Comparative proteomic analysis of oral squamous cell carcinoma and adjacent non-tumour tissue from Thailand

archives of oral biology 58 (2013) 1677–1685

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Comparative proteomic analysis of oral squamous cell carcinoma and adjacent non-tumour tissue from Thailand Pitak Chanthammachat a, Waraporn Promwikorn a, Kowit Pruegsanusak b, Sittiruk Roytrakul c, Chantragan Srisomsap d, Daranee Chokchaichamnankit d, Jisnuson Svasti d, Pleumjit Boonyaphiphat e, Singkhamanan K f, Paramee Thongsuksai e,* a

Department of Anatomy, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand Department of Otorhinolaryngology Head and Neck Surgery, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand c Genome Institute, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Pathumthani 12120, Thailand d Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand e Department of Pathology, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand f Department of Biomedical Science, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand b

article info

summary

Article history:

Objective: The study was aimed at analysing and identifying the proteins that are differen-

Accepted 4 August 2013

tially expressed in oral squamous cell carcinoma (OSCC) compared to adjacent non-tumour tissue.

Keywords:

Materials and methods: Two-dimensional (2D) sodium dodecyl sulphate–polyacrylamide gel

Oral squamous cell carcinoma

electrophoresis accompanied by mass spectrometry (matrix-assisted laser desorption/ioni-

Proteomics

sation-time-of-flight mass spectrometry and liquid chromatography–tandem mass spec-

Mass spectrometry

trometry) was used to analyse and identify the differentially expressed proteins in 10 pairs

Two-dimensional gel

of tumours and adjacent non-tumour tissues from five cases of early-stage and five cases of

electrophoresis

late-stage OSCC. The statistical differences of the protein spots were analysed by the

Immunohistochemistry

Wilcoxon signed-rank test. A validation study using immunohistochemistry and quantita-

Quantitative real-time RT-PCR

tive real-time reverse transcription-polymerase chain reaction (qRT-PCR) was performed. Results: A total of 68 proteins (63 up-regulated, five down-regulated) were differentially expressed in early-stage disease, and 39 proteins (37 up-regulated, two down-regulated) were significantly altered in late-stage disease. Among these, 14 proteins were altered in both groups. A total of 44 proteins were identified, including heat shock proteins (HSPs: Hsp90, HSPA5 and HSPA8), keratins (K1, K6A and K17), tubulin, cofilin 1, 14-3-3s and metabolic enzymes. These proteins are involved in various cellular processes essential for cell growth, survival and cell migration. The validation study on a-tubulin and 14-3-3s using

* Corresponding author. Tel.: +66 74451591; fax: +66 74212908. E-mail addresses: [email protected] (P. Chanthammachat), [email protected] (W. Promwikorn), [email protected] (K. Pruegsanusak), [email protected] (S. Roytrakul), [email protected] (C. Srisomsap), [email protected] (D. Chokchaichamnankit), [email protected] (J. Svasti), [email protected] (P. Boonyaphiphat), [email protected] (S. K), [email protected], [email protected] (P. Thongsuksai). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.08.002

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immunohistochemistry and KIAA1199 expression using real-time RT-PCR confirmed the results in proteomics analysis. Conclusions: The study identified many proteins, both known and unknown, for cancer cell processes. At least two proteins, KIAA1199 and Horf6, are novel for oral cancer. # 2013 Elsevier Ltd. All rights reserved.

1.

