Centrosome anomalies contribute to tumorigenesis, but it remains unclear how they are generated in lethal cancer phenotypes. Here, it is demonstrated that human microsatellite instable (MSI) and BRAFV600E-mutant colorectal cancers with a lethal rhabdoid phenotype are characterized by inactivation of centrosomal functions. A splice site mutation that causes an unbalanced dosage of rootletin (CROCC), a centrosome linker component required for centrosome cohesion and separation at the chromosome 1p36.13 locus, resulted in abnormally shaped centrosomes in rhabdoid cells from human colon tissues. Notably, deleterious deletions at 1p36.13 were recurrent in a subgroup of BRAFV600E-mutant and microsatellite stable (MSS) rhabdoid colorectal cancers, but not in classical colorectal cancer or pediatric rhabdoid tumors. Interfering with CROCC expression in near-diploid BRAFV600E-mutant/MSI colon cancer cells disrupts bipolar mitotic spindle architecture, promotes tetraploid segregation errors, resulting in a highly aggressive rhabdoid-like phenotype in vitro. Restoring near-wild-type levels of CROCC in a metastatic model harboring 1p36.13 deletion results in correction of centrosome segregation errors and cell death, revealing a mechanism of tolerance to mitotic errors and tetraploidization promoted by deleterious 1p36.13 loss. Accordingly, cancer cells lacking 1p36.13 display far greater sensitivity to centrosome spindle pole stabilizing agents in vitro. These data shed light on a previously unknown link between centrosome cohesion defects and lethal cancer phenotypes providing new insight into pathways underlying genome instability.
Implications: Mis-segregation of chromosomes is a prominent feature of chromosome instability and intratumoral heterogeneity recurrent in metastatic tumors for which the molecular basis is unknown. This study provides insight into the mechanism by which defects in rootletin, a centrosome linker component causes tetraploid segregation errors and phenotypic transition to a clinically devastating form of malignant rhabdoid tumor. Mol Cancer Res; 16(9); 1385–95. ©2018 AACR.
This article is featured in Highlights of This Issue, p. 1333
The century-old hypothesis on the relationship between centrosomes and cancer, formulated by the German embryologist Theodor Boveri more than 100 years ago (1, 2), remains unanswered. Centrosome abnormalities, consisting usually in increased numbers, are common in human tumors (3), and experimentally induced tetraploid cells from extra centrosomes can be critical for aneuploidy and metastatic progression of malignancy (3, 4). However, insufficient progress has been made in our knowledge on genetic defects underlying centrosome anomalies in tumorigenesis (1–4). In this scenario, the rare and lethal pathologic variant of common colorectal cancers showing rhabdoid phenotype (5–7), is of particular interest as it features recurrent mitotic anomalies of enigmatic origin (8–10). We thus hypothesized that the systematic study of rare rhabdoid colorectal cancers, could provide insights into biological mechanisms responsible for the generation of genome instability and reveal key factors for the development of aggressive disease entities. To test this idea, we performed whole-exome sequencing of two rhabdoid colorectal cancers and discovered an enrichment of centrosome anomalies and inactivation of Rootletin encoded by ciliary rootlet coiled-coil (CROCC) gene (11, 12), a structural component of the centrosome linker, which assembles and keeps the two centrioles connected. Centrosomal alterations were assessed in an expanded series of rare rhabdoid colorectal cancers and related tumors, and functionally characterized in colorectal cancer cellular models.
Materials and Methods
Materials and Methods and any associated references as a continuation of the main text are described more in detail within the Supplementary Material.
Patient and tissue cohort
This study was conducted in accordance with Declaration of Helsinki ethical guidelines. It was approved by an institutional review board, approval no. 997CESC from the Ethics Committee (Comitato Etico di Verona e Rovigo dell'Azienda Ospedaliera Universitaria Integrata) on September 7, 2016, documented by the CESC prot. 42160 on 9 September 2016, and formalized by the General Manager with deliberation no. 458 of September 16, 2016, communicated with protocol 51319 on September 23, 2016.
