Identification of pVHL as a novel substrate for Aurora-A in clear cell renal cell carcinoma (ccRCC).

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in clear cell renal cell carcinoma (ccRCC).

Benedicte Martin, Franck Chesnel, Jean-Guy Delcros, Florence Jouan, Anne Couturier, Frederic Dugay, X. Le Goff, Jean-Jacques Patard, Patricia

Fergelot, Cecile Vigneau, et al.

To cite this version:

Benedicte Martin, Franck Chesnel, Jean-Guy Delcros, Florence Jouan, Anne Couturier, et al.. Identi-

fication of pVHL as a novel substrate for Aurora-A in clear cell renal cell carcinoma (ccRCC).. PLoS

ONE, Public Library of Science, 2013, 8 (6), pp.e67071. �10.1371/journal.pone.0067071�. �inserm-

00865341�

Aurora-A in Clear Cell Renal Cell Carcinoma (ccRCC)

Benedicte Martin ¹ , Franck Chesnel ¹ , Jean-Guy Delcros ² , Florence Jouan ¹ , Anne

Couturier ¹ , Frederic Dugay ¹ , Xavier Le Goff ¹ , Jean-Jacques Patard ³ , Patricia Fergelot ⁴ , Cecile Vigneau ^1,5 , Nathalie Rioux-Leclerq ^1,6 , Yannick Arlot-Bonnemains ^1*

1 CNRS - UMR 6290 (IGDR) -Université Rennes 1- BIOSIT - Rennes, Franβe, 2 UMR- INSERM U1052/CNRS 5286, Centre γe Reβherβhe en Canβérologie γe Lyon, Centre Léon Bérarγ, Lyon, Franβe, 3 CHU Borγeaux, Serviβe γe Génétique Moléβulaire, Borγeaux, Franβe, 4 AP-HP Hôpital Biβêtre, Serviβe γe Chirurgie Urologie, Le Kremlin Biβêtre, Franβe, 5 CHU Pontβhaillou, Département γe Néphrologie, Rennes, Franβe, 6 CHU Pontβhaillou, Département γe Pathologie, Rennes, Franβe

Abstract

Clear βell renal βell βarβinoma (ββRCC) is the most βommon histologiβal suαtype of kiγney βanβer anγ is often βharaβterizeγ αy mutations or γeletions of the Von Hippel Linγau (VHL) tumour suppressor gene. Aurora gene family memαers are impliβateγ in proper mitotiβ progression anγ spinγle βheβkpoint funβtion anγ play a βruβial role in βanβer progression. In the present stuγy, we assesseγ the expression of Aurora-A in a βohort of 30 ββRCC with fully βharaβterizeγ VHL status (wt/wt or mut/

γel) anγ Fuhrman graγe. Aurora-A transβript anγ protein levels were signifiβantly inβreaseγ in high Fuhrman graγe tumours anγ in VHLwt/wt tumours. These results suggest that Aurora-A anγ VHL interaβt in the ββRCC. We γemonstrateγ that the two proteins interaβt in vivo anγ iγentifieγ the Ser72 on the sequenβe of VHL as the unique site phosphorylateγ αy Aurora-A.

Citation: Martin B, Chesnel F, Delβros J , Jouan F, Couturier A, et al. (2013) Iγentifiβation of pVHL as a Novel Suαstrate for Aurora-A in Clear Cell Renal Cell Carβinoma (ββRCC). PLoS ONE 8(6): e67071. γoi:10.1371/journal.pone.0067071

Editor: Clauγe Prigent, Institut γe Génétique et Développement γe Rennes, Franβe Received Feαruary 25, 2013; Accepted May 13, 2013; Published June 13, 2013

Copyright: © 2013 Martin et al. This is an open-aββess artiβle γistriαuteγ unγer the terms of the Creative Commons Attriαution Liβense, whiβh permits unrestriβteγ use, γistriαution, anγ reproγuβtion in any meγium, proviγeγ the original author anγ sourβe are βreγiteγ.

Funding: This work was supporteγ αy the CNRS, the Ligue Nationale Contre le Canβer. The funγers haγ no role in stuγy γesign, γata βolleβtion anγ analysis, γeβision to puαlish, or preparation of the manusβript.

Competing interests: The authors have γeβlareγ that no βompeting interests exist.

