Loss of CDCA5 Promotes Cell Apoptosis by Activating the DNA Damage Response in Clear Cell Renal Cell Carcinoma

doi:10.21203/rs.3.rs-952449/v1

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Loss of CDCA5 Promotes Cell Apoptosis by Activating the DNA Damage Response in Clear Cell Renal Cell Carcinoma

https://doi.org/10.21203/rs.3.rs-952449/v1

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Background:

Cell division cycle-associated 5 (CDCA5) protein, which is involved in cohesion, contributes to cell cycle regulation and chromosome segregation by maintaining genomic stability. Accumulating evidence indicates that CDCA5 is upregulated in many types of cancers with poor prognoses. However, it remains largely unknown about the biological function of CDCA5 in clear cell renal cell carcinoma (ccRCC).

Methods:

UALCAN databases were used to display CDCA5 expression profile and its prognosis in ccRCC patients. Further，we performed immunohistochemistry (IHC) on tissue microarray (TMA) containing 137 paired ccRCC and adjacent normal tissue samples, and evaluated correlation between CDCA5 expression and clinicalpathological parameters. Survival analysis by univariate and multivariate Cox regression were applied to identify the independent risk factors. By short hairpin RNAs (shRNA) knockdown, knockdown, a series of functional assays were conducted to determine the role of CDCA5 in ccRCC malignancy in vitro. DNA damage was analyzed by immunofuorescent staining and western blot. Orthotopic ccRCC model illuminated the function of CDCA5 in tumorgenicity in vivo.

Results:

TCGA data mining revealed that CDCA5 was more highly expressed in ccRCCs than adjacent normal tissues. Importantly, such high expression was correlated with a higher risk of distal metastasis and poorer clinical outcomes, which we further validated via immunohistochemical analysis of tissue microarrays. Functional studies showed that CDCA5 depletion significantly inhibited the proliferation and migration of ccRCC cells and suppressed the growth of xenografts in nude mice. Mechanistically, CDCA5 knockdown induced serious DNA damage with persistent accumulation of gamma-H2AX foci, resulting in G2/M cell cycle arrest and finally chromosomal instability and apoptosis. CDCA5 knockdown significantly decreased the phosphorylation of AKT, STAT3, and NF-κB, suggesting that CDCA5 plays a role in regulating the inflammatory response.

Conclusion:

ccRCC cells require CDCA5 for malignant progression, and that CDCA5 inhibition may enhance the outcomes of patients with high-risk ccRCC.

Cancer Biology

Cell division cycle-associated 5

ccRCC

Bioinformatic analysis

Tissue microarray

Prognosis

DNA damage repair.

Kidney cancer was currently the 11st leading malignancy incidence in men (254,507 cases) and the 16th common in women (148,755 cases), ranking as the 14th most common cause of cancer morality (175,098 cases) worldwide in 2018[1]. Based on intratumor heterogeneity (ITH) and biological characteristics[2, 3], genetic diversity determine varying responds to therapy, and even develop drug resistance by some key determinants including applying therapeutic pressures[4]. Thereby, it is urgent to find novel therapeutic target. Besides, except for widely used Fuhrmann grade to evaluate tumor malignancy, effective biomarker was less adopted to profile RCC.

CDCA5, also known as sororin, is a substrate of APC/Cdc20 (anaphase-promoting complex/cyclosome associated with Cdc20) [5], playing an important role in sister chromatid cohesion[6], chromatin structure[7], genome integrity[8], and DNA damage repair[9, 10]. Cohesion is a “ring structure” complex composed of some core subunits like SMC1, SMC3, RAD21 and STAG1/2[11]. CDCA5 antagonized WAPL to adjust loading and unloading of cohesion, thus stabilize cohesion-DNA interactions in the S and G2/M phases [12, 13].

In recent years, several studies demonstrated CDCA5 was upregulated with increasing tumor malignancy as an indicator of unfavorable prognosis, including breast cancer[14], hepatocellular carcinoma[15–17], colorectal cancer[18], esophageal squamous cell carcinoma[19], urothelial carcinoma[20] etc. Despite studies above, there has been no report describing the expression and function of CDCA5 in renal cell carcinoma.

