Centrosome Linker-induced Tetraploid Segregation Errors Link …download.xuebalib.com/1gxkXRB9dc5M.pdf · 8 Claudio Ghimenton2 Fortunato Lonardo13, Fulvio D’angelo14, Federica Grillo15,

1

Centrosome Linker-induced Tetraploid Segregation Errors Link 1

Rhabdoid Phenotypes and Lethal Colorectal Cancers 2

3 Andrea Remo

1†, Erminia Manfrin

2†, Pietro Parcesepe

2†, Alberto

Ferrarini

3, Hye Seung Han

4, 4

Mickys Ugnius5, Carmelo Laudanna

6, Michele Simbolo

2, Donatella Malanga

6, Duarte Mendes 5

Oliveira6, Elisabetta Baritono

1, Tommaso Colangelo

7, Lina Sabatino

8, Jacopo Giuliani

1, Enrico 6

Molinari1, Marianna Garonzi

9, Luciano Xumerle

9, Massimo Delledonne

9,10, Guido Giordano

11,12, 7

Claudio Ghimenton2 Fortunato Lonardo

13, Fulvio D’angelo

14, Federica Grillo

15, Luca Mastracci

15, 8

Giuseppe Viglietto6, Michele Ceccarelli

8,14, Vittorio Colantuoni

8, Aldo Scarpa

2,16* and Massimo 9

Pancione8,17*

. 10 11 1Pathology Unit, “Mater Salutis” Hospital AULSS9, Legnago (Verona), Italy;

2 Department of 12

Diagnostics and Public Health – Section of Pathology, University and Hospital Trust of Verona, 13 Verona, Italy;

3 Menarini Silicon Biosystems S.p.A, Bologna, Italy,

4Department of Pathology, 14

Konkuk University School of Medicine, Seoul, Korea; 5National Center of Pathology, Affiliate of 15

Vilnius University Hospital Santariskiu Clinics, Vilnius, Lithuania; 6

Department of Experimental 16 and Clinical Medicine "Gaetano Salvatore", University “Magna Grecia”,Catanzaro, Italy;

7Institute 17

for Stem-cell Biology, Regenerative Medicine and Innovative Therapies (ISBReMIT), Casa 18 Sollievo della Sofferenza-IRCCS, San Giovanni Rotondo, Italy;

8Department of Sciences and 19

Technologies, University of Sannio, Benevento, Italy; 9Functional Genomics Center, Department of 20

Biotechnology, University of Verona, Verona, Italy; 10

Personal Genomics S.r.l., Verona, Italy; 21 11

CRO Aviano National Cancer Center, Aviano, Italy; 12

Medical Oncology Unit, San Filippo Neri 22 Hospital, Rome, Italy;

13Medical Cytogenetics and Molecular Genetics Unit, AORN "Gaetano 23

Rummo," Benevento; 14

Bioinformatics Laboratory, BIOGEM scrl, Ariano Irpino, Avellino, Italy; 15

24 Department of Surgical and Diagnostic Sciences (DISC), University of Genova and S. Martino 25 Polyclinic Hospital, Genova, Italy;

16ARC-Net Centre for applied research on cancer, University 26

and Hospital trust of Verona, Verona, Italy; 17

Department of Biochemistry and Molecular Biology 27 II, Faculty of Pharmacy, Complutense University, Madrid, Spain. 28 29 † These authors contributed equally to this work. 30

31 *Correspondence should be addressed to: Massimo Pancione, ([email protected]) 32

Department of Sciences and Technologies, University of Sannio, Via Port’Arsa, 1182100 33 Benevento, Italy; Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, 34 Complutense University, Madrid, Spain. Phone: +39 0824 305157; +34913941785 Fax +39 0824 35 305147 or Aldo Scarpa, ([email protected]), ARC-NET Research Centre, Policlinico GB 36 Rossi, Piazzale L.A. Scuro, 10, Phone.: +39 045 8124043, Fax:+39 045 8127432, 37 38 Running title: Centrosome Cohesion Anomalies and Cancer via CROCC Defects 39 40 Key words: Centrosome, rhabdoid Colorectal cancer, Aneuploidy, tumor heterogeneity 41 42 DISCLOSURE OF COMPETING INTERESTS: The authors declare that they have no 43 competing interests. 44 45 46 47 48

49

50

on May 23, 2018. © 2018 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

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mailto:[email protected]


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2

Abstract 51

Centrosome anomalies contribute to tumorigenesis but it remains unclear how they are generated in 52

lethal cancer phenotypes. Here, it is demonstrated that human microsatellite instable (MSI) and 53

BRAF(V600E) mutant colorectal cancers with a lethal rhabdoid phenotype are characterized by 54

inactivation of centrosomal functions. A splice site mutation that causes an unbalanced dosage of 55

rootletin (CROCC), a centrosomal-linker component required for centrosome cohesion and 56

separation at the chromosome 1p36.13 locus, resulted in abnormally shaped centrosomes in 57

rhabdoid cells from human colon tissues. Notably, deleterious deletions at 1p36.13 were recurrent 58

in a subgroup of BRAF(V600E) mutant and microsatellite stable (MSS) rhabdoid colorectal cancers 59

but not in classical colorectal cancer or pediatric rhabdoid tumors. Interfering with CROCC 60

expression in near-diploid BRAF(V600E) mutant/MSI colon cancer cells disrupts bipolar mitotic 61

spindle architecture, promotes tetraploid segregation errors resulting in a highly aggressive 62

rhabdoid-like phenotype in vitro. Restoring near-wild-type levels of CROCC in a metastatic model 63

harboring 1p36.13 deletion results in correction of centrosome segregation errors and cell death, 64

revealing a mechanism of tolerance to mitotic errors and tetraploidization promoted by deleterious 65

1p36.13 loss. Accordingly, cancer cells lacking 1p36.13 display far greater sensitivity to 66

centrosome spindle pole stabilizing agents in vitro. These data shed light on a previously unknown 67

link between centrosome cohesion defects and lethal cancer phenotypes providing new insight into 68

pathways underlying genome instability. 69

Implications 70

Mis-segregation of chromosomes is a prominent feature of chromosome instability and intra-71

tumoral heterogeneity recurrent in metastatic tumors for which the molecular basis is unknown. The 72

present study provides insight into the mechanism by which defects in rootletin, a centrosome linker 73

component causes tetraploid segregation errors and phenotypic transition to a clinically devastating 74

form of malignant rhabdoid tumor. 75

76




3

INTRODUCTION 77

The century-old hypothesis on the relationship between centrosomes and cancer, formulated by the 78

German embryologist Theodor Boveri more than 100 years ago (1,2), remains unanswered. 79

Centrosome abnormalities, consisting usually in increased numbers, are common in human tumours 80

(3), and experimentally induced tetraploid cells from extra centrosomes can be critical for 81

aneuploidy and metastatic progression of malignancy (3,4). However, insufficient progress has been 82

made in our knowledge on genetic defects underlying centrosome anomalies in tumourigenesis (1-83

