Non-SMC condensin I complex subunit H mediates mature chromosome condensation and DNA damage in pancreatic cancer cells

Non-SMC condensin I complex subunit H (NCAPH) is a vital gene associated with chromosome stability and is required for proper chromosome condensation and segregation. However, the mechanisms through which NCAPH affects pancreatic cancer (PC) and its molecular function remain unclear. In this study, we examined the role of NCAPH in PC cells. Our results showed that NCAPH was overexpressed in clinical PC specimens (GEPIA) and cell lines. In addition, in NCAPH-knockdown cells, colony formation and proliferation were inhibited, and the cell cycle was arrested at the S and G2/M phases owing to failure of mature chromosome condensation (MCC) in poorly condensed chromosomes. Increased cell death in NCAPH-knockdown cells was found to help initiate apoptosis through the activation of caspase-3 and PARP cleavage. Furthermore, NCAPH-knockdown cells showed an increase in chromosomal aberrations and DNA damage via activation of the DNA damage response (Chk1/Chk2) signaling pathways. These data demonstrated that NCAPH played an important role in cell cycle progression and DNA damage by maintaining chromosomal stability through progression of MCC from poorly condensed chromosomes. Ultimately, NCAPH knockdown induced apoptotic cell death, which was partially mediated by caspase-dependent pathways. These findings highlight the potential role of NCAPH as a therapeutic target for PC.


Results
ncApH expression was upregulated in pc tissues and cell lines. First, we analyzed the mRNA expression of all subunits of condensin I and II complexes in PC and normal tissues using a publicly available database (The Cancer Genome Atlas [TCGA]). According to the data obtained from TCGA, the expression of NCAPH was significantly higher than that of the condensin subunits in all pancreatic adenocarcinoma (PAAD) tumor types (n = 179) compared with that in their normal tissue counterparts obtained from TCGA and that obtained from GTEx data (n = 171; Fig. 1A and Supplementary Fig. 1A). In addition, the overall survival (OS) of patients with PC with high NCAPH expression was significantly lower than that of patients with low NCAPH expression (Fig. 1A). Among all subunits of condensins I and II, NCAPH elicited the greatest difference in OS, based on its mRNA expression ( Fig. 1A and Supplementary Fig. 1A). We also examined condensin I (NCAPH, NCAPD2, NCAPG) and condensin II (NCAPH2, NCAPG2) protein levels in PC cell lines, including AsPC-1, PANC-1, MIA PaCa-2, Capan-1, and Capan-2 cells. The results showed that protein expression of condensin I rather than II was higher in various PC cell lines compared with its expression in HPDE cells, and NCAPH was markedly upregulated by approximately 10-fold ( Fig. 1B,C, and Supplementary Fig. 1B).

ncApH knockdown inhibited colony formation and proliferation in pc cells.
To evaluate the effects of NCAPH 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 using three NCAPH siRNAs specifically targeting NCAPH. Among these siRNAs, NCAPH siRNA_3 resulted in the most significant inhibition of NCAPH expression and colony formation in MIA PaCa-2 and PANC-1 cells ( Fig. 2A,B, and Supplemental Fig. 2A) and was therefore selected for subsequent studies. Indeed, NCAPH knockdown significantly suppressed the colony formation ability of several PC cell lines (e.g., AsPC-1, Capan-1, and Capan-2 cells; Fig. 2C). NCAPH knockdown significantly inhibited the proliferation of PC cell lines, such as MIA PaCa-2 and PANC-1, as demonstrated by MTT assay 5-7 days after siRNA transfection (Fig. 2D). Additionally, we performed a reconstitution experiment after NCAPH siRNA transfection. For this experiment, we constructed a lentiviral vector and NCAPH. When NCAPH was depleted, the proliferation of PC cells was significantly reduced. In contrast, Lenti-NCAPH was able to partially recover the reduced PC cell proliferation (Fig. 2E). Expression of NCAPH protein was also restored by Lenti-NCAPH (Fig. 2F). These results indicated that NCAPH played essential roles in the colony formation and proliferation of PC cells.

NCAPH knockdown arrested cells in S and G2/M phases and promoted cell apoptosis.
