Cdc6 disruption leads to centrosome abnormalities and chromosome instability in pancreatic cancer cells

Cell division cycle 6 (Cdc6) plays key roles in regulating DNA replication, and activation and maintenance of cell cycle check points. In addition, Cdc6 exerts oncogenic properties via genomic instability associated with incomplete DNA replication. This study aimed to examine the effects of Cdc6 on pancreatic cancer (PC) cells. Our results showed that Cdc6 expression was higher in clinical PC specimens (based on analysis of the GEPIA database) and cell lines, and the high Cdc6 expression was associated with poorer survival in The Cancer Genome Atlas-PC cohort. In addition, Cdc6-depleted PC cells significantly inhibited cell proliferation and colony formation, delayed G2/M cell cycle progression, and increased expression of p-histone H3 and cyclin A2 levels. These observations could be explained by Cdc6 depletion leading to multipolar and split spindles via centrosome amplification and microtubule disorganization which eventually increases chromosome missegregation. Furthermore, Cdc6-depleted PC cells showed significantly increased apoptosis, which was consistent with increased caspase-9 and caspase-3 activation. Collectively, our results demonstrated that Cdc6-depleted PC cells are arrested in mitosis and eventually undergo cell death by induced multipolar spindles, centrosome aberrations, microtubule disorganization, and chromosome instability. In conclusion, Cdc6 may be a potential biomarker and therapeutic target for PC.

Many previous studies have indicated that abnormal Cdc6 expression plays an important role in brain tumors 15 , hepatocellular carcinoma 16 , breast cancer 17 , gastric cancer 18 , lung cancer 19 , ovarian cancer 20,21 , prostate cancer 22 , mitotic slippage, and drug resistance in bladder cancer and neuroblastoma 23,24 . Moreover, Cdc6 knockdown leads to increased DNA damage in Kras mutant cells 25 ; however, the significance of Cdc6 in the progression of PC remains unknown.
In this study, we investigated whether Cdc6 expression was significantly associated with PC progression, and if it induced centrosome abnormalities, microtubule disorganization, and chromosome instability. We showed that Cdc6 may be a potential anticancer target and may help to understand the mechanism of PC progression.

Expression of Cdc6 in PC patients and cell lines.
To determine the clinical relevance of Cdc6 expression in PC, we analyzed mRNA expression of Cdc6 in clinical PC tissues from the publicly available Gene Expression Profiling Interactive Analysis (GEPIA) database. Our results showed that Cdc6 mRNA levels were significantly higher in PC tissues (n = 179) than normal tissues (n = 171) from The Cancer Genome Atlas (TCGA) and GTEx data (Fig. 1A). The overall survival (OS) and disease-free survival (DFS) data demonstrated that high Cdc6 expression contributed to significantly poorer survival in PC patients (Fig. 1B). Next, we analyzed the expression of Cdc6 in PC cell lines by western blot analysis, and observed that Cdc6 was up-regulated in various PC cell lines including AsPC-1, PANC-1, MIA PaCa-2, and Capan-1 cells compared to human umbilical vein The OS and DFS of PC patients with low or high Cdc6 expression levels were analyzed using the Kaplan-Meier method and a log rank test in PC. Median values are indicated by full lines. *P < 0.005. TCGA, The Cancer Genome Atlas; TPM, transcripts per million; OS, overall survival; DSF, disease free survival; HR, hazard ratio. (C) Cdc6 expression was detected in PC cell lines (AsPC-1, PANC-1, MIA PaCa-2 and Capan-1 cells), HUVEC, and HPDE pancreatic cells by western blotting. The Cdc6/β-actin ratio was determined by densitometric analysis using ImageJ. Error bars represent standard deviations of the means of three biological replicates. Statistical analysis was performed using t-test. **p < 0.001, ***p < 0.0001. www.nature.com/scientificreports/ endothelial cells (HUVEC) and normal human pancreatic duct epithelial (HPDE) cells (Fig. 1C). These results suggest that Cdc6 is aberrantly overexpressed in human PC cells and influences the survival of patients with PC.