Introduction

Oral cancer is a significant cancer worldwide, as it ranks as the eighth most common cancer in males with an average global incidence of 6.3 per 100,000.1 It is more prevalent in developing countries than in developed countries. The incidence of oral cancer in the southern province of Songkhla is the highest in Thailand, with an age-standardised incidence rate of 9.8 per 100,000 in males.2 Mortality from oral cancer averages less than half the incidence.1 The 5-year survival rate has only been increasing subtly during the past two decades, in contrast with the advances in treatment modality3; this figure is largely due to the advanced stages of the disease at diagnosis which, in turn, limits or causes suffering from treatment. The identification of the molecular mechanisms underlying cancer initiation and progression could aid in the development of new diagnostic and treatment options for the disease. The processes of cancer cell transformation and progression are extremely complex events involving the deregulation of a variety of genes controlling cell proliferation, differentiation and cell death.4,5 Different proteins, probably in the hundreds or thousands, may be up-regulated or down-regulated simultaneously and may conduct distinct cell functions. Therefore, the analysis of hundreds of proteins simultaneously holds great promise for accurately predicting the function of marker proteins. Recent advances in mass spectrometry (MS) technology, combined with the rapid growth in genomic databases, allows for the identification of thousands of proteins in a given cell at a time. This approach, called proteomics, is therefore, a promising technique for the identification of new biomarkers for early cancer detection or as a new target for therapeutic intervention.6 In this study, we identified proteins that were altered during tumour development and progression of oral squamous cell carcinoma (OSCC). Two MS schemes, matrixassisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography–tandem mass spectrometry (LC-MS/MS), were used to maximise protein identification efficiency. The proteins that change in early-stage tumour may be implicated as potential markers of early diagnosis or detection of disease reappearance. Likewise, the proteins that change in late-stage tumour may be candidate prognostic or predictive markers.

2.

Materials and methods

2.1.

Tissue samples

Ten cases of OSCCs from seven women and three men aged 45–82 years were included in the study. Five cases were stage I

and II (early-stage group) and five cases were stage III and IV (late-stage group). The tumours of all cases were welldifferentiated OSCCs. Fresh tissue samples of cancer and their adjacent normal mucosa were obtained at the time of surgical resection from 10 patients with OSCC at Songklanagarind Hospital, Faculty of Medicine, Prince of Songkla University, located in Songkhla, Thailand. Each tissue was sharply bisected: one half was frozen-sectioned for histopathological confirmation and the other half was stored at 80 8C until analysis. The histopathological evaluations of all cases were reviewed and confirmed by an experienced pathologist. Cancer staging was defined by the extent of the lesion (Tumour, Node, Metastasis (TNM) system) according to the AJCC Staging Manual 6th edition. The research protocol was approved by the Ethics Committee of the Faculty of Medicine, Prince of Songkla University (Reference Number SUB.EC 49/400-008).

2.2.

Protein extraction

Each sample tissue, approximately 0.8  0.8  0.4 cm3, was ground with a 300-ml lysis buffer (7 M urea, 2 M thiourea, 4% 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS), 1% dithiothreitol (DTT) and 2% immobilised pH gradient (IPG) buffer), frozen in liquid nitrogen, then thawed. The homogenate was centrifuged at 14,000 rpm at 4 8C for 15 min and the supernatants were transferred into a new microcentrifuge tube for protein purification. The protein concentration was measured with a Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Hercules, CA, USA) based on the Bradford method, using bovine serum albumin (BSA) as a standard.

2.3.

2D electrophoresis

Fifty micrograms of extracted protein was cleaned up using a 2-D Clean-Up Kit (GE Healthcare, Little Chalfont, UK) and then mixed with a rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 60 mM DTT and 0.5% IPG buffer). The sample was subjected to first-dimension electrophoresis by being rehydrated and focussed on a 13-cm IPG strip (pH 3–10) for a total of 18.8 kVh in Ettan IPGphore II (GE Healthcare, Little Chalfont, UK) at 20 8C. The focussed IPG strips were immediately continued to second-dimension electrophoresis using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS– PAGE). The IPG strip was equilibrated with an equilibration solution (75 mM Tris–HCl pH 8.8, 6 M urea, 2% SDS, 34.5% glycerol, a trace of bromophenol blue and 65 mM DTT) for 15 min, followed by a second equilibration for 15 min in the same solution containing 135 mM iodoacetamide instead of DTT. Then, the equilibrated strip was transferred to the top of a 12.5% polyacrylamide gel and held in position with molten

archives of oral biology 58 (2013) 1677–1685

0.5% agarose gel in the running buffer (25 mM Tris–HCl, 0.1% SDS, 0.192 M glycine). The gel was run at 10 mA/gel for 15 min, followed with 20 mA/gel for 4–5 h or until the dye front reached the bottom of the gel. The gel was routinely stained with silver nitrate. The stained gels were scanned with an ImageScanner (GE Healthcare, Little Chalfont, UK) at a resolution of 300 dots per inch.