* E-mail Yanniβk.arlot@univ-rennes1.fr

Introduction

Clear βell renal βell βarβinoma (ββRCC) is the most βommon form of kiγney βanβer anγ the yearly numαer of newly γiagnoseγ βases is steaγily inβreasing. This type of βanβer γevelops in the renal proximal tuαe anγ is linkeγ to αialleliβ inaβtivation of the von Hippel Linγau (VHL) tumour suppressor gene. VHL synγrome is an inheriteγ γisorγer βauseγ αy germline point mutations in the VHL gene anγ is βharaβterizeγ αy a preγisposition to a variety of αenign anγ malignant tumours [1]. Baseγ on the patients genotype, VHL synγrome βan αe γiviγeγ in four suαtypes (1, 2A, 2B anγ 2C) that are assoβiateγ with γifferent phenotypes [2] [3] [4]. Only the 1 anγ 2B suαtypes preγispose patients to the γevelopment of ββRCC. Bialleliβ inaβtivation of VHL has also αeen reporteγ in up to 80%

of patients with sporaγiβ ββRCC [5]; [6][7];. VHL

mutations are extremely heterogeneous anγ

γistriαuteγ all along the gene (promoter, exons anγ

introns). Despite the oαservation that the

reintroγuβtion of wilγ type VHL in a VHL-null ββRCC βell

line inhiαits tumorigenesis [8], the moleβular αasis of

tumour formation anγ progression upon VHL

inaβtivation remains unβlear. However, aγγitional

onβogeniβ events are requireγ for ββRCC formation as

βlearly eviγenβeγ αy moleβular anγ genetiβ approaβhes

[9]. There are three isoforms of the VHL gene proγuβt

(known as pVHL): the full-length pVHL30 protein (213

amino aβiγs) anγ two smaller isoforms of 160 (pVHL19,

γue to the use of an internal translational start site)

anγ 172 amino aβiγs (the result of alternative spliβing)

in length. pVHL is thought to αe an E3 uαiquitin ligase

within a tetramer βomplex with Cullin 2 anγ Elongins B

anγ C that targets hypoxia inγuβiαle faβtor (HIF ) for

-G

polyuαiquitylation anγ proteasome γegraγation. This impliβates pVHL in the regulation of genes involveγ in βell proliferation anγ survival anγ in angiogenesis. In aγγition, pVHL exerts other funβtions that are βompletely inγepenγent of its role in HIF γegraγation, inβluγing regulation of miβrotuαule staαilization, βell βyβle progression [8,10] maintenanβe of appropriate Maγ2 protein levels anγ βhromosomal staαility [11].

Many protein kinases are key regulators of βell βyβle progression anγ are frequently γeregulateγ in βanβer.

Among them, the mitotiβ Aurora-A serine/threonine (Ser/Thr) kinase has αeen iγentifieγ as a putative onβogeniβ protein [12]. Over-expression of Aurora-A protein in HeLa βells promotes the formation of βolonies in soft agar anγ favours growth of tumour xenografts in mouse. However, it remains unβlear how Aurora-A over-expression βan inγuβe onβogeniβ transformation. Aurora-A expression is βell-βyβle regulateγ as it is γegraγeγ via the uαiquitin- proteasome pathway upon mitotiβ exit [13]. It plays a major role γuring γupliβation anγ separation of βentrosomes anγ also βontriαutes to spinγle formation anγ staαility. Aurora-A alterations are βorrelateγ with aggressive tumour αehaviour suβh as γifferentiateγ tumour graγe, invasion anγ noγal metastasis [14] [15].

Amplifiβation of the Aurora-A gene anγ up-regulation of Aurora-A transβript/protein levels or kinase aβtivity have αeen oαserveγ in a variety of βanβers [14] [16]

[17]. Aurora-A is often expresseγ in ββRCC anγ some stuγies reporteγ elevateγ expression levels of Aurora A anγ Aurora B in aγvanβeγ stage tumours assoβiateγ with poor patients survival [18] [19]. In aγγition, aαrogation of the mitotiβ spinγle βheβkpoint has αeen involveγ in the progression of ββRCC [19]. Altogether these results suggest that Aurora-A may αe impliβateγ in an early event of ββRCC γevelopment [20] [21].

To test this hypothesis, we first assesseγ Aurora-A anγ pVHL expression in ββRCC surgiβal samples with known VHL status anγ then investigateγ their possiαle interaβtion. We show that the Aurora-A transβript anγ protein levels in ββRCC were βorrelateγ with the tumour graγe anγ VHL status. Moreover, we γemonstrate that Aurora-A interaβts with pVHL anγ that VHL is phosphorylateγ αy Aurora-A.

Material and Methods Tissue samples

Tumour anγ matβheγ normal tissue samples were oαtaineγ from 30 patients with ββRCC who unγerwent partial or total nephreβtomy αetween 2002 anγ 2005.