Cdc6 depletion inhibits cell proliferation, colony formation, and induces G 2 /M cell cycle arrest in PC cells.
To evaluate the effects of Cdc6 knockdown on the survival and growth of PC cells, we employed colony formation assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. First, we transfected PANC-1 cells with three Cdc6 siRNAs (Cdc6_1, Cdc6_2, and Cdc6_3). Cdc6_1 siRNA than Cdc6_2 siRNA or Cdc6_3 siRNA resulted in significant inhibition of Cdc6 expression of PANC-1 cells (Supplementary Fig. 1A). Also, Cdc6_1 siRNA and Cdc6_3 siRNA resulted in significant inhibition of proliferation of PANC-1 cells (Supplementary Fig. 1B). Therefore, Cdc6_1 siRNA was used in subsequent experiments. Next, the results of western blot analysis showed that Cdc6 expression was effectively reduced in Cdc6 siRNA-transfected cells for up to 96 h ( Fig. 2A and Supplementary Fig. 1C). PC cell proliferation was markedly reduced by Cdc6 depletion compared with the control (Fig. 2B), and Cdc6 knockdown significantly inhibited colony formation in PANC-1, AsPC-1, and Capan-1 cells (Fig. 2C). These results indicate that Cdc6 plays an important role in the proliferation of PC cells.
To further characterize the role of Cdc6 in the cell cycle, we analyzed the cell cycle distribution of Cdc6 siRNA-transfected PANC-1 and control-transfected cells by flow cytometry analysis with PI staining 72 h after transfection. We observed that Cdc6 depletion led to the accumulation of cells in G 2 /M phase (Fig. 2D), increased the expression of histone H3 phosphorylation (Ser10) (a mitotic marker), and maintained cyclin A2 expression (a cell cycle marker whose expression is known to increase in G 2 phase) for longer than the control (Fig. 2E). These results indicate that Cdc6 depletion increases expression of the cell cycle-related proteins p-histone H3 and cyclin A2, and induces G 2 /M cell cycle arrest.
Cdc6 depletion promotes apoptosis. To explore the effects of Cdc6 on the apoptosis of PC cells, we performed staining analysis using annexin V-fluorescein isothiocyanate (FITC), which recognizes phospholipid phosphatidylserine on the outer membrane of apoptotic cells, and propidium iodide (PI), a marker of cell membrane permeability 26 . Annexin V-FITC(+)/PI(−) staining indicated the early apoptotic cells, while annexin V-FITC(+)/PI(+) staining revealed the late apoptotic cells. The number of apoptotic cells significantly increased when transfected with Cdc6 siRNA compared with the control. As a result, the percentages of early and late apoptotic cells were significantly increased to 14.99 ± 1.72% and 52.6 ± 5.6%, respectively, after Cdc6 siRNA transfection compared with 6.1 ± 1.47% and 17.23 ± 3.08%, respectively, in the control-transfected cells (Fig. 3A,B). Caspase-9 and caspase-3 have distinct roles in the intrinsic apoptotic pathways. First, caspase-9 is activated by mitochondria-released cytochrome c, and then caspase-3 is activated 27 . The upregulation of apoptosis in Cdc6-depleted cells was confirmed by western blotting; caspase-9 and caspase-3 protein expression were observed 48 h after transfection and the cleaved form was highly expressed in Cdc6-depleted cells (Fig. 3C,D). Thus, Cdc6 depletion appears to induce cell cycle arrest in PC cells, followed by apoptotic cell death.

