Proteomics-based Strategy to Delineate the Molecular Mechanisms of

Article pubs.acs.org/jpr

Proteomics-based Strategy to Delineate the Molecular Mechanisms of RhoGDI2-induced Metastasis and Drug Resistance in Gastric Cancer Hee Jun Cho,†,# Kyoung Eun Baek,†,# In-Kyu Kim,†,# Sun-Mi Park,†,# Yeong-Lim Choi,† In-Koo Nam,† Seung-Ho Park,† Min-Ju Im,† Jong-Min Yoo,† Ki-Jun Ryu,† Young Taek Oh,† Soon-Chan Hong,‡ Oh-Hyung Kwon,§ Jae Won Kim,† Chang Won Lee,† and Jiyun Yoo*,† †

Department of Microbiology/Research Institute of Life Science, College of Natural Sciences, Gyeongsang National University, Jinju, Korea ‡ Department of Surgery/Gyeongnam Regional Cancer Center, Gyeongsang National University School of Medicine, Jinju, Korea § Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea S Supporting Information *

ABSTRACT: Rho GDP dissociation inhibitor 2 (RhoGDI2) was initially identified as a regulator of the Rho family of GTPases. Our recent works suggest that RhoGDI2 promotes tumor growth and malignant progression, as well as enhances chemoresistance in gastric cancer. Here, we delineate the mechanism by which RhoGDI2 promotes gastric cancer cell invasion and chemoresistance using two-dimensional gel electrophoresis (2DE) on proteins derived from a RhoGDI2-overexpressing SNU-484 human gastric cancer cell line and control cells. Differentially expressed proteins were identified using matrixassisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 47 differential protein spots were identified; 33 were upregulated, and 14 were downregulated by RhoGDI2 overexpression. Upregulation of SAE1, Cathepsin D, Cofilin1, CIAPIN1, and PAK2 proteins was validated by Western blot analysis. Loss-offunction analysis using small interference RNA (siRNA) directed against candidate genes reveals the need for CIAPIN1 and PAK2 in RhoGDI2-induced cancer cell invasion and Cathepsin D and PAK2 in RhoGDI2-mediated chemoresistance in gastric cancer cells. These data extend our understanding of the genes that act downstream of RhoGDI2 during the progression of gastric cancer and the acquisition of chemoresistance. KEYWORDS: RhoGDI2, gastric cancer, metastasis, invasion, cisplatin resistance, CIAPIN1, PAK2, cathepsin D

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INTRODUCTION Although the incidence and mortality of gastric cancer have steadily declined over the past several decades, it remains one of the most important malignancies because of its relatively high incidence and mortality rates. The acquisition of an invasive phenotype is a prerequisite for the metastatic spread of tumor cells, which constitutes a major cause of poor prognosis for cancer patients. Chemotherapy is commonly used for this type of cancer; however, the prognosis of advanced gastric cancer is still poor, and treatment is usually unsuccessful. Cisplatin is a common antitumor chemotherapeutic agent that is used to treat various cancers.1−5 The initial tumor response to cisplatin treatment is robust; however, the treatment efficacy decreases as the duration and number of therapy cycles increase. Cisplatin resistance is rapidly acquired, and it contributes to therapy failure.6 However, the mechanisms by which cells develop resistance to cisplatin are not fully understood. Rho GTPases, including RhoA, Rac1, and Cdc42, control a wide range of signaling pathways that regulate a variety of biological processes.7 Aberrant signaling through these proteins, © 2012 American Chemical Society

which is commonly observed in human cancers, has been implicated in facilitating virtually all aspects of the malignant phenotype.8,9 The biological activities of Rho GTPases are controlled through a tightly regulated GDP/GTP cycle, which is stimulated by guanine nucleotide exchange factors (GEFs) and terminated by GTPase-activating proteins (GAPs).10 An additional level of regulation is provided by Rho GDP dissociation inhibitors (RhoGDIs). RhoGDIs were originally identified as negative regulators of Rho GTPases because they bind to a majority of Rho GTPases in the cytoplasm, keeping them in the inactive form, and thus, preventing their interactions with target effector proteins.11,12 However, recent reports suggest that RhoGDIs may also act as positive regulators of Rho GTPase activity by conferring cues for spatial restriction, guidance, and accessibility to effectors.13 These positive interactions maintain Rho in an active form by inhibiting both the intrinsic and GTPase-activating proteinstimulated GTPase activities of the Rho GTPases. Received: November 8, 2011 Published: February 27, 2012 2355

dx.doi.org/10.1021/pr2011186 | J. Proteome Res. 2012, 11, 2355−2364

Journal of Proteome Research

Article

transferred to 12% SDS polyacrylamide slap gels (185 × 200 × 1.0 mm) for separation in the second dimension.

