Proteomics pattern associated with gingival oral squamous cell carcinoma and epulis: A case analysis

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Oraì S{ience lnternational I 5 (2O I I } 4 1 -47

Contents lists available at ScienceDirect

Oral Science

International

iournal

homepage: www.alsevier.comllocate/osi

Review

Proteomics

pattern

associated

with

gingival oral

squamous

cell

carcinoma

and

epulis:

A

case

analysis

Francesco Papaa,

Rosa

Anna Sicilianob,

Francesco

Inchingoloc,

Maria

Fiorella

Mazzeob,

Salvatore

Scacco'i, Rosa

Lippolis

d'-a _Deryrtfirent_oI_Bssic_Medical_{Sciences, IVeurosciences}_and_Sense_{Organs, Univercity Aldo Moro, Bari,}_ltaly b _lnstitille_{oJ Foad SdÉ,rces,}_Noriondl_R$eilrù_{Co&ndl rCNRì Avellino,}_ttaly

' Dewrtmenl oI lnterdisciptinary Mcdicine,lhtiversity Aldo Maro, ltalv

'r lrstifute o/Biorrwmbranes and Bioenergeric§, Ifalisn Naaional ResearrJr Council (CNR), Bari. ltaly

o

ckk td

§dss

ARTICTE

INFO

ABSTRACT

,4rticle history; Received 7 June 2Ol 7 Received in revised form

24 November 2OI 7 Accepted 5 lleccrnber 201 7 Available online 1 February 2Ol

I

Objectives: Oral squamous cell carcinoma (OSCC) is the mo$ common epithelial malignant neoplasm affecting the oral cavity. OSCC can mimic oral lesions of inflammarory origin with benign features, olten Ieading to delay in diagnosis and treatment. Early detectiorr is important to greatly increase the chances ofa successful treatment. The present study reports a proteomic analysis ofa gingival oral squamous cell carcinoma (C-OSCCI and an epulis.

Mateials and methods: Normal mucosae tissue, C-OSCC tissue, and epulis tissue as e comparative sample

of benign nature were collected and immediately frozen in liquid nitrogen. Tissue-extracted proteins were separated by two-dimensional gel electrophoresis and subjected to image analysis. Proteins that

showed a significant diffelence in the expression level in the G-OSCC tissue were identified by the

nano-ESI-HPIC-MSIMS experiment and database searchi.

Resulfs and conclusion: The proteomic analysis of C-OSCC tissue enabled the identification of proteins tìrat are potentially rehted to tlre disease; these proteins can be considered as signature molecules for

diagnostic and prognostic tumor markers.

Keywords: Biemarkers Proteomic analysis

0ral squamous ccll carcinoma

1. ?.. Contents

2.4. Samplepreparationforproteomicanalysis...,..

...,...42

2.5.

Two-rlimensional electrophoresis (2-DE).. ..

.

.. . . .. .. .. ,43

2-7.

Proteinanalysisbynano-ESI-HPLC-MS/MSexperimentsanddatabasesearching

...43

3.1. 2-DBanddataanalyses..

...-...43

AppendixA

Supptenrentarydata...

...,...46

Abhrevittions: OS(C, oral squamous ccll carcinomas; G-OSCC, gingival oral squamous cell carcinoma; 2-DE, two-dimensional electrophoresis; CllAIrS.

3-l(3-+ _{Correspondingauthorat:NarionalResearchCouncil(CHR),CloDepartmentofBasicMedicalSciences,NeuroriencesandSenseOrgans.P.zzaCiulioCesarell,TOt24}

Bari, Italy.