The objective of our study was to investigate the prognostic value of CDCA5 expression levels in ccRCC and to throw light upon tumorigenesis associated with CDCA5. Based on that, for the first time we report the expression difference between ccRCC tumor specimen and adjacent normal tissue. Furthermore, more advanced grade and stages of ccRCC exhibited higher expression of CDCA5, predicting unfavorable survival. In vivo and in vitro tests were conducted to investigate the function and underlying mechanisms of CDCA5 in malignant progression of clear-cell renal cell carcinoma.

Our findings support the oncogenic role of CDCA5 in maintaining genome stability and promoting inflammatory signals, thus promoting ccRCC survival and progression, and CDCA5 may be a novel biomarker and potential therapeutic target to ccRCC.

Databases used and bioinformatics

To identify differential expressed genes (DEGs), we resort to the cancer genome atlas (TCGA) dataset (http://genome-cancer.ucsc.edu) to screen all genes positively correlated with poor prognosis of RCC. Gene ontology and KEGG pathway enrichment analysis revealed most of these differential expressed genes are associated with cell cycle control. After tumor survival analysis of these hub genes, we found cell-division cycle-associated 5 overexpressed in ccRCC and predicted a poor prognosis in patients.

Patients and tumor specimen

From October 2012 to May 2016, total 137 patients underwent RCC resection at Chinese People’s Liberation Army (PLA) General Hospital were enrolled in this research with median followed-up period 76.93 months (range 4.43-86.06 months). Resected samples were immediately snap-frozen in liquid nitrogen, and then stored at -80°C. Paired tumor tissue and adjacent normal tissue were taken for further analysis. None of them received chemoradiotherapy or molecular targeted therapy prior to nephrectomy. Written informed consent was obtained from all participants. The study was conducted in accordance with protocol approved by the Protection of Human Subjects Committee of Chinese People’s Liberation Army (PLA) General Hospital.

All RCC specimens were staged according to the 2011 Union for International Cancer Control (UICC) TNM classification of malignant tumors. The Fuhrman nuclear grading system was used to determine the nuclear grade.

Antibodies and reagents

The following primary antibodies were applied in this experiment:

Anti-CDCA5(1:2000, ab192237, Abcam), β-tubulin(1:3000, BE0025, Easybio), Akt (11E7, 1:2000, 4685S, CST), phosphate Akt (Ser473, 1:2000, 4685S, CST), Stat3 (D3Z2G, 1:2000, 12640S, CST), Phospho-Stat3 (Tyr705, 1:2000, 9145S, CST), NF-κB (1:1000 BE3154, Easybio), Phospho-NF-κB (1:2000, 3033S, CST), Phospho-mTOR (Ser2448, 1:2000, 5536, CST), P53 (Ser2448, 1:1000, 10442-1-AP, proteintech), Phospho-Histone H2A.X (Ser139, 1:1000, 9718T, CST), Phospho-BRCA1 (Ser1524, 1:1000, 9009, CST), Cyclin B1 (1:1000, 55004-1-AP, proteintech), PARP (46D11, 1:1000, 9532, CST), BCL2 (Ser2448, 1:1000, 12789-1-AP, proteintech), Ki67(D3B5, 1:400, 9129S, CST),

Cell lines

Human normal cell line, HKC, 293T, and RCC cell lines like SN12-PM6, ACHN, Caki-1, A498, OSRC-2, 769-P, and 786-O, were purchased from the National Platform of Experimental Cell Resources for Sci-Tech (Beijing, China). All cell lines were cultured respectively in Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Media (MEM), Roswell Park Memorial Institute (RPMI) 1640 medium or McCoy’s 5A (Gibco, ThermoFisher Scientific, Shanghai, China) supplemented with 10% fetal bovine serum (FBS) (EVERY GREEN, TIANHANG biotechnology, Zhejiang, China) and 1% penicillin G sodium/streptomycin with humidified 37°C incubator containing 5% CO2. Cells were authenticated by short tandem repeats (STR) typing and tested to exclude mycoplasma contamination throughout experiments.

