supplementary Figure 2: Immunohistochemistry of SET in tumor cells and non-tumor cells. Supplement Table 1: Patient's characteristics of various saroma cases Supplement Table 2: A summary of the intensity of all protein spots. Supplement Table 3: Supportive information for protein identification. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +81-3-3542-2511. Fax: +81-3-3547-5298. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was supported by the National Cancer Center Research Core Facility and by the National Cancer Center Research and Development Fund (23-A-7 and 23-A-10). Patient's characteristics of various saroma cases.

■

REFERENCES

(1) Christopherson, W. M.; Foote, F. W., Jr.; Stewart, F. W. Alveolar soft-part sarcomas; structurally characteristic tumors of uncertain histogenesis. Cancer 1952, 5 (1), 100−111. (2) Ladanyi, M.; Lui, M. Y.; Antonescu, C. R.; Krause-Boehm, A.; Meindl, A.; Argani, P.; Healey, J. H.; Ueda, T.; Yoshikawa, H.; MeloniEhrig, A.; Sorensen, P. H.; Mertens, F.; Mandahl, N.; van den Berghe, H.; Sciot, R.; Dal Cin, P.; Bridge, J. The der(17)t(X;17)(p11;q25) of human alveolar soft part sarcoma fuses the TFE3 transcription factor gene to ASPL, a novel gene at 17q25. Oncogene 2001, 20 (1), 48−57. (3) Lazar, A. J.; Das, P.; Tuvin, D.; Korchin, B.; Zhu, Q.; Jin, Z.; Warneke, C. L.; Zhang, P. S.; Hernandez, V.; Lopez-Terrada, D.; Pisters, P. W.; Pollock, R. E.; Lev, D. Angiogenesis-promoting gene patterns in alveolar soft part sarcoma. Clin. Cancer Res. 2007, 13 (24), 7314−7321. (4) Stockwin, L. H.; Vistica, D. T.; Kenney, S.; Schrump, D. S.; Butcher, D. O.; Raffeld, M.; Shoemaker, R. H. Gene expression profiling of alveolar soft-part sarcoma (ASPS). BMC Cancer 2009, 9, 22. (5) Covell, D. G.; Wallqvist, A.; Kenney, S.; Vistica, D. T. Bioinformatic analysis of patient-derived ASPS gene expressions and ASPL-TFE3 fusion transcript levels identify potential therapeutic targets. PLoS One 2012, 7 (11), e48023. (6) Bisogno, G.; Rosolen, A.; Carli, M. Interferon alpha for alveolar soft part sarcoma. Pediatr. Blood Cancer 2005, 44 (7), 687−688. (7) Roozendaal, K. J.; de Valk, B.; ten Velden, J. J.; van der Woude, H. J.; Kroon, B. B. Alveolar soft-part sarcoma responding to interferon alpha-2b. Br. J. Cancer 2003, 89 (2), 243−245. (8) Kuriyama, K.; Todo, S.; Hibi, S.; Morimoto, A.; Imashuku, S. Alveolar soft part sarcoma with lung metastases. Response to interferon alpha-2a? Med. Pediatr. Oncol. 2001, 37 (5), 482−483. (9) Azizi, A. A.; Haberler, C.; Czech, T.; Gupper, A.; Prayer, D.; Breitschopf, H.; Acker, T.; Slavc, I. Vascular-endothelial-growth-factor (VEGF) expression and possible response to angiogenesis inhibitor bevacizumab in metastatic alveolar soft part sarcoma. Lancet Oncol. 2006, 7 (6), 521−523. (10) Mir, O.; Boudou-Rouquette, P.; Larousserie, F.; Blanchet, B.; Babinet, A.; Anract, P.; Goldwasser, F. Durable clinical activity of singleagent bevacizumab in a nonagenarian patient with metastatic alveolar soft part sarcoma. Anticancer Drugs 2012, 23 (7), 745−748. (11) Demetri, G. D.; van Oosterom, A. T.; Garrett, C. R.; Blackstein, M. E.; Shah, M. H.; Verweij, J.; McArthur, G.; Judson, I. R.; Heinrich, M. C.; Morgan, J. A.; Desai, J.; Fletcher, C. D.; George, S.; Bello, C. L.;