E-mail atidress : r.li ppolis@ibbe.cnr.ir (R. Lippolis),

https:l/doi.orgi10.l0l6151348 8643i17)30044 7

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l:. fttpc et *1. I Oral Sci.:nce lntenlational | 5 (2O18] 4I _-4V

1. Introduction

Thc

majcrity

of, caficcrs

in

thq oral caviiy arc oral sqllamous

cell

carcinomas (OSCC)

_[l],.1

multifa(torial

disease

that

arises

from the stratified squamous epithelium

lining

rhe oral mucosa [2-'a]. The tumor originates by morphological cell transformÀtion, olten through clinically benign precaucerous lesions and develops according to theil _{potential of neoplastic transfot.nration [5*9].}

Cin-gival oral squamous cell carcinom.r (C-OSCC), _{classified as.r subset}

ol

OSCC, is a relatively rare malignant carcinoma of the oral

cav-ily,

reprcsenting less than 10% of diagnoserl intt.aoral carcinomas. The etiology oiOSCC remains unknown, but

it

is esrablished that tlre rlisease is associaterl

with

a variety

ol

risì< factors,

in

partic-ular smoking and heavy alcohol use

_ll0l"

OSCC involves a series

of mutations thaf result

in

the selective growth of mutated cells replacing normal cells in a specific region

_[1i

_].Because of its close

proximiry to the teeth and periodontium, G-OSCC can mimic other

rooth-related leslons, especially those of inflammatory origin. Clin-icai presentation of G-OSCC can be quite variable, and hence,

it

is

misdiagnosed as benign tumor

or

as inftrammatory response. At

present, diagnosis of oral cancer is mainly based on clinical oral examination. Histopathology is an adjuvant technique

in

identi-tying oral tumor or malignant transformafion of oral lesions. The convenrional diegnostic methods alone are, however, not sufficient to support early diagnosis of the disease and differential diagnosis

with

respect to benign tumors _{[12,13i. The}majority

(two+hirds)

ofOSCCs are diagnosed at an advanced stage _{[14] when} progno-sis is

fairly

poor, and the overall 5-year relative surviyal rate

of

oral

and pharyngeal cancer patients

is

approxinìately 5S% _[i51.

Delay in diagnosis can still be considered a major cause of the high

morbidity

rnd

mortaliry of OSCC p"ltients. Current standards-of-care (surgery and/or radiotherapy) oiten end-up

with

devastating consequences on the appearance and firnction ol affected organs, thereby c.rusing a marked clefriment on the quality of life even in snccessfirlly treated patients. The molecular mechanism

unclerly-ing _{the pÀthogenesis ofOSCC is}relatively poorly understood and represents ,l topic olsignificant _{importance. A better}

understand-ing of the _{molecular mechanisms involvecl in the pathogenesis and} progression oFthe disease is a key to the developmentof moreeif'ec-tive tools to improveearly diagnosis aforal cancer, befterprognùsis, and quality of life of fhe patients. Discovering new rcli.1ble m.1rk-ers For OSCC _{anrl developing new diagnostic tools for its early ancl}

easy detection is [hus a relevant issue in tlre fietrl oforal pathology fesearch.

Weinberger

et

al. analyzed molecular differences

in

protein

expression among tumars thararise from different sites olthe head

and neck region, and they found considerable molecular diversify

between

diflerent

squamous cell carcinomas (SCCs) _{161.

_In

_the

present study, a proteomic analysis was conducted to analyze the cellular protein profile of a case of G-OSCC ancl to identify

pote'n-tial _{signatule molecules, Healthy tissue and non-neoplastic epulis}

tissue were taken as samples for comparison.

2.

Materials and methods 2.1. Case report

A

S8-year-old female

patient

reported

at

the

Maxillofacial

Unit

of

the

involvement were collected. Histopathological analysis of biopsy specimen revealed a malignant neoplasia olepithelial origin, which

is _{characterized by invasive proliferation of nests and cords o[} neo-plastic epithelial cells. Based on histologi(algrading, the tumor was categorized as a well-differentiated C-OSCC.

A case (S5-year-old lemale)

olepuiis

is aiso included. Epulis is

present as a sessile formation on the upper

right

mucosa gingiva near the front

olthe

_{mouth befween the canine teeth and the nrst}

premolar. Its histology is relarerl

to

fibrosing granulàtiofi tissue,

firm and rubbery, and

pile

pink in color.

