centrosome linker induced tetraploid segregation …...centrosome linker–induced tetraploid...

DNA Damage and Repair

Centrosome Linker–induced TetraploidSegregation Errors Link Rhabdoid Phenotypesand Lethal Colorectal CancersAndrea Remo1, Erminia Manfrin2, Pietro Parcesepe2, Alberto Ferrarini3, Hye Seung Han4,Ugnius Mickys5, Carmelo Laudanna6, Michele Simbolo2, Donatella Malanga6,Duarte Mendes Oliveira6, Elisabetta Baritono1, Tommaso Colangelo7, Lina Sabatino8,Jacopo Giuliani1, Enrico Molinari1, Marianna Garonzi9, Luciano Xumerle9,Massimo Delledonne9,10, Guido Giordano11,12, Claudio Ghimenton2, Fortunato Lonardo13,Fulvio D'angelo14, Federica Grillo15, Luca Mastracci15, Giuseppe Viglietto6,Michele Ceccarelli8,14, Vittorio Colantuoni8, Aldo Scarpa2,16, and Massimo Pancione8,17

Abstract

Centrosome anomalies contribute to tumorigenesis, but itremains unclear how they are generated in lethal cancerphenotypes. Here, it is demonstrated that human microsatel-lite instable (MSI) and BRAFV600E-mutant colorectal cancerswith a lethal rhabdoid phenotype are characterized by inac-tivation of centrosomal functions. A splice site mutation thatcauses an unbalanced dosage of rootletin (CROCC), a cen-trosome linker component required for centrosome cohesionand separation at the chromosome 1p36.13 locus, resulted inabnormally shaped centrosomes in rhabdoid cells fromhuman colon tissues. Notably, deleterious deletions at1p36.13 were recurrent in a subgroup of BRAFV600E-mutantand microsatellite stable (MSS) rhabdoid colorectal cancers,but not in classical colorectal cancer or pediatric rhabdoidtumors. Interfering with CROCC expression in near-diploidBRAFV600E-mutant/MSI colon cancer cells disrupts bipolarmitotic spindle architecture, promotes tetraploid segregationerrors, resulting in a highly aggressive rhabdoid-like pheno-type in vitro. Restoring near-wild-type levels of CROCC in a

metastatic model harboring 1p36.13 deletion results in cor-rection of centrosome segregation errors and cell death, reveal-ing a mechanism of tolerance to mitotic errors and tetraploi-dization promoted by deleterious 1p36.13 loss. Accordingly,cancer cells lacking 1p36.13 display far greater sensitivity tocentrosome spindle pole stabilizing agents in vitro. These datashed light on a previously unknown link between centrosomecohesion defects and lethal cancer phenotypes providing newinsight into pathways underlying genome instability.

Implications: Mis-segregation of chromosomes is a prom-inent feature of chromosome instability and intratumoralheterogeneity recurrent in metastatic tumors for whichthe molecular basis is unknown. This study providesinsight into the mechanism by which defects in rootletin,a centrosome linker component causes tetraploid segre-gation errors and phenotypic transition to a clinicallydevastating form of malignant rhabdoid tumor. Mol CancerRes; 16(9); 1385–95. �2018 AACR.

1Pathology Unit, "Mater Salutis" Hospital AULSS9, Legnago (Verona), Italy.2Department of Diagnostics and Public Health, Section of Pathology, Universityand Hospital Trust of Verona, Verona, Italy. 3Menarini Silicon Biosystems S.p.A,Bologna, Italy. 4Department of Pathology, Konkuk University School ofMedicine, Seoul, Korea. 5National Center of Pathology, Affiliate of VilniusUniversity Hospital Santariskiu Clinics, Vilnius, Lithuania. 6Department ofExperimental and Clinical Medicine "Gaetano Salvatore", University "MagnaGrecia", Catanzaro, Italy. 7Institute for Stem Cell Biology, RegenerativeMedicineand Innovative Therapies (ISBReMIT), Casa Sollievo della Sofferenza-IRCCS, SanGiovanni Rotondo, Italy. 8Department of Sciences and Technologies, Universityof Sannio, Benevento, Italy. 9Functional Genomics Center, Department ofBiotechnology, University of Verona, Verona, Italy. 10Personal Genomics S.r.l.,Verona, Italy. 11CRO Aviano National Cancer Center, Aviano, Italy. 12MedicalOncology Unit, San Filippo Neri Hospital, Rome, Italy. 13Medical Cytogeneticsand Molecular Genetics Unit, AORN "Gaetano Rummo," Benevento, Italy.14Bioinformatics Laboratory, BIOGEM scrl, Ariano Irpino, Avellino, Italy.15Department of Surgical and Diagnostic Sciences (DISC), University of Genovaand S. Martino Polyclinic Hospital, Genova, Italy. 16ARC-Net Centre for Applied

Research on Cancer, University and Hospital Trust of Verona, Verona, Italy.17Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy,Complutense University, Madrid, Spain.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

A. Remo, E. Manfrin, and P. Parcesepe contributed equally to this article.

Corresponding Authors: Massimo Pancione, Department of Sciences andTechnologies, University of Sannio, Via Port'Arsa, 1182100 Benevento, Italy.Phone: 3908-2430-5157; Fax: 3908-2430-5147; E-mail:[email protected]; Aldo Scarpa, ARC-NET Research Centre,Policlinico GB Rossi, Piazzale L.A. Scuro, Verona, Italy. Phone: 3904-5812-4043; Fax: 3904-5812-7432; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-18-0062

�2018 American Association for Cancer Research.

