Reduced cilia frequencies in human renal cell carcinomas versus neighboring parenchymal tissue.

3.6 MB·English

by  Basten, Sander G

Bastenetal.Cilia2013,2:2
http://www.ciliajournal.com/content/2/1/2
RESEARCH Open Access
Reduced cilia frequencies in human renal cell
carcinomas versus neighboring parenchymal
tissue
Sander G Basten1,2, Sven Willekers1, Joost SP Vermaat2, Gisela GG Slaats1, Emile E Voest2, Paul J van Diest3
and Rachel H Giles1*
Abstract
Background: Cilia are essential organellesinmultiple organ systems,including thekidney where they serve as
important regulators ofrenalhomeostasis. Renal nephron cilia emanate from theapical membrane ofepithelia,
extending into thelumenwhere they function inflow-sensingand ligand-dependent signaling cascades. Ciliary
dysfunction underlies renalcyst formation that is in part caused by deregulation of planar cell polarity and
canonical Wnt signaling. Renal cancer pathologies occur sporadically or inheritable syndromes caused bygermline
mutations in tumor suppressor genesincluding VHL.Importantly,Von Hippel-Lindau(VHL) patients frequently
develop complex renal cysts that can be considered a premalignant stage. One of thewell-characterized molecular
functions ofVHL is itsrequirement for the maintenance of cilia. In this study, tissue from 110renal cancer patients
who underwent nephrectomy was analyzedto determine iflower ciliary frequency is a common hallmark of renal
tumorigenesis bycomparingcilia frequencies inboth tumor and adjacent parenchymal tissue biopsies from the
same kidney.
Methods: We stained sections of human renal material using markersfor cilia. Preliminary staining was performed
using an immunofluorescent approach and a combinationof acetylated-α-tubulinand pericentrin antibodies and
DAPI. After validationof an alternative, higher throughput approach using acetylated-α-tubulin
immunohistochemistry, wecontinued to manually quantify cilia in alltissues. Nuclei were separately counted inan
automated fashion in order to determine ciliary frequencies. Similar staining and scoring for Ki67 positive cellswas
performed to exclude thatproliferation obscures cilia formation potential.
Results: Samples from renal cell carcinoma patients deposited in our hospital tissue bank were previously used to
compose a tissue microarray containing three cores of both tumor and parenchymal tissue per patient. Cilia
frequencies ina total of eighty-nineclear cell, eight papillary, five chromophobe renal cell carcinomas, two
sarcomatoid renal tumors and six oncocytomas were determined. A marked decrease ofprimary cilia across renal
cell carcinoma subtypes was observed comparedto adjacent nontumorigenic tissue.
Conclusions: Our study shows that cilia are predominantly lost in renal cell carcinomas compared to tissue of the
tumor parenchyma. These results suggestthat ciliary loss is common inrenal tumorigenesis,possibly participating
inthe sequence ofcellular events leading to malignant tumor development. Future therapies aimed at restoring or
circumventing cilia signaling might therefore aid incurrenttreatment efficacy.
Keywords: Cilia, Kidney, Clear cell renal cell carcinoma,Chromophobe RCC, Papillary RCC, Oncocytoma,Histology
*Correspondence:[email protected]
1DepartmentofNephrologyandHypertension,UniversityMedicalCenter
Utrecht,Heidelberglaan100,3584,CX,Utrecht,TheNetherlands
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Bastenetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Background oncocytoma/chromophobe(50%) neoplasms, and lessfre-
Primary cilia are small hair-like organelles that generally quently ccRCC (9%) [15]. Interestingly, the gene products
extendfromtheapicalplasmamembraneandarealmost ofthese heritablekidneycancer locihaveall been directly
ubiquitously expressed throughout the human body [1]. linkedwithciliafunction.As renalcystsare commonfea-
Cilia function as sensory organelles in response to extra- tures in VHL, TSC and BHD syndromes, a common hy-
cellular stimuli, such as fluid flow (mechanosensation) in pothesisisthatrenaltransformationoriginatesfromcystic
addition to mitogenic, morphogenic and olfactory fac- lesions comprising a pretumorigenic stage in the natural
tors. Ciliary signaling is pivotal during development and disease course. In VHL patients, direct evidence of this
organ homeostasis [2]. Dysfunctional cilia underlie the transformation has been documented [16]. This is further
development of a broad range of diseases, collectively supported by the observation that patients with acquired
known as ciliopathies. Hallmark disease syndromes that cystic kidney disease (ACKD) have an increased risk for
find their origin in cilia dysfunction include polycystic RCC with incidences ranging from 2 to 5% [13], and the
kidney disease (PKD), Bardet-Biedl syndrome (BBS), development of both cysts and RCC in mouse modelsfor
Meckel-Gruber syndrome (MKS) and nephronophthisis VHLdisease[17],TSC[18]andBHDsyndrome[19].