Plasmid construction

To generate stable expression of CDCA5 knockdown cell lines, SN12-PM6 and 786-O were infected with lentiviral vector. phU6-MCS-Ubiquitin-EGFP-IRES-puromycin plasmid (GV280, Genechem, China) was used to colon shRNA for CDCA5. Based on the sequence of CDCA5, two interference sequences were designed:

5’-CCGGCCAAAGTACCATAGCCAGTTTCTCGAGAAACTGGCTATGGTACTTTGGTTTTTG-3’ (named KD1), and 5’-CCGGGAGCAGTTTGATCTCCTGGTTCTCGAGAACCAGGAGATCAAACTGCTCTTTTTG-3’ (named KD2).

EcoR I and BamH I restriction sites were introduced. Vector and synthesized interference sequence were double-enzyme linearized by T4 DNA. The lentiviruses were incubated in 293T cells with the pSPAX2 packaging plasmid and VSVG envelope plasmid together with reconstructed plasmid. After 48h and 72h transfection, virus supernatants were filtered through a 0.45 µm syringe filter. Then it was concentrated for immediate use or frozen at −80℃.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)

Cells and tissues were lysed with TRIzol Reagent (Invitrogen, USA). Total RNA was extracted using chloroform extraction as well as isopropanol precipitation. Reverse transcription was then conducted by a cDNA synthesis kit (E6560S, New England Biolabs) at 42℃ for 15 min. Subsequent quantitative polymerase chain reaction(qPCR) analyses for each cDNA genes were quantified under specific conditions as follows: Pre-denaturation at 94℃ for 30 seconds; 40 cycles of denaturation (94℃ for 5 seconds), annealing (50℃ for 15 seconds, 72℃ for 10 seconds). PPIA was used as an internal control. They were performed on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, USA). The data were calculated with the comparative Ct ( ) method. Primers of respective gene are listed in detail (Additional file 1: Table S1).

Cell proliferation assay

Cell proliferation assay was evaluated by crystal violet assay for determining cell viability. We start to seed 786-O and SN12-PM6 cells separately in a 6-well plate. After transfection of lentivirus for 72h, cells were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 20 min. Wash the cells gently in water, and then dissolve the dye into acetic acid. Measure the optical density of collecting solution at 570nm (OD570) with a plate reader. Calculate the mean value of three independent experiments.

Cell migration assay

In cell migration assay, the Boyden chamber was available for 24-well plates. Inserts were coated with polycarbonate Transwell membrane (Corning, NY) with an pore size. Followed by refilling with 500 high concentration medium (10% FBS) into the 24-well plate, approximately cells were adding into compartments with 150 medium containing 1% FBS. After incubation for 12 hours or overnight, cells remaining on the surface above were gently removed by cotton swab. Then the chamber with migrating cells attached to the bottom surface were fixed with methanol, then stained with 0.1% violet dye. Subsequently, chambers were screened, imaged, and quantified in five random fields per well under an electronic upright microscope. All assays were performed independently three times.

Cell cycle detection

Transfected 786-O and SN12-PM6 cell lines with plasmid for 48hs, collecting cells of both CDCA5 knockdown and negative control groups were washed in PBS and fixed in ice-cold 70% ethanol overnight at -20℃. Then cells were resuspended and treated with propidium iodide (PI) staining solution (P04D17, Gene-Protein Link, China) for 24 h consistent with manufacturer’s instructions. BD FACSCalibur (BectonDickinson, Franklin Lakes, NJ, USA) flow cytometer was used to analyze percentage of the cells in cell cycle phases. Samples was assayed in triplicate and repeated thrice.

Apoptosis analysis

Cell apoptosis determination was analyzed using apoptosis detection kit (P04D03, Gene-Protein Link, China) following manufacturer’s instructions. The treated RCC cells were stained with Annexin V-fluorescein isothiocyanate (FITC) and prodidium iodide (PI) for 15 minutes at room temperature under light-proof condition. Subsequently, cells were evaluated by flow cytometry as mentioned above.