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IntroductionThe century-old hypothesis on the relationship between cen-

trosomes and cancer, formulated by the German embryologistTheodor Boveri more than 100 years ago (1, 2), remains unan-swered. Centrosome abnormalities, consisting usually in increasednumbers, are common in human tumors (3), and experimentallyinduced tetraploid cells from extra centrosomes can be criticalfor aneuploidy and metastatic progression of malignancy (3, 4).However, insufficient progress has been made in our knowledgeon genetic defects underlying centrosome anomalies in tumori-genesis (1–4). In this scenario, the rare and lethal pathologicvariant of common colorectal cancers showing rhabdoid pheno-type (5–7), is of particular interest as it features recurrent mitoticanomalies of enigmatic origin (8–10). We thus hypothesized thatthe systematic study of rare rhabdoid colorectal cancers, couldprovide insights into biological mechanisms responsible for thegeneration of genome instability and reveal key factors for thedevelopment of aggressive disease entities. To test this idea, weperformed whole-exome sequencing of two rhabdoid colorectalcancers and discovered an enrichment of centrosome anomaliesand inactivation of Rootletin encoded by ciliary rootlet coiled-coil(CROCC) gene (11, 12), a structural component of the centro-some linker, which assembles and keeps the two centrioles con-nected. Centrosomal alterations were assessed in an expandedseries of rare rhabdoid colorectal cancers and related tumors, andfunctionally characterized in colorectal cancer cellular models.

Materials and MethodsMaterials and Methods and any associated references as a

continuation of the main text are described more in detail withinthe Supplementary Material.

Patient and tissue cohortThis study was conducted in accordance with Declaration of

Helsinki ethical guidelines. It was approved by an institutionalreview board, approval no. 997CESC from the Ethics Commit-tee (Comitato Etico di Verona e Rovigo dell'Azienda Ospeda-liera Universitaria Integrata) on September 7, 2016, documen-ted by the CESC prot. 42160 on 9 September 2016, andformalized by the General Manager with deliberation no.458 of September 16, 2016, communicated with protocol51319 on September 23, 2016.

Formalin-fixed paraffin-embedded (FFPE) samples from 7cases of primary rhabdoid colorectal cancers andmatched normalcolonic mucosa were studied (cases RC1 to RC7 SupplementaryTable S1). Moreover, an independent validation series to screenthe mutational status of newly identified genes was analyzed(cases RC8 to RC12 Supplementary Table S1). FFPE samples from7 rhabdoid tumors arising in central nervous system of patientsbetween2months and19 years of agewere collected from thefilesof the AziendaOspedalieraUniversitaria Integrata (Verona, Italy).These pediatric/young adult rhabdoid tumors are indicated asrhabdoid of infancy throughout the article. Two independentdatasets of patients with classic type sporadic colorectal cancerwere analyzed (dataset A included 141 primary cancers anddataset B included 102 primary cancers).

Cell linesHuman colon cancer cell lines HCT116, HT29, CaCo-2,

LoVo, RKO, T84, DLD1, SW480, and SW620 were purchased

from ATCC. BJ human fibroblasts and G401 cells derived fromnormal foreskin and pediatric rhabdoid tumor were used as anonneoplastic control and a pure rhabdoid model, respectively.

Whole-exome sequencingWhole-exome sequencing with 100-bp paired reads was per-

formed with a HiSEQ1000 (Illumina), using 1.3 mg genomicDNA (based on fluorometric Picogreen dsDNA quantification),and enrichment for whole exome was done according to TruSeqExome Enrichment Guide (Illumina).

Functional in vitro assaysRKO cells were transiently transfected with SureSilencing con-

trol or CROCC shRNA expression plasmids KH23140P (Qiagen)containing the puromycin-resistant cassette. After selection puro-mycin (Thermo Fisher Scientific), single colonies were amplifiedand assessed for efficient CROCC silencing by qPCR andWesternblot analysis, respectively. HT29 and T84 cells were transfectedwith the full-length CROCC coding sequence or a truncated form(1–494 aa) cloned with GFP epitope or GFP alone (used ascontrol). For long-term experiments, CROCC-GFPþ cells weremaintained in 0.6 mg/mL of G418.

Statistical analysisData are presented as mean, medians, and ranges. P values of

<0.05 (two tailed) were considered to be significant. Statisticalanalyses were conducted by GeneSpring R/bioconductor v.12.5and R based package, SPSS v15, and GraphPad Prism 5.

ResultsDiscovery of CROCC mutations and centrosome anomalies

Two previously reported primary BRAFV600E-mutant rhabdoidcolorectal cancers (RC1 and RC2; refs. 9, 10), harboring MSI dueto defective DNA mismatch repair (MMR) machinery caused bypromoter methylation of the MLH1 gene, were subjected towhole-exome sequencing (WES) using DNA from FFPE-matchedtumor/normal samples (Supplementary Table S1). We detectedan exceptionally large number of somatic point mutations, 1,056and 1,078 per 106 bases for RC1 and RC2, respectively, which isconsistent with the presence of MMR defects (13–15; Fig. 1A).About twenty percent of mutations occurred within CpG dinu-cleotide context as seen in classical colorectal cancers (14). Transi-tions were more frequent than transversions (71.8% vs. 28.2%;Supplementary Fig. S1A) with a dominance of C>T/G>A, T>C/A>G transitions (Supplementary Fig. S1B), which is characteristicof the mutational signature due to alterations of MMR mechan-isms (signature 6; ref. 13). The most prevalent single-nucleotidevariants (SNV)were nonsilentmutations (14), where over 90%ofpotentially damaging mutations were missense and around 10%were splicing, stop-gain, stop-loss, or, rarely, frameshift insertionsor initiation codon mutations (Fig. 1A; Supplementary Fig. S1Cand S1D). The two rhabdoid colorectal cancer cases shared 112(10%) mutated genes (Supplementary Fig. S2A). By applyingDrGaP computational tool (16), which allows inferring cancerdriver genes, a number of potential candidate disease-causinggenes were identified (Supplementary Table S2), approximatelyhalf of which (45%) were enriched for cytoskeleton/centrosomeand microtubule biological functions (Supplementary Fig. S2Band S2C).