2.4.

Measurement of protein spots

Protein spots were detected with ImageMaster 2D Platinum software version 6.0 (GE Healthcare, Little Chalfont, UK). To eradicate the error from the different levels of stained protein, the density of each protein spot on a 2D gel was calculated as a percentage of total protein volume of the 2D gel. The differences of protein spots between tumour and non-tumour tissues were analysed by a non-parametric Wilcoxon signedrank test. The statistical significance was considered if p < 0.05. The statistical analysis was performed using the statistical package STATA version 6.0.

2.5.

Protein identification by MALDI-TOF MS

The gels were stained with Coomassie Brilliant Blue R-250 (GE Healthcare, Little Chalfont, UK) as well as silver staining for visualisation and selection for MS. The selected spots were excised, de-stained and subjected to in-gel trypsin digestion. The de-stained gels were dehydrated in 200 ml 100% acetonitrile (ACN) at room temperature for 5 min, reduced by 10 mM DTT at 56 8C for 1 h and alkylated in 100 mM iodoacetamide at room temperature in the dark for 1 h, followed by sequential washing in 200 ml ACN for 5 min, 200 ml NH4HCO3 for 10 min and 200 ml ACN for 5 min, respectively. The gel pieces were incubated in Sequencing Grade Modified Trypsin solution (trypsin 0.2 mg/sample with 20 mM NH4HCO3) on ice for 20 min; then, 20 ml of 20 mM NH4HCO3 was added and the gel pieces were incubated at 37 8C overnight. The digested peptides were extracted with 60 ml of extract solution (50% ACN and 0.1% trifluoroacetic acid (TFA)) at 37 8C for 20 min, and then the extraction steps were repeated. The solution was then stored at 80 8C for further steps. Mass spectra chromatograms of protein were obtained using a Reflex IV MALDI-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany). The extracted peptides were resuspended in 3 ml of 60% ACN and 0.1% TFA (1:1). The peptide samples were crystallised with a matrix solution (0.026 mM 2,5-dihydroxybenzoic acid (DHBA), 0.0045 mM sinapinic acid (SA), 60% ACN and 0.1% TFA) in a 1:5 ratio and spotted onto an AnchorChip plate, then left to air-dry at room temperature. The spectra were generated by the FlexControl software in the reflector mode, with a range from 900 to 2000 kDa. Bradykinin and P14R were used as the standard peptides. The peptide mass fingerprint (PMF) data were submitted to the FlexAnalysis software to search the National Centre for Biotechnology Information (NCBI) non-redundant databases using the MASCOT search engine (www.matrixscience.com, Matrix Sciences, London, UK). A Homo sapiens taxonomy confinement allowing a maximum of one missed cleavage by trypsin, followed by assignment of the

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carboxyaminomethylation and methionine oxidation, as well as peptide mass deviation was assigned by default.

2.6.