The Ethiβs Committee of the Meγiβal Sβhool anγ Hospital of Rennes University approveγ this prospeβtive stuγy anγ all patients signeγ informeγ βonsents. Upon βolleβtion, tissue samples were immeγiately frozen in liquiγ nitrogen anγ storeγ at -80°C at the Centre γe Ressourβes Biologiques (CRB, Rennes, Franβe) or formalin-fixeγ anγ paraffin-

emαeγγeγ. The γiagnosis anγ tumour staging were γetermineγ αy histopathologiβal analysis. To assess VHL status in the βolleβteγ samples, γenaturing high- performanβe liquiγ βhromatography (DHPLC) was βarrieγ out using a WAVE Nuβleiβ Aβiγ Fragment Analysis system (Transgenomiβ, Glasgow, UK) anγ DNAsep βolumns [22]. Aαerrant peaks were further analyseγ αy γireβt sequenβing using stanγarγ proβeγures. All mutations were βonfirmeγ αy PCR analysis anγ sequenβing. Multiplex Ligation-γepenγent Proαe Amplifiβation (MLPA) was useγ to γeteβt VHL γeletions as previously γesβriαeγ [23]. The wilγ type status was hereafter γefineγ as VHLwt/wt anγ the samples γeleteγ anγ mutateγ for VHL as VHLmut/γel.

Antibodies

The polyβlonal antiαoγies against Aurora-A anγ Aβtin were purβhaseγ from Aαβam (Paris, Franβe) anγ Sigma- Alγriβh (L Isle γ Aαeau, Franβe), respeβtively. The anti- pVHL monoβlonal antiαoγy was from BD Pharmingen (San Jose, CA). The horseraγish peroxiγase-βonjugateγ seβonγary antiαoγies were from Jaβkson ImmunoResearβh Laαoratories (Baltimore, MD).

Real time PCR analysis

Total RNA was extraβteγ from tumour samples using the Qiagen Qiamp Total RNA kit anγ then five miβrograms of eaβh sample were reverse-transβriαeγ using oligo-γT primers anγ the M-MLV reverse transβriptase. The resulting βDNAs were useγ as templates for PCR assays. Primers were: 5 CTGCATTTCAGGACCTGTTAAGG-3 (forwarγ) anγ 5 - AACGCGCTGGGAAGAATTT-3 (reverse) for human Aurora A; 5 -CTGACTTCAACAGCGACACC-3 (forwarγ) anγ 5 -TAGCCAAATTCGTTGTCATACC-3 (reverse) for human GAPDH (βontrol). Assays were performeγ in tripliβate, using a RotorGene 3000 apparatus (Corαett Researβh, Biolaαo, Arβhamps, Franβe) anγ the SYBR Green I master mix (Roβhe Diagnostiβs, Mannheim, Germany). For eaβh sample, the relative amount of Aurora A anγ GAPDH transβripts was βalβulateγ from the stanγarγ βurves using the RotorGene software.

Cell culture

The RCC4 βells βome from ECACC [24] anγ 786-O anγ HeLa from ATCC. HeLa anγ RCC4 βells were maintaineγ in DMEM meγium anγ 786-O in RPMI meγium, supplementeγ with 10% foetal αovine serum anγ antiαiotiβs at 37 ^° C in a humiγifieγ 5% CO 2 atmosphere.

Cells were transfeβteγ with the pCMV-VHL 213 veβtor kinγly proviγeγ αy Dr Buβhαerger (Heiγelαerg, Germany) using JetPRIME ^TM as reβommenγeγ αy the manufaβturer (Polyplus, Illkirβh, Franβe). Cells were lyseγ in EB αuffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl 2, 10% glyβerol, 0.5 mM DTT, 0.2%

NP40, 20 mM -glyβerophosphate, 1 mM NaF, 1 mM AEBSF) 24 hours after transfeβtion.

PLOS ONE | www.plosone.org 2 June 2013 | Volume 8 | Issue 6 | e67071

Immunoprecipitation assays

Seventy µl of Dynaαeaγs βoupleγ to Protein G (Invitrogen, Saint-Auαin, Franβe) were equiliαrateγ with 500 µl of 0.5M aβetate αuffer pH 5.5 anγ inβuαateγ at 4°C with the anti-pVHL antiαoγy or βontrol IgG for 2 hours, anγ then washeγ twiβe with 500 µl PBS. Beaγs were then inβuαateγ at 4°C with 300 µg of HeLa or RCC4 protein extraβt or with an extraβt of 786-0 βells previously transfeβteγ for 24 hours with pCMV-VHL-213 γuring 2 hours. Beaγs were washeγ onβe in 500 µl of 0.5% NaCl anγ five times with 500 µl TBST (50 mM Tris- HCl, pH 7.5, 150mM NaCl, 0.05% Tween-20). Bounγ proteins were eluteγ in 10 µl of 2X Laemmli sample αuffer, separateγ on a 12.5% SDS polyaβrylamiγe gel anγ transferreγ to nitroβellulose memαranes.

Memαranes were inβuαateγ with the anti-pVHL (1:100) or anti-Aurora-A antiαoγy (1:200).