The search of candidate genes in The Cancer Genome Atlas(TCGA) database (13–15) comprising 224 sequenced classical

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colorectal cancers (http://www.cbioportal.org) revealed that themajority (65%) of the candidates had a low frequency of muta-tions (�4% of cases). Among all candidate genes, only forCROCC mapping to 1p36.13 (11, 12) involved in centrosomecohesion and disjunction, no somatic mutations (0/224; 0%)were reported (Fig. 1B). Notably, 1p36 deletions are recurrent inneuroblastoma, Wilms tumor, and medulloblastoma. In our tworhabdoid colorectal cancer cases, CROCC harbored twomissensemutations, p.Ala161Ser (c.481G>T, Exon 4) and p.Val1885Ala(c.5654T>C, Exon 35), and one prominent splicing mutation atthe conserved 30 acceptor splice site (c.3705-2A>G) in the intronbetween exons 25 and26 (Fig. 1C). A reviewofmultiple colorectalcancer sequencing datasets (N ¼ 2070) revealed CROCC muta-tions in (1.4% of cases). However, although of unknown signif-icance, none of the CROCCmutations were identified as putative

driver mutations in colorectal cancer (Supplementary Fig. S2D).SMARCB1 and SMARCA4mutations, which have been associatedwith rhabdoid phenotype (6), showed a trend of mutual exclu-sivity withCROCC alterations. However, only putative truncatingdriver mutations in SMARCB1 and/or SMARCA4 correlated withtumor poor differentiation and short-timemetastatic progression.Therefore, we reasoned that the splicingmutation detected in RC1might be causally correlated with rhabdoid phenotype. Indeed,themutation reduced the strength of the physiologic acceptor site,causing a large deletion of the CROCC coding region involvingexons 23–31 (17, 18) (Supplementary Fig. S3A). RT- PCR analysis(exons 5–7 and 33–35) revealed reduced CROCC mRNA in RC1harboring the splicing mutation as compared to the normalmucosa. This suggested the alteration of the mature CROCCtranscript by the utilization of cryptic splice sites or by the

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Figure 1.

WES reveals mutations in CROCC, encoding an essential component of the centrosome linker. A, Representative rhabdoid colorectal cancer histopathologicimages from RC1 and RC2 that were subjected to WES (H&E, hematoxylin and eosin). The graph indicates the total number of somatic mutations per tumor.The circo shows the distribution of nonsilent mutations and copy number variations (CNV); the outer ring indicate the chromosomes. B, Prevalence ofalterations in the candidate genes harboring somatic mutations in both RC1 and RC2 in 224 colorectal cancers of the TCGA database. C, CROCC chromosomelocalization (1p36.13) and organization (from Ensembl, reference transcript ENST00000375541). All 37 exons are depicted as vertical bars and introns ashorizontal lines. Solid circles indicate the mutations identified in RC1 and RC2. The "proteinaceous linker" is composed of CROCC filaments (black arrow) thatphysically connect the mother (M) and daughter (D) centrioles surrounded by the pericentriolar material (PCM). At the onset of mitosis (Mi), the linker isdisassembled to support the formation of the bipolar mitotic spindle. D, Quantification of CROCC mRNA (qPCR) expression levels in tumor and adjacentnormal mucosa. Data are mean � SD; n ¼ 5 biological replicates; �� , P < 0.01, two-tailed Student t test). Representative images of CROCC immunostaining innonneoplastic colon mucosa and a fallopian tube used as control (arrow). CROCC immunopositive centrosomes are reduced in number (arrow) or mispositioned(distant/separated from the nucleus) in RC1, (inset modeled image). Scale bars are reported in each microphotograph.

Centrosome Cohesion Anomalies and Cancer via CROCC Defects

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activation of the nonsense-mediated mRNA decay, which, insome cases can eliminate aberrant mRNA transcripts (Supple-mentary Fig. S3B; ref. 17). Unexpectedly, RC2 also displayedexpression levels in tumor lower than in normal tissue, support-ing a role for defective CROCC expression in rhabdoid tumors(Fig. 1D). We next used IHC and immunofluorescence at highmagnification with an anti-CROCC antibody to analyze thecentrosomes in rhabdoid cancers and matched normal tissues(Supplementary Table S3). We found that nearly 50% of tumorcells had no CROCC immunolabeling, and the presence of cellswith a single and often abnormally shaped, larger (up to 6-foldgreater than normal), or fragmented centrosomes, suggestingthe presence of numerical and structural centrosome aberra-tions (Supplementary Fig. S3B). We also observed dramatic anduncommon cytologic defects, such as anucleated cells havinglarger centrosomes positive for CROCC associated with mitoticcatastrophe in late telophase, particularly in RC1 harboring thesplicing site mutation (Fig. 1d). We used the pericentriolarmaterial (PCM) component g-tubulin as our reference markerfor immunolabeling experiments, because it consistently colo-calizes with centriole markers, which are closely connected ininterphase by the centrosomal linker (1, 2, 11, 12). Moreover,g-tubulin has been proposed as a marker to identify spindlepoles (19, 20). We observed a remarkable loss of cell polarityin interphase nuclei and abnormal mitotic figures, many ofwhich included asymmetric bipolar or monopolar spindles.Rhabdoid cells showed a diffused staining of g-tubulin into thecytoplasm and reduced centrosomal localization, a phenome-non described in tumors with high metastatic potential (ref. 19;Supplementary Fig. S3C and S3D). Double immunofluores-cence analysis using antibodies directed against CROCC andg-tubulin confirmed these observations and revealed cellseither with fragmented/larger centrosomes or with a consistentloss of centrosome staining (Supplementary Fig. S3). Theseexperiments indicated that genetic defects in CROCC and othercentrosome components may compromise centrosome func-tion in rhabdoid colorectal cancers.