Sections of the normal buccal mucoea obtaiued from the two

patients were used as the

cortrol

tissue,

2.2.

Reagents

Immobiline

DrySrrip

_{{pH 3-i0}

and

4*7,

13cmJ,

DryStr'p,

cover

fluid,

immobitized

pH

gradienr

(lpc)

buffer,

3-[(3*cholamidopropyl)

dimerhytammoniol-1-propanesr:lfonate (CHAPS),

bronroplrenol

blue,

.tgarose,

acryl.1mide. tris,base,

glycine, sodium dodecyl sulfate

(SDS), N,N,N',N'-tetrarnethylethylenediamine (TEMED), Coomassie Bril-liant BIue R-250,

rlifhiothreitol

(Dfi),

iodoacetamide, acefic acid, and trifluoroacetic acid were purchased from CE Healthcare

(Upp-sala, Sweden)- Sequencing grade-modified trypsin was purchased from Promega (Madison, WI, USA). The remàining chemicals were of analytical grade. All bu[fers were prepared

with Milli-ewater.

2.3. Tissue callection

were obtained

selec-tively by dissection

with

special care

lor

minimal contamination

of

nonepithelial cells.

All

ihe

samples were immediately frozen

in

liquid

nitrogen and stored

in

a deep freezer

(-gO.C) for

pro-teomic analyses. The study was approved try the university ethics

committee-2.4. Sample preparotion _farproteomic analysis

Proteins were extracted

tollowing

the

protocol reported by Zhang [17]. Briefly, tissue samples of normal, ncn-neoplastic, and

malignant tissues (*2OOmg) were

gmund

in

small

pieces

in

a

morfar

with

liquid nitrogen. collected in turres, and homogenized

in

2 mL fresh lysis buffer composed

of

7

M

urea, Z

M

thiourea,

40 n]M Tris, 4% (wlv) CHAPS, 100 mM DTT, 0.5 mM

phenytmerharre-sullonyl fluoricle (PMSF), 0.5 mM ethylenediaminetetraacetic .1cid iEDTA), 1% _{TritonX-100, and 5% lpC buffer. Tissne lysate was}

fur-ther clisrupted with ;ln ultrasonir _{homogenizer and centrifi.rged at}

12,000 x g lor I O min at +4 "C. Supernatafit was precipitated by the addition of cold èc€toile (dilution rèrio 1 :12, vlv) and incubated ar

-20"C

overnight. AFrer centrifugation

at

14,OOO x g

for

.15

nlin

at +4"C, fhe pellet was resuspended in rehydration _{buffer. The}

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l:. l4pa et at.I Or«l Science lntpmationa] 15 (2$18) 4I -47 4J Braclford method (DC Prctein Assay; Bio-Rad, Hercules, CA, USA)

l1tì1. Protein samples were stored at

_-80,C.

2.5. Two-dimensional electrophoresis _t2-DE)

2-DE was carriecl ont

with

Anrersham Biosystenrs IPCplror IEF and Ettan Daltsix electrophoresis units according to the protocol previously described {191. Briefly, 25A pgoitissue-extracted

pro-teins were solubilized

in

IPC

strip

rehydration buffer

(BM

urea,

2%

_twlv)

CHAPS, ù.52 If,G buffer, 2%

(wiv)

DTT, and rrace

ol

bro-mc,phenol blue) and loaded

on

13-cm IpG strips

that

provielecl

Iinear pH gràdients

3-10

and 4-7. The

first

pH gradient is suited

for an overview pattern of total cell exfracfs; the second is usetl

fo zoom rhe specinc region

of

the gel (Fig. i

l.

lsoelectric

focus-ing was carried

out

at 20"C

by

using Ettan [soelectric Focusing sysrem (GE Healthcare)

lor

a total

ol

70kvh. After focusing, rhe

IPG strips were equilibrated for 12 min in the equililrration buffer (50 mM TrislHCl pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v), SDS) cootaining 1U D'ff and for 10 mio in the same equilibratian buffer

containing 2.5X iodoacetamide and 0.05% bromophenr:l blue. For

the second dimension, homogeneous SDS-12.5% polyacrylamide electrophoresis gels were used. Electrophoresis was carried our in

a Laemmli system [20Jar consfant current

of l5mAlgel

ar 10 ,C.