(NPHP) [3]. These syndromes have a partially overlap- During the cell cycle, both the cilium and the mitotic
ping disease spectrum and interestingly, one of the most spindle lay claim to the centrosome, rendering these pro-
frequently affected tissues is the kidney, which is charac- cesses mutually exclusive and cilia must therefore first be
terized by a high prevalence of renal cyst formation [3]. disassembled prior to cell division [20,21]. Collectively,
The tubules that make up the nephron are highly cilia- these data raise the question of whether renal cell carcin-
ted and essential for regulating cell proliferation in re- omas sustain normal ciliary frequencies. One study in a
sponse to fluid flow, as well as maintaining planar smallcohortofccRCCindeedestablishessomeinitialsup-
organization of the tissue [4]. Loss of cilia in the renal port for this hypothesis [22]. Here we analyze cilia fre-
tubules has been described to induce a switch from non- quency in renal tumor resections by immunohistoche-
canonical to canonical Wnt signaling that leads to in- mistry for cilia axonemal marker acetylated-α-tubulin. In
appropriateβ-cateninactivation,cellproliferationandloss order to efficiently and reproducibly analyze a large num-
of planar cell polarity, however there is some controversy ber of patients in standardized conditions, we used a tis-
concerningtheexactmechanismsinvolved[5].Ingeneral, sue microarray (TMA) of RCC biopsies, including eighty-
loss of ciliary function marks the initiation of cyst forma- nine ccRCC, eight pRCC, five chrRCC, two sarcomatoid
tioneventuallyseverelyimpairingrenalfunction. renal tumors and six oncocytomas [23]. The TMA addi-
Kidneycancerscanbesubcategorizedintoseveralhisto- tionally includes tissue originating from the benign renal
pathological subtypes, of which renal cell carcinoma parenchyma surrounding the tumor, which we used to
(RCC)isthemostpredominant[6].DistinctRCCpatholo- correlatetheciliaryfrequenciesinRCC.
giesareclearcell(ccRCC)withanincidenceof75%,papil-
lary (pRCC, 10 to 15%) and chromophobe (chrRCC, 5%) Methods
[7]. Closely resembling the histology of chromophobe Patientmaterialandtissuemicroarray
RCCisthesubtyperenaloncocytomawithaprevalenceof The tissues analyzed in this study were obtained from
5% [8,9]. The overall majority of kidney cancers is spor- nephrectomy samplescollectedfrompatientsattheUni-
adic and 4% is attributable to heritable cancer syndromes versity Medical Center Utrecht (UMCU) and part of the
[6]. The best-studied familial kidney cancer syndrome is UMCU biobank. All patient samples conform to the
Von Hippel-Lindau disease (VHL, MIM608537), predis- medical and ethical guidelines applied by the UMCU
posingtoccRCCdevelopmentaswellasextrarenaltumor and Dutch law. Selected tissues comprise a variety of
development[10].Interestingly,theVHLgeneisalsoinac- RCC histologies; eighty-nine ccRCC, eight pRCC, five
tivated in up to 87% of sporadic clear cell RCCs [11]. chrRCC, six oncocytomas and two sarcomatoid renal
Tuberous sclerosis (TSC) is associated with germline tumors (Table S1 in Additional file 1). The TMAs were
mutations in the TSC1/Harmatin (MIM605284) and previously constructed using an arrayer (Beecher Instru-
TSC2/Tuburin (MIM191092) genes [12]. The renal path- ments, Sun Prairie, WI, USA) and three cores of 1 mm
ology of TSC patients is predominantly benign renal in diameter from the cancer tissue, and three cores of 1
angiomyolipoma (50 to 80%), although a minority deve- mm in diameter from the healthy renal parenchyma tis-
lops ccRCC with an earlier onset of disease compared to sue taken anywhere between 1 cm and 8 cm from the
the general population (3%) [13]. Birt-Hogg-Dubé syn- edge ofthe tumor were included. Every composite paraf-
drome (BHD) is a monogenic disorder caused by muta- fin block included the same tumor and parenchyma tis-
tionsinFLCN/Folliculin[14](MIM607273).Renaltumors sue for normalization. All tissues and corresponding
isolatedfrom BHD patientsarehistologicallydiverse,pre- patientinformationusedwerewelldocumentedandpre-
dominantly consisting of chrRCC (34%) and hybrid viously categorizedforhistologicalsubtype [23].