Formalin-fixed paraffin-embedded (FFPE) samples from 7 cases of primary rhabdoid colorectal cancers and matched normal colonic mucosa were studied (cases RC1 to RC7 Supplementary Table S1). Moreover, an independent validation series to screen the mutational status of newly identified genes was analyzed (cases RC8 to RC12 Supplementary Table S1). FFPE samples from 7 rhabdoid tumors arising in central nervous system of patients between 2 months and 19 years of age were collected from the files of the Azienda Ospedaliera Universitaria Integrata (Verona, Italy). These pediatric/young adult rhabdoid tumors are indicated as rhabdoid of infancy throughout the article. Two independent datasets of patients with classic type sporadic colorectal cancer were analyzed (dataset A included 141 primary cancers and dataset B included 102 primary cancers).
Human 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 from normal foreskin and pediatric rhabdoid tumor were used as a nonneoplastic control and a pure rhabdoid model, respectively.
Whole-exome sequencing with 100-bp paired reads was performed with a HiSEQ1000 (Illumina), using 1.3 μg genomic DNA (based on fluorometric Picogreen dsDNA quantification), and enrichment for whole exome was done according to TruSeq Exome Enrichment Guide (Illumina).
Functional in vitro assays
RKO cells were transiently transfected with SureSilencing control or CROCC shRNA expression plasmids KH23140P (Qiagen) containing the puromycin-resistant cassette. After selection puromycin (Thermo Fisher Scientific), single colonies were amplified and assessed for efficient CROCC silencing by qPCR and Western blot analysis, respectively. HT29 and T84 cells were transfected with the full-length CROCC coding sequence or a truncated form (1–494 aa) cloned with GFP epitope or GFP alone (used as control). For long-term experiments, CROCC-GFP+ cells were maintained in 0.6 mg/mL of G418.
Data are presented as mean, medians, and ranges. P values of <0.05 (two tailed) were considered to be significant. Statistical analyses were conducted by GeneSpring R/bioconductor v.12.5 and R based package, SPSS v15, and GraphPad Prism 5.
Discovery of CROCC mutations and centrosome anomalies
Two previously reported primary BRAFV600E-mutant rhabdoid colorectal cancers (RC1 and RC2; refs. 9, 10), harboring MSI due to defective DNA mismatch repair (MMR) machinery caused by promoter methylation of the MLH1 gene, were subjected to whole-exome sequencing (WES) using DNA from FFPE-matched tumor/normal samples (Supplementary Table S1). We detected an exceptionally large number of somatic point mutations, 1,056 and 1,078 per 106 bases for RC1 and RC2, respectively, which is consistent with the presence of MMR defects (13–15; Fig. 1A). About twenty percent of mutations occurred within CpG dinucleotide context as seen in classical colorectal cancers (14). Transitions 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 characteristic of the mutational signature due to alterations of MMR mechanisms (signature 6; ref. 13). The most prevalent single-nucleotide variants (SNV) were nonsilent mutations (14), where over 90% of potentially damaging mutations were missense and around 10% were splicing, stop-gain, stop-loss, or, rarely, frameshift insertions or initiation codon mutations (Fig. 1A; Supplementary Fig. S1C and S1D). The two rhabdoid colorectal cancer cases shared 112 (10%) mutated genes (Supplementary Fig. S2A). By applying DrGaP computational tool (16), which allows inferring cancer driver genes, a number of potential candidate disease-causing genes were identified (Supplementary Table S2), approximately half of which (45%) were enriched for cytoskeleton/centrosome and microtubule biological functions (Supplementary Fig. S2B and S2C).
