Y-box binding protein 1/cyclin A1 axis specifically promotes cell cycle progression at G₂/M phase in ovarian cancer

Abstract

Y-box binding protein 1 (YBX1) promotes oncogenic transformation and tumor growth. YBX1 plays a role in regulation of cell cycle promotion via upregulation of cell cycle-related genes. In ovarian cancer, YBX1 also promotes tumor growth, but the mechanisms of YBX1 in cell growth and cell cycle in ovarian cancer remain not to be fully understood. Here, we investigated whether YBX1-dependent cancer cell proliferation was specifically associated with expression of cell cycle related genes in ovarian cancer. Protein and mRNA expression levels of YBX1 and cell cycle-related genes in ovarian cancer cell lines and tissues were determined by western blot analysis, immunohistochemical analysis and reverse transcription-quantitative PCR. Cell cycle analysis was performed by flow cytometry. Luciferase assay and Chromatin immunoprecipitation assay were used to investigate a transcriptional function of YBX1. YBX1 silencing induced marked growth suppression in 4 cell lines (group A), moderate suppression in 5 cell lines (group B), and no suppression in 3 cell lines (group C) among 12 ovarian cancer cell lines in culture. The YBX1 silencing induced cell cycle arrest at G₂/M phase and suppressed expression of cyclin A1 gene in group A and B cell lines, but not in group C cell lines. Cyclin A1 silencing specifically suppressed cell proliferation in group A cell lines and partially in group B cell lines, but not at all in group C cell lines. YBX1 mRNA levels were significantly correlated with cyclin A1 mRNA levels in patients with high-grade serous carcinoma. Augmented YBX1 expression plays a key role in tumor growth promotion in ovarian cancer in its close association with cyclin A1.

Introduction

The cell cycle is precisely controlled by cyclin-dependent kinases, cyclins, and checkpoint regulators in normal cells. However, in cancer cells, the cell cycle is frequently altered by mutations in genes involved in the cell cycle, and promising cancer therapeutic drugs targeting cell cycle-related proteins have recently been developed^1,2. Ovarian cancer shows abnormalities in cell cycle-related genes, including mutations in CDKN2A, overexpression of cyclin D1, and mutations in Rb. CDK4 and CDK6 inhibitors that target the cell cycle are currently in clinical use for the treatment of patients with estrogen receptor-positive metastatic breast cancer. These agents, alone or in combination with other agents, are being tested in ovarian cancer³. More than 70% of ovarian cancer cases are identified as either high-grade serous adenocarcinoma (HGSC) or ovarian clear cell carcinoma (OCCC). Bai and colleagues recently reported that approximately 20% of HGSC cases exhibit cyclin E gene amplification and often show resistance to chemotherapeutics⁴.

Y-box binding protein 1 (YBX1) is an ancestral DNA/RNA binding oncoprotein containing a cold shock domain. It is involved in the regulation of transcription, translation, DNA damage repair, and various other biological processes. YBX1 protein is mainly localized in the cytoplasm, but some is also found in the nucleus. In the nucleus, YBX1 is crucial for transcriptional regulation through the Y-box (inverted CCAAT box)⁵, while in the cytoplasm, it is responsible for mRNA stability and translation⁶. YBX1 promotes cancer cell growth, tumorigenesis, and malignant progression in various human malignancies including breast cancer, prostate cancer, and colorectal cancer^5,7–11, and enhances the expression of genes that mediate the cell cycle, cell growth, and drug resistance in cancer cells^12–18. Regarding its role in cell cycle promotion, YBX1 facilitates cell cycle progression at G₁/S^19–24 and G₂/M phases^25,26 by augmenting the expression of various genes involved in the transition from G₁ to S and/or from G₂ to M, such as cyclin A2, cyclin B, cyclin D, cyclin E, CDC6, CDC20, and CDKN1A, at the transcriptional and/or post-transcriptional levels^15,16,27.

Previous studies have reported that enhanced expression of YBX1 is closely correlated with poor outcomes and drug resistance in ovarian tumors^28–30. Drug resistance to paclitaxel and cisplatin was induced by increasing YBX1 expression in ovarian cancer^31,32. Furthermore, co-expression of cyclin A with YBX1 is associated with resistance to platinum-based chemotherapy and poor outcomes in ovarian cancer³³. Additionally, YBX1-mediated promotion of cell growth through activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated protein kinase (ERK)/p90 ribosomal S6 kinase (RSK) and/or phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathways contributes to poor outcomes in patients with ovarian cancer³⁴. Co-expression of YBX1 and cyclin B also contributes to chemoresistance in ovarian cancer¹³. Collectively, these findings suggest that targeting YBX1 and related genes may provide an effective strategy for treating ovarian cancer³⁵ as well as other malignancies in women^27,36.

Here, we investigated the mechanisms underlying distinct growth-suppressive effects of YBX1 downregulation in various cell lines. Furthermore, we have examined whether the expression levels of any cell cycle-related genes are specifically associated with YBX1-driven cell growth; analysis of this pathway may contribute to improving our understanding of the roles of YBX1 in oncogenic transformation and tumor growth. Accordingly, in the current study, we assessed whether cancer cell growth driven by YBX1 was closely associated with any specific cell cycle phase transition, and also with the expression of cyclin A, cyclin B, cyclin D, cyclin E, CDC6, and CDC20 genes.