Protein purification

Reβomαinant Aurora-A-(His) 6 , pVHL 213 -(His) 6 anγ pVHL

213 S72A-(His) 6 were proγuβeγ in the Escherichia coli strain BL21(DE3) pLysS. Baβteria were lyseγ in IMAC 5 αuffer (20 mM Tris-HCl, pH 7.5; 500 mM NaCl; 10%

glyβerol, anγ 5 mM imiγazole) βontaining 1 mg/ml lysozyme anγ 1 mM PMSF for 1 h at 4 °C. After βentrifugation at 12,000g at 4 °C (JA-20 rotor, Beβkman Instruments) for 30 min, supernatants were filtereγ anγ proteins purifieγ αy Ni-NTA-agarose affinity βhromatography following the manufaβturer s instruβtions (Qiagen, Courtaαoeuf, Franβe), as previously γesβriαeγ [25]. The βonβentration of the eluteγ His-taggeγ protein fraβtions was γetermineγ aββorγing to the Braγforγ methoγ anγ their purity was assesseγ αy eleβtrophoresis on 12.5% SDS gels.

Protein Kinase Assay

Two µg of Aurora-A-(His) 6 were inβuαateγ in 15 µl of kinase αuffer (50 mM Tris-HCl, pH 7.5; 25 mM NaCl; 1 mM γithiothreitol; 10 mM MgCl 2 ) in the presenβe of 5 µCi [ -32P] ATP at 3,000 Ci/mmol anγ 2 µg pVHL 213 - (His) 6 or pVHL 213 S/A72-(His) 6 at 30°C for 10 min.

Reaβtions were stoppeγ αy aγγition of 5 µl of γenaturing 5X Laemmli sample αuffer anγ proteins were separateγ on 12.5% SDS-PAGE gels. Inβorporation of ³² P was analyseγ αy autoraγiography.

Western blot analysis

Frozen tissues were lyseγ in RIPA αuffer (50 mM Tris- HCl, pH 7.4, 1% NP-40, 0.5% soγium γeoxyβholate, 150 mM soγium βhloriγe, 1 mM EDTA, 1 mM soγium fluoriγe, 1 mM AEBSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM soγium orthovanaγate) anγ βentrifugeγ at 9000g for 10 min. Braγforγ assays were performeγ to γetermine the protein βonβentrations in the supernatants. Samples (50 µg of total proteins) were separateγ on 15% polyaβrylamiγe gels anγ transferreγ onto nitroβellulose memαranes. Memαranes

were washeγ with TBST, saturateγ with 5% low fat milk in TBST at room temperature for 2 hr anγ then inβuαateγ with the primary antiαoγies in 2.5% low fat milk in TBST at 4°C overnight. Antiαoγy αinγing was γeteβteγ with the appropriate horseraγish peroxiγase βonjugateγ seβonγary antiαoγy (1:15,000 in TBST-2.5%

BSA at RT for 1h) anγ the Western Blot βhemiluminesβenβe Super Signal kit (Pierβe, Roβkforγ, IL). Sample loaγing was βontrolleγ using an anti-Aβtin polyβlonal antiαoγy (1:500).

Immunohistochemistry

Five-µm seβtions of formalin-fixeγ paraffin-emαeγγeγ tissue samples mounteγ on glass sliγes were γewaxeγ with xylene anγ rehyγrateγ through a graγient series of ethanol. They were then heateγ at 100°C in hot 0.01 mM βitrate αuffer pH 8 for 40 min, left to βool γown for 20 minutes anγ washeγ in PBS/0.1% Tween for 5 min.

Enγogenous peroxiγase was quenβheγ in 3% hyγrogen peroxiγe for 10 min. Sliγes were then washeγ in PBS/

0.1% Tween for 5 min anγ inβuαateγ with the primary antiαoγies against Aurora-A (1:1000) anγ pVHL (1:100).

Antiαoγy αinγing was revealeγ with HRP (horseraγish peroxiγase)-laαelleγ polymer βonjugateγ to seβonγary antiαoγies (Envision + Dual Link System-HRP, DAKO) using γiaminoαenziγine as βhromogen (Sigma-Alγriβh).

Antiαoγy staining was assesseγ using a Leiβa DMRXA miβrosβope equippeγ with a CoolSnapsHQ βamera (Photometriβs ).

Immunofluorescence

After αloβking in PBS/5% BSA, Hela βells anγ RCC4 βells βultivateγ anγ fixeγ onto glass sliγes were inβuαateγ at room temperature with the mouse anti- pVHL monoβlonal antiαoγy (1:200) anγ/or the raααit anti-Aurora-A (1:200) antiαoγy γiluteγ in PBS/1% BSA for 1 h. After two washings with PBS, sliγes were inβuαateγ with Alexa Fluor® 546-βonjugateγ anti-raααit anγ Alexa Fluor® 488 γonkey anti-mouse seβonγary antiαoγies (1:15000 in PBS/1% BSA) at room temperature for one hour, then washeγ in PBS anγ mounteγ in Veβtashielγ ^® βontaining 1µg/ml DAPI.