The search of candidate genes in The Cancer Genome Atlas (TCGA) database (13–15) comprising 224 sequenced classical colorectal cancers (http://www.cbioportal.org) revealed that the majority (65%) of the candidates had a low frequency of mutations (≤4% of cases). Among all candidate genes, only for CROCC mapping to 1p36.13 (11, 12) involved in centrosome cohesion and disjunction, no somatic mutations (0/224; 0%) were reported (Fig. 1B). Notably, 1p36 deletions are recurrent in neuroblastoma, Wilms tumor, and medulloblastoma. In our two rhabdoid colorectal cancer cases, CROCC harbored two missense mutations, p.Ala161Ser (c.481G>T, Exon 4) and p.Val1885Ala (c.5654T>C, Exon 35), and one prominent splicing mutation at the conserved 3′ acceptor splice site (c.3705-2A>G) in the intron between exons 25 and 26 (Fig. 1C). A review of multiple colorectal cancer sequencing datasets (N = 2070) revealed CROCC mutations in (1.4% of cases). However, although of unknown significance, none of the CROCC mutations were identified as putative driver mutations in colorectal cancer (Supplementary Fig. S2D). SMARCB1 and SMARCA4 mutations, which have been associated with rhabdoid phenotype (6), showed a trend of mutual exclusivity with CROCC alterations. However, only putative truncating driver mutations in SMARCB1 and/or SMARCA4 correlated with tumor poor differentiation and short-time metastatic progression. Therefore, we reasoned that the splicing mutation detected in RC1 might be causally correlated with rhabdoid phenotype. Indeed, the mutation reduced the strength of the physiologic acceptor site, causing a large deletion of the CROCC coding region involving exons 23–31 (17, 18) (Supplementary Fig. S3A). RT- PCR analysis (exons 5–7 and 33–35) revealed reduced CROCC mRNA in RC1 harboring the splicing mutation as compared to the normal mucosa. This suggested the alteration of the mature CROCC transcript by the utilization of cryptic splice sites or by the activation of the nonsense-mediated mRNA decay, which, in some cases can eliminate aberrant mRNA transcripts (Supplementary Fig. S3B; ref. 17). Unexpectedly, RC2 also displayed expression levels in tumor lower than in normal tissue, supporting a role for defective CROCC expression in rhabdoid tumors (Fig. 1D). We next used IHC and immunofluorescence at high magnification with an anti-CROCC antibody to analyze the centrosomes in rhabdoid cancers and matched normal tissues (Supplementary Table S3). We found that nearly 50% of tumor cells had no CROCC immunolabeling, and the presence of cells with a single and often abnormally shaped, larger (up to 6-fold greater than normal), or fragmented centrosomes, suggesting the presence of numerical and structural centrosome aberrations (Supplementary Fig. S3B). We also observed dramatic and uncommon cytologic defects, such as anucleated cells having larger centrosomes positive for CROCC associated with mitotic catastrophe in late telophase, particularly in RC1 harboring the splicing site mutation (Fig. 1d). We used the pericentriolar material (PCM) component γ-tubulin as our reference marker for immunolabeling experiments, because it consistently colocalizes with centriole markers, which are closely connected in interphase by the centrosomal linker (1, 2, 11, 12). Moreover, γ-tubulin has been proposed as a marker to identify spindle poles (19, 20). We observed a remarkable loss of cell polarity in interphase nuclei and abnormal mitotic figures, many of which included asymmetric bipolar or monopolar spindles. Rhabdoid cells showed a diffused staining of γ-tubulin into the cytoplasm and reduced centrosomal localization, a phenomenon described in tumors with high metastatic potential (ref. 19; Supplementary Fig. S3C and S3D). Double immunofluorescence analysis using antibodies directed against CROCC and γ-tubulin confirmed these observations and revealed cells either with fragmented/larger centrosomes or with a consistent loss of centrosome staining (Supplementary Fig. S3). These experiments indicated that genetic defects in CROCC and other centrosome components may compromise centrosome function in rhabdoid colorectal cancers.