Molecular weight markers and

pl

standards were from Bio-Rad.

The gels were stained using Coomassie Blue Colloidal dye (Sigma-Aldrich, St. Louis, MO, tlSA), which enabled quanriflcation of protein §faining intensities, allowing quantitative cornparison

of

protein expressiorl levels between the samples.

2.6.

lmagecn€llrsis

Srained gels were scanned

with

an image scanner (GE Health-care)

at 3oodpi

resolufion

to

acquire the gel inrages

that

were analyzed using tmage-Master 2D Platinum v.Éisoftware (CE Health-care),

which allows

quantitative comparison

of

spot

intensity. Relative spot volume (% vol.), i.e., digitized staining intensiry

inte-grated over the area of the individual spot divided by the sum

of

volumes of all spots in the gel and mr-rltiplied by 100, was used for

spot quanlificafion _{2I _].The m.ìrch lD numt}er was used fo identify all spots in a match. Spots that were present in all the gels of the three classes _{oFsurgical specimens and that showed differences in} their relative volume >1.7 foltl or

<-.l.7

fold

wifh

respect fo con, trol samples and

with

p < _0.05,_{using the nffo-tailed Student}_f_test,

were selected I'or further mass spectrometric analyses (i ébl€ I ).

2.7. Protein analysis by nano-ESl-HpLC-MS/MS _{expertments afid}

database searchittg

Spots oF interest were excised

irom

the 2-DE gels, and in-gel

tryptic digestion was carried out following the procedure already clescribed [ 19].

The obtained tryfitic peptides were analyzed by nano-ESI-HpLC-MSIMS _{using a Q-Exactiyeru mas§ spectrometer (Thermn Fisher} Scientific, Walrham, MA, USA) interfaced

with

an UltiMate 3OOi)

RSLCnano LC system (Thermo Fisher' Scientifi c).

Peptide mixtures were concentrated and desalted on a

trap-ping _{precolumn (Acclaim PepMap C18,300}_pm_{x 5 mm nanoViper,} 5 pm, 100À; Thermo Fisher Scientilìc), Lrsing O.05% tormic acid and

2% acetonitrile èt a flow rate of 10

pllmin.

The peptide separation was performed .rt 35',C using

a

Clg column (Acclaim Easy Spray FepMap RSLC ClB, 75prn x

l5crn

nanoviper, 3 p.nr, 100 A; Thermo Fisher Scientific)with 0.1% formic acid (HCOOH) as eluent A and 80% acetonitrile in 0.08% HCOOH as

elueni B, and a linear gi-adient was estabiishetl lrom 4K,

b

SA% B

over 30 rnin, maintained for 6 min. from 50% to g0Z B over 1 nìin, maintained for lOr.nin before column re-equilibrution ro 4% B.

Mass spectra were acquired in the m/z range of 350- 1600. Data

acquisition was performed

in

a data-dependent Full MS/ddMS2, cnabling thc acquisition of MSIMS spcctra fsr

thc

10 most intcnsc precursor ions (top 1 0) with a dynamic exclusion of 3O s. Resolution

wrs set to 70.000 For MS specrra acquisition and 'l7.5OO for MSi MS

spectra arquisition.