Orthotopic RCC tumorigenicity in nude mice

Animal experiments were carried out following Animal Research Advisory Committee Guidelines of NIH and protocols approved by the Institutional Animal Care and Use Committee of Chinese PLA General Hospital. 18 four-six-week-old male BALB/c nude immunodeficient mice, weighing 15-18 g, were obtained from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China) and bred under a special pathogen-free (SPF) grade laboratory (Cyagen, Heibei, China). After transfected with lentivirus to build stable expression of CDCA5 shRNAs and control shRNA, those cells ( ) were suspended in PBS with Matrigel (Corning, Australia). Mice were anesthetized (Pentobarbital sodium, 30 mg/kg) and then orthopedically implanted into the right subrenal capsule (n=9 in each group). At 28 days post tumor engraftment, mice were euthanized followed by injectable anesthetics. Tumor volume calculations were obtained using the formula: V = , where ‘length’ represents the longest dimension and ‘width’ represents the longest dimension[21]. Tumor size was thereafter measured with standard calipers. Tumor weight, imaging, and histological staining were conducted extracorporeally for further analysis.

Western blot

Total protein from tissues and cells were prepared in RIPA lysis buffer containing protease inhibitor cocktail (#5871, CST). Quantified protein (30 µg) was separated by 8-12% SDS/PAGE gel electrophoresis, and then transferred to 0.45 polyvinylidene fluoride membranes (Direct-Q, Millipore, MA). After blocking the membrane with 5% non-fat milk for 1 hour, they were incubated with respective primary antibody overnight. Afterwards, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h and visualized using the ECL system (Thermo Fisher Scientific, Waltham, MA).

Tissue microarray construction and immunohistochemistry of tissue

Seven slides of TMA containing 137 paired formalin-fixed paraffin-embedded (FFPE) ccRCC and adjacent normal tissue samples on each slide was constructed. IHC for CDCA5 expression in TMA was carried out by standard protocol. TMA slides were deparaffinized and rehydrated through a series of graded xylene and ethanol washes. Antigen retrieval was conducted in a 0.01 M citrate buffer for 15 min with microwave heat Induction. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Then sides were applied by incubation of rabbit anti-CDCA5 antibody overnight at 1:200, followed with incubation of secondary anti-rabbit antibody (PV-9001, ZSGB-BIO, China). After utilizing horseradish peroxidase (HRP) as the detection system, staining was visualized with chromogen 3,3’-diaminobenzidine (DAB) and hematoxylin (ZIL-9018, ZSGB-BIO, China). Each TMA spot was evaluated independently and blindly by two researchers and assigned a score on its intensity of staining: 0 (no staining, negative), 1 (<10% of malignant cells staining, weak positive), 2 (10%-50% of malignant cells staining, moderate positive), or 3 (>50% of malignant cells staining, strong positive) within carcinomatous areas. Normal mouse lung tissues were used as negative control [22].

Immunofluence analysis with confocal microscope

786-O and SN12-PM6 cells were each cultured in 3 wells of 24-well plate with coverslips. After transfection of lentivirus for 24h and screening by puromycin for another 24h, cells were fixed with 4% paraformaldehyde for 15 min followed by permeabilization with 0.3% Triton X-100 for 10 min on ice. Then, cells were blocked in 3% goat serum albumin-PBS for 30 min. Primary antibody against γH2AX (1:100, 9718s, CST) incubated with cells overnight at 4℃. Cells were then washed and incubated in secondary antibodies Alex Fluor 594 Goat Anti-Rabbit IgG (1:500, A32740, Invitrogen) for 1h at room temperature. Place the coverslip on the fluorescent mounting medium with DAPI (ZLI-9557, ZSGB-BIO, China). Slides were imaged by Leica SP5 confocal microscopy with X100 amplification and analyzed by software.

Statistical analysis.