We performed flow cytometry analysis to investigate the effects of NCAPH on cell cycle regulation and apoptosis in PC cells. Quantification of cells at the subG 1 , G 1 , S, and G 2 /M phases of the cell cycle revealed that the number of NCAPH-knockdown cells accumulated in the S and G 2 /M populations and that the subG 1 population (apoptotic cells) significantly increased compared with that in the control, whereas the G 1 cell population was decreased in a concentration-dependent manner (Fig. 3A,B). In addition, we investigated the molecular mechanisms altered by NCAPH knockdown in cell cycle regulation by evaluating the expression of cyclin A and phospho-histone H3 (Ser10) proteins, which regulate the G 2 /M phase transition, by western blotting analysis. Cyclin A and phospho-histone H3 (Ser10) levels significantly increased in NCAPH-knockdown cells 24-48 h after transfection (Fig. 3C). Collectively, these observations suggested that NCAPH knockdown caused S and G 2 /M phase arrest, which could partly explain the reduced viability and enhanced cell death in PC cells.
www.nature.com/scientificreports www.nature.com/scientificreports/ To further determine whether the cell death observed in NCAPH-knockdown cells was induced by apoptosis, apoptosis levels were investigated using the Annexin V-FITC/PI double staining technique. NCAPH knockdown by siRNA was observed in the early/late apoptosis induction effect (UR and LR quadrants; Fig. 3D). We also identified caspase-3 activity and poly-ADP ribose polymerase (PARP) cleavage events to ensure apoptotic cell death 30 and found that caspase-3 activation and PARP cleavage were significantly increased in NCAPH-knockdown cells (Fig. 3C). To confirm these results, cells were incubated with 20 μM Z-VAD-Fmk, a pan-caspase inhibitor, for 24 h, which was able to suppress the early apoptosis induced by NCAPH knockdown (LR quadrant; Fig. 3D). Additionally, protein expression of caspase-3 and cleaved PARP was also significantly decreased by Z-VAD-Fmk (Fig. 3E). These data clearly indicated the partial involvement of caspase-dependent apoptosis in NCAPH knockdown-induced cell death. ncApH was involved in progression of mature chromosome condensation (Mcc) from poorly condensed chromosomes. Accumulation of cell ratios at the S and G 2 /M phases indicated that these cells were not able to successfully undergo mitosis when the chromosomes could not be separated before the formation of two daughter cells. To better determine whether the S and G 2 /M phase accumulation previously observed The OS of patients with low or high expression levels of NCAPH was analyzed by the Kaplan-Meier method and a log rank test in PAAD. The dotted high line is a sample with a higher expression level than the median value of TPM and is considered a high expression cohort, whereas the dotted low line is a sample with a lower expression level than the median value of TPM and is considered a low expression cohort. Median values are indicated by full lines. *P < 0.005. TCGA, The Cancer Genome Atlas; PAAD, Pancreatic adenocarcinoma; TPM, transcripts per million; OS, overall survival; HR, hazard ratio. (B) NCAPH protein expression was analyzed by western blotting in HPDE cells and different PC cell lines (AsPC-1, PANC-1, MIA PaCa-2, Capan-1, and Capan-2 cells). Cell lysates were immunoblotted with the indicated antibodies. (C) Frequency of NCAPH protein expression in HPDE and PC cell lines. The NCAPH/β-actin ratio was determined by densitometric analysis using ImageJ. Error bars represent standard deviations of the means of three biological replicates. Values represent means ± SEMs. *P < 0.005, **P < 0.001. Statistical analysis was performed using one-way analysis of variance followed by Bonferroni's multiple comparison tests. (2019) 9:17889 | https://doi.org/10.1038/s41598-019-54478-3 www.nature.com/scientificreports www.nature.com/scientificreports/ in NCAPH-knockdown cells was a result of a delay or block in cell cycle progression, we investigated cell cycle progression over time after cell release from a double thymidine block (Fig. 4A). Thymidine is a pyrimidine deoxynucleoside that can be used to synchronize cells at the G 1 /S transition. Cell synchronization using double thymidine blocks in control cells allows entry into S phase (0-4 h) and promotes synchronous mitosis (G 2 /M phase: 7-8 h). NCAPH was depleted by siRNA, and then double thymidine block was used to prevent entry of cells into G 1 /S phase. Synchronized cells at different phases were harvested at 0, 4, and 8 h after release from the thymidine block (Fig. 4A). As expected, most cells in the control group had a DNA content consistent with being blocked at either G 1 or S phase followed by double thymidine treatment (Fig. 4B,C), and the number of cells with a DNA content consistent with S phase increased (22.1% to 32.9%) at 4 h and they progressed to the G 2 /M phase (8.3% to 32.9%) at 8 h after release from the second thymidine block (Fig. 4B,C). In comparison, the