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Immunohistochemistry tissue spots exported using Aperio Imagescope. Using
TMA sections of 4 μm were processed as previously Photoshop, a color selection range was matched to
described[23].Followingdeparaffinationandrehydration, hematoxylin(asindicatedinFigureS1inAdditionalfile2).
antigen retrieval was achieved by boiling in citrate buffer Thisselectionwasextractedfromtheoriginalimagetore-
for 20 minutes and the sections were allowed to cool to move background and converted and inverted to an
room temperature over one hour on the bench. Endoge- 8-bit file usingImageJand built-in macros.Next, a binary
nousperoxidaseactivitywasblockedwith3%H O for15 imagethresholdwassetbetween10and255.Theparticle
2 2
minutes before blocking with 5% horse serum for one analysiswasset for betweena25and 300pixel rangeand
hour at room temperature. Acetylated-α-tubulin antibody singularity 0 to 1.00 to identify single nuclei. To circum-
from Sigma-Aldrich (St.Louis, MO, USA) (clone 6-11B-1 venttheproblem ofaggregatednuclei,a second andthird
purified from mouse, 1:12,000) and Ki67 antibody from particle analysis was performed using 300 to 550 and 550
Dako (Glostrup, Denmark) (1:200) were used for primary to 5,000 pixel ranges to identify ‘two nuclei’ and ‘>’ re-
stainingovernightat4°C.Secondaryantibodystainingwas spectively. The events identified by the various pixel ana-
performed using anti-mouse HRP (1:100) and Power- lyses were summarized to calculate the total number of
Vision (Immunologic, Duiven,TheNetherlands) activated cells, where the event number identified ‘two nuclei’/300
withH O andstainedwithNovared.Serialsectionswere to 550 pixel range was multiplied by two, and the ‘>’/550
2 2
counterstained 15 to 30 seconds with hematoxylin and to5,000pixelrange wasmultipliedbyfive. Thelatterwas
developed for 10 minutes in running tap water. To deter- chosen as, although visual observation shows a large vari-
mine the amount of actively proliferating cells, the per- ation, the number of multicellular clusters was low and
centage of Ki67 positive cells were blindly scored by five cellswasthe average numberofcells present in these
a pathologist together with an independent researcher clusters. The number of events identified in the 550 to
(Table S1 in Additional file 1). The mean score of the 5,000 pixel range analysis was typically low; ranging from
threecoreswasused. 0 to50events, 51to100 eventswereobserved in densely
populated cores. Once the number of events obtained in
Immunofluorescence the550to5,000pixelrangeanalysisexceeded100events,
After deparaffination and rehydration, 4 μm sections were the sample was excluded as the automated nuclei count
digested in protease XXIV (Sigma-Aldrich, 0.02 mg/ml in became unreliable. To validate, the automated results
PBS, pH 7.3, 60 minutes at room temperature) [24]. After generated were overlaid on the original image for 10 ran-
washing and blocking in 1% BSA in PBS, primary anti- dom images and an error margin <10% was determined
bodies (mouse monoclonal acetylated-α-tubulin, clone 6- (TableS2inAdditionalfile3).