A validation set of 10 additional rhabdoid colorectalcancers was studied, including 3 cases (RC7, RC9, RC11) withmicrosatellite instability due to MLH1 promoter methylationand 7 cases (RC3–6, RC8, RC10, RC12)with stablemicrosatellites(Fig. 2A; Supplementary Table S1). Targeted sequencing identi-fied three CROCC mutations (p.Ser1320Ile, p.Arg1659His andp.Ala1510Thr) of unknown significance in additional 2 cases(RC9 and RC11) harboring microsatellite instability (1, 2,11, 12). Indeed, the 24 CROCC mutations identified acrosscBioportal database were more recurrent in MSI (12/24; 50%)than in MSS (6/24; 25%) colorectal cancers. Notably, CROCCmutations were classified as missense (n ¼ 22) or truncatingmutations (n ¼ 2) of unknown significance but associated withboth well- differentiated and early-stage classical colorectal can-cers. In our rhabdoid colorectal cancer dataset, 5 of the remainingcases for which sufficientmaterial was available (cases RC3–RC7)harbored loss of heterozygosity (LOH)at the1p36.13 locus,whereCROCC resides, which was associated with mRNA below normallevels (Fig. 2B). In keepingwith thefindings inRC1andRC2 cases,comparable levels of centrosome defects and a high prevalenceof bizarre mitotic figures and/or cytomorphologic aberra-tions were evident in all tumors (Fig. 2C; SupplementaryTable S3). Analysis of independent colorectal cancer databases(n ¼ 1,387) for which copy number alterations were available

(http://www.cbioportal.org) revealed no alteration at 1p36.13locus, suggesting CROCC impairment as a consequence of re-duced gene dosage (21) caused by allelic deletion. As centrosomeanomalies are intimately connected with chromosome segrega-tion errors (1, 3, 20), we assessed DNA content in tumor samples(cases RC1–RC7, Supplementary Table S3). Remarkably, we ob-served recurrent ploidy abnormalities mainly consisting of trip-loid or near-tetraploid cells ranging from 10% to 40% of tumorcells (Fig. 2D). Globally, these results indicated that centrosomedefects underlie rhabdoid colorectal cancer pathogenesis.

Centrosome and genomic profiling of rhabdoid tumors ofinfancy

Insight into genetic characterization of rhabdoid neoplasms arelimited to the so called extrarenal rhabdoid tumors arising inchildren, in which inactivating mutation and/or deletion of thechromatin remodeling gene SMARCB1 (INI1) and low mutationload have been reported (6, 22–24). We analyzed 7 cases of thistumor type, hereafter named rhabdoid of infancy, for centrosomeand molecular anomalies (Fig. 3A; Supplementary Table S4).Compared with rhabdoid colorectal cancers, analysis in pediatrictumors was associated with much higher CROCC mRNA expres-sion levels than matched normal tissues (P < 0.00001; Fig. 3B).Target sequencing identified no mutations (0/7 tumors) in theCROCC gene. Moreover, CROCC immunostaining was seen as asingle and large signal adjacent to the nuclei in almost the totalityof the cells (80%)—only 10%of cells had no centrosome staining(Fig. 3B; Supplementary Table S4). Consistent with literature, thegenetic profile of rhabdoid of infancy revealed missense or trun-cating mutations in SMARCB1 (5/7, 71%; refs. 6, 23, 24) and/orTP53 (3/7, 42%) accompanied by a near diploid DNA contentand less aggressive clinical course when compared with rhabdoidcolorectal cancers (Fig. 3C–E). This suggested that rhabdoid ofinfancy,which is characterized by SMARCB1 (INI1)mutation, didnot harbor any CROCC alteration and did not display the cen-trosomal defects observed in rhabdoid colorectal cancer. Inspec-tion of an available database from pediatric rhabdoid cells (25)confirmed that both mutations and genetic deletion affectingCROCC locuswere infrequent (2/20, 10%),whereas the transcriptprofile tended to be similar to our rhabdoid of infancy dataset(Fig. 3C). Therefore, rhabdoids arising in colorectal cancer,although morphologically indistinguishable from their pediatriccounterparts, demonstrate distinct molecular, cytogenetic, andcentrosomal aberrations.

Centrosome and CROCC expression in classical colorectalcancers

We screened 242 primary classic colorectal cancers, comprisingtwo independent series of 140 (dataset A; refs. 26, 27) and 102(dataset B; refs. 28, 29) cases for CROCC mRNA and proteinexpression (Supplementary Table S5). CROCCmRNA expressionlevels were higher in colorectal cancers than in normal colonicmucosa. However, no significant protein expression change intumor tissues compared with that in normal mucosae was detect-ed (Supplementary Fig. S4A). In cohort A, using IHC and immu-nofluorescence against CROCC and g-tubulin, we found roundand uniform centrosomes, prevalently in normal number (1–2per cell; 97/140, 69.3%), supernumerary (>2 per cell; 39/140,27.8%), and only few (<1 per cell; 4/140, 2.9%) displayedreduced centrosome labeling (Supplementary Fig. S4B). Centro-some abnormalities, particularly supernumerary centrosomes,

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were more prevalent in advanced stage (stage III–IV) than in lowstage (stage I–II) lesions (Supplementary Fig. S4C). In cohort B,which was enriched for stage III–IV tumors (79.5% of cases), weconfirmed a high prevalence of supernumerary (60/102; 59%;refs. 1, 3, 20) or defective (11/102; 11%) centrosomes associatedto poorer clinical course than those expressing a normal pattern[31/102; 30%, HR ¼ 0.30; 95% confidence interval (CI),0.21–0.81; P < 0.0001] in keeping with the notion that numericalcentrosomal abnormalities are more common in invasive cancers(1, 3, 20; Supplementary Fig. S4C). To independently validatethe pattern of gene expression changes detected in our datasets,we analyzed the patient-matched tumor–normal expression dataavailable from the TCGA (14) and three independent datasets

GSE20916 (30), GSE41258 (31), andGSE30540 (32), of classicalcolorectal cancer (Supplementary Fig. S4D). CROCC mRNA wasupregulated in colorectal cancer compared with normal only inTCGA database. The analysis of other datasets revealed a hetero-geneous expression pattern, while CROCC upregulation com-pared with normal control did not reach statistical significance.The analysis of GSE30540 (32) dataset, for which both transcrip-tomic data and degree of chromosome instability (CIN) wereavailable, revealed that CROCC expression levels tended to belower in CIN-high than in CIN-low tumors (SupplementaryFig. S4D). These data suggested the possibility that imbalancedgenetic defects at CROCC locus may be related to marked anom-alies in the fidelity of chromosome segregation.