A validation set of 10 additional rhabdoid colorectal cancers was studied, including 3 cases (RC7, RC9, RC11) with microsatellite instability due to MLH1 promoter methylation and 7 cases (RC3–6, RC8, RC10, RC12) with stable microsatellites (Fig. 2A; Supplementary Table S1). Targeted sequencing identified three CROCC mutations (p.Ser1320Ile, p.Arg1659His and p.Ala1510Thr) of unknown significance in additional 2 cases (RC9 and RC11) harboring microsatellite instability (1, 2, 11, 12). Indeed, the 24 CROCC mutations identified across cBioportal database were more recurrent in MSI (12/24; 50%) than in MSS (6/24; 25%) colorectal cancers. Notably, CROCC mutations were classified as missense (n = 22) or truncating mutations (n = 2) of unknown significance but associated with both well- differentiated and early-stage classical colorectal cancers. In our rhabdoid colorectal cancer dataset, 5 of the remaining cases for which sufficient material was available (cases RC3–RC7) harbored loss of heterozygosity (LOH) at the 1p36.13 locus, where CROCC resides, which was associated with mRNA below normal levels (Fig. 2B). In keeping with the findings in RC1 and RC2 cases, comparable levels of centrosome defects and a high prevalence of bizarre mitotic figures and/or cytomorphologic aberrations were evident in all tumors (Fig. 2C; Supplementary Table S3). Analysis of independent colorectal cancer databases (n = 1,387) for which copy number alterations were available (http://www.cbioportal.org) revealed no alteration at 1p36.13 locus, suggesting CROCC impairment as a consequence of reduced gene dosage (21) caused by allelic deletion. As centrosome anomalies are intimately connected with chromosome segregation errors (1, 3, 20), we assessed DNA content in tumor samples (cases RC1–RC7, Supplementary Table S3). Remarkably, we observed recurrent ploidy abnormalities mainly consisting of triploid or near-tetraploid cells ranging from 10% to 40% of tumor cells (Fig. 2D). Globally, these results indicated that centrosome defects underlie rhabdoid colorectal cancer pathogenesis.
Centrosome and genomic profiling of rhabdoid tumors of infancy
Insight into genetic characterization of rhabdoid neoplasms are limited to the so called extrarenal rhabdoid tumors arising in children, in which inactivating mutation and/or deletion of the chromatin remodeling gene SMARCB1 (INI1) and low mutation load have been reported (6, 22–24). We analyzed 7 cases of this tumor type, hereafter named rhabdoid of infancy, for centrosome and molecular anomalies (Fig. 3A; Supplementary Table S4). Compared with rhabdoid colorectal cancers, analysis in pediatric tumors was associated with much higher CROCC mRNA expression levels than matched normal tissues (P < 0.00001; Fig. 3B). Target sequencing identified no mutations (0/7 tumors) in the CROCC gene. Moreover, CROCC immunostaining was seen as a single and large signal adjacent to the nuclei in almost the totality of the cells (80%)—only 10% of cells had no centrosome staining (Fig. 3B; Supplementary Table S4). Consistent with literature, the genetic profile of rhabdoid of infancy revealed missense or truncating mutations in SMARCB1 (5/7, 71%; refs. 6, 23, 24) and/or TP53 (3/7, 42%) accompanied by a near diploid DNA content and less aggressive clinical course when compared with rhabdoid colorectal cancers (Fig. 3C–E). This suggested that rhabdoid of infancy, which is characterized by SMARCB1 (INI1) mutation, did not harbor any CROCC alteration and did not display the centrosomal defects observed in rhabdoid colorectal cancer. Inspection of an available database from pediatric rhabdoid cells (25) confirmed that both mutations and genetic deletion affecting CROCC locus were infrequent (2/20, 10%), whereas the transcript profile tended to be similar to our rhabdoid of infancy dataset (Fig. 3C). Therefore, rhabdoids arising in colorectal cancer, although morphologically indistinguishable from their pediatric counterparts, demonstrate distinct molecular, cytogenetic, and centrosomal aberrations.