4). In this scenario, the rare and lethal pathological variant of common colorectal cancers showing 84

rhabdoid phenotype (5-7), is of particular interest as it features recurrent mitotic anomalies of 85

enigmatic origin (8-10). We thus hypothesized that the systematic study of rare rhabdoid colorectal 86

cancers (RC), could provide insights into biological mechanisms responsible for the generation of 87

genome instability and reveal key factors for the development of aggressive disease entities. To test 88

this idea, we performed whole exome sequencing of two RC and discovered an enrichment of 89

centrosome anomalies and inactivation of ciliary rootlet coiled-coil (CROCC) gene (11,12), a 90

structural component of the centrosome linker which assembles and keeps connected the two 91

centrioles. Centrosomal alterations were assessed in an expanded series of rare RCs and related 92

tumours, and functionally characterized in colorectal cancer cellular models. 93

94 95 MATERIALS AND METHODS 96 97 Materials and Methods and any associated references as a continuation of the main text are 98

described more in detail within the supplementary material. 99

Patient and tissue cohort. 100

This study was conducted in accordance with Declaration of Helsinki ethical guidelines. It was approved by 101

an institutional review board, approval n. 997CESC from the Ethics Comittee (Comitato Etico di Verona e 102

Rovigo dell’Azienda Ospedaliera Universitaria Integrata) on 7 September 2016, documented by the CESC 103




4

prot. 42160 on 9 September 2016 and formalized by the General Manager with deliberation n. 458 of 16 104

September 2016, communicated with protocol 51319 on 23 September 2016. 105

Formalin-fixed paraffin-embedded (FFPE) samples from 7 cases of primary rhabdoid colorectal 106

cancers (RC) and matched normal colonic mucosa were studied (cases RC1 to RC7 Supplemental 107

Table 1). Moreover, an independent validation series to screen the mutational status of newly 108

identified genes was analyzed (cases RC8 to RC12 Supplemental Table 1). FFPE samples from 7 109

rhabdoid tumours arised in central nervous system of patients between 2 months and 19 years of age 110

were collected from the files of the Azienda Ospedaliera Universitaria Integrata, Verona, Italy 111

These pediatric/young adults rhabdoid tumours are indicated as Rhabdoid of infancy (RI) 112

throughout the article. Two independent datasets of patients with classic type sporadic colorectal 113

cancer were analyzed: Dataset A including 141 primary cancers and Dataset B including 102 114

primary cancers. 115

Cell lines. 116

Human colon cancer cell lines HCT116, HT29, CaCo-2, LoVo, RKO, T84, DLD1, SW480 and 117

SW620 were purchased from American Type Culture Collection (ATCC). BJ human fibroblasts and 118

G401 cells derived from normal foreskin and pediatric rhabdoid tumour were used as a non-119

neoplastic control and a pure rhabdoid model, respectively. 120

Whole-Exome Sequencing. 121

Whole-exome sequencing with 100-bp paired reads was performed with a HiSEQ1000 (Illumina), 122

using 1.3 µg genomic DNA (based on fluorometric Picogreen dsDNA quantification) and 123

enrichment for whole exome according to TruSeq Exome Enrichment Guide (Illumina). 124

Functional in vitro assays 125

RKO cells were transiently transfected with SureSilencing control or CROCC shRNA expression 126

plasmids KH23140P (Qiagen) containing the puromycin resistance cassette. After selection 127

puromycin (Thermofisher), single colonies were amplified and assessed for efficient CROCC 128

silencing by quantitative PCR (qPCR) and western blot, respectively. HT29 and T84 cells were 129




5

transfected with the full-length CROCC coding sequence or a truncate form (1–494aa) cloned with 130

GFP epitope or GFP alone (used as control). For long-term experiments CROCC-GFP+ cells were 131

maintained in 0.6 mg/ml of G418. 132

Statistical analysis 133

Data are presented with mean, medians and ranges. The P values were calculated two sided. 134

Statistical analyses were conducted by GeneSpring R/bioconductor v.12.5 and R based package, 135

SPSS v15 and GraphPad Prism 5. 136

137

138

RESULTS 139

Discovery of CROCC mutations and centrosome anomalies 140

Two previously reported primary BRAFV600E

mutant RCs (RC1 and RC2) (9,10), harboring 141

microsatellite instability due to defective DNA mismatch repair (MMR) machinery caused by 142

promoter methylation of the MLH1 gene, were subjected to whole exome sequencing (WES) using 143

DNA from formalin-fixed paraffin-embedded (FFPE) matched tumour/normal samples 144

(Supplemental Table 1). We detected an exceptionally large number of somatic point mutations, 145

1056 and 1078 per 106

bases for RC1 and RC2, respectively, which is consistent with the presence 146

of MMR defects (13-15) (Figure 1a). About 1/5 of mutations occurred within CpG dinucleotide 147

context as seen in classical colorectal cancers (14). Transitions were more frequent than 148

transversions (71.8% vs 28.2%, Supplemental Fig. 1a) with a dominance of C>T/G>A, T>C/A>G 149

transitions (Supplemental Fig. 1b) which is characteristic of the mutational signature due to 150

alterations of MMR mechanisms (signature 6) (13). The most prevalent single nucleotide variants 151

(SNVs) were non-silent mutations (14), where over 90% of potentially damaging mutations were 152

missense and around 10% were splicing, stop-gain, stop-loss or, rarely, frameshift insertions or 153

initiation codon mutations (Figure 1a and Supplemental Fig. 1c,d). The two RC cases shared 112 154

(10%) mutated genes (Supplemental Fig. 2a). By applying DrGaP computational tool (16), which 155




6

allows to infer cancer driver genes, a number of potential candidate disease-causing genes were 156

identified (Supplemental Table 2), ~half of which (45%), were enriched for 157

cytoskeleton/centrosome and microtubule biological functions (Supplemental Fig. 2b,c). 158

The search of candidate genes in the cancer genome atlas (TCGA) database (13-15) 159

comprising 224 sequenced classical colorectal cancers (http://www.cbioportal.org) revealed that the 160

majority (65%) of the candidates had a low frequency of mutations (≤4% of cases). Strikingly, 161

among these, only for CROCC gene mapping to 1p36.13 (11,12) involved in centrosome cohesion 162

and disjunction, no somatic mutations (0/224; 0%) were reported (Figure 1b). Notably, 1p36 163

deletions are recurrent in neuroblastoma, Wilms' tumor and medulloblastoma. In our two RC cases, 164

CROCC harbored two missense mutations, p.Ala161Ser (c.481G>T, Exon 4) and p.Val1885Ala 165

(c.5654T>C, Exon 35), and one prominent splicing mutation at the conserved 3’ acceptor splice site 166