Materials and methods

Patient characteristics

From 2007 to 2019, 101 patients with histologically proven cancer were treated at the Department of Obstetrics and Gynecology, Kyushu University (n = 62) and the Department of Obstetrics and Gynecology, The Jikei University School of Medicine (n = 39). This study was approved by the institutional review board at each hospital (Kyushu University, # 622-00; The Jikei University School of Medicine, 32–379 (10467)). This study conforms to the principles of the Declaration of Helsinki, and informed consent was obtained from all patients. Patient demographics are summarized in Supplementary Table S1.

Cell culture

TOV-21G, TOV-112D, OVCAR-3, and SK-OV-3 cells were purchased from American Type Culture Collection (Manassas, VA, USA). OVSAHO cells (JCRB1046) were purchased from Japanese Collection of Research Bioresources (Osaka, Japan). ES2, JHOC-5, and MCAS cells were kindly provided by Dr. Kiyoko Kato (Department of Obstetrics and Gynecology, Kyushu University). OVISE, OVMANA, OVTOKO, and KOC-7C cells were kindly provided by Dr. Hiroaki Itamochi (Department of Obstetrics and Gynecology, Iwate Medical University School of Medicine). JHOC-5, OVTOKO, TOV-21G, KOC-7C, OVISE, and OVMANA cells were derived from OCCC; ES2, OVCAR3, SK-OV-3, and OVSAHO cells were derived from HGSC; MCAS cells were derived from mucinous cystadenocarcinoma; and TOV-112D cells were derived from endometrial carcinoma. JHOC-5 cells were maintained in Dulbecco’s modified Eagle medium (DMEM)/F12 (cat. no. 11320–033; Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 0.1 mM MEM nonessential amino acid solution (Thermo Fisher Scientific Inc., MA, USA). KOC-7C cells were maintained in DMEM (cat. no. #05919; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% FBS. TOV-21G, TOV-112D, OVCAR-3, SK-OV-3, OVSAHO, ES2, MCAS, OVISE, OVMANA, and OVTOKO cells were maintained in RPMI (cat. no. #05918; Nissui Pharmaceutical Co., Ltd.) supplemented with 10% FBS. All cell lines were maintained in an atmosphere of 5% CO₂ and passaged for 6 months or less. The cell lines were not further tested or authenticated by the investigators. In the current study, we classified the ovarian cancer cell lines into three groups based on the inhibitory effect of YBX1 siRNA treatment on cell growth for 5 days. Group A includes cells with more than 50% inhibition, group B includes cells with 50% to 10% inhibition, and group C includes cells with less than 10% inhibition. ES2, JHOC-5, MCAS, and OVTOKO cells are in group A, OVCAR3, TOV-112D, TOV-21G, SK-OV-3, and KOC-7C cells are in group B, and OVSAHO, OVISE, and OVMANA cells are in group C.

Cell proliferation assay

All cell lines were seeded at 5–10 × 10³ cells/well in 24-well plates and counted using a CDA-1000 particle counter (Sysmex Corporation, Hyogo, Japan) at 1, 3, and 5 days after small interfering RNA (siRNA) transfection. Results are expressed as means ± standard deviations of triplicate wells.

Reagents

Antibodies against p-YBX1 (cat. no. #2900), cyclin A2 (cat. no. #67955), cyclin B1 (cat. no. #4138), cyclin D1 (cat. no. #55506), cyclin E (cat. no. #4129), and CDC6 (cat. no. #3387), PKCα (cat. no. #59754), p-PKCα/β II (cat. no. #9375), AKT (cat. no. #9272) p-AKT (cat. no. #4060), and CREB (cat. no. #9197) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against YBX1 (cat. no. ab76149), cyclin A1 (cat. no. ab270940), and β-actin (cat. no. ab8227) were purchased from Abcam (Cambridge, UK). Antibodies against α-tubulin (cat. no. T6074) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against CDC20 (cat. no. NB100-59828) were purchased from Novus Biologicals (Centennial, CO, USA). Antibodies against GAPDH (cat. no. 2275-pc100) were purchased from Trevigen (Gaithersburg, MD, USA).

Transfection of siRNA

RNA-interference assays were performed using siRNA. The siRNAs corresponding to the nucleotide sequences of YBX1 (s9732 and s9731), CCNA1 (s17021), and CCNA2 (s2512) were purchased from Thermo Fisher Scientific. Cells were transfected with siRNA duplexes using Lipofectamine RNAiMAX and Opti-MEM (cat. no. 31985–070; Thermo Fisher Scientific Inc.) according to the manufacturer’s recommendations. As a negative control, cells transfected with Stealth RNAi siRNA Negative Control (cat. no. 12935300) were used.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from human ovarian tumor tissue or ovarian cancer cell lines using ISOGEN (cat. no. 311–02501; Nippon Gene Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions. The RNA concentration was assessed by spectrophotometry at 260 nm using NanoDrop 1000 (Thermo Fisher Scientific Inc.). The sets of primers and TaqMan probes for YBX1 (Hs00358903_g1), cyclin A1 (Hs00171105_m1), cyclin A2 (Hs00996788_m1), cyclin B1 (Hs00259126_m1), cyclin D1 (Hs00277039_m1), cyclin E1 (Hs01026536_m1), CDC6 (Hs00154374_m1), CDC20 (Hs00426680_mH), and RNA18S5 (Hs03928985_g1) were purchased from Thermo Fisher Scientific. RT-qPCR was performed using iTaq Universal Probes One-Step Kit (cat. no. 1725141; Bio-Rad Laboratories, Hercules, CA, USA) in a CFX Connect Real-Time PCR Detection System (cat. no. 1855201J1; Bio-Rad Laboratories). Each 10 µL reaction mix contained 20 ng extracted RNA, 5 µL 2 × iTaq PCR reaction mix, 0.25 µL iScript reverse transcriptase, and 0.5 µL 20 × primer and probes. The thermal cycling conditions were as follows: 50 °C for 10 min and 95˚C for 3 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 15 s. The expression of cell cycle-related genes was presented as relative ratios calculated using the ΔΔCq method³⁷. mRNA data were normalized to RNA18S5.