Centrosome and CROCC expression in classical colorectal cancers
We screened 242 primary classic colorectal cancers, comprising two independent series of 140 (dataset A; refs. 26, 27) and 102 (dataset B; refs. 28, 29) cases for CROCC mRNA and protein expression (Supplementary Table S5). CROCC mRNA expression levels were higher in colorectal cancers than in normal colonic mucosa. However, no significant protein expression change in tumor tissues compared with that in normal mucosae was detected (Supplementary Fig. S4A). In cohort A, using IHC and immunofluorescence against CROCC and γ-tubulin, we found round and uniform centrosomes, prevalently in normal number (1–2 per cell; 97/140, 69.3%), supernumerary (>2 per cell; 39/140, 27.8%), and only few (<1 per cell; 4/140, 2.9%) displayed reduced centrosome labeling (Supplementary Fig. S4B). Centrosome abnormalities, particularly supernumerary centrosomes, were more prevalent in advanced stage (stage III–IV) than in low stage (stage I–II) lesions (Supplementary Fig. S4C). In cohort B, which was enriched for stage III–IV tumors (79.5% of cases), we confirmed a high prevalence of supernumerary (60/102; 59%; refs. 1, 3, 20) or defective (11/102; 11%) centrosomes associated to poorer clinical course than those expressing a normal pattern [31/102; 30%, HR = 0.30; 95% confidence interval (CI), 0.21–0.81; P < 0.0001] in keeping with the notion that numerical centrosomal abnormalities are more common in invasive cancers (1, 3, 20; Supplementary Fig. S4C). To independently validate the pattern of gene expression changes detected in our datasets, we analyzed the patient-matched tumor–normal expression data available from the TCGA (14) and three independent datasets GSE20916 (30), GSE41258 (31), and GSE30540 (32), of classical colorectal cancer (Supplementary Fig. S4D). CROCC mRNA was upregulated in colorectal cancer compared with normal only in TCGA database. The analysis of other datasets revealed a heterogeneous expression pattern, while CROCC upregulation compared with normal control did not reach statistical significance. The analysis of GSE30540 (32) dataset, for which both transcriptomic data and degree of chromosome instability (CIN) were available, revealed that CROCC expression levels tended to be lower in CIN-high than in CIN-low tumors (Supplementary Fig. S4D). These data suggested the possibility that imbalanced genetic defects at CROCC locus may be related to marked anomalies in the fidelity of chromosome segregation.
CROCC depletion impairs mitosis and induces rhabdoid-like features
We next sought a genetic basis for the relation between CIN and rhabdoid phenotype by examining WES and transcriptomic data from The Cancer Cell Line Encyclopedia (CCLE; ref. 25). Unexpectedly, data from a collection of 60 colorectal cancer cell lines revealed that the deletions at 1p36.13 locus tended to be more prevalent in CIN-high (23.6%, 9/38) compared with CIN-low (9.1%, 2/22) cells (Supplementary Fig. S5A). However, cell lines with 1p36.13 deletion displayed neither rhabdoid phenotype nor BRAF mutations. As expected, compared with cells retaining 1p36.13 locus, those harboring the deletion revealed a gene expression signature significantly enriched for pathways implicated in chromosomal instability (refs. 33, 34; Supplementary Fig. S5B and S5C). In a panel of colorectal cancer cells, we then confirmed that both CROCC mRNA and protein expression levels were concordant and higher in CIN-low than in CIN-high cell lines (P < 0.05, Supplementary Fig. S6A). CIN-low cells showed centrosomes stained for CROCC and γ-tubulin that were structurally indistinguishable from those in normal human fibroblasts BJ, consistent with literature (ref. 35; Supplementary Fig. S6A and S6B). In line with this, CIN-high displayed a higher frequency of micronuclei and nuclear γH2AX foci than CIN-low cells (ref. 29; Supplementary Fig. S6C and S6D). This suggested that CROCC might be a CIN suppressor gene and its deletion in CIN-low/BRAF-mutant cell lines might be permissive for abnormal phenotypes. Therefore, we reasoned that RKO cells, sharing a near-diploid karyotype, BRAFV600E mutation/MSI, and alterations in microtubule/centrosomal components with rhabdoid colorectal cancer (14), could be an excellent system to explore CROCC silencing in vitro. We found that the clone sh4, hereafter named CROCCKD, provided a stable and consistent knockdown of CROCC transcript to more than 75% and protein to 3.3-fold lower then RKO cells transfected with control vector (shCon), achieving nearly comparable