Three human RhoGDIs have been identified until date: RhoGDI1 (also referred to as RhoGDI or RhoGDI-α), RhoGDI2 (Ly-GDI or D4GDI, or RhoGDI-β), and RhoGDI3 (RhoGDI-γ). RhoGDI1 is ubiquitously expressed in all mammalian organs,14 whereas RhoGDI3 is expressed in the brain, lungs, kidneys, testes, and pancreas.15,16 By contrast, RhoGDI2 is preferentially expressed in the hematopoietic tissues and in the B- and T-lymphocytes.17,18 However, several studies have demonstrated that RhoGDI2 is also frequently overexpressed in multiple types of human cancers and can regulate cancer progression, especially augmenting aggressive phenotypes such as cancer cell invasion and metastasis.19−21 In addition, our recent work demonstrated that RhoGDI2 is associated with the acquisition of resistance to chemotherapeutic agents such as cisplatin in gastric cancer cells, a hallmark of aggressive cancers.22 In this study, we aimed to define the molecular mechanisms by which RhoGDI2 could induce gastric cancer cell invasion and cisplatin resistance. A two-dimensional gel electrophoresis (2-DE)-based proteomic approach was deployed to screen for proteins that are differentially expressed between RhoGDI2overexpressing SNU-484 gastric cancer cells, which have enhanced invasive potential and chemoresistance, and control cells. Five differentially expressed proteins were confirmed by immunoblotting, and their roles in gastric cancer cell invasion and chemoresistance were tested using small interference RNA (siRNA)-mediated inactivation. Our study implicates CIAPIN1 and PAK2 as essential factors that are responsible for enhanced invasiveness. In addition, cathepsin D and PAK2 are required for acquiring enhanced cisplatin resistance in RhoGDI2overexpressing SNU-484 gastric cancer cells.

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Protein Visualization and Image Analysis

The protein spots were visualized with a modified silver staining23 and Coomassie Brilliant Blue staining.24 For silver staining, gels were fixed in methanol:acetic acid:water (50:12:38) for 1.5 h, followed by washing twice in 50% ethanol for 20 min. Gels were pretreated for 1 min in 0.02% Na2S2O3 solution. After washing with deionized water, proteins were stained in a solution containing 0.2% AgNO3 and 0.075% (v/v) formalin for 20 min. Gels were developed in a solution containing 0.06% (v/v) formalin, 2% Na2CO3, and 0.0004% Na2S2O3 and the developing reaction was stopped using 1% acetic acid. For Coomassie blue staining, gels were fixed in 30% ethanol containing 2% phosphoric acid for 20 min and rinsed three times in 2% phosphoric acid. Gels were then equilibrated in a solution containing 18% ethanol, 2% phosphoric acid, and 15% ammonium sulfate for 30 min. Finally, Coomassie brilliant G-250 was added to a concentration of 1%. The image of the stained gel was processed using the Image-Master highresolution flatbed scanner (Bio-Rad, GS-710 Calibrated Imaging Densitometer). The resulting gel image files were normalized with PD-Quest software (Bio-Rad). During the normalization, 1 000 000 pixels were assigned to all detected protein spots on each 2-D gel in proportion to each spot’s intensity. Proteins were accepted as differentially accumulated when they displayed a significant change by pairwise t test at a significance level of 95% (P < 0.05). Protein Identification by MALDI-TOF/MS

To identify the protein spots, in-gel digestion of protein spots on silver or Coomassie stained gels was performed as previous described.25 After staining was completed, slab gels were washed twice with water for 10 min. The spots of interest were excised with a scalpel, cut into pieces, and put into 1.5 mL microtube. The excised gels were washed twice with water for 15 min and twice with water/acetonitrile (1:1 v/v) for 15 min. The solution volume was approximately twice the gel volume. After removal of liquid, the gel particles were treated with acetonitrile for 5 min followed by rehydration in 0.1 M NH4HCO3 for 5 min. Acetonitrile was added to give a 1:1 (v/ v) mixture of 0.1 M NH4HCO3/acetonitrile and the mixture was incubated for 15 min. Total liquid was removed and the gel particles were dried in a vacuum concentrator. The dried gel particles were swollen in 10 mM DTT/0.1 M NH4HCO3, and incubated for 45 min at 56 °C to reduce the peptides. After chilling the tubes to room temperature and removing the liquid, 55 mM iodoacetamide in 0.1 M NH4HCO3 was added. The tubes were incubated for 30 min at room temperature in the dark to S-alkylate the peptides. After the reaction, the iodoacetamide solution was removed, and the gel particles were washed with a digestion buffer containing 0.1 M NH4HCO3, 5 mM CaCl2, and 12.5 ng/μL of trypsin for 45 min on ice. Excess liquid was removed and 20 μL of digestion buffer without trypsin was added. After overnight digestion at 37 °C, 25 mM NH4HCO3 was added, and then the tube was incubated for 15 min. An equal volume of acetonitrile was added and the tube was incubated for a further 15 min. The supernatant was recovered, and the extraction was repeated twice with 5% formic acid/acetonitrile (1:1 v/v). The three extracts were pooled and dried in a vacuum concentrator. Target preparation was carried out by solution phase nitrocellulose method.26 CHCA was used as a matrix and