Luciferase assay

Cells were seeded into 12-well plates, and the next day, cells were transfected with reporter vector (400 ng), phRL-TK vector (50 ng), and siRNA (6 µM) using Lipofectamine LTX reagent (cat. no. 15338–100; Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. Firefly and Renilla luciferase activities were measured 48 h after transfection using the Dual-luciferase reporter assay system (cat. no. E1910; Promega, Madison, WI, USA). The firefly luciferase activity was normalized to Renilla luciferase activity.

Chromatin immunoprecipitation (ChIP) assay

ChIP assay was performed as described previously³⁸. Briefly, cells were fixed with formaldehyde, achieving a final concentration of 1%, and subsequently decrosslinked by 125 mM glycine. The cells were then lysed in SDS lysis buffer (50 mmol/L Tris–HCl, 10 mmol/L EDTA, 1% SDS, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 1 mmol/L sodium orthovanadate) and sonicated. The soluble chromatin was incubated with 2 µg anti-YB-1 antibody or IgG antibody, and immunoprecipitated. The immunoprecipitated DNA was treated with RNase A and proteinase K, followed by purification using the Qiaquick PCR purification kit (cat. no. 28106; QIAGEN, Hilden, Germany). Purified DNA was dissolved in 50 µl H₂O, and 1 µl DNA was used for PCR analysis with GoTaq Green Master mix (cat. no. M7128; Promega) and the following primer pairs: CyclinA1#1, 5′-GGCTTGTCAGTCACCTATGC-3′ (forward) and 5′-GCAGAGGAGATGTGGGGATT-3′ (reverse); CyclinA1#2, 5′-AAACAGTCCCTTCCAAAGCC-3′ (forward) and 5′-GACCTGCTCACCTGACTCG-3′ (reverse). The thermal cycling conditions were as follows: 95 °C for 2 min, followed by 35 cycles at 95 °C for 30 s, 56 °C for 30 s, and 72 ˚C for 30 s, with a final extension at 72 °C for 5 min. PCR products were analyzed on 3% agarose gels and stained with ethidium bromide.

The cancer genome atlas (TCGA) dataset

TCGA data included a cohort of 316 patients with serous ovarian cancer obtained from the cBioPortal for Cancer Genomics (http://www.cbioportal.org)³⁹. Using Spearman’s rank correlation coefficient, we examined the positive and negative associations between YBX1-correlated genes. In addition, by ranking genes in order of Spearman’s rank correlation coefficients for associations between YBX1 and those genes, the importance of the association between YBX1 and those genes was investigated.

Cell cycle analysis

For cell cycle analysis, cells were seeded in 60 mm dishes, incubated for 3 days, and then treated with 2 nM siRNAs for 3 days. Cell cycle analysis was conducted using a BD Pharmingen FITC BrdU Flow Kit (cat. no. 559619; BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions, and cell cycle distribution was determined using a FACS-Calibur instrument (cat. no. 342973; BD Biosciences).

Subcellular protein fractionation

Subcellular fractionation was performed according to the manufacturer’s instructions using the Subcellular Protein Fractionation Kit for Cultured Cells (cat. no. 78840; Thermo Fisher Scientific Inc.).

Statistical analysis

Experimental results were expressed as mean ± SD. All calculations (Student's t tests, Mann–Whitney U tests and Pearson correlation coefficients) were done using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA) and Microsoft Excel software. A P value of less than 0.05 was considered significant.

Results

YBX1 silencing suppressed the growth of some ovarian cancer cell lines in vitro

Enhanced YBX1 expression predicts poor outcomes in patients with various tumors including ovarian cancer^15,16,27,28. In this study, we found that higher YBX1 mRNA expression was correlated with shorter survival periods in patients with HGSC based on the Kaplan–Meier plotter database (Fig. 1A) and GEO datasets (GDS3297; Fig. 1B). Therefore, we investigated the role of YBX1 in the proliferation of ovarian cancer cells in culture. Among 12 ovarian cancer cell lines, doubling times varied from 18 to 60 h (Supplementary Figure S1). The cells harbored various mutations in PIK3CA, PTEN, KRAS, BRAF, ARID1A, TP53, and BRCA1/2 genes, and the cyclin E gene was also amplified in OVCAR3 cells⁴⁰. Western blot analysis demonstrated that the expression and phosphorylation of YBX1 also varied among the ovarian cancer cell lines (Fig. 1C). We next assessed whether silencing of YBX1 using siRNA could influence the growth rates of the 12 cell lines, finding that YBX1 silencing reduced the growth rate in some cells. YBX1 protein and mRNA levels were almost completely reduced in all cell lines after transfection with YBX1 siRNA#1 (Fig. 1D and Supplementary Figure S2). Figure 1E shows cell growth rates on days 1, 3, and 5 after transfection with or without YBX1 siRNA#1. ES2, JHOC-5, MCAS, and OVTOKO cell lines showed either almost complete growth suppression or non-logarithmic growth when YBX1 was silenced. OVCAR3, TOV-112D, TOV-21G, SK-OV-3, and KOC-7C cell lines showed reduced but still logarithmic growth as compared with the control following YBX1 siRNA transfection. Furthermore, OVSAHO, OVISE, and OVMANA cell lines showed no growth suppression following YBX1 siRNA transfection. Figure 1F summarizes the effects of YBX1 silencing on the cell growth suppression rate. We classified ovarian cancer cell lines into three groups based on the effect of YBX1 siRNA treatment on cell growth. Group A cell lines (ES2, JHOC-5, MCAS, and OVTOKO) showed more than 50% inhibition of cell proliferation compared to controls, while group B cell lines (OVCAR3, TOV-112D, TOV-21G, SK-OV-3, and KOC-7C) showed a weaker than group A but more than 10% inhibition of cell proliferation. On the other hand, cell lines of group C (OVSAHO, OVISE, and OVMANA) showed less than 10% inhibition after 5 days.