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Centrosome anomalies characterize colorectal cancer with rhabdoid phenotype. A, Histopathologic images from a subset of five additional prototypical rhabdoidcolorectal cancers (RC), in which are evident rounded eosinophilic cytoplasmic inclusions, eccentric nuclei, and prominent nucleoli. Scale bar, 20 mm H&Eimages. B, Mutations for selected driver genes and CpG island methylation (CIMP) profile accompanied by loss of heterozygosity analysis at 1p36.13 locus.CROCCmRNA (qPCR) expression levels in tumors and adjacent normalmucosa. Data aremean� SEM; (n¼ 5 biological replicates; � , P <0.05; ��, P <0.01; two-tailedStudent t test). C, Representative IHC analysis from case RC5: Cytokeratin-18 (CK18) marks intermediate filaments in an anucleated cell (arrow); Ki67reveals abnormal chromosome structures (arrow); CROCC marks a multinucleated cell (arrow), anucleated cell (arrow) or it appears fragmentedin a mitotic cell (monopolar spindle, arrow). Scale bar, 10 mm. Right, quantification of the centrosome phenotypes against CROCC observed in all RCs(n ¼ 2 experiments, >500 cells/sample). D, Cytogenetic abnormalities (tetraploid signals, arrows) observed by FISH using the centromeric chromosomeprobes illustrated. Scale bars, 20 and 40 mm. Left, ploidy pattern in all RCs (for chromosomes 1, 12, and 17, n ¼ 2 experiments, >500 cells per sample).Right, quantification of cells with polyploidy measured as ratio of triploid and tetraploid on diploid cells for each tumor.






CROCC depletion impairs mitosis and induces rhabdoid-likefeatures

Wenext sought a genetic basis for the relation betweenCIN andrhabdoid phenotype by examining WES and transcriptomic datafrom The Cancer Cell Line Encyclopedia (CCLE; ref. 25). Unex-pectedly, data from a collection of 60 colorectal cancer cell linesrevealed that the deletions at 1p36.13 locus tended to be moreprevalent in CIN-high (23.6%, 9/38) compared with CIN-low(9.1%, 2/22) cells (Supplementary Fig. S5A). However, cell lineswith 1p36.13 deletion displayed neither rhabdoid phenotype nor

BRAF mutations. As expected, compared with cells retaining1p36.13 locus, those harboring the deletion revealed a geneexpression signature significantly enriched for pathways impli-cated in chromosomal instability (refs. 33, 34; Supplementary Fig.S5B and S5C). In a panel of colorectal cancer cells, we thenconfirmed that both CROCCmRNA and protein expression levelswere concordant and higher in CIN-low than in CIN-high celllines (P < 0.05, Supplementary Fig. S6A). CIN-low cells showedcentrosomes stained for CROCC and g-tubulin that were struc-turally indistinguishable from those in normal human fibroblasts

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Centrosome and cytogenetic aberrations comparison between colorectal and pediatric rhabdoid tumors. A, Representative hematoxylin and eosin (H&E) imagesof pediatric rhabdoid tumors. Scale bar, 50 mm. B, IHC and interphase FISH analysis for the indicated markers (right). Note that centrosomes are single, larger,uniform in size and close to the nuclei (black arrowhead). Left, quantification of CROCC IHC and centromeric (CEN) signals (Chr 1 and Chr 17) in pediatricrhabdoid tumors, (>500 cells per tumor) were evaluated, percentages represent mean values from three independent investigators. Quantification of CROCCmRNA (qPCR) expression levels. Each circle represents the mean value of five biological replicates from a single lesion (�� , P < 0.01, two-tailed Student t test).C, CROCC expression in pediatric rhabdoid–derived cancer cell lines according to copy number alterations and mutational load (Novartis/broad cancer celllines encyclopedia). D, The panel shows the distribution of nonsilent missense or truncating mutations for the indicated pathways in rhabdoid colorectal cancer(RC) and rhabdoid of infancy. E, Kaplan–Meier overall survival curve for rhabdoid of infancy (age class 2 months–19 years) and rhabdoid colorectal cancers(age class 49–83 years). The P value is obtained by the log-rank test is reported in the graph.

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BJ, consistent with literature (ref. 35; Supplementary Fig. S6A andS6B). In line with this, CIN-high displayed a higher frequency ofmicronuclei and nuclear gH2AX foci than CIN-low cells (ref. 29;Supplementary Fig. S6C and S6D). This suggested that CROCCmight be a CIN suppressor gene and its deletion in CIN-low/BRAF-mutant cell lines might be permissive for abnormal phe-notypes. Therefore, we reasoned that RKO cells, sharing a near-diploid karyotype, BRAFV600E mutation/MSI, and alterations inmicrotubule/centrosomal components with rhabdoid colorectalcancer (14), could be an excellent system to explore CROCCsilencing in vitro. We found that the clone sh4, hereafter namedCROCCKD, provided a stable and consistent knockdown ofCROCC transcript to more than 75% and protein to 3.3-foldlower then RKO cells transfected with control vector (shCon),achieving nearly comparable levels to those seen in vivo (Supple-mentary Fig. S7A). Previous studies have demonstrated thatCROCC knockdown in nontransformed cells causes centriolesplitting and increases centrosome separation (11, 12). By usingg-tubulin and centrin as reference markers, we observed a con-sistent PCM fragmentation after CROCC depletion, whichresulted in abnormal chromosome segregation and higher fre-quency ofmonopolar spindles as comparedwith control (Fig. 4A;Supplementary Fig. S7B). Importantly, monopolar spindles dis-played larger or "fragmented" centrosomes, which accounted for85% of the abnormal phenotype (Fig. 4A–C; Supplementary Fig.S7C). Thus, BRAF-mutant/MSI colorectal cancer cell lines, inwhich centrosome and microtubule stability is damaged bygenetic hypermutation,CROCCdepletionmaydetermine amajorimpact in the progression ofmitotic errors (36–38). Consistently,an increased frequency of micronuclei (median 11% CROCCKD