2260



Article

Huang, X.; Baum, C. M.; Casali, P. G. Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial. Lancet 2006, 368 (9544), 1329− 1338. (12) Stacchiotti, S.; Tamborini, E.; Marrari, A.; Brich, S.; Rota, S. A.; Orsenigo, M.; Crippa, F.; Morosi, C.; Gronchi, A.; Pierotti, M. A.; Casali, P. G.; Pilotti, S. Response to sunitinib malate in advanced alveolar soft part sarcoma. Clin. Cancer Res. 2009, 15 (3), 1096−1104. (13) Stacchiotti, S.; Negri, T.; Zaffaroni, N.; Palassini, E.; Morosi, C.; Brich, S.; Conca, E.; Bozzi, F.; Cassinelli, G.; Gronchi, A.; Casali, P. G.; Pilotti, S. Sunitinib in advanced alveolar soft part sarcoma: evidence of a direct antitumor effect. Ann. Oncol. 2011, 22 (7), 1682−1690. (14) Hanash, S. Progress in mining the human proteome for disease applications. OMICS 2011, 15 (3), 133−139. (15) Balgley, B. M.; Guo, T.; Zhao, K.; Fang, X.; Tavassoli, F. A.; Lee, C. S. Evaluation of archival time on shotgun proteomics of formalinfixed and paraffin-embedded tissues. J. Proteome Res. 2009, 8 (2), 917− 925. (16) von Lindern, M.; van Baal, S.; Wiegant, J.; Raap, A.; Hagemeijer, A.; Grosveld, G. Can, a putative oncogene associated with myeloid leukemogenesis, may be activated by fusion of its 3′ half to different genes: characterization of the set gene. Mol. Cell. Biol. 1992, 12 (8), 3346−3355. (17) Li, M.; Makkinje, A.; Damuni, Z. The myeloid leukemiaassociated protein SET is a potent inhibitor of protein phosphatase 2A. J. Biol. Chem. 1996, 271 (19), 11059−11062. (18) Seshacharyulu, P.; Pandey, P.; Datta, K.; Batra, S. K. Phosphatase: PP2A structural importance, regulation and its aberrant expression in cancer. Cancer Lett. 2013, 335 (1), 9−18. (19) Kondo, T.; Hirohashi, S. Application of highly sensitive fluorescent dyes (CyDye DIGE Fluor saturation dyes) to laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE) for cancer proteomics. Nat. Protoc. 2006, 1 (6), 2940− 2956. (20) Perrotti, D.; Neviani, P. Protein phosphatase 2A: a target for anticancer therapy. Lancet Oncol. 2013, 14 (6), e229−e238. (21) Perrotti, D.; Neviani, P. Protein phosphatase 2A (PP2A), a drugable tumor suppressor in Ph1(+) leukemias. Cancer Metastasis Rev. 2008, 27 (2), 159−168. (22) Yasui, H.; Hideshima, T.; Raje, N.; Roccaro, A. M.; Shiraishi, N.; Kumar, S.; Hamasaki, M.; Ishitsuka, K.; Tai, Y. T.; Podar, K.; Catley, L.; Mitsiades, C. S.; Richardson, P. G.; Albert, R.; Brinkmann, V.; Chauhan, D.; Anderson, K. C. FTY720 induces apoptosis in multiple myeloma cells and overcomes drug resistance. Cancer Res. 2005, 65 (16), 7478− 7484. (23) Nagahara, Y.; Ikekita, M.; Shinomiya, T. T cell selective apoptosis by a novel immunosuppressant, FTY720, is closely regulated with Bcl-2. Br. J. Pharmacol. 2002, 137 (7), 953−962. (24) Matsumura, M.; Tsuchida, M.; Isoyama, N.; Takai, K.; Matsuyama, H. FTY720 mediates cytochrome c release from mitochondria during rat thymocyte apoptosis. Transpl. Immunol. 2010, 23 (4), 174−179. (25) Kayton, M. L.; Meyers, P.; Wexler, L. H.; Gerald, W. L.; LaQuaglia, M. P. Clinical presentation, treatment, and outcome of alveolar soft part sarcoma in children, adolescents, and young adults. J. Pediatr. Surg. 2006, 41 (1), 187−193. (26) Soft Tissue Tumor Registry in Japan, 2010. (27) Kondo, T.; Hirohashi, S. Application of highly sensitive fluorescent dyes (CyDye DIGE Fluor saturation dyes) to laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE) for cancer proteomics. Nat. Protoc. 2007, 1 (6), 2940− 2956. (28) Cervoni, N.; Detich, N.; Seo, S. B.; Chakravarti, D.; Szyf, M. The oncoprotein Set/TAF-1beta, an inhibitor of histone acetyltransferase, inhibits active demethylation of DNA, integrating DNA methylation and transcriptional silencing. J. Biol. Chem. 2002, 277 (28), 25026−25031. (29) Piazza, R.; Valletta, S.; Winkelmann, N.; Redaelli, S.; Spinelli, R.; Pirola, A.; Antolini, L.; Mologni, L.; Donadoni, C.; Papaemmanuil, E.; Schnittger, S.; Kim, D. W.; Boultwood, J.; Rossi, F.; Gaipa, G.; De