Fig. 1.

Growth inhibition in ovarian cancer cell lines following transfection with YBX1 siRNA#1. (A) Kaplan–Meier analysis of patients with HGSC showing high and low expression of YBX1 (Kaplan–Meier plotter database). (B) Comparison of 5-year survival rates in patients with HGSC showing high and low expression of YBX1 mRNA (GEO dataset accession no. GDS3297). **p = 0.00165. (C) Expression levels of YBX1 in 12 cell lines, as assessed by western blot analysis. (D) Western blot analysis showed that treatment with YBX1 siRNA#1 for 3 days inhibited the expression of YBX1 in all cell lines. (E–F) Cell growth was examined on days 1, 3, and 5 after transfection with YBX1 siRNA#1 (E). Based on cell growth inhibition levels after transfection with siRNA#1 for 5 days, the 12 cell lines were classified into 3 groups: marked inhibition (group A), moderate inhibition (group B), and no inhibition (group C) (F). Student’s t-tests were used to compare differences between groups. ^∗p < 0.05; ^∗∗p < 0.01. Data were represented as mean ± standard deviation for 3 independent experiments. Black, control siRNA; gray, YBX1 siRNA#1.

We next investigated whether the dependence of cell proliferation on YBX1 affects drug sensitivity to cisplatin, paclitaxel, and SU056, a YBX1 inhibitor (Supplementary Table S2). Almost all cell lines of groups A, B, and C showed similar sensitivity to cisplatin. Paclitaxel sensitivity was similar for these cell lines except for OVTOKO (group A) and KOC-7C (group B). KOC-7C showed much less sensitivity to paclitaxel (0.37 µmol/L). There was no correlation between sensitivity to cisplatin or paclitaxel and YBX1-dependent proliferation. On the other hand, OVSAHO and OVMANA cell lines of group C and KOC-7C of group B were less sensitive to SU056 than all group A cell lines and two group B cell lines. Cell lines with YBX1-dependent proliferation were more sensitive to SU056, indicating that YBX1-dependent proliferation is associated with drug sensitivity.

PKCα is highly expressed in cells that are dependent on YBX1 for proliferation

RPPA (Reverse Phase Protein Array) data from the MD Anderson Cell Lines Project (MCLP) showed that PKCα expression was higher in ES2 and OVTOKO in group A than in OVSAHO, OVISE and OVMANA in group C (Supplementary Table S3)⁴¹. Previous studies have reported that YBX1 is phosphorylated by PKCα and phosphorylation of YBX1 induces its translocation to the nucleus^27,42. Consistent with RPPA data, we confirmed higher expression and phosphorylation of PKCα in group A and B cell lines than in group C cell lines (Supplementary Figure S3A). The pPKCα/PKCα ratios were also lower in OVCAR3 and KOC-7C in group B and three cell lines in group C. However, nuclear YBX1 was expressed at similar levels in both group A and group C cell lines (Supplementary Figure S3B). These results suggest that PKCα is highly expressed in group A cells, but is not associated with nuclear translocation of YBX1.

YBX1 silencing induced cell cycle arrest at G₂/M and in part at G₁/S

In Fig. 1, YBX1 silencing reduced cell proliferation, predicting and alteration in cell cycle regulation. We next performed FACS analysis for cell cycle tracing after transfection of the 12 ovarian cancer cell lines with YBX1 siRNA#1. FACS analysis (Supplementary Figure S4) showing the distribution of all cell lines in G₀/G₁, S, and G₂/M phases is presented in Fig. 2A, B, and C, respectively. All 4 cell lines in group A and 1 cell line in group B significantly showed G₂/M arrest when transfected with YBX1 siRNA#1, and 3 of the 9 cell lines in groups A and B showed reduced cell populations in S phase. By contrast, no cell cycle arrest was observed when group C cell lines were transfected with YBX1 siRNA#1.

Fig. 2.

Effects of YBX1 siRNA#1 on the cell cycle. (A–C) Percentages of cells in G₀/G₁ (A), S (B), and G₂/M (C) phases following transfection with YBX1 siRNA #1 for 3 days. The percentages were determined by gating the FACS plot (Supplementary Figure S4). Student’s t-tests were used to compare differences between control siRNA and YBX1 siRNA#1 transfection. ^*p < 0.05; ^**p < 0.01. Error bars indicate standard deviations for 3 independent experiments.