MATERIALS AND METHODS

Cell Cultures

Human gastric cancer cell lines SNU-484, SNU-719, and MKN-28 were obtained from the Korean Cell Line Bank (Seoul, Korea) and maintained in RPMI-1640 medium (Gibco) supplemented with 10% FBS and antibiotics. The SNU-484 and SNU-719 cells stably transfected with RhoGDI2 and MKN-28 cells stably transfected with shRNA-expressing lentiviral vector for targeting RhoGDI2 were described in our previous report, respectively.21,22 Two-Dimensional Gel Electrophoresis

For sample preparation, cells were harvested when 80% of confluency was attained, then washed three times with ice-cold PBS. The cell pellets were suspended in lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, and protease inhibitor cocktail (Roche). The lysates were homogenized and centrifuged at 12 000g for 15 min. The supernatant was collected as the protein sample. Protein concentrations were determined using the modified Bio-Rad protein assay kit. Fifty micrograms of protein was loaded onto a 18 cm IPG strips at pH 4−7 NL (Amersham Biosciences, Immobiline DryStrip) and focused using an PROTEIN IEF Cell (Bio-Rad) at 250 V for 15 min, at 10,000 V for 30 h, and 10,000 V for 60.000 V h. Current was limited to 50 μA per strip, and temperature maintained at 20 °C for all IEF steps. Strips were then placed in an equilibration solution (6 M urea, 2% SDS, 30% glycerol, 50 mM Tris−HCl, pH 8.8) containing 1% DTT for 10 min with shaking at 50 rpm. The strip gels were then transferred to equilibration solution containing 2.5% iodoacetamide and shaken for an additional 10 min. The strips were then 2356



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Figure 1. Proteomic comparison between RhoGDI2-overexpressing SNU-484 and control cells. (A) Representative images of 2-DE gels of the proteins extracted from human gastric cell line SNU-484(Mock) and RhoGDI2-overexpressing SNU-484(GDI2-4). Data are the representative images of more than three individual experiments. (B) Functional annotation of proteins differentially regulated by RhoGDI2 identified on this screen. Functional classification was performed according to the Human Protein Reference Database.

dry milk and probed with the appropriate primary antibodies. The bound antibodies were visualized with a suitable secondary antibody conjugated with horseradish peroxidase using enhanced chemiluminescence reagents (ECL, Amersham Bioscience).

internal peptide calibrants including bradykinin (monoisotopic mass, 904.468 Da), fibrinopeptide B (1570.677 Da), and neurotensin (1672.971 Da) were used for size estimation. The tryptic peptides were analyzed in a Voyger-DE STR MALDITOF mass spectrometer (PerSeptive Biosystems). The acquired peptide mass fingerprints were used to search through the Swiss-Prot Protein Data Base with the Mascot software (www.matrixscience.com). After removal of known contamination peaks, such as keratin and autoproteolysis peaks, the following search parameters were used in all mascot searches: human species, monoisotopic peptide masses, tolerance of one missed cleavage, and a maximum error tolerance of 50 ppm, carbamidomethylation and oxidation of methionine as fixed and variable modification. Protein scores greater than 66 were considered as significant (p < 0.05).

RNA Interference Experiments

siRNA oligo duplexes for targeting SAE1, COF1, CIAPIN1, CATD, and PAK2 were purchased from Bioneer (Daejeon, Korea). The sequence of each siRNA is shown in Supporting Information Table 1. Transient transfection of each siRNA oligo duplex was accomplished using siLentFect Reagent (BIORAD). After incubation for 48 h, the cells were harvested and efficiency of each siRNA oligo duplex was confirmed by Western blot analysis. Invasion Assay