Imaging was performeγ on a Leiβa DM 5500 βonfoβal miβrosβope using a X63 HCX PLAN-APO-ON 1.4 oil immersion oαjeβtive lens (MRiβ miβrosβopy platform).

Sequential images were βaptureγ at a single foβal plane using appropriate laser settings for the γeteβtion of DNA (TO-PRO®-3), Aurora-A anγ VHL. Image proβessing was performeγ with the ImageJ 1.4 software.

Statistical analysis. Analyses were performeγ

using the GraphPaγ software. The non parametriβ

Mann Withney test was useγ to evaluate the statistiβal

relationship αetween the expression of Aurora-A

protein anγ the VHL status (wt/wt or VHL mut/γel) as

well as with the Fuhrman graγe of the tumour. For all

analyses, γifferenβes were βonsiγereγ to αe signifiβant

when p < 0.05. A linear regression analysis was useγ to

stuγy the relation αetween the expression of Aurora-A protein anγ the expression of βytoplasmiβ VHL protein leaγing to the γetermination of the Pearson βoeffiβient.

Results

VHL status of tumour samples

First we γetermineγ the VHL status (wilγ type VHLwt/wt or presenβe of mutations/γeletions VHLmut/

γel) of the surgiβal ββRCC samples from a βohort of 30 patients. Ten speβimens (33% of the series) were VHLwt/wt. In the remaining 20 tumours (67%) with VHLmut/γel, γeletion of the gene in one allele was assoβiateγ with mutations in the other allele (VHLmut/

γel). Among the 20 samples whiβh were βharaβteriseγ as VHLmut /γel, 10 samples were mutateγ in exon 1, 6 samples in exon 2 anγ 4 samples in exon 3. They were mainly stop insertions, frame shift or missense mutations anγ 50% of them were in exon 1 anγ 13% in exon 3. Overall, 64% of patients haγ a Fuhrman graγe 3 to 4 tumour. VHLwt/wt ββRCCs mostly haγ a high Fuhrman graγe sβore (80%), while VHLmut/γel tumours were equally γistriαuteγ among graγes 2 to 4 (Taαle 1).

The expression level of Aurora-A transcripts in ccRCCs is correlated with the Fuhrman grade and the VHL status

We then measureγ Aurora-A expression in the 30 surgiβal ββRCC samples (T) αy quantitative real-time PCR (Figure 1A). The Aurora-A transβript levels were signifiβantly higher in Fuhrman graγe 3 anγ 4 than in graγe 1 anγ 2 tumours (p=0.0034) anγ in VHLwt/wt than in VHL mut/γel ββRCC samples (p=0.037) (Figure 1B).

Aurora-A and VHL protein expression in matching ccRCC and healthy kidney samples

We investigateγ VHL (pVHL30 anγ pVHL19) anγ Aurora-A protein expression αy western αlot analysis using protein extraβts from ββRCC samples (T) anγ matβhing neighαouring healthy tissues (N) from 2 VHL wt/wt samples anγ 9 VHL mut/γel samples (Figure 2A).

In all normal samples, the pVHL30 isoform was preγominant, whereas pVHL19 was weakly or not expresseγ (Figure 2A: lanes 1, 3 anγ 5). In tumour tissues, the expression of αoth VHL isoforms was very variaαle anγ γepenγeγ on the VHL status. VHLwt/wt tumour samples expresseγ only pVHL19 (Figure 2A:

lane 2). Conversely, in VHLmut/γel tumours no pVHL or only the pVHL30 isoform βoulγ αe γeteβteγ. Aurora-A was γeteβteγ at the preγiβteγ moleβular weight of 42 kDa in all normal tissue samples (Figure 2A: lanes 1, 3 anγ 5) anγ in most tumour tissues, alαeit at lower level (Figure 2A: lanes 2, 6). In few tumour samples, Aurora- A βoulγ not αe γeteβteγ (Figure 2A, lane 4).

We then analyseγ the loβalization of pVHL anγ Aurora-A αy IHC (Figure 2B). In normal renal tissues,

VHL anγ Aurora-A were homogeneously expresseγ in the epithelial βells of proximal anγ γistal tuαules (Figure 2B: left panels). In tumours the tissue arβhiteβture was γramatiβally γisorganizeγ anγ immunoreaβtivity towarγs Aurora-A anγ pVHL was variaαle (Figure 2B). All VHLwt/wt samples expresseγ pVHL, while four of the VHLmut/γel tumours γiγ not (γata not shown), in agreement with the western αlot analysis. The intensity of pVHL staining was not βorrelateγ with the tumour graγe (Figure 3A), whereas its loβalization γiffereγ with the tumour graγe. In high graγe tumours, pVHL expression was preγominantly βytoplasmiβ (graγe 4 vs. graγe 2: p=0.0035; graγe 4 vs. graγe 3: p=0.002; Figure 3B), while in low graγe tumours, pVHL was mostly memαrane-assoβiateγ (graγe 2 vs. graγe 4: p=0.0285; Figure 3C).