Proteomic analysis

was

performed using Thermo Proteome Discoverer'fM platform (version 2.1.0.8'l ; Thermo Fisher Scientific),

interlacerl

with

the SEOUEST HT Search Engine server (l,lniversify olWashington, USAJ used for protein identificarion. The param-efers used for the database searches were as follows: Swiss-pr.ot human protein database (v2016-04-13) and a contaminent protein dafabase (Confaminantcombi nedcon taminanf_fi nal fasta. provided

lry

the manufacturer), trypsin as proteolyric enzyme, up

fo

rwo missed cleavage, carbamidomethyl as fixed modification for cys-teine resirlues, oxiclation of methianine residues ancl formation

of

pyroglutemic acid of N-terminal glutamine residues as dynamic modification, 20 ppm mass tolerance for precursor ion ancl 0.02 Da mass tolerance for MS/MS fragments. Results were filtered for high sonfident peptides and proteins (False Discovery Rate, O.O1%).

3.

Resultsanddiscussion 3.1. Z-DE untl tlutu unoiyses

tJifferential 2-DE analysis of fhe proteome of C-OSCC tissue sanÌ-ples was perlormed. Healthy mucosa and a clinically benign epulis lesion were used as control and comparative samples, respectively. Fig. 1 A and B show rhe representative 2-DE gek of the fhree samples in the pH range 3-10 and4-7.

Proteomic naps of control, epulis, and C-OSCC samples showed seveml hundreds ÒFwell-resolved protein spofs distributed over a

wide range of pl values and molecular masses. The overall position anrl the nuffiber of protein spots observed

wis

practically sinrilar

in

the 2-DE rnaps oF rhe control _§A7

*24).

epulis (489

t32),

and G-OSCC samples (481

*37).

'fhe _{percEntage of}_{matches between}_gels

from the same class and

fmm fhe three different clesses was similar (around GB%)

wifhouf

statistically significant dilferences berween the classes. To reduce

the possibility of false

positiv$,

only t['re protein spots

systemat-ically present in all the gels

oi

each class were considered in the analysis (Flg.2).

Image analysis led to highlight spots whose relative volume var-ied in the 2-DE maps obtained lrorn fhe analysis of the proteomes extracted from C-OSCC _{or epulis tissues compared}_{to the}_control

sample. These spots contained

diilerentialty

expressed proteins

that could lle relaied to the patholùgic conditions.

In particular, proteins present in 23 spots were overexpressed in the C-OSCC _{sample {in comparison to}rhe control sample) and 9 of those were also overexpressed in the epulis sample. Moreover,

exhìbited opposite trend in the expression level in the two samples.

These results confirmed that epulis tissue showed a lower number of _{alterations in protein expression level compared to the C-OSCC.}

i.2.

Protein identificatian

Protein spots showing significant diflerences in theirvslumes in

C-OSCC ancl epulis tissue samples. Às conÌpared to norn"ìal tissue, wereexcised lrom the gelsand analyzed by nano-ESI-Hpl-C-MS/MS experiments. These experirnents resulted

in

tlre identification

of

3.1 proteins _{whose exprÈssion level was changed}

_in

_the_C-OSCC

sample.The expression level of 1 1 of these proteins was also altered in the epulis sample.

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§

Tnble I

Spot

lD'

Ac(es.

No,\

Protein name

_Cov.

_{Pep. MW'fkDal}_pt. _{Cene ID} _Chrom._ScorÈ

SEQUEST

HT

Categories Fold changee

Epul C.oSCC 342 Jbfi 433 450 573 549 556 567 570 614 628 631

u7

648 650 {i92 696 700 767 768 783 820 826 833 t177 884 890 891 933 999 1009 p55072 r42679 p20s9 I r,1 r r42-l P1080§ rs2§75 p08670 t}l4868 12338 r Q8NBS9-1 014745 I08670 p08670 P08670 rt6733-l Ii68032 I,60709 p29508 P4859,1 0602r