vs. 1% ShCon cells P¼ 0.0003) and gH2AX nuclear foci (median43% CROCCKD vs. 18% ShCon cells P ¼ 0.011) were observed(Fig. 4C; Supplementary Fig. S7D). Metaphase karyotypingrevealed that CROCC deficiency leads to an increased numberof tetraploid (4N) cells (median 13.3% CROCCKD vs. 3.51%ShCon cells, P ¼ 0.001) characterized by prominent and largernuclei than diploid (2N) cells. Consistently, analysis of centro-meric probes in intephase nuclei confirmed tetraploidy (Fig. 4C).The number of CROCC-deficient cells was reduced in G0–G1 orG2–M phases when compared with the wild-type population (by26%–45%, FACS analysis), suggesting an impaired cell-cycleprogression as a consequence of misaligned chromosomes(1–3, 33; Supplementary Fig. S7D). In contrast, cells grown underreplication stress conditions (serum deprivation) resulted inhigher proliferation rate than control cells (ref. 34; Fig. 4D). Moststrikingly, CROCC-deficient cells exhibited all cardinal signs ofrhabdoid features (8–10), displaying huge nuclei pushed to theperiphery of the cells with single or multiple large nucleoliassociated with eosinophilic cytoplasmic inclusions and largecellular protrusions resembling the morphology observed in vivo(Fig. 4D; Supplementary Fig. S8A). These features resulted in theactivation of prometastatic genes involved in epithelial mesen-chymal transition accompanied by a dramatic change of spindle-shaped morphology (4, 7) consistent with the enhanced meta-static potential of rhabdoid phenotype (Supplementary Fig. S8Aand S8B). Expression of exogenous GFP-tagged CROCC(1–2018 aa) rescued these phenotypic changes induced by deple-tion of endogenous CROCC (Supplementary Fig. S8B; ref. 11). Inline with previous results (11, 12), we observed no alteration incell-cycle profile or aberrant phenotypic changes after CROCCdepletion in nontransformed BJ cells. Therefore, CROCC deple-

tion in BRAF-mutant near-diploid cancer cells induces tetraploi-dization and rhabdoid phenotype in vitro.

Tolerance to mitotic errors and tetraploidization promoted by1p36.13 deletion

As colorectal cancer cells with driver mutations inCROCC havenot been reported, to test the hypothesis that CROCC impactstumor growth and centrosome-relatedmitotic errors, we analyzedmetastatic colorectal cancer T84 cells harboring an allelic deletionat 1p36.13 locus (25). Although T84arewell-differentiated cancercells and do not show rhabdoid morphology, they, however,exhibit some of the characteristics detected in RKO CROCC-depleted cells. Consistent with reduced CROCC endogenousactivity, we observed an increased rate of micronuclei, tetraploid,or near-tetraploid cells and recurrent mitotic errors resultingessentially in "monopolar spindles," which were more recurrentunder replication stress conditions (Fig. 5A; Supplementary Fig.S8C and S8D).We then investigated the localization of CROCC inthe centrosome by immunofluorescence. Almost half of the cells(40%) revealed a faint CROCC signal, which was consistentlyaccompanied by atypical g-tubulin aggregates prevalently in cellswith mitotic anomalies (Fig. 5A). Most strikingly, such aberra-tions were rarely, if ever, detected in pediatric rhabdoid G401 orcolon cancer cell lines with an intact 1p36.13 locus (Supplemen-tary Fig. S8C and S8D). Therefore, we transfected CROCC-GFPand GFP alone (control) into T84 cells. Restoration of CROCCdetermined adramatic decrease of cell viability (12days later, 0%)as comparedwith control plasmid. Similarly, we detected a highernumber of G0–G1 cells than control (41% vs. 26%; P ¼ 0.0018;Fig. 5B and C). Gain of CROCC conferred a flat/adherent phe-notype and formation of filament-like structures colocalizingwith g-tubulin resulting in an expression of mesenchymal geneslower than in control (ref. 4; Supplementary Fig. S9A). Accord-ingly,wedetected a4-folddecrease of tetraploid cells, and reducedgH2AX foci from 59% to 22% with respect to control cells (Fig.5C) raising the possibility that the centrosome spindle poleintegrity is strongly affected by 1p36.13 deletion. To see whetherT84 cells are sensitive tomitotic drugs,wemined the data from theGenomics of Drug Sensitivity in Cancer project (Sanger panel). Asshown in (Supplementary Fig. S9B), among 221molecules tested,IGF1R inhibitor (linsitinib) and Epothilone B, a microtubule-stabilizing agent, were significantly effective in T84 lines. Accord-ingly, we observed a significant difference in the sensitivity toEpothilone B in 1p36.13-deleted cells as comparedwith cells withan intact 1p36.13 locus. Similar results were not reproducedcomparing CIN-low and CIN-high colorectal cancer cell lines(Supplementary Fig. S9B). We used another cell line HT29 withan intact 1p36.13 locus to test CROCC restoration. Similarly toT84, we observed a significant decrease of micronuclei in HT29-CROCC-GFPþ cells compared with control. Although gain ofCROCC in HT29 increased the cell death, it appeared an essentialgene only for T84 cell survival lacking 1p36.13 (SupplementaryFig. S9C). This supported the hypothesis that a reduced CROCCdosage promotes defects in spindle-assembly checkpoint. Whenwe repeated the experiments using aGFP-tagged truncated formofCROCC (1–494 aa; ref. 11), we observed that this mutant failedto rescue the aberrant growth phenotype and mitotic errors(Supplementary Fig. S9D). Consistent with previous findings(11), we did not detect phenotypic changes in nontransformedBJ cells transduced with the full-length CROCC construct. Thus,we conclude that DNA segregation errors resulting from impaired






centrosome function are driven by reduced CROCC dosage at1p36.13 locus.