Protein identification by LC-MS/MS

The de-stained gel pieces were treated with a 50 ml reduction buffer (10 mM DTT, 1 mM ethylene diamine tetraacetic acid (EDTA) and 0.1 M NH4HCO3) at 45 8C for 45 min, then alkylated with a 50 ml alkylation buffer (100 mM iodoacetamide and 0.1 NH4HCO3) at room temperature in the dark for 30 min, thoroughly washed with 50 ml of 50 mM Tris–HCl, pH 8.5 in 50% ACN, followed by drying in a speed vacuum. The dried gel pieces were treated by Sequencing Grade Modified Trypsin solution (trypsin 0.3 mg/sample with 0.1% acetic acid, 0.05 mM Tris–HCl, pH 8.5, 10% ACN and 1 mM calcium chloride) at 37 8C overnight. The reaction was stopped by 25 ml of 2% TFA at 60 8C for 30 min. The gel pieces were placed in 40 ml extract buffer (0.05 mM Tris–HCl, pH 8.5 and 1 mM CaCl2) at 30 8C for 10 min. The equivalent volume of ACN was added and incubated at 30 8C for 10 min. The gel pieces were treated with 40 ml of a mixture of 5% formic acid and 100% ACN in a ratio 1:1 at 30 8C for 10 min and dried in a speed vacuum. LC-MS/MS analysis was performed using a capillary LC system coupled to a Micromass quantitative-time-of-flight (QTOF) mass spectrometer (Waters, Manchester, UK), equipped with a Z-spray ion source working in the nanoelectrospray mode. The digested peptides were concentrated and desalted on a 300 mm internal diameter (ID)  150 mm length C18 column. Six millilitres of peptide samples were introduced to the LC system and separated by two eluents, including eluent A (3% ACN and 0.1% formic acid in H2O) and eluent B (0.1% formic acid in 97% ACN). The elution was performed using the following gradient: 0 min 93% A, 7% B; 35 min 50% A, 50% B; 45 min 20% A, 80% B; 49 min 20% A, 80% B; 50 min 93% A, 7% B; 60 min 93% A, 7% B. The partial peptide sequences (PPSs) obtained were searched with ProteinLynx screening SWISS-PROT and NCBI software. For some proteins that were difficult to identify, the data were analysed by the MS/MS ion search tool on the MASCOT search engine using an NCBI non-redundant protein sequence database. The variable modifications (phospho(ST)), mass values (mono-isotopic), protein mass (unrestricted) and peptide mass tolerance (1.2 Da) and fragment mass tolerance (0.2 Da) were included in the search parameters. A maximum of one trypsin missed cleavage was allowed. Protein identifications were considered to be corrected when the protein score was greater than the MOlecular Weight SEarch (MOWSE) score ( p < 0.05).

2.7.

Immunohistochemistry

Immunohistochemical (IHC) staining for a-tubulin and 14-33s was performed on formalin-fixed, paraffin-embedded sections. The 4-mm-thick sections were deparaffinised with xylene and rehydrated in graded alcohol. Antigen was retrieved in Tris–EDTA buffer, pH 9, in a pressure cooker at 95 8C for 4 min. The slides were treated with 3% H2O2 to block endogenous peroxidase activity and incubated with 3% normal horse serum for 30 min to block nonspecific proteins. Then, they were incubated with a ready-to-use antibody

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against a-tubulin (clone DM1A, NeoMarkers, Fremont, CA, USA) and 14-3-3s (clone 5D7, Santa Cruz Biotechnology, CA, USA; dilution 1:800). The antibody reactions were revealed using the EnVisionTM system (DakoCytomation, Glostrup, Denmark), followed by colour development using diaminobenzidine and counterstained with haematoxylin. Immunostaining was evaluated under light microscopy. Strong immunoreactivity (2+ or 3+) was considered positive staining and the percentage of positively stained cells was estimated overall.

2.8. Quantitative real-time reverse transcriptionpolymerase chain reaction To determine the relative expression level of the KIAA1199 gene, the amplicons from 15 independent cases of OSCC were compared to adjacent normal tissue from the same patient by using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) following the manufacturer’s protocols. Complementary DNA (cDNA) was reverse transcripted from 500 ng RNA by using the iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) in a total volume of 20 ml and incubated at 42 8C for 30 min. The sequences of the primers for KIAA1199 were forward 50 -GGCGACCATCCCTGACAATTCC-30 , reverse 50 CTGCCTTTGAAGCCAACGAAT-30 and those for the 18S ribosomal RNA (18S rRNA) gene, which was used as internal control, were forward 50 -GCTTAATTTGACTCAACACGGGA-30 and reverse 50 -CGTTCGTTATCGGAATTAACCA-30 . All PCR reactions were performed under the CFX96TM system (Bio-Rad Laboratories Inc., Hercules, CA, USA) by using SsoFastTM EvaGreen (Bio-Rad Laboratories Inc., Hercules, CA, USA). A master mix containing 1 ml cDNA and 50 ng primers in 19 ml SsoFast EvaGreen buffer was prepared according to the manufacturer’s instruction. PCR cycle conditions consisted of initial denaturation for 10 min at 95 8C, followed by 45 cycles of