Expression of cyclin A1 is highly influenced by YBX1 silencing in ovarian cancer cell lines

Some cell cycle-related genes were previously reported to be well correlated with YBX1 in various cancer cell lines^15,16,27. Therefore, we examined whether cyclin A (A1 and A2), cyclin B1, cyclin D1, cyclin E1, and CDC6 were associated with YBX1 in ovarian cancer (Fig. 3A). In addition, we also searched other genes associated with YBX1 based on RNA-seq data from a cohort of patients with HGSC (TCGA). The results showed that YBX1 expression was closely correlated with the expression of cell proliferation-, cell division-, and DNA replication-related genes (Supplementary Figure S5A). Of these genes, the top correlated gene was CDC20 (Supplementary Figure S5B). We further selected CDC20 as a gene associated with YBX1 and the cell cycle, in addition to the above 6 cell cycle-related genes. The YBX1 gene was amplified in 8% of HGSC cases, and the CDC20 gene, which was localized at 1p34.2 (approximately 800 kb away from YBX1), was amplified in 7% of HGSC cases (Supplementary Figure S5C), suggesting that the close association of YBX1 and CDC20 was in part related to co-amplification of both genes in chromosome 1 in cases of HGSC. By contrast, the other 5 genes related to the cell cycle were localized in other chromosomes (Fig. 3A).

Fig. 3.

Effects of YBX1 siRNA on the expression levels of YBX1, cyclin A1, cyclin A2, cyclin B1, cyclin D1, cyclin E1, CDC6, and CDC20 mRNAs in ovarian cancer cell lines. (A) The eight selected genes are listed. Correlations of YBX1 with other genes are presented according to Spearman scores based on gene ontology analysis (TCGA database, n = 316) of HGSC, and the chromosomal locations of the 8 genes are indicated. The right panel of the illustrated model shows the sites of action of 7 cell cycle-related genes in the cell cycle. (B) RT-qPCR analysis showing mRNA expression levels of cell cycle-related genes in ovarian cancer cells. Student’s t-tests were used to compare differences between YBX1 siRNA#1-transfection groups and the control group. ^*p < 0.05; ^**p < 0.01. Error bars indicate standard deviations for 3 independent experiments. (C) RT-qPCR analysis showing mRNA expression levels of 7 genes in ovarian cancer cell lines following transfection with YBX1 siRNA#1 for 3 days. Expression levels were expressed as ratios relative to the expression in the corresponding negative control based on 3 independent assays. Student’s t-tests were used to compare differences between YBX1 siRNA#1-transfection groups and the control group. ^*p < 0.05; ^**p < 0.01. Error bars indicate standard deviations for 3 independent experiments. A heatmap plot of cell cycle-related genes from the comparison of the YBX1 siRNA#1-transfection group and the control group was presented. High expression levels in the YBX1 siRNA#1-transfection group are indicated in red, and low expression levels are indicated in blue. (D) JHOC-5 cells were transfected with luciferase reporter constructs containing the 1166 bp 5′-flanking region of the cyclin A1 gene with YBX1 siRNA#1 for 2 days. The firefly luciferase activity was normalized to the Renilla luciferase activity. Student’s t-tests were used to compare differences between YBX1 siRNA#1-transfection groups and the control group. ^**p < 0.01. Error bars indicate standard deviations for 3 independent experiments. (E) Schematic representation of potential YBX1 binding sites (Black boxes) and primer locations (arrows) used for ChIP assay in the promoter region of Cyclin A1 gene. (F) The YBX1 binding to the cyclin A1 promoter by ChIP assay in JHOC-5 cells.

The expression levels of cyclin A1 and cyclin E1 were highly variable among the 12 cell lines, and those of cyclin A2, cyclin B1, cyclin D1, CDC6, and CDC20 showed approximately fivefold differences in expression (Fig. 3B). Among the 7 genes tested in this analysis, cyclin A1, but not cyclin A2, was downregulated in all cell lines in group A, and only slightly downregulated if at all, in cell lines in group B following transfection with YBX1 siRNA#1 (Fig. 3C). Cyclin D1 expression was moderately reduced in 7 of the 9 cell lines in groups A and B, but not at all in group C cell lines (Fig. 3C). Together, these findings demonstrated that YBX1 knockdown suppressed cyclin A1 expression and moderately suppressed cyclin D1 expression in group A and B cell lines, but had little effect on the expression of cell cycle-related genes in group C cell lines.

We further confirmed the differential effects of YBX1 silencing by YBX1 siRNA#1 on cell growth and the expression of cell cycle-related genes using YBX1 siRNA#3. Consistent with YBX1 siRNA#1 transfection studies, transfection with YBX1 siRNA#3 induced growth inhibition in cell lines in groups A and B (Supplementary Figure S6A and B). However, there was no inhibition of cell growth in 3 cell lines in group C following transfection with YBX1 siRNA#3 (Supplementary Figure S6C). Furthermore, transfection with YBX1 siRNA#3 induced G₂/M arrest in 4 cell lines in groups A and B, and reduced S phase population in 5 cell lines in group A (Supplementary Figure S6D–G). Among the 7 cell cycle-related genes, cyclin A1 was most markedly downregulated in 8 cell lines in groups A and B, while the expression levels of other genes were only slightly reduced, if at all, in a few cell lines of groups A and B (Supplementary Figure S7), consistent with our data for transfection with YBX1 siRNA#1.

To investigate whether YBX1 induces transcriptional activation of the cyclin A1 gene directly, we carried out luciferase assay and ChIP assay. Luciferase assay revealed that cyclin A1 promoter activity was downregulated by transfection with YBX1 siRNA in JHOC-5, SK-OV-3, but not in OVSAHO (Fig. 3D, Supplementary Figure S8A). Putative Y-box-like elements were located in the 5′-flanking region of the cyclin A1 gene (Fig. 3E). We also analyzed the binding of YBX1 to the promoter of the cyclin A1 gene by ChIP assay. The ChIP assay confirmed that YBX1 directly interacted with the 5′-flanking region of the cyclin A1 gene in JHOC-5, OVTOKO, but not in OVSAHO (Fig. 3F, Supplementary Figure S8B). These results suggest that YBX1 transcriptionally regulates cyclin A1 expression, except for group C cells.