levels to those seen in vivo (Supplementary Fig. S7A). Previous studies have demonstrated that CROCC knockdown in nontransformed cells causes centriole splitting and increases centrosome separation (11, 12). By using γ-tubulin and centrin as reference markers, we observed a consistent PCM fragmentation after CROCC depletion, which resulted in abnormal chromosome segregation and higher frequency of monopolar spindles as compared with control (Fig. 4A; Supplementary Fig. S7B). Importantly, monopolar spindles displayed larger or “fragmented” centrosomes, which accounted for 85% of the abnormal phenotype (Fig. 4A–C; Supplementary Fig. S7C). Thus, BRAF-mutant/MSI colorectal cancer cell lines, in which centrosome and microtubule stability is damaged by genetic hypermutation, CROCC depletion may determine a major impact in the progression of mitotic errors (36–38). Consistently, an increased frequency of micronuclei (median 11% CROCCKD vs. 1% ShCon cells P = 0.0003) and γH2AX nuclear foci (median 43% CROCCKD vs. 18% ShCon cells P = 0.011) were observed (Fig. 4C; Supplementary Fig. S7D). Metaphase karyotyping revealed that CROCC deficiency leads to an increased number of tetraploid (4N) cells (median 13.3% CROCCKD vs. 3.51% ShCon cells, P = 0.001) characterized by prominent and larger nuclei than diploid (2N) cells. Consistently, analysis of centromeric probes in intephase nuclei confirmed tetraploidy (Fig. 4C). The number of CROCC-deficient cells was reduced in G0–G1 or G2–M phases when compared with the wild-type population (by 26%–45%, FACS analysis), suggesting an impaired cell-cycle progression as a consequence of misaligned chromosomes (1–3, 33; Supplementary Fig. S7D). In contrast, cells grown under replication stress conditions (serum deprivation) resulted in higher proliferation rate than control cells (ref. 34; Fig. 4D). Most strikingly, CROCC-deficient cells exhibited all cardinal signs of rhabdoid features (8–10), displaying huge nuclei pushed to the periphery of the cells with single or multiple large nucleoli associated with eosinophilic cytoplasmic inclusions and large cellular protrusions resembling the morphology observed in vivo (Fig. 4D; Supplementary Fig. S8A). These features resulted in the activation of prometastatic genes involved in epithelial mesenchymal transition accompanied by a dramatic change of spindle-shaped morphology (4, 7) consistent with the enhanced metastatic potential of rhabdoid phenotype (Supplementary Fig. S8A and S8B). Expression of exogenous GFP-tagged CROCC (1–2018 aa) rescued these phenotypic changes induced by depletion of endogenous CROCC (Supplementary Fig. S8B; ref. 11). In line with previous results (11, 12), we observed no alteration in cell-cycle profile or aberrant phenotypic changes after CROCC depletion in nontransformed BJ cells. Therefore, CROCC depletion in BRAF-mutant near-diploid cancer cells induces tetraploidization and rhabdoid phenotype in vitro.
Tolerance to mitotic errors and tetraploidization promoted by 1p36.13 deletion
As colorectal cancer cells with driver mutations in CROCC have not been reported, to test the hypothesis that CROCC impacts tumor growth and centrosome-related mitotic errors, we analyzed metastatic colorectal cancer T84 cells harboring an allelic deletion at 1p36.13 locus (25). Although T84 are well-differentiated cancer cells and do not show rhabdoid morphology, they, however, exhibit some of the characteristics detected in RKO CROCC-depleted cells. Consistent with reduced CROCC endogenous activity, we observed an increased rate of micronuclei, tetraploid, or near-tetraploid cells and recurrent mitotic errors resulting essentially in “monopolar spindles,” which were more recurrent under replication stress conditions (Fig. 5A; Supplementary Fig. S8C and S8D). We then investigated the localization of CROCC in the centrosome by immunofluorescence. Almost half of the cells (40%) revealed a faint CROCC signal, which was consistently accompanied by atypical γ-tubulin aggregates prevalently in cells with mitotic anomalies (Fig. 5A). Most strikingly, such aberrations were rarely, if ever, detected in pediatric rhabdoid G401 or colon cancer cell lines with an intact 1p36.13 locus (Supplementary Fig. S8C and S8D). Therefore, we transfected CROCC-GFP and GFP alone (control) into T84 cells. Restoration of CROCC determined a dramatic decrease of cell viability (12 days later, 0%) as compared with control plasmid. Similarly, we detected a higher number of G0–G1 cells than control (41% vs. 26%; P = 0.0018; Fig. 5B and C). Gain of