(c.3705-2A>G) in the intron between exons 25-26 (Figure 1c). A review of multiple CRC 167

sequencing datasets (N=2070) revealed CROCC mutations in (1,4% of cases). However, although 168

of unknown significance none of the CROCC mutations was identified as putative driver mutations 169

in colorectal cancer (Supplemental Fig. 2d). SMARCB1 and SMARCA4 mutations, which have 170

been associated with rhabdoid phenotype (6), showed a trend of mutual exclusivity with CROCC 171

alterations. However, only putative truncating driver mutations in SMARCB1 and/or SMARCA4 172

correlated with tumour poor differentiation and short-time metastatic progression. Therefore, we 173

reasoned that the splicing mutation detected in RC1 might be causally correlated with rhabdoid 174

phenotype. Indeed, the mutation reduced the strength of the physiologic acceptor site, causing a 175

large deletion of the CROCC coding region involving exons 23-31 (17,18) (Supplemental Fig. 3a). 176

Independent RT-PCR-derived products spanning exons 5-7 and 33-35 showed that the expression 177

of CROCC in the tumour samples RC1 containing the splicing mutation was reduced, thus 178

suggesting the alteration of the mature transcript by the utilization of cryptic splice sites or by the 179

activation of the nonsense-mediated mRNA decay pathway, which impair transcripts harboring 180

large deletions (Supplemental Fig. 3b) (17). Unexpectedly also RC2, displayed expression levels 181



http://www.cbioportal.org/


7

in tumour lower than in normal tissue, supporting a role for defective CROCC expression in 182

rhabdoid tumours (Figure 1d). We next used immunohistochemistry and immunofluorescence at 183

high magnification with an anti-CROCC antibody to analyze the centrosomes in rhabdoid cancers 184

and matched normal tissues (Supplemental Table 3). We found that nearly 50% of tumour cells 185

had no CROCC immunolabelling, and the presence of cells with a single and often abnormally 186

shaped, larger (up to 6-fold greater than normal) or fragmented centrosomes, suggesting the 187

presence of numerical and structural centrosome aberrations (Supplemental Fig. 3b). We also 188

observed dramatic and uncommon cytological defects, such as anucleated cells having larger 189

centrosomes positive for CROCC associated to mitotic catastrophe in late telophase particularly in 190

RC1 harboring the splicing site mutation (Figure 1d). We used the pericentriolar material (PCM) 191

component γ-tubulin, as our reference marker for immunolabelling experiments, because it 192

consistently colocalizes with centriole markers which are closely connected in interphase by the 193

centrosomal linker (1,2,11,12). Moreover, γ-tubulin has been proposed as a marker to identify 194

spindle poles (19,20). We observed a remarkable loss of cell polarity in interphase nuclei and 195

abnormal mitotic figures many of which included asymmetric bipolar or monopolar spindles. 196

Rhabdoid cells showed a diffuse staining of γ-tubulin into the cytoplasm and reduced centrosomal 197

localization, a phenomenon described in tumors with high metastatic potential (19) (Supplemental 198

Fig. 3c,d). Double immunofluorescence analysis using antibodies directed against CROCC and γ-199

tubulin confirmed these observations and revealed cells either with fragmented/larger centrosomes 200

or with a consistent loss of centrosome staining (Supplemental Fig. 3). These experiments 201

indicated that genetic defects in CROCC and other centrosome components may compromise 202

centrosome function in RC. 203

A validation set of 10 additional rhabdoid colorectal cancers was studied, including 3 cases 204

(RC7, RC9, RC11) with microsatellite instability due to MLH1 promoter methylation and 7 cases 205

(RC3-6, RC8, RC10, RC12) with stable microsatellites (Figure 2a and Supplemental Table 1). 206

Targeted sequencing identified three CROCC mutations (p.Ser1320Ile, p.Arg1659His and 207




8

p.Ala1510Thr) of unknown significance in additional 2 cases (RC9 and RC11) harboring 208

microsatellite instability (1,2,11,12). Indeed, the 24 CROCC mutations identified across cbioportal 209

database were more recurrent in MSI (12/24; 50%) than in MSS (6/24; 25%) CRCs. Notably, 210

CROCC mutations were classified as missense (n=22) or truncating mutations (n=2) of unknown 211

significance but associated with both well- differentiated and early-stage classical CRCs. In our RC 212

dataset, 5 of the remaining cases for which sufficient material was available (cases RC3 to RC7) 213

harbored loss of heterozygosity (LOH) at the 1p36.13 locus, where CROCC resides, which was 214

associated to mRNA below normal levels (Figure 2b). In keeping with the findings in RC1 and 215

RC2 cases, comparable levels of centrosomes defects and a high prevalence of bizarre mitotic 216

figures and/or cytomorphologic aberrations were evident in all tumours (Figure 2c and 217

Supplemental Table 3). Analysis of independent CRC databases (N=1387) for which copy number 218

alterations were available (http://www.cbioportal.org) revealed no alteration at 1p36.13 locus, 219

suggesting CROCC impairment as a consequence of reduced gene dosage (21) caused by allelic 220

deletion. As centrosome anomalies are intimately connected with chromosome segregation errors 221

(1,3,20), we assessed DNA content in tumour samples (cases RC1 to RC7, Supplemental Table 222

3). Remarkably, we observed recurrent ploidy abnormalities mainly consisting of triploid or near-223

tetraploid cells ranging from 10% to 40% of tumour cells (Figure 2d). Globally, these results 224

indicated that centrosome defects underlie RC pathogenesis. 225

226

Centrosome and genomic profiling of rhabdoid tumours of infants 227

Insight into genetic characterization of rhabdoid neoplasms are limited to the so called 228

extrarenal rhabdoid tumours arising in children, in which inactivating mutation and/or deletion of 229

the chromatin remodelling gene SMARCB1 (INI1) and low mutation load have been reported (6,22-230

24). We analyzed 7 cases of this tumour type, hereafter named Rhabdoid of infant (RI), for 231

centrosome and molecular anomalies (Figure 3a and Supplemental Table 4). Compared to RCs, 232

analysis in pediatric tumours was associated with much higher CROCC mRNA expression levels 233



http://www.cbioportal.org/


9

than matched normal tissues (P<0.00001; Figure 3b). Target-sequencing identified no mutations (0 234

of 7 tumours) in the CROCC gene. Moreover, CROCC immunostaining was seen as a single and 235

large signal adjacent to the nuclei in almost the totality of the cells (80%), only 10% of cells had no 236

centrosome staining (Figure 3b and Supplemental Table 4). Consistent with literature, the 237

genetic profile of RI revealed missense or truncating mutations in SMARCB1 (5/7, 71%) (6,23,24) 238

and/or TP53 (3/7, 42%) accompanied by a near diploid DNA content and less aggressive clinical 239

course when compared to RCs (Figure 3c-e). This suggested that RIs, which are characterized by 240

SMARCB1 (INI1) mutation, did not harbor any CROCC alteration and did not display the 241

centrosomal defects observed in RC. Inspection of an available database from pediatric rhabdoid 242

cells (25) confirmed that both mutations or genetic deletion affecting CROCC locus were infrequent 243

(2/20, 10%), whereas the transcript profile tended to be similar to our RI dataset (Figure 3c). 244