cell cycle profile for NCAPH-knockdown cells showed an increase in cells with a DNA content consistent with S phase (26.8% to were transfected with three different siRNA constructs to knockdown NCAPH expression, and the expression levels of NCAPH protein were measured 48 h later by western blotting. Cell lysates were immunoblotted with the indicated antibodies. (B) Colony formation assays were performed on MIA PaCa-2 and PANC-1 cells transfected with the control and three different NCAPH siRNAs (NCAPH siRNA_1, siRNA_2, and siRNA _3). (C) Colony formation assays were performed in AsPC-1, Capan-1, and Capan-2 cells with control siRNA and NCAPH siRNA_3. The colonies were imaged and counted. Values represent means ± SEMs. Statistical analysis was performed using unpaired t-tests. ***P < 0.0001. (D) MTT assays were performed to determine proliferation of control and NCAPH-knockdown PC cells for 5 or 7 days. The curve was based on biological triplicates with values expressed as means ± SEMs. *P < 0.005, ***P < 0.0001. Statistical analysis was performed using two-way analysis of variance with Bonferroni's multiple comparison tests. (2019) 9:17889 | https://doi.org/10.1038/s41598-019-54478-3 www.nature.com/scientificreports www.nature.com/scientificreports/  Values represent means ± SEMs. *P < 0.005, ***P < 0.0001. Statistical analysis was performed using one-way analysis of variance followed by Bonferroni's multiple comparison tests. 62.7%) at 4 h, with a significant proportion of cells maintained at G 2 /M phase (5.4% to 65.8%) at 8 h after release from the second thymidine block (Fig. 4B,C). However, when we performed these experiments on HPDE cells, no differences in cell cycle progression between control siRNA and NCAPH siRNA treatment were observed, as observed in PC cells (Supplemental Fig. 3A). These results indicated that NCAPH may influence the S and G 2 /M phases in PC cells.
Next, to determine when structural chromosome aberrations occurred during cell division, we examined chromosomal breaks, segmentation, and aberrations in control and NCAPH-knockdown cells. In this experiment, thymidine block was performed in control or NCAPH-knockdown MIA PaCa-2 cells, and the chromosomal structure of cells released for 3 h was confirmed by metaphase chromosome spreading assays. Surprisingly, knockdown of NCAPH did not lead to the progression of MCC from poorly condensed chromosomes (Fig. 4D). Furthermore, in NCAPH-knockdown cells, only 9.7% of cells progressed to MCC from poorly condensed chromosomes, as compared with 45.8-54.1% of control cells (Fig. 4E). These results indicated that NCAPH was vital for the progression of MCC from poorly condensed chromosomes. ncApH knockdown promoted chromosomal aberrations and DnA damage. In Xenopus egg extracts, as condensins I and II are forced to be smaller, chromosomes become shorter and thicker. Condensin I is involved in lateral compaction, and condensin II is involved in axial shortening 31 . Additionally, in chicken DT40 cells, mitotic chromosomes are wide and short owing to depletion of condensin I, and chromosomes of condensin II-depleted cells appear to be more extended and lack axial stiffness 32 . To elucidate how mitotic chromosome structures are affected by NCAPH knockdown, we performed chromosome spreading assays in MIA PaCa-2 and HeLa cells. Similar to the previous report, shortening and thickening of chromosomes was observed in both types of cells ( Supplementary Fig. 4A). However, upon specifically staining with anti-NCAPH antibodies and 4′,6-diamidino-2-phenylindole (DAPI) in MIA-PaCa-2 cells, NCAPH was detectable along the chromatid axis in cells of the control group but not in cells of the NCAPH-knockdown group, and the twisted and segmented chromosome morphology was observed in the NCAPH-knockdown group ( Supplementary Fig. 4B). When measuring the number of structural chromosome aberrations in NCAPH-knockdown cells compared with those in control cells, we observed a significant increase (23.7% versus 75.2%, respectively; Supplementary  Fig. 4C). To define chromosomal structures more clearly, we divided the state of the chromosomal structures into normal or abnormal chromosome condensations and classified them as mild, severe, or segmentation. The abnormal chromosome condensation (mild and severe) and segmentation type chromosome morphology were increased in NCAPH-knockdown cells (Fig. 5A,B). Additionally, we sought to determine whether the structural chromosome aberrations in NCAPH-knockdown cells were associated with DNA damage responses. To establish the presence of DNA damage, we monitored the appearance of DNA damage foci using antibodies detecting phosphorylated H2AX at S139 (phospho-H2AX), a marker of DNA double-strand breaks (DSBs). Western blot and immunofluorescence analyses showed that the levels of phospho-H2AX were higher in NCAPH-knockdown cells than in control cells (Fig. 5C-E). Moreover, phospho-H2AX was more abundant in NCAPH-knockdown cells than in control cells.