11B-1(Sigma-Aldrich)1:12,000,andrabbitpolyclonalanti-
pericentrin (Novus Biologicals, Littleton, CO, USA) 1:100) Statistics
wereincubatedfor60minutesatroomtemperature.After ForFigure1B,wecomparedthenormalizedciliafrequen-
repeated washing in PBS, secondary antibodies are incu- cies of randomly chosen tumor tissues. Using a paired
bated for an additional 60 minutes at room temperature: t-test analysis at 95% confidence (Prism5) we determined
goat anti-mouse conjugated to Cy5 (Millipore, Bedford, P<0.0001andr=0.9552(n=20).ForFigure2F,ciliafre-
MA, USA) (1:100) and goat anti-rabbit-Cy3 (Life Tech- quencies were calculated as a percentage of cilia events
nologies, Carlsbad, CA, USA) (1:100). Sections were compared to nucleievents. The averaged cilia frequencies
washed again repeatedly, incubated in DAPI (diluted ofthreecoresectionswecomparedbetweenparenchymal
1:5,000 in PBS) for 15 minutes and after a final round of tissue and tumor tissue. Using paired t-test analyses at
washesinPBS,mountedwithFluoromountG.Stainedsec- 95% confidence (Prism5), the cilia frequencies of ccRCC,
tions are stored in the dark at 4°C until confocal imaging oncocytoma and chrRCC populations were determined
withaZeissLSM70063xobjective(CarlZeissMicroscopy, at P <0.0001 (n = 89), P = 0.0078 (n = 6) and P = 0.0444
Jena, Germany). Z-stacks covering 4 μm were taken until (n=5)respectively.
approximately300nucleiwerescoredblindlyforcilia.
Results
Ciliacountingandautomatednucleicounting Classificationofrenaltumors
Acetylated-α-tubulin-stained TMA sections were scanned A collection of renal tumor biopsies and tissue of the
at high resolution and analyzed using Aperio Imagescope tumor parenchyma, recovered 1 cm to 8 cm adjacent to
(Aperio, Vista, CA, USA) [25]. Manual cilia counts were the tumor lesion, was used to compile aTMA. Sections
made from scans using a 40X digital zoom. The number were previously well-documented and classified accor-
ofciliacountedwasrandomlydouble-checkedbyanaddi- ding to WHO standards by pathologists [23]. Both the
tional researcher. Hematoxylin and eosin (H&E)-stained tumor andparenchymaltissuewererepresentedbythree
TMA sections were similarly scanned and individual cores 1 mm in diameter. A total of eighty-nine clear cell
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various pathologies indicates there to be an underestima-
tion error that is <10% for ccRCC and chrRCCandonco-
cytoma (Figure S1C in Additional file 2, Table S2 in
Additional file 3). pRCC cell nuclei are often intensely
stained and aggregated, as the automated analysis
exceeded our predetermined parameter threshold (see
Methods) and rendered the automated quantification
unreliable, we excluded these samples for automated
nuclei count. It is important to emphasize that the
acetylated-α-tubulin-stained TMAs that we use to
count cilia frequencies were not suitable for automated
nuclei count as the immunohistochemistry (IHC) inter-
feres with the nuclear hematoxylin signal; we therefore
used corresponding hematoxylin and eosin-stained
TMA sections checked for matching morphology and
cellcounts.
Ciliafrequencyinrenaltumorcores
TMAsectionswerestainedbyimmunofluorescencewith
acetylated-α-tubulin andpericentrin,establishedmarkers
for the axoneme and base of the cilium, respectively.
This approach allows sensitive high-resolution images of
cilia to be captured and scored using confocal z-stack
imaging (Figure 1A); however, for comparing a large
Figure1Immunofluorescentanalysisofciliainrenaltissues.
number of patientssystematically ina single experiment,
(A)Sections(4μm)ofrenalparenchymaltissuesandtumortissues
we found that the imaging time required for each core
werestainedwithDAPI,acetylated-α-tubulin(Ac-tub)and
pericentrin(PCNT)tomarkcellnucleiandcilia.Presentedimagesare (approximately 1 hour per patient) negatively affected
maximalprojectionsofconfocalimagesoftypicalparenchymal subsequent sample signals through indirect bleaching,
tissueandarepresentativeccRCC.Scalebars20μm.(B)Normalized warming and aging. We therefore validated immu-
ciliafrequenciesofrenaltumors,shownarepairedquantificationsof
nofluorescent high-resolution immunostaining in 20
n=20samples.Theplotcomparesthetwociliaquantification
random patients with standard immunochemistry for
methodologiesdescribed;datawasobtainedbyimmunofluorescent
(IF)confocalimageacquisitionorscoringofimmunohistochemical acetylated-α-tubulin. Standard IHC for acetylated-α-
(IHC)stainedsections.Statisticsweredeterminedbyperforminga tubulin could obtain more data through semi-automatic
pairedt-testata95%confidenceinterval. processing; however, it was not clear that the lesser sen-
sitivity to signal and 3D processing would produce cil-
RCC (ccRCC) of which ten were obtained from VHL iary frequency data comparable to immunofluorescence.