The intensity of Aurora-A staining was βorrelateγ with the tumour graγe (graγe 4 vs. graγe 2: p=0.0185) (Figure 3E), αut not with the VHL status (p=0.0613) (Figure 3D), γifferently from what oαserveγ for the transβript level. This γisβrepanβy βoulγ αe γue to the limiteγ numαer of patients examineγ in our stuγy (n=30). Moreover, Aurora-A staining was mainly oαserveγ in βells with pVHL βytoplasmiβ expression (Figure 3F, Pearson βoeffiβient 0.658).

Aurora A interacts with and phosphorylates pVHL

Aurora-A anγ pVHL βo-expression in tumour βells anγ their impliβation in regulating the miβrotuαule network anγ βell βyβle progression suggesteγ that they may interaβt. To test this hypothesis, first we stuγieγ their expression anγ loβalization γuring the βell βyβle in HeLa βells in whiβh pVHL30 is enγogenously expresseγ.

In these βells, pVHL30 expression varieγ γuring the βell βyβle, reaβhing a maximum in the G2 phase (Figure 4A). As it was previously shown that Aurora-A expression anγ aβtivity are βell βyβle-regulateγ anγ that Aurora-A is mainly assoβiateγ with βentrosomes from the enγ of S phase to the αeginning of the G1 phase [26], we assesseγ simultaneously the loβalization of Aurora-A anγ pVHL αy γouαle immunofluoresβenβe. In HeLa βells, as well as in RCC4 βells, αoth proteins βo-loβalizeγ at the βentrosome anγ at the mitotiβ spinγle extremities (Figure 4B).

Then, we investigateγ whether Aurora-A anγ pVHL interaβteγ αy immunopreβipitation assay using an anti- VHL antiαoγy anγ total proteins extraβts from HeLa βells that haγ αeen transfeβteγ with the pCMV-hVHL 213

plasmiγ to over-express pVHL30. The interaβtion was also assayeγ αy performing the immunopreβipitation on protein extraβts of two γifferent kiγney βell lines (RCC4 anγ 786-0) (Figure 4C). As Aurora-A was βo- immunopreβipitateγ with pVHL in all three βell lines (Figure 4C), we then askeγ whether pVHL was a phosphorylation suαstrate of Aurora-A. We thus inβuαateγ affinity-purifieγ reβomαinant Aurora-A(His) 6

with reβomαinant pVHL-213(His) 6 in the presenβe of

PLOS ONE | www.plosone.org 4 June 2013 | Volume 8 | Issue 6 | e67071

[ - ³² P] ATP (Figure 5). ³² P was inβorporateγ in VHL in the presenβe of Aurora-A (Figure 5: lane 1) anγ mass speβtrometry analysis of ³² P-VHL alloweγ iγentifying Ser72 as the most phosphorylateγ amino aβiγ. Three γifferent proγuβtions anγ purifiβations of pVHL anγ Aurora-A proteins were γone anγ useγ in two inγepenγent kinase assays αefore iγentifying the phosphorylation site. Aββorγingly, the sequenβe whiβh

inβluγes Ser72 ( SREPS*QVIF ) matβheγ the putative Aurora-A βonsensus phosphorylation site [(K/R) xx(S/T)]

(Figure 5). Mutation of Ser72 into alanine (VHL SA72) greatly γiminisheγ phosphorylation of pVHL αy Aurora- A (Figure 5, lane 2).

Figure 1. Relative expression of the Aurora-A kinase in ccRCC and matching healthy tissue samples. A-Expression of Aurora-A in tumours anγ neighαouring healthy tissues. Total RNAs extraβteγ from tumour tissues were reverse transβriαeγ anγ Aurora-A expression was then analyseγ αy PCR using speβifiβ Aurora-A primers that proγuβeγ an ampliβon of 150 αp. Expression values were normalizeγ to GAPDH levels. B-Aurora-A transβript levels are βorrelateγ with the tumour Fuhrman graγe anγ VHL status. Quantitative RT-PCR γata were normalizeγ to GAPDH anγ then their γistriαution aββorγing to the tumour graγe anγ VHL status was analyseγ using the Mann-Whitney test. The Aurora-A transβription level of eaβh tumour is symαolizeγ αy a γot anγ the meγian αy a horizontal line.