DiscussionOur understanding of the molecular architecture and function

of centrosomal linker components in physiologic and pathologicprocesses remain rudimentary. Besides (Rootletin), multipleproteins including C-NAP1 (CEP250), CEP68, and LRRC45have been implicated in centrosome linker formation and func-tion. CROCC is able to maintain centrosome cohesion in partthrough inhibition of VHL-mediated Cep68 degradation (36). It

has recently been proposed that a vast network of repeatingCROCC units with C-Nap1 as ring organizer and CEP68 asfilament modulator forms the centrosome linker structure (37).We show here that genetic deletion in CROCC, leads to centro-some anomalies resulting in tetraploid DNA segregation errors,providing insights into mechanism by which genome instabilitycontributes to lethal cancers for which no therapies are available(Fig. 5D). In addition, we show that rhabdoid colorectal cancersare not genetically related to their pediatric counterparts (22–24),in which we find recurrent SMARCB1 gene alterations but noevidenceof centrosomeanomalies. Previous studies have revealedthat driver genes implicated in human cancer (3, 4) can promote

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CROCC depletion induces rhabdoid phenotype exacerbating DNA segregation errors. A, Images of RKO cells with stable CROCC depletion (CROCC KD)showing that in mitosis there is an abnormal spindle formation, "monopolar spindles," as compared with control (CON, left). Large (white arrow) orfragmented (arrow) centrosomes are shown. Cells are stained using immunofluorescence antibodies as indicated (aTUB, anti-a-tubulin antibody; gTUB,anti-b-tubulin antibody) and nuclei are stained with DAPI (4,6-diamidino-2-phenylindole). Scale bar, 5 mm. Below is a schematic illustration of cells withaberrant spindles (85%). B, Anaphase bridges (white arrow), multinucleated (arrow), multilobulated nucleus (arrow), and fragmented centrosomes(arrows) associated with loss of cell polarity and large micronuclei (white arrow) in CROCC-depleted cells. Scale bar, 5 mm. C, The top left graph shows thepercentage of micronuclei and monopolar spindles (>250 cells per cell line, triplicate experiments, �� , P < 0.01; �� , P < 0.001 Mann–Whitney U test).Representative images of metaphase chromosome spreads and cells stained with DAPI and anti-centromere antibody (ACA). Scale bar, 10 mm. Thebottom left graph shows the quantification of ploidy content at metaphase. Data are mean � SEM; n ¼ 5 biological replicates; �� , P < 0.01, two-tailed Student ttest). The bottom right graph shows tetraploid on diploid cells ratio. D, The top graphs report the survival assay with serum supplementation or underreplication stress condition "serum deprivation." Error bars represent mean � SEM of five independent experiments (� , P < 0.05; �� , P < 0.01; �� , P < 0.001,two-tailed Student t test. Below are representative cytomorphologic changes showing large polygonal cells and eccentric round nuclei with prominentnucleoli (black arrow) and eosinophilic hyaline cytoplasmic inclusions (arrow).

Remo et al.





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CROCC abrogates centrosome-related mitotic errors in 1p36.13-deleted cancer cells.A, Images of T84 cells with a large micronucleus (white arrow) and significantlyreduced CROCC staining (enlarged in insets), anaphase bridges (arrow), or monopolar spindle (arrow) associated to deficient or fragmented g-tubulin dots(enlarged in insets). Scale bar, 5 mm. The top right graph reports the percentage of anaphase showing segregation errors and micronuclei in T84 cells withserum supplementation or serum deprivation (SD) at 12 hours. Error bars represent mean � SEM; � , P < 0.05, two-tailed Student t test). B, Representativeimages of T84 cells transfected with full-length human CROCC-GFP or GFP alone, immunostained for g-tubulin (enlarged in insets). The graph on the right showsthe survival of T84 transfected with CROCC and matched control cells maintained in neomycin selection (0.6 mg/mL) for the indicated time. Viability wasassessed by a colony formation assay. The GFP vector was used as a control. Cells were fixed, stained, and photographed after 6 and 12 days of culture. C, Theleft graph reports flow cytometry analysis after 6 days. Error bars represent mean � SEM of five independent experiments; �� , P < 0.01; �� , P < 0.001,two-tailed Student t test). Tetraploid ondiploid cells ratio after 6 days quantifiedbymetaphases spreads (16 independent experiments for each condition; �� , P<0.01,two-tailed Student t test). The right graph shows the percentage of gH2AX foci in prometaphase (>250 cells per cell line, ��, P < 0.001, Mann–WhitneyU test). D, Schematic representation of rhabdoid colorectal cancer progression. In colorectal cancer with defective microtubule functions, BRAFV600E

mutation depletion of CROCC causes defective centrosome structure, abnormal mitotic progression, and lethal cancer phenotypes.






centrosome overduplication (2, 20), which through wholegenome doubling facilitates chromosomal instability, especiallyinmetastatic tumors (4, 38). However, an important drawback ofthese studies (3, 4) is that themechanism of tetraploidization andunderlying biological causes has remained unresolved. We pro-vide evidence that centrosome linker genes might be altered dueto imbalanced genetic defects, interfering with protein complexesrequired for the correct assembling of spindle functions in colo-rectal cancer cells (39).