denaturation at 95 8C for 10 s, annealing 60 8C for 10 s and extension at 72 8C for 10 s. After amplification, the melting curves were analysed to distinguish specific amplicons from nonspecific ones and primer dimers. The results of real-time PCR were shown as Ct values, Ct being the threshold cycle of the PCR where the amplicon was initially detected. The mean Ct value of each sample was the average value from two independent experiments. The normalised Ct (DCt) values were determined by subtracting the mean Ct value of the internal control gene from the mean Ct value of the test gene. High DCt values represent low expression. The significance of the difference between the DCt values of the cancerous tissue and the normal one was tested by the Wilcoxon signed-rank test.

3.

Results

Approximately 400–600 spots were detected across all gels and 488 spots were analysed through the ImageMaster 2D software. For the early-stage group, 63 protein spots were up-regulated and five spots were down-regulated, while in the late-stage group, 37 protein spots were up-regulated and two spots were down-regulated in tumours compared to the adjacent non-tumour tissue (Fig. 1). Among these, 14 spots were significantly altered in both groups. The 55 differentially expressed protein spots were excised for protein identification and 44 proteins were identified by either MALDI-TOF MS or LC-MS/MS (Table 1). The identified proteins function in various important cellular processes, including protein synthesis, cell proliferation, cell differentiation, cell migration, metabolism, immune defence and signal transduction. A validation study using immunostaining for a-tubulin and 14-3-3s was evaluated in six cases used in 2D analysis and in 12 independent OSCC cases. In all cases, the tumour stained diffusely (mean percentage 82.2% for a-tubulin and 81.7% for

Fig. 1 – 2D-SDS–PAGE gels of normal tissue (A) and tumour (B) of a representative case using IPG strip pH 3-10 in the isoelectric focusing and 12.5% SDS–PAGE gel with silver staining. Successfully identified proteins are labelled numerically.

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Table 1 – Proteins identified by mass spectrometry. Spot IDa

Tumour groupc

FC_Ed

FC_L

25

E, L, A

3.091

2.82

98

18

E, L, A

7.064

4.11

71.08/5.37

104

26

L, A

7.644

8.74

59.24/6

53.6/5.62

111

30

A

3.100

2.25

gij119628379

53.55/8.15

53.71/6.85

35

15

E, A

4.652

4.48

gij83318444 gij16507237

70.16/5.42 64.84/5.44

68.61/5.11 72.4/5.07

80 258

26 44

E, A E, A

2.844 4.218

1.77 1.61

gij11935049 gij46812692 gij4557701 gij307358 gij14389309 gij5174735 gij116875840 gij119579887 gij4507641 gij109156944

34.41/867 46.79/7.77 39.9/5.38 51.36/5.73 47.32/5.55 46.13/5.37 34.49/9.49 34.36/6.97 23.36/4.94 20.59/5.22

66.2/8.16 60.32/7.59 48.61/4.97 53.96/5.43 50.55/4.96 50.26/4.79 33.74/8.53 31.42/5.48 22.49/5.46 19.97/4.79