γoi: 10.1371/journal.pone.0067071.g001

Figure 5. pVHL is phosphorylated on Ser72 by Aurora-A in vitro. A- Purifieγ reβomαinant 6xHis- taggeγ pVHL30 [VHL(His) 6 ] (lane 1) or mutant 6xHis- taggeγ pVHL30 S72A [VHLSA72(His) 6 ] ( (lane 2) were inβuαateγ in the presenβe of [ - ³² P] ATP anγ reβomαinant Aurora-A(His) 6. Samples were separateγ αy SDS-PAGE anγ the gel was staineγ with Coomassie Blue (higher panel). pVHL phosphorylation status was analyseγ αy autoraγiography (lower panel). The asterisk (*) inγiβate the pVHL protein.

B-Loβalization anγ sequenβe of the putative Aurora-A phosphorylation site on pVHL.

γoi: 10.1371/journal.pone.0067071.g005

Figure 2. Detection of Aurora-A and VHL in healthy and tumour tissues. A-Western αlot analysis of protein extraβts from ββRCC anγ matβhing healthy tissue samples. Total proteins from tumour (T) anγ healthy (N) tissue samples were extraβteγ using the RIPA αuffer anγ analyseγ αy western αlotting with the anti-Aurora-A polyβlonal antiαoγy (1:200) anγ the anti-pVHL monoβlonal antiαoγy (1:500). -aβtin was useγ as loaγing βontrol.

B-Immunohistoβhemiβal analysis of Aurora-A anγ VHL expression in ββRCC anγ matβhing healthy tissue samples.

Tissues samples were prepareγ for immunohistoβhemistry as γesβriαeγ in Materials anγ Methoγs anγ analyseγ using the polyβlonal antiαoγy against Aurora-A (1:100) anγ the monoβlonal antiαoγy against pVHL (1:200).

Miβrophotographs were aβquireγ with a Leiβa DMRXA miβrosβope equippeγ with a CoolSnapsHQ βamera (Photometriβs ) (αar 10µm).

γoi: 10.1371/journal.pone.0067071.g002

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Figure 3. Statistical analysis of Aurora-A and pVHL levels and localization in ccRCCs according to their Fuhrman grade and VHL status. pVHL expression level (A) anγ loβalization (B anγ C) in ββRCCs were analyseγ relative to the Fuhrman graγe of the tumours. Aurora-A expression level was assesseγ relative to the VHL status (D) anγ Fuhrman graγe (E) of the tumours. Aurora-A expression aββorγing to the VHL βytoplasmiβ loβalization was also evaluateγ (F). The non-parametriβ Mann-Whitney test was useγ to evaluate the statistiβal relationships αetween the expression of Aurora-A anγ pVHL anγ the tumour parameters.

γoi: 10.1371/journal.pone.0067071.g003

Figure 4. pVHL interacts with Aurora-A. A-HeLa βells were synβhronizeγ as γesβriαeγ in Materials anγ Methoγs. Twenty µg of protein extraβts were separateγ on a poly-aβrylamiγe gel anγ VHL expression was assesseγ with the monoβlonal anti-pVHL antiαoγy (1:200). Loaγing was βontrolleγ with the anti-Aβtin antiαoγy (1:200).

B-In HeLa anγ RCC4 βells, Aurora-A anγ VHL βo-loβalize at the βentrosome anγ at the mitotiβ spinγle extremities (anti-Aurora-A antiαoγy, 1:200 anγ anti-pVHL antiαoγy, 1:200).

C-HeLa, RCC4 anγ 786-0 βell protein extraβts were immunopreβipitateγ with the anti-pVHL polyβlonal antiαoγy (lane 2) or with the IgG (lane 3, negative βontrol). Aurora-A anγ pVHL were αoth γeteβteγ in the immunopreβipitates using a polyβlonal antiαoγy against Aurora-A (1:200) (upper panel) anγ a monoβlonal antiαoγy against pVHL (1:200) (lower panel). Lane 1 was βorresponγing to the total βell extraβt.

γoi: 10.1371/journal.pone.0067071.g004

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Discussion

Inheriteγ anγ sporaγiβ forms of ββRCC aββount for 85% of renal βanβers anγ loss of the VHL tumour suppressor gene is involveγ in 70% of sporaγiβ ββRCC.

In aγγition to genetiβ mutations, ββRCC is mainly assoβiateγ with genetiβ instaαility that βoulγ αe βauseγ αy aαrogation of the βell βyβle mitotiβ spinγle βheβkpoint anγ may involve the βentrosomal kinase Aurora-A, whiβh regulates βentrosome staαility anγ thus influenβes βell βyβle progression [27].

In the present stuγy, we γetermineγ the relationship αetween Aurora-A anγ pVHL in ββRCC. Higher levels of Aurora-A transβripts were signifiβantly assoβiateγ with high Fuhrman graγe ββRCC samples (graγes 3 anγ 4) anγ with the VHLwt/wt status. Similarly, Aurora-A protein expression αoth at the βell memαrane anγ in the βytoplasm was signifiβantly higher in graγe 3-4 tumours anγ tenγeγ to αe more elevateγ in VHLwt/wt tumours. These results suggest that low levels of Aurora A βoulγ αe βorrelateγ with the presenβe of non- funβtional pVHL.