Therefore, rhabdoids arising in colorectal cancer although morphologically indistinguishable from 245

their pediatric counterparts demonstrate distinct molecular, cytogenetic and centrosomal 246

aberrations. 247

248

Centrosome and CROCC expression in classical colorectal cancers 249

We screened 242 primary classic colorectal cancers (CRCs), comprising two independent 250

series of 140 (dataset A) (26,27) and 102 (dataset B) (28,29) cases for CROCC mRNA and protein 251

expression (Supplemental Table 5). CROCC mRNA expression levels were higher in CRCs than in 252

normal colonic mucosa. However, no significant protein expression change in tumour tissues compared to 253

that in normal mucosae was detected (Supplemental Fig. 4a). In cohort A, using 254

immunohistochemistry and immunofluorescence against CROCC and γ-tubulin, we found round 255

and uniform in size centrosomes, prevalently in normal number (1-2 per cell; 97/140, 69.3%), 256

supernumerary (>2 per cell; 39/140, 27.8%) and only few (<1 per cell; 4/140, 2.9%) displayed 257

reduced centrosome labeling (Supplemental Fig. 4b). Centrosome abnormalities, particularly 258

supernumerary centrosomes were more prevalent in advanced stage (stage III-IV) than in low stage 259




10

(stage I-II) lesions (Supplemental Fig. 4c). In cohort B which was enriched for stage III-IV 260

tumours (79.5% of cases), we confirmed a high prevalence of supernumerary (60/102; 59%) 261

(1,3,20) or defective (11/102; 11%) centrosomes associated to poorer clinical course than those 262

expressing a normal pattern (31/102; 30%, HR=0.30; 95% CI (0.21-0.81); P<0.0001) in keeping 263

with the notion that numerical centrosomal abnormalities are more common in invasive cancers 264

(1,3,20) (Supplemental Fig. 4c). To independently validate the pattern of gene expression changes 265

detected in our datasets, we analyzed the patient-matched tumour-normal expression data available 266

from the TCGA (14) and three independent datasets GSE20916 (30), GSE41258 (31), GSE30540 267

(32) of classical colorectal cancer (Supplemental Fig. 4d). CROCC mRNA was up-regulated in 268

CRC compared to normal only in TCGA database. The analysis of other datasets revealed an 269

heterogeneous expression pattern, while CROCC up-regulation compared to normal control not 270

reached statistical significance. The analysis of GSE30540 (32) dataset, for which both 271

transcriptomic data and degree of chromosome instability (CIN) were available, revealed that 272

CROCC expression levels tended to be lower in CIN-high than in CIN-low tumours (Supplemental 273

Fig. 4d). These data suggested thus the possibility that imbalanced genetic defects at CROCC locus 274

may be related to marked anomalies in the fidelity of chromosome segregation. 275

276

CROCC depletion impairs mitosis and induces rhabdoid-like features 277

We next sought a genetic basis for the relation between CIN and rhabdoid phenotype by 278

examining whole exome sequencing and transcriptomic data from The Cancer Cell Line 279

Encyclopedia (CCLE) (25). Unexpectedly, data from a collection of 60 CRC cell lines revealed that 280

the deletions at 1p36.13 locus tended to be more prevalent in CIN-high (23.6%, 9 of 38) compared 281

to CIN-low (9.1%, 2 out of 22) cells (Supplemental Fig. 5a). However, cell lines with 1p36.13 282

deletion displayed neither rhabdoid phenotype nor BRAF mutations. As expected, compared to 283

cells retaining 1p36.13 locus, those harboring the deletion revealed a gene-expression signature 284

significantly enriched for pathways implicated in chromosomal instability (33,34) (Supplemental 285




11

Fig. 5b,c). In a panel of CRC cells, we then confirmed that both CROCC mRNA and protein 286

expression levels were concordant and higher in CIN-low than in CIN-high cell lines (P<0.05, 287

Supplemental Fig. 6a). CIN-low cells showed centrosomes stained for CROCC and γ-tubulin that 288

were structurally indistinguishable from those in normal human fibroblasts BJ, consistent with 289

literature (35) (Supplemental Fig. 6a,b). In line with this, CIN-high displayed a higher frequency 290

of micronuclei and nuclear γH2AX foci than CIN-low cells (29) (Supplemental Fig. 6c,d). This 291

suggested that CROCC might be a CIN-suppressor gene and its deletion in CIN-low/BRAF mutant 292

cell lines might be permissive for abnormal phenotypes. Therefore, we reasoned that RKO cells, 293

sharing a near-diploid karyotype, BRAF(V600E)

mutation/MSI and alterations in 294

microtubule/centrosomal components with RC (14), could be an excellent system to explore 295

CROCC silencing in vitro. We found that the clone sh4, hereafter named CROCCKD

, provided a 296

stable and consistent knockdown of CROCC transcript to more than 75% and protein to 3.3-fold 297

lower then RKO cells transfected with control vector (shCon), achieving nearly comparable levels 298

to those seen in vivo (Supplemental Fig. 7a). Previous studies have demonstrated that CROCC 299

knockdown in non-transformed cells causes centriole splitting and increases centrosome separation 300

(11,12). By using γ-tubulin and centrin as reference markers, we observed a consistent PCM 301

fragmentation after CROCC depletion which resulted in abnormal chromosome segregation and 302

higher frequency of monopolar spindles as compared to control (Figure 4a and Supplemental Fig. 303

7b). Importantly, monopolar spindles displayed larger or “fragmented” centrosomes which 304

accounted for 85% of the abnormal phenotype (Figure 4a-c and Supplemental Fig. 7c). Thus, 305

BRAF-mutant/MSI CRC cell lines in which centrosome and microtubule stability is damaged by 306

genetic hypermutation, CROCC depletion may determine a major impact in the progression of 307

mitotic errors (36-38). Consistently, an increased frequency of micronuclei (median 11% 308

CROCCKD

versus 1% ShCon cells P=0.0003) and γH2AX nuclear foci (median 43% CROCCKD

309

versus 18% ShCon cells P=0.011 were observed (Figure 4c, and Supplemental Fig. 7d). 310

Metaphase karyotyping revealed that CROCC deficiency leads to an increased number of tetraploid 311




12

(4N) cells (median 13.3% CROCCKD

versus 3.51% ShCon cells P=0.001) characterized by 312

prominent and larger nuclei than diploid (2N) cells. Consistently, analysis of centromeric probes in 313

intephase nuclei confirmed tetraploidy (Figure 4c). The number of CROCC-deficient cells was 314

reduced in G0/G1 or G2/M phases when compared to the wild-type population (by 26–45%, FACS 315

analysis), suggesting an impaired cell cycle progression as a consequence of misaligned 316

chromosomes (1-3,33) (Supplemental Fig. 7d). By contrast, cells grown under replication stress 317

conditions (serum deprivation) resulted in higher proliferation rate than control cells (34) (Figure 318