patients, eight papillary RCC (pRCC), five chromophobe Prior to manually counting cilia, parameters were estab-
RCC (chrRCC), six oncocytomas and two sarcomatoid lished by which putative cilia were to be included or
renal tumors and their corresponding parenchymal tis- excluded: 1) size; the cilium needed to be a thin struc-
suewere analyzed. ture, intensely stained broad structures were considered
background; 2) elongation; the cilium needed to be a
Analysisoftotalcellnumbersinnormalandcancertissue continuous thin extending structure, square or dot-like
In order to generate a percentage ofciliation per sample, structures were excluded. Because higher cilia counts
the number of cells composing each core section was overall were observed using the immunofluorescent
determined. For this, we developed a methodology to stainings, we compared normalized data when the par-
automatically count the number of nuclei per core (for enchymal tissue ciliation frequency was set to 100%. In
an extended description see the Methods section). In this analysis, we observe an extremely strong correlation
brief, a colorimetric selection is set to match the nuclear (r = 0.9552) between ciliaryfrequencies in all 20 samples
markerhematoxylinandextractedtodepletebackground. comparingboth techniques(Figure 1B).
Subsequently, images are further processed and analyzed We therefore felt confident that an approach involving
usingImageJparticleanalysistodeterminethetotalnuclei IHC was justified and would yield relevant results in a
number per core (Figure S1A, B in Additional file 2). greater number of patients that we could compare in a
Overlaying the identified nuclear events from the auto- single experiment. Both staining protocols clearly mark
mated analysis with the original core for 10 samples of cilia in luminal spaces in the normal tissues present on
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Figure2Determinationofciliafrequency.(A)Acetylated-α-tubulin-stainedrenaltissuesectionobtainedfromtheparenchymaoftumortissue
showsthepresenceofcilialiningtubularstructures.Structuresencircledingreenareidentifiedascilia,redencircledstructuresareconsidered
aberrantacetylated-α-tubulinstainingorlacksufficientclarity.Stromalorsupportivetissuecellsbetweentubulestypicallydonothavecilia.
(B)Acetylated-α-tubulin-stainedsectionoftissueobtainedfromtypicalandabnormalparenchymaltissueandatypicalccRCC.(C-E)Hematoxylin
andeosin(H&E)andacetylated-α-tubulinstainingsofatypicaloncocytoma,chromophobeRCC(chrRCC)andsarcomatoidrenaltumor.(F)Scatter
plotandmedianofciliafrequencyinparenchymal(P,green)tissuescomparedtomatchedRCCsubtypes(T,red).Significantpopulationsusing
t-test(95%confidence)areindicatedwithasterisks.Allimages1mmcoreandoutlinedmagnifications.
the TMA (Figures 1A and 2A). The TMA contains both (Figure2C),n=5chrRCC(Figure2D)andn=2sarcoma-
tumortissueandtheparenchymasurroundingthetumor; toidrenaltumor(Figure2E)samplesthatwereincludedin
three corespertissuewerescored.Allcoresweremanua- the cilia frequency analysis (Table S1 in Additional file 1).