I

P5289s P4733A P07355-2 p04083 P54920 P48556 QeY4E8 pl r 476- t Q9HOU4 P20340- l I\2&"7 Q0r46S pri989l P02042 QgUBC3 Pl 3646 pl 3646 89.77 51.479 75.473 70.854 6r.016 55.892 53.619 57.1 53.1 32 47.599 38.845 53,6 r g 53.619 53.h19 47.139 41.992 41 .?1 44.537 44.*2s 35.S97 36.712 36.83 40.386 38.60 33.211 39.587 1 r 2.348 22.663 22.157 23.578 30.7s9 15.1 55 16,13 16.045 53.5A2 49.557 49.557 VCP FGG MX1 HSPAS HSPDl FCB vtM DARS WARS TXNDC5 SLCSA3RI VIM VIM VIM ENOl ACTCl ACTB SERPJNB3 SERPIN84 AKR1 B1O AKR1C? AKRI C3 ANXAZ ANXAI NAPA PSMDS USP'I5 RABlA RAB1 I] RAB6A APOAI FABPs HBCI HBD CRNN KR.T13 KRT1 J +4.45 +5.29 *1.95 *1.72 +1.93 +1.99 *10.8 *15^4V *60.56

Transitronal endoplasmic reticulum ATpa$e Fibrinogen gamma chain

Interferon,induced CTP-binding prorein Mxl

Heat shock cognate 7 I kDa prorein

60 kDa heat shock prÒtein, mitÒchÒndrial Fibrinogen-beta clrain

Vimentin

Asp.trLate tRNA ligase, cyroplasmic Tryptophen-rRNA ligase, cytoplasmic

Thioredoxin domain-contaj ning protein 5

Ne(+)/H(r) exchange regularory cofacror NHE-RF I

Vinrentin Vimentin Vimentin

Alpha-enolase

Artin, alpha cardiac muscle 1

Actin, cytoplasmic l

Serpin 83 Serpin 84

Aldo-keto reductase family I member Bl0

Aldo-keto reduftese family I member C2 Aldo-keto reducrase family I member C3

Isof, 2 oiAunexin A2 Annexin A1

AIpha-soluble nsf attachment protein

265 protÈasome non-ATPase regulatory sub. 8

Ubiquitin carboxyl terminal hydroiase i 5

Ras-related prrJtein Rab-1 A Ras-reiated protein Rab-l B

Ras-related proteirr Rab-6A ApolipoprÒrèin A-l

Fattv acid-binding protein, Èpideriltèl

HemÒ91Òbin sub, gamma-'t Hernoglobin suh. delta

_-3.74

'

+2.27

*

_-2.98

*

+2.8? 5.26 5,62 5.83 5.52 5.47 Q11 5.1 2 6.55 6.23 5.97 5.77 5.12 5,12 5.72 7.35 5,39 5.48 6"81 6.2r 7.84 7.49 7.94 8.37 7.O2 s.36 7.68 5.22 6.2r 5.73 5.s4 5.7ti 7.At 7.2 8.05 6.1 4.96 4.S6 !) 4 21 l1 2 4 l0 2 l4 6 l7 10 l0 l0 1 ì5 7 l8 18 7 10 l0 I5

I

1g 1§ 12 I.I 11 l1 8 t1 'tr 1 r7 17

PrÒtein processing-related proteins

Blood-borne glycoproteins

RNA processing-telated proteins

Stress response-related proreins

Stress rl'sponse-related proteins

Blood-borne glycoproteins

Cytoskeleron structural proteins

RNA prccessing-related proteins

Enzyme

Disulfi de isomerase pfotein

RNA prÒcessing-related _fjroteirìs Cytoskeleton structural proteins

Cytoskeleron stt'uctural pl oteins

(ytoskeleton structural proteins

Enzyme

Cytoskeletan structural proteins

CysteÌne protease inhibitors

Cysteine protease inhibitors

Enzyme Enzyme Enzyme

Ca2' - and phospholipid-binding protÈins Ca?'- and phospholipid-birlding proteins

Protein pr ocessing-related pl oteins Componefit of the 26§ proteasfime Enzyme

Protei n processing-relÀted proreins Protein processing-related proteins Prolein processing-related proteins

Lipid-binding proreins Lipid-binding proreins

lnvolvÈd ìn the oxygen transport Involved in the oxygen transport Calcium-binding protein

Cytoskeleron stru(tural proteins

! § fr

:

5 § è

§

I

\

'

Spot ldentification NumbÈr b Arcession Number.