Next, we determined whether G 2 /M phase cell cycle arrest was caused by DNA damage induced by NCAPH knockdown. The results showed that the levels of phospho-Chk1 and phospho-Chk2, which are DNA damage markers, were increased 24 and 30 h after NCAPH knockdown (Fig. 5F). In NCAPH-knockdown cells, the signals of phospho-Chk2 increased significantly compared with the signals of phospho-Chk1 (Fig. 5F). Moreover, to further define the role of ATM kinase in Chk2 phosphorylation in cell cycle defects induced by NCAPH knockdown, cell cycle changes were compared with those in cells when treated with thymidine double blocks alone or with a second thymidine block using an ATM inhibitor (KU-55933, ATMi). The result showed that the G 2 /M phase arrest in NCAPH-knockdown cells was significantly reduced by the ATM inhibitor (Fig. 5G). These results demonstrated that knockdown of NCAPH induced structural chromosome aberrations, which were associated with DNA damage, through the Chk1/Chk2 signaling pathway in PC cells.

Discussion
In this study, we reached the following conclusions: (1) NCAPH was upregulated in PC tissues and cell lines; (2) knockdown of NCAPH induced cell cycle arrest and cell apoptosis; (3) NCAPH was important for proper progression of MCC from poorly condensed chromosomes; and (4) NCAPH knockdown induced DNA damage though the Chk1/Chk2 signaling pathway. To the best of our knowledge, this is the first report demonstrating that chromosomal aberrations and DNA damage were induced in NCAPH-knockdown PC cells through the Chk1/ Chk2 signaling pathway.
Recent studies have evaluated the relationships between condensin complexes and cancers [25][26][27][28][29] . For example, NCAPH knockdown inhibits proliferation and invasion of CC cells 29 . In hepatocellular carcinoma, lack of NCAPG reduces cell viability, increases apoptosis, and arrests cells in the S phase of the cell cycle 26 . In this study, we focused on NCAPH because its expression was increased in PC tissues and cell lines. Although NCAPH has been shown to be associated with cell invasion and proliferation in CC, its functional role in PC had not yet been evaluated. We found that cell cycle arrest and cell apoptosis were increased by NCAPH knockdown, and apoptosis occurred via the intrinsic apoptotic pathway with loss of mitochondrial potential, activation of caspase-3, and generation of the cleaved form of PARP-1, a typical caspase-3 substrate 33 . In addition, lack of NCAPD2, another subunit of condensin I, also inhibited colony formation by PC cells (Supplemental Fig. 2B,C), confirming that the condensin complex played an important role in PC growth.
To ensure proper chromosome condensation and segregation in mitosis, condensin II promotes the separation of replicated chromosomal loci and initiates structural reorganization of replicated chromosomes during S phase 34 . However, our study showed that when NCAPH (condensin I) was absent, cells were arrested in the S and G 2 /M phase of the cell cycle. Importantly, normal maintenance of the S phase in cell cycle progression could www.nature.com/scientificreports www.nature.com/scientificreports/ affect the structure of mitotic chromosomes. For example, when mammalian cells in the G 2 phase are fused with G 1 -phase cells, all cells enter mitosis synchronously. However, the DNA from G 1 -phase cells passes through S phase faster than expected, resulting in less condensed mitotic chromosomes 35 . Importantly, when the G 1 -phase nucleus is fused with the mitotic nucleus, the detected chromosomes showed insufficiently condensed and much longer than normal chromatids 35,36 . These experiments have demonstrated that activities in mitotic cells result in condensation of interphase chromatin but that the structure of the condensed chromosomes is deficient. Moreover, lack of B-Myb, an Myb family transcription factor that plays an essential role during S phase, decreases S-phase progression and increases chromosomal fragmentation and other aberrations 37 . In any case, the completion of DNA replication is associated with proper chromosome condensation. Based on our observations after the knockdown of NCAPH, we hypothesize that NCAPH may have a major role in facilitating correct cell cycle progression through the S and G 2 /M phases and accompanying chromosome condensation. NCAPH knockdown resulted in chromosomal fragmentation and DNA damage responses, which affected the progression of MCC from poorly condensed chromosomes. We demonstrated that NCAPH-knockdown cells were arrested at S and G 2 /M phases of the cell cycle and blocked the progression of MCC from poorly condensed chromosomes, preventing the completion of proper chromosome condensation of PC cells. These findings suggested that NCAPH played an important role in the progression of MCC from poorly condensed chromosomes during the cell cycle process, revealing a novel mechanism through which progression of MCC from poorly condensed chromosomes is regulated by NCAPH.