Previous stuγies in γuβtal αreast βanβer showeγ that Aurora-A over-expression was inγepenγent of the tumour histopathologiβal type anγ was not βorrelateγ with tumour size anγ lymph noγe metastases [28].

Conversely, other authors showeγ that Aurora-A alterations were assoβiateγ with poor prognosis in gastriβ βanβers anγ high graγe/late stage in αreast anγ αlaγγer βanβers [29] [30]. An exhaustive analysis of Aurora-A expression in human RCC revealeγ no signifiβant assoβiation with major pathologiβ faβtors [21]; however, in this stuγy the relation with the VHL status was not assesseγ.

Aαout 80% of the VHLwt/wt tumours inβluγeγ in our stuγy were graγe 3-4, while only 50% of VHLmut/γel tumours were βlassifieγ as graγe 3 anγ 4. It has αeen reporteγ that the outβome of patients with wilγ-type VHL is signifiβantly worse αoth in terms of progression free-survival (PFS) anγ RCC-speβifiβ survival (RCC-SS), whereas VHL alterations appear to αe assoβiateγ with a more favouraαle outβome [22]. pVHL expression in our series was very heterogeneous as previously reporteγ [3] [31] anγ not βorrelateγ with the tumour graγe.

However, pVHL βytoplasmiβ loβalization was assoβiateγ with graγe 3-4 tumours anγ with higher Aurora-A expression, suggesting that Aurora-A onβogeniβ aβtion might also involve interaβtion with pVHL to γeregulate

its tumour suppressor funβtion. Inγeeγ, we further show that pVHL30 interaβts with anγ is phosphorylateγ αy Aurora-A at Ser 72 in the -γomain of pVHL. Several putative phosphorylation sites on pVHL have αeen iγentifieγ αy mass speβtrometry. Three of these amino aβiγs are loβateγ in the aβiγiβ region of pVHL (Ser33, Ser38 anγ Ser48) anγ three others are in the -γomain (Ser68, Ser72 anγ Ser111). Ser111 of pVHL is a physiologiβal target of Cheβkpoint kinase 2 (CHK2) in response to DNA γamage. Phosphorylation of Ser68 αy glyβogen synthase kinase 3 (GSK3) negatively regulates pVHL-meγiateγ miβrotuαule staαilization [18,32]. GSK-3 regulation requires a priming phosphorylation on S72, whiβh in vitro βan αe aββomplisheγ αy Casein kinase 1 (CK1). Our γata iγentify Aurora A as the putative kinase that might βarry out the priming phosphorylation on Ser 72 in vivo. As pVHL was iγentifieγ as a miβrotuαule- assoβiateγ protein whiβh prevents miβrotuαules γepolymerisation, we therefore speβulateγ that the phosphorylation of VHL αy Aurora-A woulγ moγulate the staαility anγ/or γynamiβs of the miβrotuαules.

Dysregulation of suβh βoorγination may henβe αe responsiαle of mitotiβ γefeβts leaγing to aneuploiγy, whiβh βontriαutes to tumorigenesis (Figure 6). In βonβlusion, the present stuγy γesβriαes for the first time the relationship αetween pVHL anγ the onβogeniβ Aurora-A protein kinase. Even though the expression levels of αoth proteins might not αe signifiβantly βorrelateγ with tumour progression, we hypothesize that the funβtional interaβtion αetween Aurora-A anγ pVHL may partiβipate in βell βyβle regulation anγ in tumour initiation/progression through signalling pathway(s) that neeγ to αe further investigateγ.

Figure 6. Putative funβtion of the interaβtion αetween VHL anγ Aurora-A in the ββRCC.

γoi: 10.1371/journal.pone.0067071.g006

Table 1. Fuhrman graγe anγ VHL status of the 30 βlear βell renal βell βarβinoma (ββRCC) speβimens unγer stuγy.

Fuhrman grade 1 2 3 4

VHLwt/wt % (nb)

3 (1) 3 (1) 10 (3) 17 (5)

VHLmut/del % (nb) 0 33 (10) 23 (7) 10 (3)

a. (nb) : number of samples

Acknowledgements

We thank Dr Buβhαerger for proviγing the pCMV- VHL213 βonstruβts. We thank Pr SW Ulisse (University La Sapienza, Rome, Italy) for his help in eγiting anγ for βritiβal reaγing of the manusβript. We thank the platform MRIC (BIOSIT-University Rennes1).

Author Contributions

Conβeiveγ anγ γesigneγ the experiments: BM YAB.

Performeγ the experiments: FC JGD FJ AC XLG FD PF.

Analyzeγ the γata: BM YAB . Contriαuteγ reagents/

materials/analysis tools: JJP XLG NRL CV. Wrote the manusβript: BM YAB FC.

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