4d). Most strikingly, CROCC-deficient cells exhibited all cardinal signs of rhabdoid features (8-10), 319

displaying huge nuclei pushed to the periphery of the cells with single or multiple large nucleoli 320

associated with eosinophilic cytoplasmic inclusions and large cellular protrusions resembling the 321

morphology observed in vivo (Figure 4d and Supplemental Fig. 8a). These features resulted in 322

the activation of pro-metastatic genes involved in epithelial mesenchymal transition accompanied 323

by a dramatic change of spindle-shaped morphology (4,7) consistent with the enhanced metastatic 324

potential of rhabdoid phenotype (Supplemental Fig. 8a,b). Expression of exogenous green 325

fluorescent protein (GFP)-tagged CROCC (1–2018aa) rescued these phenotypic changes induced by 326

depletion of endogenous CROCC (Supplemental Fig. 8b) (11). In line with previous results 327

(11,12), we observed no alteration in cell cycle profile or aberrant phenotypic changes after CROCC 328

depletion in non-transformed BJ cells. Therefore, CROCC depletion in BRAF-mutant near-diploid 329

cancer cells induces tetraploidization and rhabdoid phenotype in vitro. 330

331

Tolerance to mitotic errors and tetraploidization promoted by 1p36.13 deletion 332

As colorectal cancer cells with driver mutations in CROCC have not been reported, to test the 333

hypothesis that CROCC impacts tumour growth and centrosome-related mitotic errors, we analyzed 334

metastatic colorectal cancer T84 cells harboring an allelic deletion at 1p36.13 locus (25). Although 335

T84 are well-differentiated cancer cells and do not show rhabdoid morphology, they however 336

exhibit some of the characteristics detected in RKO CROCC-depleted cells. Consistent with 337




13

reduced CROCC endogenous activity, we observed an increased rate of micronuclei, tetraploid or 338

near-tetraploid cells and recurrent mitotic errors resulting essentially in “monopolar spindles, which 339

were more recurrent under replication stress conditions (Figure 5a and Supplemental Fig. 8c,d). 340

We then investigated the localization of CROCC in the centrosome by immunofluorescence. 341

Almost half of the cells (40%) revealed a faint CROCC signal, which was consistently accompanied 342

by atypical γ-tubulin aggregates prevalently in cells with mitotic anomalies (Figure 5a). Most 343

strikingly, such aberrations were rarely, if ever, detected in pediatric rhabdoid G401 or colon cancer 344

cell lines with an intact 1p36.13 locus (Supplemental Fig. 8c,d). Therefore, we transfected 345

CROCC-GFP and GFP alone (control) into T84 cells. Restoration of CROCC, determined a 346

dramatic decrease of cell viability (12 days later, 0%) as compared to control plasmid. Similarly, 347

we detected a higher number of G0/G1 cells than control (41% vs 26%; P=0.0018, Figure 5b,c). 348

Gain of CROCC conferred a flat/adherent phenotype and formation of filament-like structures co-349

localizing with γ-tubulin resulting in an expression of mesenchymal genes lower than in control (4) 350

(Supplemental Fig. 9a). Accordingly, we detected a 4-fold decrease of tetraploid cells, and reduced 351

γH2AX foci from 59% to 22% with respect to control cells (Figure 5c) raising the possibility that 352

the centrosome spindle pole integrity is strongly affected by 1p36.13 deletion. To see if T84 cells 353

are sensitive to mitotic drugs, we mined the data from the Genomics of Drug Sensitivity in Cancer 354

project (Sanger panel). As shown in (Supplemental Fig. 9b), among 221 molecules tested, IGF1-R 355

inhibitor (linsitinib) and Epothilone B a microtubule stabilizing agent, were significantly effective 356

in T84 lines. Accordingly, we observed a significant difference in the sensitivity to Epothilone B in 357

1p36.13 deleted cells as compared to cells with an intact 1p36.13 locus. Similar results were not 358

reproduced comparing CIN-low and CIN-high CRC cell lines (Supplemental Fig. 9b). We used 359

another cell line HT29 with an intact 1p36.13 locus to test CROCC restoration. Similarly to T84, 360

we observed a significant decrease of micronuclei in HT29-CROCC-GFP+ cells compared to 361

control. Although gain of CROCC in HT29 increased the cell death, it appeared an essential gene 362

only for T84 cell survival lacking 1p36.13 (Supplemental Fig. 9c). This supported the hypothesis 363




14

that a reduced CROCC dosage promotes defects in spindle-assembly checkpoint. When we repeated 364

the experiments using a GFP-tagged truncated form of CROCC (1–494aa) (11), we observed that 365

this mutant failed to rescue the aberrant growth phenotype and mitotic errors (Supplemental Fig. 366

9d). Consistent with previous findings (11), we did not detect phenotypic changes in non-367

transformed BJ cells transduced with the full-length CROCC construct. Thus, we conclude that 368

DNA segregation errors resulting from impaired centrosome function are driven by reduced 369

CROCC dosage at 1p36.13 locus. 370

371

DISCUSSION 372

Our understanding of the molecular architecture and function of centrosomal linker 373

components in physiological and pathological processes remain rudimentary. Besides CROCC 374

(Rootletin), multiple proteins including C-NAP1 (CEP250), CEP68 and LRRC45 have been 375

implicated in centrosome linker formation and function. CROCC is able to maintain centrosome 376

cohesion in part through inhibition of VHL-mediated Cep68 degradation (36). It has recently been 377

proposed that a vast network of repeating CROCC units with C-Nap1 as ring organizer and CEP68 378

as filament modulator forms the centrosome linker structure (37). We show here that genetic 379

deletion in CROCC, leads to centrosome anomalies resulting in tetraploid DNA segregation errors, 380

providing insights into mechanism by which genome instability contributes to lethal cancers for 381

which no therapies are available (Figure 5d). In addition, we show that rhabdoid colorectal cancers 382

are not genetically related to their pediatric counterparts (22-24), in which we find recurrent 383

SMARCB1 gene alterations but no evidence of centrosome anomalies. Previous studies have 384

revealed that driver genes implicated in human cancer (3,4) can promote centrosome over-385

duplication (2,20) which through whole genome doubling facilitates chromosomal instability, 386

especially in metastatic tumours (4,38). However, an important drawback of these studies (3,4) is 387

that the mechanism of tetraploidization and underlying biological causes have remained unresolved. 388

We provide evidence that centrosome linker genes might be altered due to imbalanced genetic 389




15

defects, interfering with protein complexes required for the correct assembling of spindle functions 390

in colorectal cancer cells (39). 391

Recently, factors involved in the stabilization and nucleation of microtubules around 392

kinetochores have been described in BRAF mutant colorectal cancer cells, highlighting the potential 393

to make these tumours vulnerable to microtubule-destabilizing anticancer drugs (40). Other studies 394

have showed that the centrosomal linker genes and microtubule motor proteins cooperate to keep 395

unlinked centrosomes in relative close proximity (41). Therefore, cumulative defects in these 396

pathways may result in spindle perturbations, providing an explanation for the observed mitotic 397

errors after CROCC depletion. The frequency of CROCC mutations in other tumors with MSI is 398

unknown. However, exploration of cbioportal database revealed a prevalence of CROCC mutations 399

in cancers with high mutational load. In contrast, 1p36.13 deletions appeared to be characteristic of 400

liver, skin or uterine carcinosarcoma with high levels of genomic instability. 401