Antibodies and Western Blot Analysis

Cell invasion was performed using QCM 24-Well Cell Invasion Assay kit (Chemicon) following the manufacturer’s instruction. 2.5 × 105 cells in 250 μL of medium were placed in the insert and allowed to invade for 20 h. The lower chamber was filled with 500 μL of appropriated media containing 20% FBS. After incubation, cells/media remaining on the top side of insert were removed by pipetting. Invasion chamber insert was transferred into a clean well containing 225 μL of prewarmed Cell Detachment Solution, and incubated for 30 min at 37 °C. The insert was removed from the well. 75 μL of Lysis Buffer/Dye

Rabbit anti-PARP and anti-PAK2 antibodies were purchased from Cell Signaling Technology. Mouse anti-CIAPIN1, rabbit anti-COF1, and goat anti-SAE1 antibodies were purchased from Abcam. Mouse anti-CATD antibody was purchased from Santa Cruz Biotechnology. Mouse anti-α-tubulin antibody was purchased from Sigma. Cell lysates were separated by 7.5−15% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Amersham Bioscience). Subsequently, the membrane was incubated in TBST supplemented with 5% nonfat 2357



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Table 1. Differentially Expressed Proteins between RhoGDI2-Overexpressing SNU-484(GDI2-4) and Control SNU-484(Mock) Cells accession numbera

expression in SNU-484(GDI24)/SNU-484(Mock)

theoretical Mr/pI

peptides match/total

sequence coverage (%)

Mascot scoreb

56382/6.6

7/14

17

96

27.0 ↑

36508/5.7 21677/5.3

19/36 8/17

53 33

145 100

9.3 ↑ 7.5 ↑

adenine phosphoribosyltransferase cytidine monophosphate (UMPCMP) kinase 1 SUMO1 activating enzyme subunit 1 inositol monophosphatase endoplasmic reticulum protein 29

19477/5.8

6/13

37

90

7.4 ↑

22223/5.4

5/13

31

73

7.2 ↑

38450/5.2

19/36

43

131

5.5 ↑

30189/5.2 28994/6.8

11/27 7/19

39 26

93 72

5.1 ↑ 4.8 ↑

30991/5.5

9/30

35

69

4.7 ↑

22276/5.3

7/48

41

69

4.4 ↑

29644/5.7

9/36

38

68

4.1 ↑

P07339

dimethylarginine dimethylaminohydrolase 1 ubiquitin-conjugating enzyme E2K dimethylarginine dimethylaminohydrolase 2 cathepsin D

44553/6.1

9/19

23

71

4.0 ↑

P23528

cofilin 1

18371/8.3

6/20

45

73

3.9 ↑

Q6FI81

cytokine-induced apoptosis inhibitor 1 hexosaminidase B (beta polypeptide) immunity-related GTPase family, Q1 splicing factor, arginine/serinerich 9 tumor protein D52

33583/5.4

7/28

26

77

3.8 ↑

63112/6.3

16/36

23

80

3.6 ↑

62718/4.8

10/27

22

104

3.5 ↑

25542/8.7

8/31

37

78

3.5 ↑

19863/4.9

7/16

31

104

3.2 ↑

33825/5.3 36442/6.3

6/12 6/20

25 21

84 70

3.2 ↑ 3.1 ↑

P08670

spermidine synthase alcohol dehydrogenase (Aldehyde reductase) vimentin

53521/5.1

12/51

27

89

3.1 ↑

Q15181 P78417 P18206

inorganic pyrophosphatase glutathione transferase omega-1 vinculin

32660/5.5 27566/6.2 87445/5.6

7/19 7/11 18/28

25 27 16

79 99 134

3.1 ↑ 2.9 ↑ 2.9 ↑

P15531 Q15056

17149/5.8 27254/6.9

8/24 7/24

59 23

120 70

2.9 ↑ 2.9 ↑

Q99614

nucleoside diphosphate kinase A eukaryotic translation initiation factor 4H tetratricopeptide repeat protein 1

33526/4.8

7/17

22

75

2.8 ↑

P49720

proteasome subunit beta type 3

22949/6.1

7/22

37

66

2.7 ↑

Q13177

P21 protein (Cdc42/Rac)activated kinase 2 RAN binding protein 1

58005/5.7

8/17

19

86

2.7 ↑

23310/5.2

7/15

32

88

2.6 ↑

retinal dehydrogenase 1 eukaryotic translation initiation factor 3 subunit 12 calpain small subunit 1

54731/6.3 25060/4.8

17/27 8/21

35 36

184 92

2.5 ↑ 2.5 ↑

28316/5.0

8/29

31

87

2.5 ↑

MAD1 mitotic arrest deficientlike 1 leucine-rich PPR motifcontaining protein FK506-binding protein 10