Suppressive effect of YBX1 silencing on protein expression of cyclin A1

We next examined the effects of YBX1 silencing on the expression of cell cycle-related genes, cyclin A1, cyclin B1, cyclin D1, cyclin E1, CDC6, and CDC20 by western blot analysis, and found that YBX1 silencing markedly decreased cyclin A1 (Fig. 4). All 12 cell lines showed various levels of protein expression for 6 cell cycle-related genes. In particular, the protein expression levels of cyclin A1, cyclin D1, and cyclin E1 were highly variable among the cell lines (Fig. 4A), consistent with the observed mRNA levels of these genes (Fig. 3B). Almost all cell lines in groups A and B showed apparent reductions in cyclin A1 expression, when treated with YBX1 siRNA#1 or #3, but not in group C cell lines (Fig. 4B). There was a reduction in expression of cyclin A1 only in OVISE cells of group C by YBX1 siRNA#3, but its underlying mechanism remains unclear.

Fig. 4.

Effects of YBX1 knockdown by siRNA#1 and siRNA#3 on protein expression of cell cycle-related genes. (A) Expression of cell cycle-related genes as assessed by western blot analysis. (B) Effects of YBX1 siRNAs (siRNA#1 and siRNA#3) on the expression of cell cycle-related genes in cell lines following transfection with YBX1 siRNAs for 3 days. α-tubulin was used as a loading control.

Suppressive effect of cyclin A1 and A2 silencing on cell growth and the cell cycle

Among the cell cycle-related genes analyzed in this study, cyclin A1 was found to be the most susceptible to YBX1 silencing, suggesting the close regulatory relationship between cyclin A1 and YBX1 expression. We further examined whether cyclin A1 silencing induced distinct effects on cell growth and cell cycle arrest in cancer cell lines. We showed that silencing of cyclin A1 induced cell growth inhibition and G₂/M arrest in group A and B cells in Fig. 5. Cyclin A1 silencing using specific siRNA induced growth inhibition of cell lines in groups A and B but no inhibition of group C cell lines (Fig. 5A and Supplementary Figure S9A). Additionally, FACS analysis showed that cyclin A1 silencing caused G₂/M phase arrest in 4 of 9 cell lines in groups A and B (Fig. 5B, C, and D, and Supplementary Figure S9B). By contrast, no G₂/M arrest was observed in all group C cell lines. Thus, cyclin A1 and YBX1 promoted the G₂/M phase in group A and B cell lines.

Fig. 5.

Effects of cyclin A1 knockdown by cyclin A1 siRNA on cell growth, and the expression levels of YBX1 and cell cycle-related genes in HGSC and OCCC tumors. (A) Cell growth of ovarian cancer cell lines on days 1, 3, and 5 after transfection with cyclin A1 siRNA. Student’s t-tests were used to compare differences between control- and cyclin A1 siRNA-transfected cells. ^*p < 0.05; ^**p < 0.01. Error bars indicate standard deviations for 3 independent experiments. (B–D) Percentages of cells in G₀/G₁ (B), S (C), and G₂/M (D) phases following transfection with cyclin A1 siRNA for 3 days. Percentages were determined by gating the FACS plot. Student’s t-tests were used to compare differences between control- and cyclin A1 siRNA-transfected cells. ^*p < 0.05; ^**p < 0.01. Error bars indicate standard deviations for 3 independent experiments. (E) Expression levels of YBX1 and cyclin A1, as assessed by RT-qPCR analysis in HGSC (n = 50) and OCCC tumors (n = 51). The statistical significance was determined using the Mann–Whitney U tests. (F, G) Correlation of YBX1 mRNA expression levels with the mRNA expression of cyclin A1 in HGSC (n = 50) (F) and OCCC tumors (n = 51) (G). The statistical significance of the correlations was determined using Pearson’s correlation coefficients and p values.

Cyclin A2 is also known to play a key role in G₂/M transition and progression⁴³. We next examined whether cyclin A2 silencing induced distinct effects on cell growth and cell cycle in ovarian cancer cell lines (Supplementary Figure S10A–F). Cyclin A2 silencing induced growth inhibition and cell cycle arrest at G₂/M in almost all cell lines in groups A, B, and C (Supplementary Figure S10B and F). These results indicated that both cyclin A1 and cyclin A2 regulate the G2/M phase, but cyclin A1 is specifically involved in the G2/M phase transition controlled by YBX1 in ovarian cancer. It is suggested that cyclin A1 plays an important role in proliferation promoted by YBX1.

YBX1 expression correlates with cyclin A1 expression in HGSC samples

We analyzed mRNA expression levels of YBX1 and cyclin A1 genes in HGSC and OCCC specimens (Fig. 5E). HGSC tumors (n = 50) showed significantly (p = 0.0053) higher YBX1 mRNA levels than OCCC tumors (n = 51). The expression level of cyclin A1 (p < 0.0001) was significantly higher in HGSC samples than in OCCC samples.

We finally assessed whether the expression level of cyclin A1 was associated with that of YBX1 in tumor specimens of HGSC (Fig. 5F) and OCCC (Fig. 5G). In HGSC specimens, YBX1 mRNA levels were significantly (p < 0.0001) correlated with the mRNA levels of cyclin A1 (Fig. 5F). By contrast, YBX1 levels were not correlated with cyclin A1 levels in OCCC specimens (Fig. 5G). Together, YBX1 expression level was high and correlated with the expression level of cyclin A1 in HGSC tumors, but not in OCCC tumors.