Our findings underline that in CIN negative cancer cells with functionally compromised 402

centrosomes (i.e BRAF mutant CRC cells), CROCC depletion leads to monopolar spindle DNA 403

segregation defects exacerbating mitotic errors and promoting rhabdoid morphology. Therefore, 404

upregulation of CROCC in classical CRC particularly in MSI tumours, may provide a mechanism 405

of protection to potentially deleterious genetic changes (39,40). CROCC restoration in a metastatic 406

model with 1p36.13 deletion confirmed its role as a biological barrier against mitotic errors. In 407

agreement with this, colon cancer cells with 1p36.13 deletion display have increased sensitivity in 408

vitro to microtubule stabilizing agents used in pediatric tumors (42). However, we were unable to 409

demonstrate the detailed molecular mechanism by which independent CROCC defects promote 410

gross mitotic errors. In addition, other factors not present in our current models can influent 411

rhabdoid pathogenesis specially in MSS tumours. In fact, CROCC deletion was recurrent in CIN-412

High cancer cells without rhabdoid characteristic, supporting the concept that rhabdoid traits are 413

highly heterogeneous as consequence of multiple dysregulated developmental pathways. The RC 414

patients described in our study, presented lethal clinical outcomes with an average postoperative 415




16

survival of only 7 months. Therefore, the recurrent CROCC genetic deletions identified in these 416

patients may be associated with the poor prognosis. From this point of view, identifications of new 417

molecular subgroups cannot be excluded. For example, we don’t know whether CROCC deletion is 418

a unfavorable prognostic only in BRAF mutant tumours or other subtypes with SMARC gene 419

mutations (SMARCB1, SMARCA4). So far, mutations in centrosome genes like CEP57, CEP135 420

and PLK4 kinase, have been only described in rare genetic disorders with genomic instability such 421

as microcephaly and Seckel syndrome (43,44). 422

Overall, our data uncover a mechanism by which defects of critical centrosomal components 423

cause unequal DNA segregation that contributes to the ongoing genetic heterogeneity in rare and 424

aggressive colon cancers. Our findings link for the first time centrosomal cohesion defects and 425

genomic instability, prompting for studies addressing how genetic centrosome anomalies are 426

connected with key pathways involved in safeguarding the integrity of the human genome. 427

428

429

430

431

432

ACKNOWLEDGMENTS: We thank, L. Cerulo, Department of Sciences and Technologies, 433

University of Sannio, Benevento, Italy; and G. Falco, Department of Biology, University of Naples, 434

Federico II, Naples, Italy for commenting on the molecular/clinical aspects of the manuscript and 435

for helpful discussions; Roberta Maestro (CRO, Aviano, Italy) for her kind gift of the BJ human 436

skin fibroblasts and G401 cells and for helpful discussions; Erich Nigg for his kind gift and 437

suggestions about clone 6150861 pEGFP Rootletin; ARC-NET Research Centre core imaging 438

facility for assistance with microscopy. T.C. is supported by a fellowship from Associazione 439

Italiana Ricerca sul Cancro (AIRC) (project code: 19548). This work was supported by Department 440

Funds of Mater Salutis Hospital, FUR and the Italian Ministry of University and Research (MiUR) 441

to M.P., and AIRC 5x1000 (n. 12182) to AS. 442

443

444

445

446

447




17

448

449

450

References 451

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https://www.ncbi.nlm.nih.gov/pubmed/28089774










19

565 566 567 568 569 570 571 572 573 FIGURE LEGENDS 574 575 576 Fig. 1 Whole exome sequencing reveals mutations in CROCC, encoding an essential component of the centrosome 577 linker. 578 a Representative rhabdoid colorectal cancers histopathological images from RC1 and RC2 that were subjected to whole exome 579 sequencing (H&E, haematoxylin and eosin). The graph indicates the total number of somatic mutations per tumor. The circo shows 580 the distribution of non-silent mutations and copy number variations (CNVs) as indicated by the diverse colours; the outer ring 581 indicate the chromosomes. b Prevalence of alterations in the candidate genes harboring somatic mutations in both RC1 and RC2 in 582 224 colorectal cancers of the TCGA database. c CROCC chromosome localization (1p36.13) and organization (from Ensembl, 583 reference transcript ENST00000375541). All 37 exons, are depicted as green vertical bars and introns as horizontal lines. Solid 584 circles indicate the mutations identified in RC1 and RC2. The “proteinaceous linker” is composed of CROCC filaments (black 585 arrow) that physically connect the mother (M) and daughter (D) centrioles surrounded by the pericentriolar material (PCM). At the 586 onset of mitosis (Mi) the linker is disassembled to support the formation of the bipolar mitotic spindle. d Quantification of CROCC 587 mRNA (qPCR) expression levels in tumour and adjacent normal mucosa. Data are mean ± standard deviation (s.d); n=5 biological 588 replicates; **P<0.01, two-tailed Student’s t-test). Representative images of CROCC immunostaining in non-neoplastic colon mucosa 589 and a fallopian tube used as control (red arrow). CROCC immunopositive centrosomes are reduced in number (black arrow) or 590 mispositioned (distant/separated from the nucleus) in RC1, (inset modeled image). Scale bars are reported in each microphotograph. 591

592

593

594

595 Fig. 2 Centrosome anomalies characterize colorectal cancer with rhabdoid phenotype. 596 a Histopathological images from a subset of 5 additional prototypical rhabdoid colorectal cancers (RC), in which are evident rounded 597 eosinophilic cytoplasmic inclusions, eccentric nuclei and prominent nucleoli. Scale bar, 20 µm H&E images. b Mutations for 598 selected driver genes and CpG island methylation (CIMP) profile accompanied by loss of heterozygosity analysis at 1p36.13 locus. 599 CROCC mRNA (qPCR) expression levels in tumours and adjacent normal mucosa, Data are mean ± standard error of the mean 600 (s.e.m); (n=5 biological replicates, P*<0.05, **P<0.01, two-tailed Student’s t-test). c Representative immunohistochemical analysis 601 from case RC5: Cytokeratin-18 (CK18) marks intermediate filaments in an anucleated cell (red arrow); Ki67 reveals abnormal 602 chromosome structures (black arrow); CROCC marks a multinucleated cell (black arrow), anucleated cell (red arrow) or it appears 603 fragmented in a mitotic cell (monopolar spindle, green arrow). Scale bar, 10 μm. Right, quantification of the centrosome phenotypes 604 against CROCC observed in all RCs (n=2 experiments, >500 cells/sample). d Cytogenetic abnormalities (tetraploid signals, red 605 arrows) observed by fluorescence in situ hybridization (FISH) using the centromeric chromosome probes illustrated. Scale bars, 20 606 and 40 µm. Left, ploidy pattern in all RCs (for chromosomes 1, 12 and 17, n=2 experiments, >500 cells per sample). Right, 607 quantification of cells with polyploidy measured as ratio of triploid and tetraploid on diploid cells for each tumour. 608 609 610 611 612 613 Figure 3. Centrosome and cytogenetic aberrations comparison between colorectal and pediatric rhabdoid tumors. a 614 Representative haematoxylin and eosin (H&E) images of pediatric rhabdoid tumors. Scale bar, 50 µm. b Right, 615 immunohistochemical and interphase FISH analysis for the indicated markers. Note that centrosomes are single, larger, uniform in 616 size and close to the nuclei (black arrowhead). Left, quantification of CROCC immunohistochemistry (IHC) and centromeric (CEN) 617 signals (Chr 1 and Chr 17) in pediatric rhabdoid tumors, (>500 cells per tumor) were evaluated, percentages represent mean values 618 from three independent investigators. Quantification of CROCC mRNA (qPCR) expression levels. Each circle represents the mean 619 value of five biological replicates from a single lesion, **P<0.01, two-tailed Student’s t-test. c CROCC expression in pediatric 620 rhabdoid derived cancer cell lines according to copy number alterations and mutational load (Novartis/broad cancer cell lines 621 encyclopedia). d The panel shows the distribution of non-silent missense or truncating mutations for the indicated pathways in 622