83068/5.7

11/29

15

72

0.490 ↓

145203/5.5

8/14

6

68

0.470 ↓

64246/5.4

11/35

23

104

0.469 ↓

50977/5.4

7/18

22

84

0.450 ↓

P05091 P07195 P30626 P07741 P30085 Q9UBE0 P29218 P30040 O94760 P61086 O95865

P07686 Q8WZA9 Q13242 P55327 P19623 P14550

P43487 P00352 Q9UBQ5 P04632 Q9Y6D9 P42704 Q96AY3 P61978

protein name aldehyde dehydrogenase, mitochondrial precursor L-lactate dehydrogenase B sorcin

heterogeneous nuclear ribonucleoprotein K

2358

biological function signal transduction metabolism signal transduction metabolism nucleic acid metabolism metabolism metabolism protein metabolism metabolism protein metabolism metabolism protein metabolism cell growth or maintenance cell growth or maintenance metabolism innate immune response protein metabolism cell growth or maintenance metabolism metabolism cell growth or maintenance metabolism metabolism cell growth or maintenance metabolism protein metabolism signal transduction protein metabolism signal transduction signal transduction metabolism protein metabolism protein metabolism regulation of cell cycle nucleic acid metabolism protein metabolism nucleic acid metabolism



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Table 1. continued accession numbera

a

protein name

theoretical Mr/pI

peptides match/total

sequence coverage (%)

Mascot scoreb

expression in SNU-484(GDI24)/SNU-484(Mock)

P67936

tropomyosin 4

28391/4.7

13/30

31

100

0.450 ↓

P62258

14-3-3 protein epsilon

29174/4.6

15/34

54

116

0.380 ↓

Q92599

septin-8

55626/5.9

10/19

21

75

0.375 ↓

P07951

tropomyosin beta

32851/4.7

15/43

34

76

0.370 ↓

P11021

72334/5.1

10/22

18

107

0.280 ↓

P14618 Q9BTT6 P31947

78 kDa glucose-regulated protein precursor pyruvate kinase M2 leucine-rich repeat containing 1 14-3-3 protein sigma

57806/8.0 59242/4.9 27774/4.7

8/13 6/12 12/20

14 16 34

91 71 116

0.270 ↓ 0.158 ↓ 0.116 ↓

Q13813

spectrin alpha chain

284542/5.2

13/17

6

89

0.090 ↓

Q13976

protein kinase, cGMP-dependent, type I

76234/5.7

8/13

16

87

0.007 ↓

biological function cell growth or maintenance signal transduction signal transduction cell growth or maintenance protein metabolism metabolism unknown activity signal transduction cell growth or maintenance signal transduction

Swiss-Prot database accession number are referenced. bA score of more than 66 is significant (p < 0.05).

Figure 2. RhoGDI2 upregulates the expression of SAE1, CATD, COF1, CIAPIN1, and PAK2. (A) Detailed 2-DE images of 5 upregulated protein spots in RhoGDI2-overexpressing SNU-484(GDI2-4) cells compared with SNU-484(Mock) cells. (B) Western blot analyses of 5 proteins upregulated in RhoGDI2-overexpressing SNU-484(GDI2-4 and GDI2-7) and SNU-719 (GDI2-1 and GDI2-8) cells. Data are the representative images of more than three individual experiments. (C) Densitometric analysis of the Western blot bands for 5 proteins, normalized to α-tubulin. Quantification was performed using the ImageJ software.

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apoptosis inhibitor 1 (CIAPIN1), and p21 protein-activated kinase 2 (PAK2), previously implicated in cancer cell proliferation, metastasis, and apoptosis, and performed Western blot analyses to determine the level of these proteins in RhoGDI2-overexpressing gastric cancer cells. Consistent with the results of 2-DE and imaging analysis (Figure 2A), expression of SAE1, CATD, COF1, CIAPIN1, and PAK2 was significantly upregulated in RhoGDI2-overexpressing SNU484(GDI2-4 and GDI2-7) and SNU-719(GDI2-1 and GDI2-8) cells compared with control (Mock) cells (Figure 2B). We also confirmed that expression of these proteins were markedly downregulated in RhoGDI2-depleted MKN-28(shGDI2-1 and shGDI2-2) cells when compared to control cells (Supporting Information Figure 1). Taken together, these results suggest that these proteins might be direct targets for RhoGDI2.

solution (CyQuant GR Dye 1:75 with 4X Lysis Buffer) was added into each well and incubated 15 min at room temperature. 200 μL of mixture was transferred into a 96-well plate and assessed with a fluorescence plate reader using 480/ 520 nm filter set. Apoptosis Detection

Apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay using the In Situ Cell Death Detection Kit, Fluorescein (Roche Applied Science, Germany) following the manufacturer’s instruction. Cisplatin-treated or nontreated cells were washed with cold PBS and fixed with 4% paraformaldehyde. Fixed cells were permeabilized and stained using the TUNEL reaction mixture in the dark. The cells were then stained with 1 μg/mL DAPI solution for 5 min at room temperature in the dark and observed under a fluorescence microscope. The apoptosis rate was quantified by the TUNELpositive rate.