CROCC conferred a flat/adherent phenotype and formation of filament-like structures colocalizing with γ-tubulin resulting in an expression of mesenchymal genes lower than in control (ref. 4; Supplementary Fig. S9A). Accordingly, we detected a 4-fold decrease of tetraploid cells, and reduced γH2AX foci from 59% to 22% with respect to control cells (Fig. 5C) raising the possibility that the centrosome spindle pole integrity is strongly affected by 1p36.13 deletion. To see whether T84 cells are sensitive to mitotic drugs, we mined the data from the Genomics of Drug Sensitivity in Cancer project (Sanger panel). As shown in (Supplementary Fig. S9B), among 221 molecules tested, IGF1R inhibitor (linsitinib) and Epothilone B, a microtubule-stabilizing agent, were significantly effective in T84 lines. Accordingly, we observed a significant difference in the sensitivity to Epothilone B in 1p36.13-deleted cells as compared with cells with an intact 1p36.13 locus. Similar results were not reproduced comparing CIN-low and CIN-high colorectal cancer cell lines (Supplementary Fig. S9B). We used another cell line HT29 with an intact 1p36.13 locus to test CROCC restoration. Similarly to T84, we observed a significant decrease of micronuclei in HT29-CROCC-GFP+ cells compared with control. Although gain of CROCC in HT29 increased the cell death, it appeared an essential gene only for T84 cell survival lacking 1p36.13 (Supplementary Fig. S9C). This supported the hypothesis that a reduced CROCC dosage promotes defects in spindle-assembly checkpoint. When we repeated the experiments using a GFP-tagged truncated form of CROCC (1–494 aa; ref. 11), we observed that this mutant failed to rescue the aberrant growth phenotype and mitotic errors (Supplementary Fig. S9D). Consistent with previous findings (11), we did not detect phenotypic changes in nontransformed BJ 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 at 1p36.13 locus.
Our understanding of the molecular architecture and function of centrosomal linker components in physiologic and pathologic processes remain rudimentary. Besides (Rootletin), multiple proteins including C-NAP1 (CEP250), CEP68, and LRRC45 have been implicated in centrosome linker formation and function. CROCC is able to maintain centrosome cohesion in part through inhibition of VHL-mediated Cep68 degradation (36). It has recently been proposed that a vast network of repeating CROCC units with C-Nap1 as ring organizer and CEP68 as filament modulator forms the centrosome linker structure (37). We show here that genetic deletion in CROCC, leads to centrosome anomalies resulting in tetraploid DNA segregation errors, providing insights into mechanism by which genome instability contributes to lethal cancers for which no therapies are available (Fig. 5D). In addition, we show that rhabdoid colorectal cancers are not genetically related to their pediatric counterparts (22–24), in which we find recurrent SMARCB1 gene alterations but no evidence of centrosome anomalies. Previous studies have revealed that driver genes implicated in human cancer (3, 4) can promote centrosome overduplication (2, 20), which through whole genome doubling facilitates chromosomal instability, especially in metastatic tumors (4, 38). However, an important drawback of these studies (3, 4) is that the mechanism of tetraploidization and underlying biological causes has remained unresolved. We provide evidence that centrosome linker genes might be altered due to imbalanced genetic defects, interfering with protein complexes required for the correct assembling of spindle functions in colorectal cancer cells (39).
Recently, factors involved in the stabilization and nucleation of microtubules around kinetochores have been described in BRAF-mutant colorectal cancer cells, highlighting the potential to make these tumors vulnerable to microtubule-destabilizing anticancer drugs (40). Other studies have showed that the centrosomal linker genes and microtubule motor proteins cooperate to keep unlinked centrosomes in relative close proximity (41). Therefore, cumulative defects in these pathways may result in spindle perturbations, providing an explanation for the observed mitotic errors after CROCC depletion. The frequency of CROCC mutations in other tumors with MSI is unknown. However, exploration of cBioportal database revealed a prevalence of CROCC mutations in cancers with high mutational load. In contrast, 1p36.13 deletions appeared to be characteristic of liver, skin, or uterine carcinosarcoma with high levels of genomic instability.