I l1l@tiql

holÈula! @i3nt,

(

_{lhe..tiol lshctri.}

_polàr,

, _Tlè

_r[id

_Àonz6È]

_tiÉ

_tndic.le

(5)

f. l\pfl ea ql. _I_$ralScience lnternarion1l l5 (20r8) 4I -4f

EFIi3 Tiss C-oSCC lise,

A

u :s l@

t

rij

r pH - ^-lo cfrtrol lisE

t:

. - $,r{À* i 3 _--,--- pH -- 10 Egulis Ykgc

--a+

--t.3

-:-'

;-l'

'rL,

lrr

I

--

_+l)--.,

. pH (l OSCC f irB. ]F,'*

_""

_l"*;

-

.

:-'ll.-j*"-C,

Codtrol lis* ' q.1! .'r,i.rr'*.

;;,*,

H§"'

*l

'{l*.,,

""if*."

*l

"*l

7 .da

-

tu-'

_&:ò

a

*

t

It

*§.

t 1-,--- ,,-- _É{ 7 1- FH_-. , {- Él-7 Fig,1, (A)Representarive2-DEmapsoftherotalproreinexrractsfromcontrol,epulis,andG-OSCCtissuesanrples(linearlIrGpH3-18).Prot€inspotsthatshowdifferent

rclative volunes in the G-OSCC ànd epulis samples compared to the eontrol s.rmple were indicnted by match tD number. proteins presenr in the§e spDts were idenrined by nano-ESI-HPLC-MSIMS analysis and database search and are listed in Table 1. (B) Representative 2-DE maps of the totÀl protein extracts from control, epulis" and C-OSCC

matcl] lll number. Irrotcins present in these spots were identifierl by nano-t-iSl-lll'LC-MS/MS analysis and database search and are listed in lahie l.

*t'

*.|

a

ln

particulnr, fibrinogen gamma chain (sÈot 366), heat shock cognate 71 kDa (spùt 450), fibrinogen beta chain (spot 54S), 265 proteasome non-ATPase regulatory protein (spot 820), hemoglobin subunit gamma 'l (spot 890), and hemoglobin subunit delta (spot

891) were significarrtly up-reguiated

in

both G-OSCC arld epilis (Table 1).

Notably, proteomic studies

of

epulis tissues are

still

scarcei

hence, the present study might provide first insighfs on this

inflam-rnatory status also in relation to 6-osCC development.

In C-OSCC, a 14-fold increased expression of members

oi

the Aldo-keto reductase family(AI(R), AKR-I-810, AKR-1-C2, and AKR-1 -C3, was detecred. The AKR enzymes reduce a variety ofaldehydes

andlor

ketones

to

their

corresponding alcohols using NADH or

NADPH _{l22l and}are involved in intraceilular detoxification andcar-cinogenesis processes _{123-26\. Enhanced expression}of AKR-1 -B10

has been detected in many types of tqmors 123,24). Silencing of the AKR-t-810 gene was found to

inhibit

growth of colorectal cancer [27] and SCC _[2sJ.AKR-I-C2 apFears to be associated

with

disease progression in prostate cancer [2Si"

The expression level

of

tryptophan-tRNA tigase, cytopla§mic

tryptophan aminoaril t-RNA synthase (WARS), was also signif-icantiy increased

(1z-fold)

in

C-OSCC, thus confirming recently published results that, in Àddition to its well-characrerized angio-static function in endothelial cells. reported a key role ofthis protein in OSCC progression and invasi',,eness [30].

t

,t I

The large overexpression

of

the

fhree members

of

the AKR family profeins and of WARS

in

G-OSCC is

likely

involved

in

lhe development anel progression of this oral cancer. As the amount of these proteins was not altered in rhe epulis tissue (comPared to the cùntro1 sanrple, see Table 1 _),tliese Èroteins could be regarded as specific Lliomarkers

for

monitoring the

development

of

this tumor. Among the protein processing-related proteins, about five-fold iocrease was observed in the expression level r:f alpha-soluble NSF attechment protein (a-SNAP) and RAS-related proteins,

RAB-14, RAB-IB and RAB-64,

in

lhe

C-OSCC extract.