Effect of Identified Proteins on RhoGDI2-overexpressing Gastric Cancer Cell Invasion

Since RhoGDI2 is known to promote gastric cancer cell invasion,21 we first examined whether the proteins identified in our screen contribute to RhoGDI2-mediated gastric cancer cell invasion. To this end, RhoGDI2-overexpressing SNU-484(GDI2-4) cells were transfected with individual target genespecific siRNAs, and the effect of siRNA-based knockdown was determined by Western blot analysis. As shown in Figure 3A,

Statistical Analysis

We performed statistical analysis using the unipolar, paired Student t-test. The significance of the data was accepted when the P value was less than 0.05.

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RESULTS

Identification of Differentially Expressed Proteins in RhoGDI2-Overexpressing Gastric Cancer Cells

Previously, we demonstrated that overexpression of RhoGDI2 in a SNU-484 human gastric cancer cell line, in which endogenous RhoGDI2 is not normally expressed, enhances cancer cell invasion and inhibits cisplatin-induced apoptosis.21,22 To understand how RhoGDI2 enhances cancer cell invasion and confers resistance to cisplatin-induced apoptosis in gastric cancer cells, we compared the protein expression profiles between RhoGDI2-overexpressing SNU-484(GDI2-4) cells and control SNU-484(Mock) cells. Comparative 2-DE and PDQuest imaging analysis revealed 92 spots with differential intensity, with p-values less than 0.05. We successfully identified 47 of these 92 differentially expressed spots with at least a 2.5fold change by MALDI-TOF-MS, including 33 upregulated proteins and 14 downregulated proteins when SNU-484(GDI24) cells were compared with SNU-484(Mock) cells (Figure 1A). Forty-seven differentially expressed proteins are annotated in Table 1. The proteins identified were grouped according to the biological processes in which they are likely to play a role (Figure 1B), such as metabolism (31%), protein metabolism (21%), signal transduction (19%), cell growth and/or maintenance (17%), nucleic acid metabolism (6%), regulation of cell cycle (2%), innate immune response (2%), and unknown activity (2%), following recommendations from the Human Protein Reference Database (http://www.hprd.org/).27 Most the proteins deregulated by RhoGDI2 overexpression are involved in cellular metabolism or signal transduction activities. These results suggest that the proteins required for cellular metabolism and signal transduction may play an important role in conferring aggressive phenotypes in RhoGDI2-overexpressing gastric cancer cells.

Figure 3. CIAPIN1 and PAK2 are required for RhoGDI2-mediated gastric cancer cell invasion. (A) Representative immunoblot of five candidate proteins from RhoGDI2-overexpressing SNU-484(GDI2-4) cells transfected with their respective target gene-specific siRNA. (B) Effect of depletion of five candidate genes on the in vitro invasive ability of RhoGDI2-overexpressing SNU-484(GDI2-4) cells. Invasion activity of each cell was measured in vitro with the Boyden chamber after 20 h and is represented as a % of control. Data are mean ± SD of three individual experiments, each in triplicate. *, P < 0.01, as determined by paired Student t test.

the expression level of target proteins was markedly reduced by their respective siRNAs but not by control siRNA. We previously reported that SNU-484(GDI2-4) cells exhibit significantly increased invasiveness compared with SNU484(Mock) control cells (Figure 3B).21 siRNA-mediated depletion of either CIAPIN1 or PAK2 markedly suppressed cancer cell invasion of RhoGDI2-overexpressing SNU-484(GDI2-4) cells, whereas depletion of SAE1, COF1, or CATD

Validation of Differentially Expressed Proteins by Western Blot Analysis

To validate our mass spectrometry results, we selected 5 candidates: SUMO activating enzyme subunit 1 (SAE1), Cathepsin D (CATD), Cofilin 1 (COF1), cytokine-induced 2360



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Figure 4. CATD and PAK2 are required for RhoGDI2-mediated cisplatin resistance in gastric cancer cells. (A) Representative images of TUNEL staining of RhoGDI2-overexpressing SNU-484(GDI2-4) cells after cisplatin treatment (10 μg/mL) for 24 h with the depletion of the target genes indicated (left). Histogram shows the ratio of TUNEL-positive, cisplatin-treated (10 μg/mL for 24 h), RhoGDI2-overexpressing SNU-484(GDI2-4) cells after depletion of the indicated target genes (right). Data are mean ± SD of three individual experiments, each in triplicate. *, P < 0.01, as determined by paired Student t test. (D) Representative immunoblot for PARP cleavage after cisplatin treatment in RhoGDI2-overexpressing SNU484(GDI2-4) cells with the depletion of the indicated target genes.