Our findings underline that in CIN-negative cancer cells with functionally compromised centrosomes (i.e., BRAF-mutant colorectal cancer cells), CROCC depletion leads to monopolar spindle DNA segregation defects exacerbating mitotic errors and promoting rhabdoid morphology. Therefore, upregulation of CROCC in classical colorectal cancer particularly in MSI tumors, may provide a mechanism of protection to potentially deleterious genetic changes (39, 40). CROCC restoration in a metastatic model with 1p36.13 deletion confirmed its role as a biological barrier against mitotic errors. In agreement with this, colon cancer cells with 1p36.13 deletion display have increased sensitivity in vitro to microtubule-stabilizing agents used in pediatric tumors (42). However, we were unable to demonstrate the detailed molecular mechanism by which independent CROCC defects promote gross mitotic errors. In addition, other factors not present in our current models can influence rhabdoid pathogenesis, especially in MSS tumors. In fact, CROCC deletion was recurrent in CIN-high cancer cells without rhabdoid characteristics, supporting the concept that rhabdoid traits are highly heterogeneous as consequence of multiple dysregulated developmental pathways. The patients with rhabdoid colorectal cancer described in our study presented lethal clinical outcomes with an average postoperative survival of only 7 months. Therefore, the recurrent CROCC genetic deletions identified in these patients may be associated with the poor prognosis. From this point of view, identifications of new molecular subgroups cannot be excluded. For example, we do not know whether CROCC deletion is an unfavorable prognostic only in BRAF-mutant tumors or other subtypes with SMARC gene mutations (SMARCB1, SMARCA4). So far, mutations in centrosome genes like CEP57, CEP135, and PLK4 kinase, have been only described in rare genetic disorders with genomic instability such as microcephaly and Seckel syndrome (43, 44).
Overall, our data uncover a mechanism by which defects of critical centrosomal components cause unequal DNA segregation that contributes to the ongoing genetic heterogeneity in rare and aggressive colon cancers. Our findings link for the first time centrosomal cohesion defects and genomic instability, prompting for studies addressing how genetic centrosome anomalies are connected with key pathways involved in safeguarding the integrity of the human genome.
Disclosure of Potential Conflicts of Interest
M. Garonzi is an employee at Menarini Silicon Biosystems. M. Delledonne has ownership interest (including patents) and is a consultant/advisory board member for Personal Genomics Srl. No potential conflicts of interest were disclosed by the other authors.
Conception and design: A. Remo, P. Parcesepe, M. Pancione
Development of methodology: A. Remo, P. Parcesepe, E. Baritono, F. Lonardo
Acquisition 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. Pancione
Analysis 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. D′angelo, M. Ceccarelli, V. Colantuoni, A. Scarpa, M. Pancione
Writing, review, and/or revision of the manuscript: A. Remo, P. Parcesepe, J. Giuliani, L. Xumerle, G. Giordano, F. Grillo, G. Viglietto, A. Scarpa, M. Pancione
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E. Manfrin, H.S. Han, G. Giordano, F. Grillo, M. Pancione
Study supervision: A. Remo, A. Scarpa, M. Pancione
Other (performed the majority of in vitro experiments): T. Colangelo
Other (conducted some of the experiments to analyze CROCC expression in several CRC cell lines and was incharge of the cell lines used in the laboratory): L. Sabatino
The 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 the molecular/clinical aspects of the manuscript and for helpful discussions, Roberta Maestro (CRO, Aviano, Italy) for her kind gift of the BJ human skin fibroblasts and G401 cells and for helpful discussions, Erich Nigg for his kind gift and suggestions about clone 6150861 pEGFP Rootletin, and ARC-NET Research Centre core imaging facility for assistance with microscopy. T. Colangelo is supported by a fellowship from Associazione Italiana Ricerca sul Cancro (AIRC; project code: 19548). This work was supported by Department Funds of Mater Salutis Hospital, FUR, and the Italian Ministry of University and Research (MiUR; to M. Pancione), and AIRC 5 × 1000 (no. 12182; to A. Scarpa).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.