82 119 808 47 363 896 39 31 47 60

21 33 50 18 34 57 23 10 33 31

E, E, E, E, E, E, A A A E,

DIV/0e 4.304 102.41 4.99 2.81 3.08 3.82 2.38 5.06 3.46

1.69 1.52 4.21 2.17 1.76 1.77 2.46 2.26 2.03 1.38

gij119615219

56.14/7.78

55.14/6.01

35

8

E, A

4.04

4.26

gij5107637 gij4503545

21.64/8.65 15.19/5.52

24.52/7.01 17.05/5.08

88 19

24 23

E, A E, A

3.22 15.79

1.40 1.67

gij1263196

54.82/8.22

64.94/6.39

53

19

E, L, A

4.18

3.72

gij4505641

30.7/4.75

29.09/4.57

36

16

E, A

DIV/0

2.56

gij29825627

73.55/8.37

35.46/7.77

38

24

L, A

2.85

2.05

gij78101133

25.54/4.52

30.03/5.79

36

23

E, A

4.66

1.75

gij119613095 gij5031635

59.36/8.25 15.69/9.25

58.58/6.41 18.72/8.22

92 166

19 33

E, L, A E, A

2.43 8.27

1.95 3.02

gij5454052 gij219669

23.28/4.74 46.21/8.25

27.88/4.68 41.43/7.53

550 31

66 23

E A

8.31 4.19

0.96 2.76

gij4557581 gij4505059

12.77/7.63 37.87/8.49

15.5/6.6 35.56/7.42

109 34

26 21

E, A E, L, A

7.41 DIV/0

1.80 6.48

Energy synthesis Transketolase 31 Pyruvate kinase 3 32 Enolase 1 33 34 Phosphoglycerate kinase 1 L-Lactate dehydrogenase 35 Triosephosphate isomerase 36 37 Aldehyde dehydrogenase 4

gij37267 gij189998 gij4503571 gij4505763 gij5031857 gij999892 gij57161701

60.95/8.97 50.81/9.04 41.3/8.52 35.96/9.32 31.96/9.4 22.18/8.49 15.23/9.13

68.44/7.9 58.45/7.95 47.49/7.01 44.99/8.30 36.95/8.44 26.81/6.51 16.65/8.8

162 353 837 351 337 299 41

38 58 68 51 34 48 32

E, E, E, E, E, L, A

3.17 8.65 2.15 2.56 3.79 1.45 6.84

2.05 2.92 1.89 2.27 3.46 2.28 7.32

Immune defense Proteasome activator 38

gij5453990

23.97/6.84

28.88/5.78

151

29

E

13.098

1.07

Protein name

Protein synthesis 1 Eukaryotic translation elongation factor 2 Translation elongation factor 2 1g 3 Heat shock 70 kDa protein 8 isoform 1 Heat shock 70 kDa protein 8 4 isoform 2 5 Chaperonin containing TCP1, subunit 6A Stress response Heat shock protein 90 kDa 6 Heat shock 70 kDa protein 5 7 Cell proliferation/differentiation Keratin 1 8 Keratin 6A 9 Keratin 17 10 11 Peripherin Tubulin, alpha 6 12 Tubulin, beta 2 13 tRNA splicing endonuclease 14 Protein phospatase 1F 15 Tumour protein D52 16 Translationally controlled 17 tumour associated protein 18 Aminopeptide puromycin sensitive 19 Karyopherin beta-2 Translation initiation factor 20 5A isoform B 21 AICR formyltransferase/IMP cyclohydrolase 22 Proliferating cell nuclear antigen Cell migration Squamous cell carcinoma 23 antigen 1 (Serpin B3) 24 Tumour necrosis factor-a converting enzyme 25 WD repeat domain 1 Cofilin 1 26 Signal transduction 14-3-3s 27 Pertussis toxin-insensitive G 28 protein 29 Fatty acid binding protein 5 Tumour-associated calcium 30 signal transducer 1 precursor

BMSb

Sequence coverage (%)

96.25/6.41

81

41.06/7.96

50.43/6.25

gij5729877

66.39/8.48

gij5729877

Accession no.

Observed MW./pI

Theoretical MW./pI

gij4503483

78.58/8.55

gij119594432

A A L, A L, A A A

A

L, A A A A A A

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Table 1 (Continued ) Spot IDa 39 40

Others 41 42 43 44 a b c d e

BMSb

Sequence coverage (%)

FC_Ed

FC_L

13.32/8.39

28

15

E

DIV/0

1.52

11.99/8.23

9.4/8.93

34

77

L, A

DIV/0

DIV/0

gij340234 gij32493203

34.65/5.12 14.93/9.55

35.09/4.7 17.42/9.6

46 34

20 44

E E

6.559 DIV/0

1.11 0.88

gij3318841 gij11992405

22.65/8 40.5/9.68

25.01/6 57.51/9.28

111 51

31 21

A A

2.220 DIV/0

1.94 9.85

Accession no.