RhoGDI2-overexpressing SNU-484(GDI2-4) cells (Figure 4B). Taken together, these results suggest that the upregulation of CATD and PAK2 expression confers resistance to cisplatininduced apoptosis in RhoGDI2-overexpressing gastric cancer cells.

has only moderate effects (Figure 3B). These results suggest that the upregulation of CIAPIN1 and PAK2 is critical for RhoGDI2-mediated gastric cancer cell invasion. Role of Identified Proteins in Conferring Chemoresistance on RhoGDI2-Overexpressing Gastric Cancer Cells

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Since RhoGDI2 can protect gastric cancer cells against apoptosis induced by various chemotherapeutic agents,22 as well as enhance gastric cancer cell invasion, we next tested the effects of the five proteins identified on our screen on RhoGDI2-mediated chemoresistance in gastric cancer cells. We examined the consequences of target gene-specific siRNAmediated depletion of protein expression on cisplatin-induced apoptosis in RhoGDI2-overexpressing SNU-484(GDI2-4) cells. As reported previously, cisplatin-induced apoptosis is significantly attenuated in RhoGDI2-overexpressing SNU-484(GDI24) cells when compared with control SNU-484(mock) cells (Figure 4A).22 Interestingly, depletion of CATD or PAK2 significantly increased cisplatin-induced apoptosis in RhoGDI2overexpressing SNU-484(GDI2-4) cells, whereas depletion of SAE1, COF1, or CIAPIN1 only increased apoptosis minimally (Figure 4A). Consistent with these results, depletion of CATD or PAK2 increased cisplatin-induced PARP cleavage in

DISCUSSION In contrast to RhoGDI1, which is ubiquitously expressed in all mammalian organs,14 RhoGDI2 is expressed preferentially in the hematopoietic tissues and in the B- and T-lymphocytes.17,18 Thus, many studies have focused on understanding the function of RhoGDI2 in the regulation of lymphocyte activation, survival, and responsiveness. However, accumulating evidence shows that RhoGDI2 is also frequently expressed in multiple types of human tumor cells, suggesting that RhoGDI2 may play a role in the progression of human cancer.19−21 In fact, RhoGDI2 is associated with advanced stage ovarian and gastric cancers,19,21 and with enhanced migration, invasion, or metastasis of breast and gastric cancer cells.20,21 In addition, RhoGDI2 contributes to the development of resistance to apoptosis induced by chemotherapeutic agents, such as cisplatin, etoposide, and staurosporin in gastric cancer cells.22 Given the increasing evidence linking RhoGDI2 expression 2361



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metastasis of cancer cells by virtue of their ability to degrade the extracellular matrix by themselves or by activating latent precursor forms of other proteolytic enzymes involved in the degradation of extracellular matrix.47−49 Elevated expression and secretion of cathepsin D has been noticed in many cancers, and increased cathepsin D expression is correlated with a poor prognosis in several types of malignancies.50−53 In contrast to previous reports, cathepsin D had moderate effect on RhoGDI2-mediated gastric cancer cell invasion in our study. However, we find that cathepsin D mainly contributes to the cisplatin resistance displayed by RhoGDI2-overexpressing gastric cancer cells. Consistent with our results, Berchem et al. demonstrated that cathepsin D overexpression prevents tumor apoptosis, and that this activity of cathepsin D is dependent on its catalytic function.54 Our results suggest that cathepsin D mainly contributes to RhoGDI2-mediated cisplatin resistance in gastric cancer. We also examined the possible pathways that lead to RhoGDI2-induced CIAPIN1, CATD, and PAK2 expression. Recently, we demonstrated that phospholipase C-gamma (PLCγ) is activated in RhoGDI2-overexpressing SNU-484 cells and it is required for RhoGDI2-mediated cisplatin resistance and cancer cell invasion in gastric cancer.55 To determine whether PLCγ is required for RhoGDI2-induced CIAPIN1, CATD, and PAK2 expression, we examined the expression levels of these proteins in PLCγ-depleted SNU484(GDI2-4) and control cells, but failed to see any difference (data not shown). Alternatively, ongoing studies in our laboratory have revealed that Rac1, but not RhoA or Cdc42, is activated in RhoGDI2-overexpressing gastric cancer cells. Therefore, we are now examining whether activation of Rac1 is involved in RhoGDI2-induced CIAPIN1, CATD, and PAK2 expression. This report describes the first application of a proteomic approach to elucidate the molecular pathways associated with RhoGDI2 in gastric cancer cells. This proteomic technique is a powerful method for the identification of the molecular mechanisms associated with metastasis/invasion and chemoresistance in RhoGDI2-expressing gastric cancer cells. In summary, we identified CIAPIN1, CATD, and PAK2 as novel targets for RhoGDI2-induced cancer cell invasion and chemoresistance in RhoGDI2-expressing gastric cancer cells. These findings provide new insights into our understanding of the molecular mechanisms of RhoGDI2-mediated gastric cancer malignancy and cisplatin resistance.