20

rhabdoid colorectal cancer (RC) and rhabdoid of infants (RI). e Kaplan-Meier overall survival curve for RI (age class 2 months–19 623 years) and RC (age class 49-83 years). The p value is obtained by the log-rank test is reported in the graph. 624 625 626 627 628 629 630 631 632 633

634 Fig. 4 CROCC depletion induces rhabdoid phenotype exacerbating DNA segregation errors. a Images of RKO cells 635 with stable CROCC depletion (CROCC KD) showing that in mitosis there is an abnormal spindle formation - “monopolar spindles” - 636 as compared to control (CON, left panel). Large (white arrow) or fragmented (green arrow) centrosomes are shown. Cells are stained 637 using immunofluorescent antibodies with different colors as indicated (αTUB, anti-alpha-tubulin antibody; γTUB, anti-beta-tubulin 638 antibody) and nuclei are stained with DAPI (49,6-diamidino-2-phenylindole). Scale bar, 5 µm. Below is a schematic illustration of 639 cells with aberrant spindles (85%). b anaphase bridges (white arrow), multinucleated (green arrow), multilobulated nucleus (red 640 arrow) and fragmented centrosomes (blue arrows) associated with loss of cell polarity and large micronuclei (white arrow) in 641 CROCC depleted cells. Scale bar, 5 µm. c The upper left graph shows the percentage of micronuclei and monopolar spindles (>250 642 cells per cell line, triplicate experiments, P**<0.01,***P<0.001 Mann–Whitney U test). Representative images of metaphase 643 chromosome spreads and cells stained with DAPI and anti-centromere antibody (ACA). Scale bar, 10 µm. The lower left graph 644 shows the quantification of ploidy content at metaphase. Data are mean ± standard error of the mean (s.e.m); (n=5 biological 645 replicates**P<0.01, two-tailed Student’s t-test). The lower right graph shows tetraploid on diploid cells ratio. d The upper graphs 646 report the survival assay with serum supplementation or under replication stress condition “serum deprivation”. Error bars represent 647 mean ± s.e.m, of five independent experiments *P<0.05, **P<0.01, ***P<0.001, two-tailed Student’s t-test. Below are 648 representative cytomorphological changes showing large polygonal cells and eccentric round nuclei with prominent nucleoli (black 649 arrow) and eosinophilic hyaline cytoplasmic inclusions (red arrow). Haematoxylin and eosin (H&E) pictures. 650 651 652 653 654 655 656 657 658 Figure 5. CROCC abrogates centrosome-related mitotic errors in 1p36.13 deleted cancer cells. a Images of T84 cells 659 with a large micronucleus (white arrow) and significantly reduced CROCC staining (enlarged in insets), anaphase bridges (green 660 arrow) or monopolar spindle (red arrow) associated to deficient or fragmented γ-tubulin dots (enlarged in insets). Scale bar, 5 μM. 661 The upper right graph reports the percentage of anaphase showing segregation errors and micronuclei in T84 cells with serum 662 supplementation or serum deprivation (SD) at 12h. Error bars represent mean ± standard error of the mean (s.e.m)*P<0.05, two-663 tailed Student’s t-test). b Representative images of T84 cells transfected with full-length human CROCC-GFP or GFP alone, 664 immunostained for γ-tubulin (red, enlarged in insets). The graph on the right shows the survival of T84 transfected with CROCC and 665 matched control cells maintained in neomycin selection (0.6 µg ml-1) for the indicated time. Viability was assessed by a colony 666 formation assay. The GFP vector was used as a control. Cells were fixed, stained, and photographed after 6 and 12 days of culture. c 667 The left graph reports flow cytometry analysis after 6 days. Error bars represent mean ± s.e.m, of five independent experiments, 668 **P<0.01, ***P<0.001, two-tailed Student’s t-test). Tetraploid on diploid cells ratio after 6 days quantified by metaphases spreads 669 (16 independent experiments for each condition, **P<0.01, two-tailed Student’s t-test). The right graph shows the percentage of 670 𝑦H2AX foci in prometaphase (>250 cells per cell line, ***P<0.001, Mann–Whitney U test). d Schematic representation of rhabdoid 671 colorectal cancer (CRC) progression. In CRC with defective microtubule functions, BRAF (V600E) mutation depletion of CROCC 672 causes defective centrosome structure, abnormal mitotic progression and lethal cancer phenotypes. 673 674 675 676 677 678 679




Somatic mutations

Gain

Nonsynomous SNV

Splicing

Stopgain

Stoploss

Frameshift insertions

Init Codon

CNVs

a

Loss

c

b d

Phosphorylation

and

displacement

G2 Mi

Kinases

Missense

c.481G>T

Exon 4

Splicing

c.3705-2A>G

Exons 25-26

Missense

c.5654T>C

Exon 35

D D

PCM PCM

Proteinaceus linker

CROCC

M

CROCC Structure

Chr 1: 16,921,950-16,972,979 (1p36.13)

Fw strand

Tot_51kb

M

RC2

RC1

0

400

800

1200

RC1 RC2

Somatic

coding mutations

Nu

mb

er

ofm

uta

tio

ns

Rhabdoid Colorectal Cancers

RC2 RC1

50µm

Rhabdoid Colorectal Cancer

RC1

1056 1078

CROCC 0%

KIF6 3%

DNAH12 2%

DNAH7 5%

ENAH 1%

EPPK1 4%

DMD 15%

NEXN 2%

SPIRE2 1%

SPTBN4 5%

TTN 37%

MCM3AP 5%

MUS81 0.5%

ERCC6 5%

PFAS 0.5%

PML 1%

PSMD3 3%

CDC27 3%

CTDP1 1%

KIAA1543 1%

Copy Gain Missense Mutation

Copy Loss Truncating Mutation

Colorectal Cancer TCGA data set N=224

20µm

20µm

20µm

Figure 1

a

50µm

RC2

CR

OC

C

im

munola

belli

ng

Fallopian Tube

20 µm

Control

Normal mucosa NM1

CR

OC

C

im

munola

belli

ng

pooled exons 5-7 and 33-35

0

100

200

300

400R

ela

tive C

RO

CC

m

RN

A l

evels

**

*

NM1 NM2 RC1 RC2

Mitotic

catastrophe

Centrosome

failure




a

c

RC5

k

20 µm 40 µm

RC4

j

20 µm Chr 12 Chr 17

RC1

a

10 µm

Cyto

log

ical

an

om

alie

s

d

Figure 2

0 Negative

1 Single

2 Together

2 Separate

00.10.20.30.40.50.6

RC

1R

C2

RC

3R

C4

RC

5R

C6

RC

7

Cells

with >

2 s

ignals

(adju

ste

d r

atio)