Observed MW./pI

Theoretical MW./pI

B cell anitibody heavy chain variable region Imminoglobulin M heavy chain

gij106897453

13.98/8.61

gij3851242

Vimentin Cervical cancer 1 protooncogene Horf6 KIAA1199

The spot numbers correspond to those on the 2D images shown in Fig. 1. BMS, based MOWSE score. Group of tumours showing significant different protein spots in 2D gel: E, early-stage group; L, late-stage group; A, all cases. Fold change of protein spots: FC_E, fold change in early-stage group; FC_E, fold change in late-stage group. Divided by zero.

14-3-3s), while the adjacent non-tumour mucosa showed focal staining (mean percentage 21.7% for a-tubulin and 20.5% for 14-3-3s) which were significantly different ( p < 0.05, Wilcoxon signed-rank test) (Fig. 2). The KIAA1199 messenger RNA (mRNA) expression in cancerous tissue was significantly higher than that in normal tissue ( p < 0.05) (Fig. 3).

4.

Tumour groupc

Protein name

Discussion

Proteomics, a study of global protein expressions within a cell, tissue or organism, has become one of the promising approaches for the discovery of candidate disease biomarkers

and new drug development. Using two platforms of mass spectrometry, MALDI-TOF MS and LC-MS/MS, 44 significantly overexpressed proteins in OSCC were identified. These proteins function in various crucial processes relating to cancer cell growth, survival and metastasis. The present study used two MS schemes, MALDI-TOF MS and LC-MS/MS, to enhance the degree of success in protein identification. The two methods analyse different types of data. Analysis of PMFs by MALDI-TOF MS involves the determination of the masses of all peptides in the digest, while PPSs directly analysed by MS/MS examine the amino acid sequence of the peptides.7,8 The advantage of MALDI-TOF MS is that it is technically less demanding and, therefore, it is

Fig. 2 – Immunohistochemistry analysis for a-tubulin (panel A) and 14-3-3s (panel B) demonstrated focal positive staining in the basal layer of adjacent non-tumour mucosa and diffuse strong cytoplasmic staining in the tumour cells. The mean percentage of positive staining was graphical summarised in the right column.

archives of oral biology 58 (2013) 1677–1685

Fig. 3 – Box plots showing log-transformed normalised expression levels of KIAA1199 in normal and tumour tissues.

suitable for large-scale sample analysis. The technical drawback is that it is susceptible to ‘signal suppression’, a phenomenon in which certain analytes are preferentially ionised and the other compounds may become undetectable.9 By contrast, PPS analysis by LC-MS/MS is technically more complicated and time consuming. The protein samples must be mixed with many chemicals prior to injection into the LCMS/MS system by electrospray ionisation (ESI). However, it has a high sensitivity and specificity. The identified proteins are proteins whose functions relate to cellular proliferation and include protein synthesis proteins, stress-response proteins, proliferation-related proteins and proteins and enzymes in energy and metabolic pathways. These findings support the highly proliferative properties of cancer cells. Most proteins have been previously reported in head and neck squamous cell carcinoma (HNSCC) or OSCC proteomic studies and include heat shock 70 kDa protein 5, Hsp90, squamous cell carcinoma antigen 1, atubulin, b-tubulin, various type of keratins, cofilin, 14-3-3s and various metabolic enzymes.10–16 However, a few proteins have not been reported previously, in particular, Horf6 and KIAA199. In our study, three members of the HSP family, including Hsp90, HSPA5 and HSPA8, were found up-regulated. Hsp90 and HSPA5 are among the most common HSPs related to cancer cell growth, mostly through an anti-apoptotic property.17 Hsp90 and HSPA5 have been reported to be overexpressed in many tumour types, including HNSCC.13,16,18 In contrast to most heat-inducible HSPs, HSPA8 is constitutively expressed. It binds to nascent polypeptides to facilitate correct protein folding. Overexpression of HSPA8 in cancer has been reported in few studies19 and the tumourigenic pathway with which HSPA8 interacts has not been reported. Consistent with other studies, various keratins including keratin 1 (K1), keratin 6A (K6A) and keratin 17 (K17) were found up-regulated.10,13,15,16 K1 is a major keratin expressed during terminal differentiation and keratinisation.20 K6 is normally expressed in stratified squamous epithelia and it is thought to