When DNA damage occurs, cells spend more time undergoing DNA repair processes or apoptosis, particularly if the damage is severe. Therefore, cells have maintained genome integrity by developing complex machinery that includes DNA-damage responses, which detect and repair DNA damage. Generally, there are two types of DNA damage, DSBs and single-strand breaks (SSBs). DSBs are rapidly detected by the Mre11/Rad50/NBS1 complex, which subsequently promotes the activation of ataxia telangiectasia mutated kinase by autophosphorylation 38 . SSBs are sensed by the Rad9/Hus1/Rad1 complex, which activates ataxia telangiectasia and Rad3-related kinase 39 . Following this sensing step, Chk1 and Chk2 amplify the signals from the sensors, phosphorylating a variety of effectors 40 . Additionally, Chk1 monitors S phase replication and mitosis entry 41,42 , and Chk2 triggers the G 1 /S checkpoint and apoptosis 43 . In particular, SMC2 recruitment to mitotic chromosomes is controlled by DNA damage response checkpoints, such as Chk2, which facilitates effective repair of genomic damage 44 . Based on these findings, our data clearly indicated that NCAPH plays a critical role in the response to DNA damage through the increased phosphorylation of Chk1/Chk2 kinases. This finding was confirmed by the results of ATM treatment, which reduced the arrest of cells in G 2 /M phase caused by NCAPH knockdown because ATM is responsible for the phosphorylation of Chk2.
In conclusion, the results of this study confirmed that NCAPH knockdown induced chromosomal aberrations and DNA damage through the Chk1/Chk2 signaling pathway, ultimately resulting in selective cell death through partially caspase-dependent apoptotic signaling in PC cells. Based on our findings, we hypothesize that NCAPH may be associated with human carcinogenesis and could be a potential therapeutic target for human cancer. Further studies are needed to test this hypothesis.

Methods
Database analysis. Expression levels of the subunits of condensin I complex in PC and normal pancreas were achieved from Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia.cancer-pku.cn/) 45 , which analyzed 179 PAAD tumors and 171 normal samples from TCGA tumor samples with paired adjacent TCGA normal samples and GTEx normal samples. Transcript per million (TPM) values were calculated, and the expression levels of genes were presented using the Log 2 (TPM + 1) scale. The cutoff values were 1 for |Log 2 FC| and 0.01 for the P value. The OS of patients with PC was also analyzed. cell culture and siRnA knockdown. MIA PaCa-2 (American Type Culture Collection [ATCC] CRL-1420; ATCC, Manassas, VA, USA) and PANC-1 (ATCC CRL-1469) human PDAC cell lines were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM). Human PDAC cell lines (AsPC-1, Capan-1, and Capan-2) were grown in RPMI medium. Noncancerous immortalized HPDE cells were obtained from Joo Kyung Park, MD (Samsung Medical Center, Seoul, South Korea). HPDE cells were grown in Defined K-SFM medium. All cell culture media contained 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Life Technologies, Grand Island, NY, USA). To knockdown NCAPH expression, the cells were transfected with siRNA using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Three different NCAPH siRNAs were prepared 46 . The three NCAPH siRNAs and control siRNA were purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan) cell proliferation and viability assay. Cell proliferation and viability assays were performed using MTT solution. MIA PaCa-2 and PANC-1 cells were seeded into 12-well plates at a density of 1 × 10 4 cells/well or 8 × 10 4 cells/well. After adhesion for 24 h, the cells were transfected with NCAPH siRNA or control siRNA and incubated for 5 or 7 days. For colony formation assays, cells were seeded into 6-well plates at a density of 500-1000 cells/well and incubated for 14 days. The colonies were then fixed in 100% methanol, stained with 10% crystal violet, and counted. Each assay was performed in triplicate. chromosome spreading assay. Cells were treated with colcemid (0.1 µg/mL) for 4 h and then harvested.
After treatment with 0.075 M KCl, cells were fixed with a drop of freshly made fixative (methanol/acetic acid [3:1]) and placed on glass slides. The slides were dried at room temperature, stained with DAPI (100 ng/mL), and visualized under a confocal microscope (Zeiss LSM710, software ZEN; Zeiss, Germany).