Cdc6 depletion induces centrosome and spindle abnormalities in PC cells.
According to previous research, Cdc6 localizes to the centrosome during the S and G 2 phases of the cell cycle, while a lack of Cdc6 has been shown to cause centrosome over-duplication in U2OS cells 13,14 . Hence, we examined whether Cdc6 depletion induced centrosome over-duplication by transfecting PANC-1 cells with Cdc6 siRNA and staining them with pericentrin (centrosome marker). Immunofluorescence confocal microscopy revealed that two normal centrosomes were found in control cells, whereas two or more abnormal centrosomes were found in Cdc6 siRNA-transfected cells (Fig. 4A). In addition, to investigate the effect of cell death on over-duplication with the centrosome, immunofluorescence analysis was performed using cleaved caspase-3 (apoptosis marker) and pericentrin antibodies. As a result, we could observe an increase in the expression of the cleaved caspase-3 antibody in cells with increased centrosomes (Supplemental Fig. 2A). Since centrosomes play an important role in microtubule organization, we analyzed severe spindle abnormalities in mitotic cells following Cdc6 depletion in PC cells. We divided spindle abnormalities into multipolar and split spindles. Multipolar spindles have at least three half-spindles and/or astral structures, while split spindles have two pair of centrioles, but at least one pair of centrioles separated by ≥ 2 μm. We measured a 7.3% incidence of multipolar spindles and a 3.5% incidence of split spindles in Cdc6-deficient mitotic cells. Overall, we observed a 10.8% occurrence of all spindle abnormalities, which was a four times greater incidence compared to the control (Fig. 4B). These data suggest that Cdc6 depletion interferes with normal centrosome division, leading to spindle abnormalities.
Cdc6 depletion leads to chromosome missegregation. We observed that Cdc6-deficiency in cells lead to an increase in DNA content 4 N or even greater suggesting polyploidy cells (Fig. 2D). In order to analyze the effect of Cdc6 on the formation of polyploidy cells, control siRNA-and Cdc6 siRNA-transfected PC cells were analyzed by flow cytometry after 96 h. As expected, Cdc6-deficient PC cells induced polyploidy (Fig. 5A). Next, using a metaphase chromosome spreading analysis to measure the number of chromosomes, we determined that aneuploidy (more than 70 chromosomes) occurred in 52.46% of Cdc6-deficient PC cells. On the other hand, in control siRNA-transfected PC cells, aneuploidy was observed in only 19.72% of the cells (Fig. 5B). Moreover, we examined whether defects in chromosome segregation in Cdc6-depleted PC cells might be due to abnormalities in the attachment between microtubules and the kinetochore by immunostaining with CREST and α-tubulin antibodies. In the control siRNA PC cells, α-tubulin was normally attached to CREST. However, in most of the Cdc6-depleted PC cells, α-tubulin extended in several directions rather than toward both ends and was not attached to CREST, indicating that Cdc6 depletion caused chromosome segregation error (Fig. 5C). As a result of analyzing the disorganized microtubules of cells dissociated into misaligned chromosomes, multipolar www.nature.com/scientificreports/ metaphase, and lagging chromosomes in the total metaphase cells, the number of misaligned chromosomes increased 5.6-fold in Cdc6-deficient PC cells compared to that in control siRNA cells. Multipolar metaphase was observed to increase more than 3.7-fold and lagging chromosome increased more than 7.1-fold in Cdc6deficient PC cells compared to that in control siRNA cells (Fig. 5C). Thus, these data demonstrate that Cdc6 depletion causes multipolar spindles and leads to chromosome instability.

Discussion
Cdc6 has been reported as a potential therapeutic target in many cancer types, and studies on its role in various cancers have been conducted 21,28,29 . However, the role of Cdc6 in PC has not been reported, and the mechanisms involved are unclear. In this study, we showed that Cdc6 was upregulated in PC cell lines and high expression of Cdc6 is related to clinical outcomes. Cdc6 depletion not only inhibited cell proliferation but also resulted in G 2 /M cell cycle arrest with upregulation of p-histone H3 and cyclin A2 in PC cells. Similar to our results, Cdc6 downregulation inhibited cell proliferation, DNA synthesis, epithelial-mesenchymal transition (EMT), migration, and invasion in human colorectal cancer 30 . Also, Cdc6 was reported to be highly expressed in osteosarcoma patients and osteosarcoma cell lines, and proliferation was inhibited in MG63 cells due to the deficiency of Cdc6 29 . However, in osteosarcoma cells, G 1 cell cycle arrest was observed, and the expression of cyclin D1 and cyclin A2 was decreased due to Cdc6 deficiency. This difference in results indicates that the inhibition of cell proliferation occurs due to the deficiency of Cdc6 in PC cells and osteosarcoma but the mechanisms underlying these effects are different. Moreover, we found that Cdc6 depletion induces a series of abnormal events, including centrosome over-duplication, multipolar spindle formation, and chromosome instability. These results are similar those reported in a previous study that Cdc6 and Plk4 are co-localized at the centrosome and have an important role in efficient centrosome duplication during the cell cycle 13 . Cdc6 depletion promoted apoptotic cell death with increased activation of caspase-3 and caspase-9. These data suggest that Cdc6 can play an important role in the proliferation, cell cycle, and death of PC cells. To the best of our knowledge, this is the first report show that Cdc6 contributes to PC cell progression. Centrosome duplication must be tightly regulated to avoid multipolar spindle assembly and genome instability during each cell cycle 31 . The duplicated centrosome is separated during the previous stages of mitosis and moves to the opposite pole, functioning as a mitotic spindle pole for chromosome separation. Later in mitosis, they are separated into individual daughter cells using chromosome segregation. If centrosome duplication and function are not controlled, they may lead to multipolar spindle formation, aneuploidy, asymmetric cell division and cell polarity destruction 32 . According to a recent study, Cdc6 is located at the centrosome during the S and G 2 phases of the cell cycle 33 . The function of centrosomal Cdc6 is to inhibit recruitment of PCM proteins such as γ-tubulin, pericentrin, CDK5RAP2, and Cep192 to the centrosome. Other studies have shown that Cdc6 controls centrosome duplication regardless of the mobilization of the PCM protein to the centrosome 34 . In addition, the interaction of Sas-6 and Cdc6, which is regulated by Plk4 phosphorylation of Cdc6, inhibits over-duplication 13 . Consistently, we observed that the number of centrosomes increased due to Cdc6 deficiency, thereby increasing cell aneuploidy.
In conclusion, our study shows that Cdc6 depletion in PC cells inhibit cell proliferation, induces G 2 /M arrest, increases centrosome over-duplication leading to multipolar spindles and microtubule disorganization, chromosome instability, and ultimately, encourages cell death. Thus, Cdc6 may serve as a promising therapeutic target for treating PC.