20 µm

1.0

0.8

0.6

0.4

0.2

0.0

H&

E

b

CE

N

sig

nal (%

)

0

1

2

>2

1.0

0.8

0.6

0.4

0.2

0.0

RC3 RC4 RC5 RC6 RC7

RC1 RC2 RC3 RC4 RC5 RC6 RC7

CIMP

BRAFV600E

MSI

RAS

TP53

APC

SMARCB1

proximal

median

distal 1p36.1

3

LO

H

Altera

tion

s

Absent

Present

Absent

Present

*

0

100

200

300

400

500

600

3 4 5 6 7 3 4 5 6 7

Rela

tive C

RO

CC

mR

NA

le

ve

ls

Normal Tumor

**

**

**

**

plo

idy

Dapi CEN HER2

CK18 CROCC CROCC

CR

OC

C

imm

unola

belli

ng

(%

)

RC5

KI67

Dapi CEN HER2

Chr 17

Dapi CEN MDM2

CROCC IHC signals




a

Amplification

Mutation

mRNA Up

mRNA Down

Del

Pediatric Rhabdoid (N=20)

Copy Number

Alterations

Mutation

load

mRNA

Figure 3 c

RI 4 0 Negative

1 Single

2 Together

2 Separate

Rhabdoid of infants (RI)

1 2 3

4

7 5 6

b

e

**

RI

CE

N

sig

nal (%

)

RC

1.0

0.8

0.6

0.4

0.2

0.0

1.0

0.8

0.6

0.4

0.2

0.0

0

1

2

>2

RI

800

600

400

200

0

CR

OC

C

imm

unola

belli

ng (

%)

H&

E

50 µm

20µm

20µm

40µm Rela

tive

CR

OC

C

tran

script le

vels

CR

OC

C

DA

PI

CE

N C

hr

1

SM

AR

CB

1

RI

Wnt/β-catenin APC

CTNNB1

MAPK

KRAS

NRAS

HRAS

EGFR

BRAF

ERBB2

ERBB4

PI(3)K

PIK3CA

PTEN

AKT1

Genome

integrity

TP53

ATM

MLH1

Chromatin

modifiers

SMARCB1

EZH2

Others

CDKN2A

CDH1

MET

GNAQ

RET

SMAD4

FBXW7

IDH1

MPL

GNAS

PTPN11

RB1

CSF1R

HNF1A

FGFR1

JAK2

NPM1

SMO

FGFR2

JAK3

NOTCH1

SRC

FGFR3

IDH2

PDGFRA

STK11

FLT3

KDR

ALK

GNA11

KIT

VHL

RI RC

45%

5%

10%

0%

0%

d

MIssesnse

Truncating

Mutations

Pathways

1.0

0.8

0.6

0.4

0.2

0.0

0 30 60 90

RC

RI

P=0.048

log-rank test

Overa

ll surv

ival

Time (months)

median (months)

6 (n=7)

20 (n=7)

CROCC

CROCC IHC signals




c

d

CON CROCC KD

H&

E

20 µm

10 µm

10 µm

Figure 4

a DAPI α-TUB

DAPI α-TUB γ-TUB DAPI α-TUB CROCC

DAPI α-TUB γ-TUB

CON CROCC KD

5 µm

meta

ph

ase

2N 4N

ACA DAPI

ACA DAPI

CON CROCC KD

10 µm **

b

e

CROCC KD

DAPI γ-TUB

DAPI 5um

DAPI γ-TUB CENTRIN DAPI α-TUB γ-TUB

a b

Time (h)

50

40

30

20

10

0

inte

rphase

Segre

ga

tio

n e

rrors

(%

)

n>

100 p

er

cell

line

Micronuclei Monopolar

spindle

CROCC

KD

***

**

CON CROCC

KD

CON

0

0.1

0.2

0.3

0.4

0.5

0 12 24 36 72

OD

(570 n

m)

CON

CROCC KD

0

0.2

0.4

0.6

0.8

1

0 12 24 36 72

OD

(570 n

m)

serum deprivation serum

*

METAPHASE PROMETAPHASE ANAPHASE

Abnormal spindles (85%) Bipolar

0

20

40

60

80

100

N 2N 3N 4N

Meta

ph

ases (

%)

CON n=15 CROCC KD n=18

**

0

0.04

0.08

0.12

0.16

0.2

Tre

trap

loid

y (

%)

4N/2N ratio

NS

** **

** *

*

**

** ***

** **

NS

*

*

*

DAPI γ-TUB CENTRIN




0

10

20

30

40

50

PI

CO

UN

T (

%)

T84

a DAPI γ-TUB α-TUB

020406080

100

T84n=43

T84 SDn=68

Mitotic e

rrors

(%

)

Aanaphase aberrations

Micronuclei* DAPI CROCC

ANAPHASE

0

20

40

60

80

100

0 2 4 6 8 10 12

T84GFP+

T84-CROCC/GFP+

T84-C

RO

CC

/GF

P+

CROCC-GFP DAPI CROCC γ-TUB

GF

P+

CE

LLS

(%

)

Time (Days)

** ** ***

***

50 µm

5 µm

b

***

c

Cells

with ≥

3yH

2A

X

foci (%

)

1.0

0.8

0.6

0.4

0.2

0.0 0

0.04

0.08

0.12

0.16

0.2

4N

/2N

(%

)

MSI

BRAF

(V600E)

MSS

CROCC

reduced

dosage

GROSS MITOTIC

ERRORS

CENTROSOME

COHESION

FAILURE

RHABDOID

d

MIT

OT

IC E

RR

OR

S

CRC WITH VULNERABLE

MICROTUBULE FUNCTION

***

Figure 5

DAPI γ-TUB α-TUB

**

T84/GFP+ T84-CROCC/GFP+

6 days 12 days

ANAPHASE




Published OnlineFirst May 21, 2018.Mol Cancer Res Andrea Remo, Erminia Manfrin, Pietro Parcesepe, et al. Link Rhabdoid Phenotypes and Lethal Colorectal CancersCentrosome Linker-induced Tetraploid Segregation Errors

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Centrosome Linker-induced Tetraploid Segregation Errors Link …download.xuebalib.com/1gxkXRB9dc5M.pdf · 8 Claudio Ghimenton2 Fortunato Lonardo13, Fulvio D’angelo14, Federica Grillo15,