Discussion

In our current study, among the 12 ovarian cancer cell lines, YBX1 silencing induced growth inhibition in 9 cell lines (groups A and B), and no growth inhibition in 3 cell lines (group C). Furthermore, SU056, a YBX1 inhibitor, also suppressed cell growth of group A cell lines more strongly than group C, except for one cell line, OVISE (Supplementary Table S2). All cell lines in group A and two cell lines in group B showed G₂/M phase arrest and reduced S phase populations following transfection with YBX1 siRNAs (Table 1). By contrast, the cell cycle distributions in group C cell lines were not at all affected by YBX1 silencing. Thus, these findings indicated that the growth of ovarian cancer cell lines could be dependent or independent of YBX1 in close association with the specific cell cycle phases (Table 1).

Table 1.

Classification of ovarian cancer cell lines into 3 groups based on cell growth dependence on YBX1.

Group	Cell line	Cell growth suppression1,2)				Cell cycle arrest3)
		siYBX1 #1	siYBX1#3	siCyclinA1	siCyclinA2	siYBX1#1	siYBX1#3	siCyclinA1	siCyclinA2
A	ES2	+  +	+  +	+	+  +	G2/M	G2/M	G2/M	G2/M
	JHOC-5	+  +	+  +	+  +	+  +	G2/M	G1	G2/M	S, G2/M
	MCAS	+  +	+	+	+  +	G2/M	G2/M	NC	S, G2/M
	OVTOKO	+  +	+  +	+	+  +	G2/M	G1	G2/M	G2/M
B	OVCAR3	+	+	+	+  +	G2/M	G2/M	G2/M	G2/M
	TOV-112D	+	+	+	+  +	S	G1, G2/M	NC	S, G2/M
	TOV-21G	+	+	+	+	NC	NC	NC	S, G2/M
	SK-OV-3	+	+	+	+  +	NC	NC	NC	S, G2/M
	KOC-7C	+	+	+	+	NC	NC	NC	S
C	OVSAHO	±	±	±	+	NC	NC	NC	S, G2/M
	OVISE	±	±	±	+  +	NC	NC	NC	G2/M
	OVMANA	±	±	±	+	NC	NC	NC	G2/M

1) Survival rato of siRNAs-treated cell lines to siCtl-treated cell lines on day 5 after siRNAs treatment.

2) The effect on growth inhibition compared to the control group on day 5 was calculated and expressed as follows: +  + : > 50%; + : > 10%; ± : < 10%

3) Phases with significantly increased population in the cell lines after 72 h of siRNA treatment compared to the siCtl-treated cell lines; NC: No change of cell cycle phases by transfected siRNA.

Previous studies reported that the high expression of YBX1 is associated with cisplatin and paclitaxel resistance in ovarian cancer^31,32. In our present study, however, the sensitivity to cisplatin and paclitaxel was not appreciably different among all cell lines of groups A, B, and C (Supplementary Table S2). Since there was no marked difference in the expression levels of YBX1 among the cell lines in groups A, B, and C (Fig. 1C), YBX1-induced drug resistance to cisplatin or paclitaxel and YBX1-dependence on cell proliferation might be mediated through different mechanisms in cancer cells. Concerning the cell cycle arrest at G₂/M phase by YBX1 silencing, Ban and colleagues reported that YBX1 is overexpressed in nasopharyngeal cancer, and also that high expression of YBX1 positively associates with expression of G₂/M checkpoint-related genes, resulting in promotion of cancer cell growth⁴⁴. Mehta et al. reported that phosphorylated YBX1 enters the nucleus in G₂/M phase⁴⁵. Jürchott et al. previously reported that cyclin A2 and cyclin B1 expression plays key roles in promoting G₂/M phase progression via YBX1 through its interaction with the Y-box in its regulatory promoter region¹⁹. Furthermore, Cybulski et al. also demonstrated that cyclin A2 expression is correlated with YBX1 expression in ovarian cancer³³. Rivera et al. previously reported that cyclin A1 is associated with apoptosis, G₂/M arrest, and mitotic catastrophe in ovarian cancer cells⁴⁶. These studies suggest that YBX1 plays key roles in cell growth promotion and transcription of cell cycle-related genes at G₂/M phase, consistent with our present study.

In this study, we first found that cyclin A1 expression was most prominently suppressed by YBX1 knockdown in group A and B cell lines (Figs. 3 and 4). Moreover, cell growth inhibition and cell cycle arrest at G₂/M phase were induced by cyclin A1 silencing in group A and B cell lines (Fig. 5). YBX1-dependent cell growth and G₂/M transition could be spurred in the close collaboration with cyclin A1 (Table 1). On the other hand, cell growth inhibition by G₂/M arrest was observed by cyclin A2 silencing in all group A, B, and C cell lines (Table 1 and Supplementary Figure S10). Cyclin A2 silencing markedly induced cell cycle arrest at S and G₂/M phases (Supplementary Figure S10). Although the expression of cyclin A2 was not at all affected by YBX1 silencing (Fig. 3), cyclin A2 also plays an important role in cell growth and cell cycle promotion in ovarian cancer cell. Previous studies demonstrated that cyclin A2 plays a key role in tumor growth and progression by other human malignancies^44,47,48. However, it has been reported that cyclinA1, but not cyclinA2, is involved in apoptosis in non-small cell lung cancer cells⁴⁶, and that the Cyclin A1/CDK1 complex phosphorylates Rb and p53 more strongly than the Cyclin A2/CDK1 complex⁴⁹. These studies suggest that cyclin A1 and cyclin A2 play different roles in the cell cycle of various cancer cell lines. Furthermore, Ye et al. pointed out that since the oscillation in cyclin A1 protein levels during the cell cycle may blunt the prediction of response to therapeutic drugs, it is reasonable to explore the regulatory mechanisms of cyclin expression to identify cyclin regulators that are constantly expressed⁵⁰. Understanding the mechanism of cell proliferation by cyclin A1 via constitutively expressed YBX1 may provide a specific therapy for ovarian cancer.