Database analysis. Expression levels of Cdc6 in PC and normal pancreases were compared by Gene
Expression Profiling Interactive Analysis (GEPIA) (https ://gepia .cance r-pku.cn/) 35 of 179 PC tissues and 171 normal tissues from Cancer Genome Atlas (TCGA) 36 and Genotype-Tissue Expression (GTEx) normal samples 37,38 . Transcripts per million (TPM) were determined, and gene expression levels were presented using a log 2 (TPM + 1) scale. The cutoff values were 1 for |Log 2 FC| and 0.01 for the P value, and the OS of patients with PC was also assayed. www.nature.com/scientificreports/  www.nature.com/scientificreports/ RNAse A, stained with 50 µg/mL PI, and analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA). To assess apoptosis, the cells were double stained with an FITC Annexin V apoptosis detection kit (BD Biosciences) and analyzed according to the manufacturer's instructions.
Western blotting. Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology Inc., Danvers, MA, USA), a protease inhibitor cocktail (Sigma-Aldrich), and phenylmethylsulfonyl fluoride (PMSF, Cell Signaling Technology). Protein concentration was measured using the bicinchoninic acid (BCA) protein assay reagent (Pierce-Thermo scientific, Rockford, IL, USA). Equal amounts of protein from each cell lysate were separated on sodium dodecyl sulfate (SDS) polyacrylamide gels, transferred onto nitrocellulose (NE) membranes, and reacted with antibodies against p-histone H3 ser10 (Thermo Fisher Scientific, Waltham, MA, USA), cyclin A2 (Cell Signaling Technology), caspase-3 (Cell Signaling Technology), or caspase-9 (Cell Signaling Technology). The membranes were then washed with TBST (Tris-buffered saline, 0.1% Tween 20), incubated with HRP-conjugated anti-mouse IgG (The Jackson Laboratory, Bar Harbor, ME, USA) or anti-rabbit IgG (Cell Signaling Technology) secondary antibodies, and the target proteins were detected with ECL western blotting detection reagents (Amersham-GE Healthcare Life Sciences, Malborough, MA, USA). Total protein loading amounts and intensity were quantified using β-actin (Cell Signaling Technology) as the loading control.
After treatment with 0.075 M KCl and incubation at 37 °C, the cells were fixed with a dropwise application of a freshly-prepared methanol/acetic acid (3:1) solution and placed on glass slides. Slides were dried at room temperature, stained with DAPI (100 ng/mL), and mounted with ProLong Gold Antifade (Invitrogen). Images were captured using a ZEISS LSM 710 confocal microscope and processed using ZEN software (ZEISS International, Oberkochen, DE).

Statistical analysis.
All data represent average values obtained in form three independent experiments.
Results were presented as means ± standard errors of means (SEMs). Statistical analyses were performed using GraphPad Prism software (version 5.0, GraphPad Software Inc., San Diego, CA, USA). Statistical comparisons were made using two-tailed unpaired t-tests and two-way analysis of variance (ANOVA). Results were considered significant when the p-value was *p < 0.005, **p < 0.001 or ***p < 0.0001. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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