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be a marker for hyper-proliferative keratinocytes.21 K17 is induced upon stress, injury or inflammation. As most normally stratified squamous epithelia lack K17, its presence may be regarded as neo-expressive during tumourigenesis. The high expression of cytokeratin 17 (CK17) may also relate to tumour differentiation as all samples used in this study are well-differentiated OSCCs, consistent with the study of Kitamura et al.,22 who reported a significantly higher expression of CK17 in well-differentiated tumour compared to moderately/poorly differentiated OSCCs. Our results revealed overexpression of both a-tubulin and b-tubulin in tumours compared to normal tissue, which was confirmed by immunohistochemistry in clinical samples. Expression of a-tubulin or b-tubulin in HNSCC or OSCC proteomic studies has also been reported by others.10,14,16 aTubulin and b-tubulin are dimer components of microtubules, the major cytoskeletal elements required for cell division, intracellular transport, cell migration, etc.23 Microtubules have long been considered an ideal target for anticancer drugs and they are found in a wide variety of compounds currently in clinical use and in development.24 In addition, different isotypes of tubulin have been increasingly reported to be a predictive marker for chemoresistance in different malignancies, including HNSCC.25 Overexpression of 14-3-3s in tumour compared to nontumourous mucosa was strongly confirmed in our validation study. The protein 14-3-3s is an intracellular phosphoserinebinding protein which regulates different signalling processes related to carcinogenesis. It is one of few proteins that are consistently reported to be overexpressed in OSCC proteomics studies.26–28 It has been shown to be a significant independent prognostic factor for poor survival.29 Therefore, 14-3-3s seems to be a promising biomarker in OSCC. We found at least two proteins rarely reported in cancer, Horf6 and KIAA1199. Horf6 is a novel member of the antioxidant enzyme class.30 However, its expression or function linked to cancer has not been demonstrated. KIAA1199 is a novel protein described recently to be highly expressed in the inner ear and mutations of the gene may be associated with hearing loss.31 Its potential role in cancer has recently been reported in colon cancer and gastric cancer. It is overexpressed in colonic adenoma compared with normal mucosa and its function may be linked to the Wnt signalling pathway, an important signalling pathway involved in colon tumourigenesis.32 In gastric carcinoma, the high expression of KIAA1199 is significantly associated with lymph node metastasis.33 Our validation study using realtime RT-PCR confirmed the overexpression of KIAA1199 in OSCC compared to normal oral mucosa, and this is the first report in OSCC/HNSCC. However, further studies are needed to confirm the result and to explore its exact functional role in oral cancer. In conclusion, our proteomic study using 2D-SDS–PAGE and MALDI-TOF MS and LC-MS/MS identified a number of proteins, most of which are known to play roles in various cellular processes important for cancer cell growth, survival and metastasis. At least two proteins are novel for OSCC, Horf6 and KIAA1199; the latter protein was validated by real-time RT-PCR. Further studies are needed to evaluate their roles as potential biomarkers in oral cancer.

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Funding 13.

This work was supported by Prince of Songkla University, Thailand. 14.

Competing interests None declared.

Ethical approval The research protocol was approved by the Ethics Committee of the Faculty of Medicine, Prince of Songkla University. Reference Number SUB.EC 49/400-008.

15.

16.

17.

18.

Acknowledgements We thank the Department of Anatomy, Faculty of Science, for 2D gel facilities and Dr. Pritsana Raungrut for the 14-3-3s antibody. We specially thank Associate Professor Chidchanok Leethanakul for critical comments on the results. This work was supported by Prince of Songkla University.

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