Data are generally expressed as the mean±standard deviation (SD) from experiments independently performed at least three times. Measurement data between groups were compared by one-way ANOVA, while the chi-square test or Fisher’s exact test were applicable to categorical variables. Comparisons of continuous values or abnormally distributed data were carried out either using Student’s t test or the Mann-Whitney U test. The overall survival analysis was calculated using Kaplan-Meier and further compared by log-rank tests. The cox proportional hazards regression model was applied to identify the risk factors. Value of P <0.05 was indicative of statistical significance. Statistical calculation was performed using SPSS 23.0 (SPSS Inc., Chicago, IL, USA)

Upregulated CDCA5 is correlated with aggressive status in ccRCC

TCGA analysis though online database UALCAN (http://ualcan.path.uab.edu/index.html) revealed that CDCA5 was upregulated in primary ccRCC tumor compared with adjacent normal tissue (Fig. 1A). Increasing expression transcript of CDCA5 correlated well with Fuhrman grade and advanced TNM stages (Fig. 1B, C). Besides, it even elevated in patients with lymph node metastasis (Fig. 1D), indicating an expression dependency on aggressive status in ccRCC. As for survival analysis (Fig. 1E, F), CDCA5 high expression predicted poorer prognosis than those patients with reduced expression (p<0.001). Stratified survival analysis with tumor grade identified the high expression with grade 4 tumor group showed the worst OS outcome (p<0.001).

Clinicopathological Characteristics and clinical significance

To investigate the malignant impact of CDCA5, Western-blot analyses of cultured human ccRCC cell lines demonstrated higher CDCA5 expression especially in 786-O and SN12-PM6 than others (Fig. 2A). As for ccRCC tissues, we performed RT-qPCR to quantified mRNA expression levels of 21 paired tumor and adjacent normal tissues, and found CDCA5 is overexpressed in ccRCC samples (Fig 2B).

Furthermore, protein expression characteristics was validated with 137 ccRCC patients’ paired tissues from our center by IHC in tissue microarray (Fig 2C). Integral optic density (IOD) values of IHC presented CDCA5 staining was increased along with advanced Fuhrman grade in ccRCC tissues (Fig 2D). Strong associations with high CDCA5 expression are primary tumor size (p=0.006), distant metastasis (p＜0.001), TNM stages (p＜0.001) and Fuhrman grade (p＜0.001) (Table 1), which show a good correspondence with TCGA analysis.

Table 1. Correlation between CDCA5 expression and clinical characteristics in ccRCC patients

Characteristics	Numbers (n=137)	CDCA5-Low (n=85)	CDCA5-High (n=52)	χ2 value	P value
Age (years)
≤60	104	66	38	0.369	0.544
>60	33	19	14
Sex
Male	103	66	37	0.729	0.393
Female	34	19	15
BMI
＜28	101	62	39	0.071	0.79
≥28	36	23	13
Primary tumor size
T1	111	75	36	7.578	0.006*
T2-T4	26	10	16
Lymph node metastasis
N1	6	1	5	3.656	0.056
N0	131	84	47
Distant metastasis
M1	13	1	12	15.557	＜0.001*
M0	124	84	40
TNM stage
I-II	116	82	34	21.686	＜0.001*
III-IV	21	3	18
Furhman Grade
I-II/Low	113	80	33	20.984	＜0.001*
III-IV/High	24	5	19
Hematuria
Present	13	7	6	0.41	0.522
Absent	124	78	46
Surgical approach
Transperitoneal	33	19	14	0.369	0.544
Retroperitoneal	104	66	38
Resection
Partial	49	34	15	1.747	0.186*
Radical	88	51	37
Chronic Disease
Present	52	31	21	0.21	0.647
Absent	85	54	31

* Significance

Moreover, with following up time 76.933±0.372 months, Kaplan-Meier survival analysis showed high-expressed CDCA5 correlated with poorer 5-year disease-free survival and overall survival (Fig 2F). Multivariate cox regression stepwise analysis (Additional file 1: Table S2), which those significant factors were briefly displayed as forest plot (Fig 2E), confirmed high CDCA5 expression remained an independent risk factor both for both OS (P = 0.025, hazard ratio (HR) = 11.296, 95% confidence interval (CI) = 1.36–93.793) and DFS (P = 0.019, HR = 6.679, 95% CI = 1.368–32.597).

Collectively, results above confirmed that CDCA5 expression may serve as a malignant prognostic factor for OS and DFS in ccRCC patients.