In

recent years,

critical roles of a-SNAP [3I ] and Rab CTPases _{[32] in}tumor devel-opmenl and their recognition as polenfial therapeutic targets have emerged.

tn

particular. a recent proteomic study also identified a

R.as-related prolein, Rab-2A isoform {ftAB2A). as a sigflature protein ot

O§CC and found the protein to beoverexpressed in OSCC[33]. About

flve-told increase of Serpin 83 and Serpin 84 levels was also found in the C-OSCC sample. Elevated expression ofthese §erinelcysteine protease inhibitors

is

reported

to

contritlute

to

cancer

develop-ment _[34].C-OSCC tissue extracts showed, however, decrease in

the expression level of proteins like

ubiquitin

carboxyl-terminal lrydrolase (USPI _),apolipoprotein

AI

{APOAI ), Na*/H* exchange regulatory cofactor NHE-RF1 (SLC9A3R1 ), and in particular cornulin

(CRNN) and keratin-type cytoskeletal 13 (CRKI]3). a u

-*d.-::-.qlù",

t r '! t

(6)

F. ?cpa er al. I ArdSrience httemstionel tS {2A18} 4l -47

LFft

"M E

E

4OT

È §

f,

. I ,.*t, .; .i

:-.

'fififis*nursu

ÉiiÈEEiEEi

_E3t

ÈEEEÉEÉ"nÉ;'

_::1;IÉrÉ

È?EE5E:E

_5e'!;::EÉ .;!-?n

g€€Ei;ÈÉ

9nÉÉ1

§?x

u

fr

t

,

É I

r

2 I + e 6il iila ,r@14 !!bt!d

CRNN, which belongs to the protein family forming the cornfield cell envelope, is an important barrier of the mucosa and skin.

lt

are

the

intermediate

filament-forming

proteins

of

epithelial cells that have an importanf role in cell protection

tiom

mechanical and _{non-mechanical stressors [37.381. Several}

stud-ies have provided evidence for alteration of keratin expression in squamous tumor tissue of the oral

cavi[t

and

their

involvemenf

ia

tumorigenesis regulation, cancer

cell

invasion, and metasta-sis _[39-43].These findings suggest thaf keratins may have a role as diagnostic tumor markers [441. The expression levels of CRNN and CRKT13

were

also

found

to

be

significantly decreased in preneoplastic tumors

with

a high risk

ol

malignant progression

[4s1.

In conclusion, the present study showed that proteomic analysis

of C-OSCC tissue enabled the idefitification of putative

biomark-ers such as aldo-keto reductase enzymes (AKR-IBIO, AKR-IC3, and AKR-I C2), tryptophen-tRNA ligase cyroplasmic (WARS), cornulins

(CRNN), and keratin type I cytoskeletal 13 (l(RTt3) rhat coukl be

useful to monifor 05CC development. These results could also lre

relevant in developing specific diagnostic tools sr.ritable to

discrim-inate C-O§CC tissues from epulis tissues lor the early diagnosis

of

this pathology.

Acknowledgement

F.P. was supported by a research grant

lrom

the Universify

of

BARI. 7W 'm CFEi@ pÉEsg inhlba106 t26 lnayÉ

Appendix

A"

Supplementary data

Supplementary

dafa

associafed

wirh

this

article

can

be found,

in

the online

version,

at

https:/ldoi.argt't}-1.O"t61s1348-8643{17FAA44-7.

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