Recently, factors involved in the stabilization and nucleationof microtubules around kinetochores have been described inBRAF-mutant colorectal cancer cells, highlighting the potentialto make these tumors vulnerable to microtubule-destabilizinganticancer drugs (40). Other studies have showed that thecentrosomal linker genes and microtubule motor proteinscooperate to keep unlinked centrosomes in relative close prox-imity (41). Therefore, cumulative defects in these pathwaysmay result in spindle perturbations, providing an explanationfor the observed mitotic errors after CROCC depletion. Thefrequency of CROCC mutations in other tumors with MSI isunknown. However, exploration of cBioportal databaserevealed a prevalence of CROCC mutations in cancers withhigh mutational load. In contrast, 1p36.13 deletions appearedto be characteristic of liver, skin, or uterine carcinosarcoma withhigh levels of genomic instability.

Our findings underline that in CIN-negative cancer cells withfunctionally compromised centrosomes (i.e., BRAF-mutantcolorectal cancer cells), CROCC depletion leads to monopolarspindle DNA segregation defects exacerbating mitotic errorsand promoting rhabdoid morphology. Therefore, upregulationof CROCC in classical colorectal cancer particularly in MSItumors, may provide a mechanism of protection to potentiallydeleterious genetic changes (39, 40). CROCC restoration in ametastatic model with 1p36.13 deletion confirmed its role as abiological barrier against mitotic errors. In agreement with this,colon cancer cells with 1p36.13 deletion display have increasedsensitivity in vitro to microtubule-stabilizing agents used inpediatric tumors (42). However, we were unable to demon-strate the detailed molecular mechanism by which independentCROCC defects promote gross mitotic errors. In addition, otherfactors not present in our current models can influence rhab-doid pathogenesis, especially in MSS tumors. In fact, CROCCdeletion was recurrent in CIN-high cancer cells without rhab-doid characteristics, supporting the concept that rhabdoid traitsare highly heterogeneous as consequence of multiple dysregu-lated developmental pathways. The patients with rhabdoidcolorectal cancer described in our study presented lethal clinicaloutcomes with an average postoperative survival of only 7months. Therefore, the recurrent CROCC genetic deletionsidentified in these patients may be associated with the poorprognosis. From this point of view, identifications of newmolecular subgroups cannot be excluded. For example, we donot know whether CROCC deletion is an unfavorable prog-nostic only in BRAF-mutant tumors or other subtypes withSMARC gene mutations (SMARCB1, SMARCA4). So far, muta-tions in centrosome genes like CEP57, CEP135, and PLK4

kinase, have been only described in rare genetic disorders withgenomic instability such as microcephaly and Seckel syndrome(43, 44).

Overall, our data uncover a mechanism by which defects ofcritical centrosomal components cause unequal DNA segregationthat contributes to the ongoing genetic heterogeneity in rare andaggressive colon cancers. Our findings link for the first timecentrosomal cohesiondefects and genomic instability, promptingfor studies addressing how genetic centrosome anomalies areconnected with key pathways involved in safeguarding the integ-rity of the human genome.

Disclosure of Potential Conflicts of InterestM. Garonzi is an employee at Menarini Silicon Biosystems. M. Delledonne

has ownership interest (including patents) and is a consultant/advisory boardmember for Personal Genomics Srl. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: A. Remo, P. Parcesepe, M. PancioneDevelopment of methodology: A. Remo, P. Parcesepe, E. Baritono, F. LonardoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Remo, E. Manfrin, U. Mickys, D. Malanga,E. Baritono, E. Molinari, M. Delledonne, G. Giordano, C. Ghimenton, F. Grillo,L. Mastracci, V. Colantuoni, A. Scarpa, M. PancioneAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Remo, P. Parcesepe, A. Ferrarini, C. Laudanna,M. Simbolo, D. Malanga, D.M. Oliveira, M. Garonzi, M. Delledonne,F. Lonardo, F. D0angelo, M. Ceccarelli, V. Colantuoni, A. Scarpa, M. PancioneWriting, review, and/or revision of the manuscript: A. Remo, P. Parcesepe,J. Giuliani, L. Xumerle, G. Giordano, F. Grillo, G. Viglietto, A. Scarpa,M. PancioneAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E. Manfrin, H.S. Han, G. Giordano, F. Grillo,M. PancioneStudy supervision: A. Remo, A. Scarpa, M. PancioneOther (performed the majority of in vitro experiments): T. ColangeloOther (conducted some of the experiments to analyze CROCC expressionin several CRC cell lines and was incharge of the cell lines used in thelaboratory): L. Sabatino

AcknowledgmentsThe authors thank L. Cerulo, Department of Sciences and Technologies,

University of Sannio (Benevento, Italy) and G. Falco, Department of Biology,University of Naples, Federico II (Naples, Italy) for commenting on themolecular/clinical aspects of the manuscript and for helpful discussions,Roberta Maestro (CRO, Aviano, Italy) for her kind gift of the BJ human skinfibroblasts and G401 cells and for helpful discussions, Erich Nigg for his kindgift and suggestions about clone 6150861 pEGFP Rootletin, and ARC-NETResearch Centre core imaging facility for assistance with microscopy. T. Colan-gelo is supported by a fellowship from Associazione Italiana Ricerca sul Cancro(AIRC; project code: 19548). This work was supported by Department Funds ofMater Salutis Hospital, FUR, and the ItalianMinistry of University and Research(MiUR; to M. Pancione), and AIRC 5 � 1000 (no. 12182; to A. Scarpa).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 23, 2018; revised March 6, 2018; accepted May 1, 2018;published first May 21, 2018.

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2018;16:1385-1395. Published OnlineFirst May 21, 2018.Mol Cancer Res Andrea Remo, Erminia Manfrin, Pietro Parcesepe, et al. Rhabdoid Phenotypes and Lethal Colorectal Cancers

induced Tetraploid Segregation Errors Link−Centrosome Linker

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