Martini, G. P.; di Celle, P. F.; Jang, H. G.; Fantin, V.; Bignell, G. R.; Magistroni, V.; Haferlach, T.; Pogliani, E. M.; Campbell, P. J.; Chase, A. J.; Tapper, W. J.; Cross, N. C.; Gambacorti-Passerini, C. Recurrent SETBP1 mutations in atypical chronic myeloid leukemia. Nat. Genet. 2013, 45 (1), 18−24. (30) Ivaska, J.; Nissinen, L.; Immonen, N.; Eriksson, J. E.; Kahari, V. M.; Heino, J. Integrin alpha 2 beta 1 promotes activation of protein phosphatase 2A and dephosphorylation of Akt and glycogen synthase kinase 3 beta. Mol. Cell. Biol. 2002, 22 (5), 1352−1359. (31) Janssens, V.; Goris, J.; Van Hoof, C. PP2A: the expected tumor suppressor. Curr. Opin. Genet. Dev. 2005, 15 (1), 34−41. (32) Switzer, C. H.; Cheng, R. Y.; Vitek, T. M.; Christensen, D. J.; Wink, D. A.; Vitek, M. P. Targeting SET/I(2)PP2A oncoprotein functions as a multi-pathway strategy for cancer therapy. Oncogene 2011, 30 (22), 2504−2513. (33) Feschenko, M. S.; Stevenson, E.; Nairn, A. C.; Sweadner, K. J. A novel cAMP-stimulated pathway in protein phosphatase 2A activation. J Pharmacol. Exp. Ther. 2002, 302 (1), 111−118. (34) Neviani, P.; Santhanam, R.; Trotta, R.; Notari, M.; Blaser, B. W.; Liu, S.; Mao, H.; Chang, J. S.; Galietta, A.; Uttam, A.; Roy, D. C.; Valtieri, M.; Bruner-Klisovic, R.; Caligiuri, M. A.; Bloomfield, C. D.; Marcucci, G.; Perrotti, D. The tumor suppressor PP2A is functionally inactivated in blast crisis CML through the inhibitory activity of the BCR/ABLregulated SET protein. Cancer Cell 2005, 8 (5), 355−368. (35) Matsuoka, Y.; Nagahara, Y.; Ikekita, M.; Shinomiya, T. A novel immunosuppressive agent FTY720 induced Akt dephosphorylation in leukemia cells. Br. J. Pharmacol. 2003, 138 (7), 1303−1312. (36) Perrotti, D.; Neviani, P. ReSETting PP2A tumour suppressor activity in blast crisis and imatinib-resistant chronic myelogenous leukaemia. Br. J. Cancer 2006, 95 (7), 775−781. (37) George, S.; Merriam, P.; Maki, R. G.; Van den Abbeele, A. D.; Yap, J. T.; Akhurst, T.; Harmon, D. C.; Bhuchar, G.; O’Mara, M. M.; D’Adamo, D. R.; Morgan, J.; Schwartz, G. K.; Wagner, A. J.; Butrynski, J. E.; Demetri, G. D.; Keohan, M. L. Multicenter phase II trial of sunitinib in the treatment of nongastrointestinal stromal tumor sarcomas. J. Clin. Oncol 2009, 27 (19), 3154−3160.

2261


Proteomics Identified Overexpression of SET Oncogene Product and

Recommend Documents