lly counted and the number of cilia per core was norma- Sections of pRCC proved unsuitablefor automated nuclei
lized to the number of nuclei as determined by the countingandonvisualinspectionofacetylated-α-tubulin-
automated analysis, generating a percentage of ciliation stained sections they appeared generally overstained and
per core (Table S1 in Additional file 1). The ciliation per- were not quantified. Of note, careful visual inspection of
centage was averaged for all three cores, we excluded pRCC suggest this subtype to similarly exhibit reduced
coresthateithergeneratednucleinumbersconsideredun- cilia numbers, although on occasion tubular structures
reliable or showed visually aberrant acetylated-α-tubulin can be identified thatappeartocontain cilia (FigureS2in
staining. In total, theTMA contained n = 89 sporadic or Additionalfile4).Wenextcomparedtheaverage ciliation
VHL-associated ccRCC (Figure 2B), n = 6 oncocytoma frequencies of parenchymal tissue (P, in green) to their
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matched RCC subtype (T, in red) in a scatter plot Based on these observations we posed the question: to
(Figure 2E). Overall, ccRCC, oncocytoma, chrRCC and which extent is cilia frequency globally affected in RCC
sarcomatoid renal tumors show a marked reduction in samples? We analyzed cilia frequency in renal tissue sec-
cilia frequency. A number of parenchymal samples ap- tions present in triplicate on aTMA of 110 patients, in-
peartohaveciliafrequenciesinthesamerangeastumor cluding RCC tissue and tissue obtained from the tumor
tissue samples, visual examination of these samples indi- parenchyma, and observed a severe reduction of cilia
cate that this parenchymal tissue was rather abnormal frequency in the various RCC subtypes. Our data sup-
and contains either excessive stromal/supportive tissue ports, extends and confirms that the low ciliary fre-
ortumorcells(Figure2B). quency characteristic of renal cysts remains an evident
characteristic ofmost renal tumors[22]. Potential effects
ReducedciliationinRCCsubtypesisindependentofcell on cilia function could not be analyzed in this approach,
proliferation hence we cannot exclude whether cilia function in the
Cilia retraction occurs prior to cell duplication to allow normal tissue is also affected. Furthermore, the paren-
for centrosome duplication and spindle pole formation, chymal tissue is likely also stressed and may very well be
in early stages of the new cell cycle ciliogenesis is nonrepresentative of normal kidney function. Future
restored but restricted to cells that exit the cell cycle analysis of downstream targets of signaling pathways
[26]. To ensure that the reduced cilia numbers are a common to cilia, such as the GLI3 and TCF/LEF tran-
characteristic of oncogenic transformation and exclude scription factors downstream of respectively the Shh and
the possibility that this is due to an increase in prolifera- Wnt-pathways, might shed light on impaired cilia func-
tion, we performed antibody staining using the widely tion inpretumorigenic tissue.
accepted proliferation marker Ki67. Three core tumor Theunderlyingprincipleofciliaresorptionexhibitedby
cores were blindly scored to determine the percentage of tumor cells and the putative necessity remains a highly
positively stained cells; results were averaged per sample. debated topic. One possibility is that the loss of cilia pro-
Except for the sarcomatoid tumor showing 23%, nearly motes, or at least contributes to, an environment rende-
all tumor samples had proliferation indexes, <5% (excep- ring these cells more susceptible to mitogenic cues that
tions were a single VHL/ccRCC with 12% and a single initiate proliferation. The mechanism ismuchmore com-
chrRCC with 7.5%), which is too low to solely account plex though, as classic ciliopathies (PKD, BBS, MKS,
fortheobservedlossofcilia(TableS1inAdditionalfile1). NPHP) do predispose to renal cysts, yet renal tumors are
onlyrarelyobserved[20].Thebestcharacterizedfunctions
Discussion of VHL, TSC1, TSC2 and FLCN are also not directly
The effect of oncogenic transformation on cilia stability related to cilia regulation, and RCC formation firstly
is tissue-specific and depends on the underlying muta- dependsonderegulatedmetabolicsignaling[37].Thebest
tion spectrum. It has been previously shown that cilia characterized function for VHL is inactivation of HIF
are less stable in early stages of melanoma and pancre- transcriptionfactorsinnormoxicconditionsthroughpro-
atic ductal carcinoma development [27,28]. However, teasomaldegradation[38].Asaresultofalteredmetabolic
certain tumor types appear to depend on proper cilia pathways,kidneycancerstypicallyshowaWarburgeffect;
formation, such as hedgehog-dependent basal cell car- where the use of glycolysis is promoted over mitochon-
cinomas and medulloblastomas [29,30]. A number of drial oxidative phospholylation. All genetic factors con-
tumor suppressor genes that have established roles in tributing to hereditary kidney cancer (including MET, FH
renal tumorigenesis, namely VHL, TSC1, TSC2 and andSDH)describedtodateplayregulatoryrolesinmeta-
FLCN, also predispose to renal cyst formation, a com- bolic pathways regulated by nutrient-, oxygen-, iron- and
mon hallmark of ciliopathy syndromes. Indeed, these energy-sensing. However, TSC1, TSC2 and FLCN have
proteins have been shown to exert molecular activity to- establishedrolesinenergy-andnutrient-sensingpathways
wards cilia function. VHL interacts with the essential through AMPK (5’-adenosine monophosphate-activated
cilia microtubule motor complex kinesin-2 [31] and loss kinase) and downstream mTOR [37], and it is becoming
of cilia in mouse tissues is observed upon loss of Vhlh evident that mTOR signaling and LKB1, an upstream
(the mouse ortholog of VHL) and either Pten [32] or kinase of AMPK, are both directly linked to cilia [39,40].