CDCA5 knockdown inhibits the proliferation and migration

Since TCGA analysis indicate expression of CDCA5 correlated with ccRCC progression and nodal metastasis, we get down to investigating biological function of CDCA5 in ccRCC cell lines. After transfecting shRNA targeting CDCA5 and negative control shRNA respectively into SN12-PM6 and 786-O, knockdown CDCA5 significantly hinder the cell proliferation. Cell viability was dramatically decreased in both cell lines by crystal violate assay (P<0.05; Fig. 3A). Furthermore, western-blot analyses confirmed reduced expression of CDCA5 protein with the two lentiviral shRNAs in both SN12-PM6 and 786-O cells (Fig. 3C). As CDCA5 significantly affects ccRCC cell proliferation, we thus evaluate its effect on invasion and migration. Transwell assays demonstrated downregulation CDCA5 impaired migrative capability in vitro compared with the control group (Fig. 3B). These results indicted CDCA5 is vital to maintain cell proliferation and migration ability.

Downregulation CDCA5 induced cell cycle arrest in G2/M phase

To further investigate underlying mechanism on cell proliferation inhibition, we resort to cell cycle analysis to clarify which phase arrest followed by CDCA5 knockdown. Flow cytometry analyses revealed CDCA5 downregulation decreased cell distribution in G0/G1 phase while increasing cell proportion in G2/M phase in both SN12-PM6 and 786-O cell lines (P<0.05; Fig 4A).

Besides, immunofluorescence of phosphate histone 3 staining demonstrated the decreasing number of positive cells in shCDCA5 groups compared with increasing positive ratio in shNC group (Fig 4B). Meanwhile, the expression levels of G2/M-related proteins p53 (Fig. 5C) and CyclinB1 (Fig. 6B) change correspondently by CDCA5 knockdown. Cyclin B1, essential for G2/M transition, decreased in both cell lines, while p-p53 was activated especially in SN12-PM6 in shCDCA5 group (Fig. 5C). These data above confirmed CDCA5 functioned in cell cycle regulation in G2/M checkpoint

CDCA5 Inhibition improves DNA damage response (DDR)

As CDCA5 is a subtract of cohesion to maintain genome stability, we further validate whether downregulation of CDCA5 could trigger DDR. Phosphorylation of H2AX, which is a biomarker of double strand breaks, was found to increase foci formation numbers in immunofluorescence after loss of CDCA5 in 786-O (Fig. 5A) and SN12-PM6 (Fig. 5B). Meanwhile, we foundγ-H2AX positive cells in both cell lines take large proportion in shCDCA5 groups (p<0.05). The increment ofγ-H2AX expression was detected likewise by western blotting compared with the control, which was in accordance with the activation of phosphate p53(Ser15) especially in shCDCA5 SN12-PM6 cells (Fig. 5C). Taken together, loss of CDCA5 trigger sequential activation of DDR.

CDCA5 knockdown enhances RCC apoptosis

As response to DNA damage and cell cycle arrest, we thus investigate the cell fate towards apoptosis. Effect of CDCA5 on cell apoptosis was analyzed by flow cytometry using Annexin V-FITC staining. The result demonstrated that CDCA5 knockdown promoted both early and late apoptosis in shCDCA5 groups compared in the control groups. The late apoptosis was prominent according to flow cytometry analysis (p<0.001, Fig 6A). In 786-O cells, average percentage of apoptotic cells (early and late apoptosis) in shCDCA5-1 was 3.3-fold increase while shCDCA5-2 was 1.6-fold increase. Besides, as for SN12-PM6, the total apoptotic cells in shCDCA5-1 were 5.2-fold increase while shCDCA5-2 was 4.1-fold increase compared with the control (Fig 6A).

Consistently, cell apoptosis -related proteins levels altered in Western blot. Downregulation of CDCA5 notably abrogated the expression of the antiapoptotic proteins BCL-2 by western blot. And downstream proteins like poly (ADP-ribose) polymerase (PARP) were activated that cleaved PARP presented upregulation in CDCA5-knockdown cells (Fig. 6B). Based on these results, shCDCA5 show a strong effect on apoptosis in ccRCC cells.