with gastric cancer progression and chemoresistance, it is important to elucidate the molecular mechanism by which RhoGDI2 regulates the aggressive features of tumor cells. In this study, we used comparative high-throughput proteomic analysis to screen for downstream target proteins potentially responsible for RhoGDI2-induced gastric cancer invasion and cisplatin resistance. Western blotting and loss-of-function analysis validate CIAPIN1 and PAK2 as novel downstream targets of RhoGDI2-mediated gastric cancer cell invasion. Similarly, CATD and PAK2 appear to act downstream of RhoGDI2 to mediate cisplatin resistance in gastric cancer cells. PAKs are well-known effector proteins for the small GTPases Rac and Cdc42.28 Six PAK proteins are classified into two groups: Group 1 (PAK1-3) and Group 2 (PAK4-6).29−31 PAKs reportedly regulate various cellular processes, including cell morphology, motility, survival, cell cycle progression, and cell transformation.29−32 PAKs are activated by various extracellular signals, and through their rapidly expanding lists of binding partners and kinase substrates, they exert regulatory control over essential biological processes. Elevated PAK2 activity is associated with several human cancers and appears to promote a more invasive and malignant phenotype.33−35 Furthermore, recent research suggests that elevated PAK2 activity supports anchorage-independent survival and growth, as well as resistance to anticancer drug-induced cell death in human breast cancer cells, thereby contributing to the malignant phenotype of cancer cells.36 In this study, we showed that depletion of PAK2 expression significantly inhibits cancer cell invasion, as well as cisplatin-induced apoptosis in RhoGDI2overexpressing gastric cancer cells. These results suggest that PAK2 protein is a critical mediator of RhoGDI2-mediated gastric cancer cell invasion and cisplatin resistance. CIAPIN1 is cell death-defying factor, which was isolated as a molecule that confers resistance to apoptosis.37 Several studies suggest that CIAPIN1 suppresses apoptosis induced by a variety of stimuli, including death receptor activation, growth factor starvation, ionizing radiation, viral infection, and genotoxic damage. Hao et al. reported that CIAPIN1 is a multidrug resistance (MDR)-related molecule in gastric cancer cells.38 Subsequently, Li et al. found that stable expression of CIAPIN1 in human gastric cancer and leukemia cell lines protects these cells from the cytotoxic effects of adriamycin.39,40 CIAPIN1 is a known mediator of the RAS signaling pathway and potentially exerts its function by upregulating the expression of Bcl-XL and Jak2.41 In contrast to previous reports, we observed no discernible effects of CIAPIN1 depletion on RhoGDI2-mediated cisplatin resistance in gastric cancer cells in this study. However, our results indicate that CIAPIN1 might act as a downstream target of RhoGDI2mediated gastric cancer cell invasion; CIAPIN1 depletion markedly reduces RhoGDI2-overexpressing gastric cancer cell invasion. Consistent with our results, many recent studies suggest that CIAPIN1 is associated with malignant tumors in hepatocarcinoma and colon, breast, and gastric cancers.42−45 In conclusion, our results suggest that CIAPIN1 contributes to RhoGDI2-mediated gastric cancer cell invasion but not chemoresistance. Cathepsins are lysosomal hydrolases that degrade proteins in the lysosome. Cathepsins can be divided into three subgroups according to their active site amino acid: cysteine (B, C, H, F, K, L, O, S, V, and W), aspartic (D and E), and serine (G) Cathepsins.46 Cathepsins, in particular, the aspartic protease Cathepsin D, have been implicated in the invasion and

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Table 1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 82-55-772-1327. Fax: 82-55-759-5199. E-mail: yooj@ gsnu.ac.kr. Author Contributions #

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest. 2362



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ACKNOWLEDGMENTS This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2007-331-E00023), Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0003247 and 20110010805), grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A080285), and National R&D Program for Cancer Control, Ministry of Health, Welfare and Family affairs, Republic of Korea (0820050).

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