The cell growth dependence on YBX1 may be attributable to whether YBX1 could promote cyclin A1 gene expression during G₂/M phase. In group A and B cells, YBX1 induced the cyclin A1 promoter activity (Fig. 3D and F). While transcriptional activation of cyclin A1 by YBX1 occurs in group A cells, this transcriptional activation does not appear in group C cells, suggesting that YBX1 exerts its function only in group A and B cells. YBX1 phosphorylation and nuclear translocation induce transcriptional activation of YBX1, and PKCα has the ability to phosphorylate YBX1 at S102^27,42. We demonstrated that the expression and phosphorylation of PKCα were higher in group A cell lines than in group C cell lines (Supplementary Figure S3A). Activation of PKCα downregulates cyclinA1 expression and induces G₂/M arrest in lung cancer cells⁵¹. In this study, there was no difference in YBX1 phosphorylation and nuclear localization between group A and group C cell lines (Fig. 1C and Supplementary Figure S3B). Phosphorylation of YBX1 at S102 by PKCα may not be associated with YBX1-dependent proliferation in ovarian cancer cells. However, these studies suggest that the close association between PKCα and YBX1 may affect the promotion of cyclin A1 expression and cell proliferation by YBX1. Further study should be required to understand how the expression of cyclin A1 and cyclin A2 could be differentially regulated in correlation with YBX1 on a molecular basis. We demonstrated higher expression of YBX1 and cyclin A1 in HGSC than in OCCC tumors and correlation of YBX1 and cyclin A1 in HGSC, not in OCCC tumors. However, we could not observe any difference in the effects of YBX1 on the cyclin A1 expression between HGSC-derived and OCCC-derived cancer cells in vitro. Some cell lines have been reported to have mutations and mRNA expression profiles that differ from tumor samples from HGSC and OCCC patients⁵². It has been pointed out that SK-OV-3 is widely considered to be a good model for HGSC although no histological subtype was specified⁵², and ES2 cells, reportedly derived from OCCC, appeared more like HGSC than OCCC⁵³. In the current study, we classified ES2 cells as HGSC. Since the actual origin of the above cell lines is not clear, further studies with cells of determined origin are required to distinguish the function of YBX1 in HGSCs and OCCC in vitro.

Expression of cyclin E1 as well as cyclin A2 was not affected by YBX1 silencing in ovarian cancer cells (Figs. 3C and 4). Three cell lines in group C showed the highest expression levels of cyclin E1 among all cell lines (Fig. 4A). RPPA analysis also showed increased protein expression levels of cyclin E1 in these three cell lines as compared to cell lines in group A (Supplementary Table S3). It has been reported that enhanced expression of cyclin E1 is associated with poor outcomes in patients with ovarian cancer^54,55. Although YBX1 is not involved in cyclin E1 expression in ovarian cancer cells in this study, enhanced cyclin E1 mRNA expression may facilitate ovarian tumor growth and progression independent of YBX1.

YBX1 is associated with tumor progression in ovarian tumors, and targeting YBX1 and its activation pathway may contribute to the further development of anticancer therapeutics^15,16,27. Our findings supported that YBX1 played pivotal roles in cell growth promotion in ovarian cancer cells, and also that this cell growth dependence on YBX1 is attributable to the promotion of G₂/M transition in collaboration with cyclin A1 but not cyclin A2. However, we could not clarify the key proteins that predict cell growth dependence on YBX1. Further study should be required to understand the mechanisms underlying how YBX1 could promote ovarian cancer growth through elucidation of the association between YBX1 and cyclin A1 on a molecular basis. In addition, we did not investigate whether YBX1-dependent proliferation is associated with malignant progression, such as tumor growth using an animal model. The results from in vitro experiments may not fully reflect the biological behavior of cells in vivo. Future studies in animal models and clinical samples are needed to validate our findings and to understand the role of YBX1-dependent growth in malignant tumor progression and tumor growth in vivo. Identification of cell cycle regulatory targets associated with YBX1 in ovarian cancer may provide novel insights to facilitate the development of cancer therapy targeting YBX1.

Author contributions

Y. M., D. K., T. M., T. Shibata., M. O., and M. K. conceived and designed the experiments. Y. M., D. K., T. M., T. Shibata, A. K, and K. Kanazawa. performed the experiments. A. K., J. A., N. Y., A. O., H. I., T. Sugiyama, A. T., S. N., N. T., and K. Kato contributed reagents/materials/analysis tools. Y. M, D. K., T. M., M. O. and M. K. wrote and edited the paper. All authors reviewed the manuscript.

Funding

This research was supported by JSPS KAKENHI Grant Number, 24K09909 to Y.M.

Data availability

The data generated in the present study may be found in Gene Expression Omnibus (GEO) at GDS3297.

Competing interests

The authors declare no competing interests.

Ethical approval and consent to participate

This study was approved by the institutional review board at each hospital (Kyushu University, # 622-00; The Jikei University school of Medicine, 32–379 (10467)). This study conforms to the principles of the Declaration of Helsinki and informed consent was obtained from all patients.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72174-9.