CDCA5 knockdown suppresses tumorigeneses in orthotopic model of RCC

In vivo experiment, to illuminate the function of CDCA5 in tumorgenicity, we inject shCDCA5-2 and shNC SN12-PM6-luciferase cells orthotopically into the right kidney of nude mice. Intratumoral green fluorescent protein (GFP) fluorescence were detected after administration of D-luciferin at 4 weeks of xenograft model. Strong luciferace signal were obtained in the mice implanted with cells transfected with control vector, whereas reduced signal intensity was observed in the shCDCA5 group (Additional file 1: Fig S1). Besides, SN12-PM6 xenograft transduced with sh-CDCA5 significantly inhibit tumor growth (Fig 7A, B) with decreased tumor volume and tumor weight (Fig 7B, C).

Then tumors were resected and embedded for H&E stanning and IHC (Fig 7D). IHC analysis confirmed shCDCA5 tumor displayed significantly limited Ki-67 expression vs. shNC tumor (p<0.001, Fig 7D, E). Orthotopic model validated shCDCA5 could suppress tumor growth in vivo.

CDCA5 regulated ccRCC behavior via the DDR

DNA double strand breaks were triggered initially by downregulation of CDCA5. In response to it, sequential alternative expression of downstream signaling pathways were investigated by western blot. The prominent change is inflammatory response related transcription factors Stata3 and NF-κB. We observed that Stat3, p-Stat3, and NF-κB expression were suppressed in CDCA5-knockdown cells (Fig 6B). Besides, compared with scramble control cells, the expression of total AKT, p-AKT and p-mTOR was inhibited in shCDCA5-1 and shCDCA5-2 cells (Fig 6B，Additional file 2: The original data). However, no significant difference in ERK expression was observed. Taken together, these results suggest that CDCA5 mainly involved in DNA damage and had functional interaction with inflammatory response by inhibition STAT3 and NF-κB.

With prevalent incidence in urologic cancer, ccRCC still lacks of effective biomarkers and show refractory response to many chemotherapies. Development of genomic instability is enabling characteristics of cancer[23], which frequently results from genes alterations in DNA damage repair and chromatin remodeling[24]. CDCA5, as an essential component of cohesion, mediate the regulation of chromatin segregation in metaphase-to-anaphase transition, and maintain the genome stability. Prior studies have noted overexpressed CDCA5 in many types of cancer progression[18, 19, 25–27]. However, none of studies so far have investigated the expression and function of CDCA5 in ccRCC.

In this study, we observed the upregulation of CDCA5 in paired ccRCC tissue vs. adjacent normal tissue. and its upregulation level was positively correlated with larger tumor size, distant metastasis, advanced TNM stages and higher Fuhrman grade. The results displayed that high-expressed CDCA5 in ccRCC tissues was correlated with poor survival. These clinicopathological characteristics were consistent with TCGA analysis.

In vitro study found downregulation of CDCA5 inhibited proliferation, migration and promoted the apoptosis of SN12-PM6 and 786-O cells. Moreover, shCDCA5 suppresses tumorigeneses in orthotopic implantation model of ccRCC. So shCDCA5 suppresses the progression and promotes the apoptosis of ccRCC cells, contributing to longer DFS and OS survival. Moreover, univariate and multivariate Cox regression analyses identified CDCA5 overexpression is an independent indicator for poorer prognosis of DFS and OS. Thus, in accord with previous studied[18, 26–31], our results supported the oncogenic role of CDCA5 in ccRCC progression which make CDCA5 possible to be a therapeutic target for ccRCC patients.