Gsk3β [33]. We recently presented data concerning the Combined, RCC seems to be a two-step process in which
role ofFLCN incilia formation show transiently reduced both the primary cilium function needs to be impaired as
cilia formation in FLCN-depleted renal cells [34,35]. In well as modification of the metabolic pathways and un-
contrast, loss of Tsc1 or Tsc2 enhances cilia length in couplingregulationbyenergy,nutrients,oxygenandiron.
mouse embryonic fibroblasts (MEFs) [36], and intri- Therapies aimed at restoring, stabilizing or molecularly
guingly, clinically there is a relatively low frequency of circumventing cilia function might therefore be a suitable
renal cyst and RCC formation in TSC patients [12]. additiontocurrent treatment strategiesin anendeavor to
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preventearlyeventssuchascystformationandsubsequent RCC:renalcellcarcinoma;TMA:tissuemicroarray;TSC:tuberoussclerosis;
tumorigenicprogressionfromthispremalignantstate[41]. UMCU:UniversityMedicalCenterUtrecht;VHL:VonHippel-Lindaudisease.
Competinginterests
Conclusions Theauthorsdeclarethattheyhavenocompetinginterests.
The crucial role of primary cilia in regulating renal
Authors’contribution
homeostasis and the links to cell proliferation and SBandRGdesignedtheresearchandSB,SW,GSandRGcarriedoutthe
polarization are intriguing facets in light of renal cancer research.SB,SW,GS,JVandRGanalyzedthedata.SB,EV,PDandRGwrote
thepaper.Allauthorsreadandapprovedthefinalmanuscript.
research. In addition, findings that associate renal tumor
suppressor proteins as ciliary functions regulators sug-
Acknowledgements
gest that renal tumor cells preferentially are nonciliated. Theauthorswouldliketothankallpatientsforcontributingandsigning
We therefore addressed the status of this organelle in consenttotheuseoftissueforresearchpurposes,theUMCUtissue
databankfortissueprocessingandmanagement,theDepartmentof
RCC tissue. In this study, we included material from 110
Pathology(UMCU)forTMAconstruction,sectioningandscanning,Frits
patients of various RCC subtypes and compared three Meeuwsenfortechnicalassistance.Thisresearchwassupportedbygrants
cores of RCC tissue to parenchymal renal tissue from fromtheEuropeanCommunity'sSeventhFrameworkProgrammeFP7/2009
undergrantagreementnumber:241955,SYSCILIAandtheNetherlands
the same patient. A severe reduction of cilia frequency
OrganizationforScientificResearchtoRHG(NWOVidi-917.66.354).
in the various RCC subtypes is observed, which is inde-
pendent of proliferation. Our results suggest that cilia Authordetails
1DepartmentofNephrologyandHypertension,UniversityMedicalCenter
loss is a common event in renal tumorigenesis and im-
Utrecht,Heidelberglaan100,3584,CX,Utrecht,TheNetherlands.
pliesthatcilialossispartofasequenceofeventsleading 2DepartmentofMedicalOncology,UniversityMedicalCenterUtrecht,
torenal malignant tumor development. Universiteitsweg100,3584,CG,Utrecht,TheNetherlands.3Departmentof
Pathology,UniversityMedicalCenterUtrecht,Heidelberglaan100,3584,CX,
Utrecht,TheNetherlands.
Additional files
Received:1August2012Accepted:8January2013
Published:31January2013
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