Furthermore, depletion of CDCA5 dramatically induced DNA damage response. In response to DNA damage, recruitment of γ-H2AX were enriched in damage sites, while it stabilized and activated the expression of tumor suppressor p53[32]. In turn, activated p53 could cause several potential tumor suppressive effects, like cell proliferation inhibition, cell cycle arrest and apoptosis. There are amounts of cancer related to the impairments of DNA damage response. Mechanistic defects are able to exploit as potential therapeutic approaches accordingly, like genotoxic agents and PARP inhibitors. Genome sequencing of ccRCC have reveals comprehensive mutations underpinnings in tumorigenesis, including VHL, PBRM1, BAP1 and SETD2 etc[3]. PBRM1 involves in formation SWI/SNF chromatin remodeling complex, while SETD2 function as histone methyltransferase to facilitate homologous recombination in DNA double-strand break and chromatin remodeling[24, 33]. By antagonizing Wapl’s ability to dissociate cohesin from DNA[12], the recruitment of CDCA5 is continuously required for sister chromatid cohesion maintenance, which involve DNA looping and transcriptional regulation[8, 34]. Molecules that involved in chromatin remodeling offered an alternative view on ccRCC treatment.

Under DNA damages, the checkpoint can prevent cell initiating mitosis resulting in cell cycle arrest[35]. Downregulation of CDCA5 cause cell cycle arrest in G2/M phase by activation p-p53-serine 15, accumulation of p21 and the depletion of cyclinB1. p-histone H3 is cell cycle specific markers that allow us to determine the nature of cell cycle-progression manner[36]. Flow cytometry and decreased expression of p-Histone-H3 in IF confirmed the presence of cell cycle arrest at G2/M checkpoint.

In the present study, we showed that knocking-down CDCA5 could decrease Stat3, p-Stat3, NF-κB, that mainly weaken the inflammatory response. Correspondingly, another inflammation related pathway like AKT, p-AKT, p-mTOR were inactivated by CDCA5 downregulation, consistent with the findings in bladder cancer that CDCA5 function through PI3K/AKT/mTOR pathway[25]. Taken together, in response to DNA damage after knockdown CDCA5, sequential cellular process occurred including DNA damage response, cell cycle arrest, and the promotion of cell apoptosis. Bcl-2 itself function prominently in blocking apoptosis[37]. And PARP regulates several biological functions mainly in cell survival and cell-death programmes[38]. PARP is predominantly activated in DDR to cleaved PARP and induction of target downstream regulator like p53[39]. Our study showed downregulating CDCA5 significantly promoted apoptosis, which were evidenced by suppressed expression of Bcl-2, increased activation of PARP to cleaved PARP, increased expression of p-p53, and reduction of inflammatory transcription factors like NF-κB and Stat3.

In conclusion, our study portrait CDCA5 overexpression in ccRCC and correlate with TNM stages. Upregulation of CDCA5 predicted poor survival outcome. Meanwhile, inhibition of CDCA5 decrease cell proliferation and malignancy in vitro and in vivo. It is likely transcribed by E2F1 and function through cell cycle pathway. CDCA5 may represent a prognostic factor for patients, and could be a prospective therapeutic target for cancer treatment.

CDCA5: Cell division cycle-associated 5; ccRCC, clear cell renal cell carcinoma; TCGA: The cancer genome atlas; PCR: Polymerase chain reaction; shRNA: Short hairpin RNA; TMA, tissue microarray; IHC: Immunohistochemistry; PARP: poly (ADP-ribose) polymerase; DDR: DNA damage response;

Acknowledgements

Not applicable.

Author Contributions: XH, YH and XZ conceptualized and designed the experiments. XH, ZL and TW performed experiments, analyzed and visualized data. HW and HF assisted with flow cytometry and animal experiments. HF followed up and generated tables. SD, SW, DS, and CW collected the clinical data. ST, ZL, and HW were in charge of data mining and curation. XH and YH prepared the manuscript. HL, BW, and XZ reviewed and revised the paper. XZ, XM, BW, and HL provided clinical insight, secured funding and supervised research.

Funding

This research was funded by National Nature Science Foundation (Grand No. 81972389 and 81770790)

Data Availability Statement

The bioinformatics analysis is achieved online from the UALCAN database (http://ualcan.path.uab.edu/index.html). The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

Deidentified archived TMA were used. The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the IRB of PLA General Hospital.

Consent for publication

All authors have reviewed the manuscript and agreed to publish this article.

Competing interests

The authors declare that they have no competing interests.

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Loss of CDCA5 Promotes Cell Apoptosis by Activating the DNA Damage Response in Clear Cell Renal Cell Carcinoma

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