Histone acetyltransferase CSRP2BP promotes the epithelial–mesenchymal transition and metastasis of cervical cancer cells by activating N-cadherin

doi:10.21203/rs.3.rs-2504056/v1

Download PDF

Research Article

Histone acetyltransferase CSRP2BP promotes the epithelial–mesenchymal transition and metastasis of cervical cancer cells by activating N-cadherin

https://doi.org/10.21203/rs.3.rs-2504056/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 17 Oct, 2023

Read the published version in Journal of Experimental & Clinical Cancer Research →

You are reading this latest preprint version

Background: Dysregulated epithelial–mesenchymal transition (EMT) is involved in cervical cancer metastasis and is associated with histone acetylation. However, the underlying molecular mechanisms of histone acetylation in cervical cancer EMT and metastasis are still elusive.

Methods: Here, we systematically investigated the expression patterns of histone acetylation genes and their correlations with EMT pathway in cervical cancer. The expression of histone acetyltransferase CSRP2BP among cervical cancer tissues and cell lines was detected using Western blotting and immunohistochemistry analyses. The effect of CSRP2BP on cervical cancer cell proliferation and tumorigenicity were determined by cell growth curve, EdU assay, flow cytometryand xenotransplantation assays. Wound healing assays, transwell migration assays and pulmonary metastasis model were used to evaluate the effect of CSRP2BP on the cellinvasion, metastasis of cervical cancer cells in vivo and in vitro. RNA-seq, Chromatin immunoprecipitation (ChIP), co-immunoprecipitation (Co-IP) and luciferase reporter were further applied to research for the roles and molecular mechanisms of CSRP2BP in promoting cervical cancer EMT and matastasis.

Results: We prioritized a top candidate histone acetyltransferase, CSRP2BP, as a key player in cervical cancer EMT and metastasis. The expression of CSRP2BP was significantly increased in cervical cancer tissues and high CSRP2BP expression was associated with poor prognosis. Overexpression of CSRP2BP promoted cervical cancer cell proliferation and metastasis both in vitro and in vivo, while knockdown of CSRP2BP obtained the opposite effect. In addition, CSRP2BP-promoted resistant to cisplatin chemotherapy. Mechanistically, CSRP2BP mediated histone 4 acetylation at lysine sites 5 and 12, and cooperate with the transcription factor SMAD4, bound to the SEB2 domain of the N-cadherin gene promotor region and upregulated N-cadherin transcription. Consequently, CSRP2BP involved cervical cancer cell EMT and matastasis dependent on activating N-cadherin.

Conclusions: These findings reveal for the first time the involvement of histone acetyltransferase CSRP2BP in cervical cancer metastasis partially through promoting the EMT process and imply that CSRP2BP could be a prognostic marker and a potential therapeutic target for combating cervical cancer metastasis.

Cervical cancer

CSRP2BP

SMAD4

N-cadherin

EMT

Metastasis

Cervical cancer is the fourth most common gynecological tumour and the second leading cause of cancer-related death in women worldwide, with a particular emphasis in low-income and middle-income countries [1]. In 2020, an estimated 604 000 new cases of cervical cancer were diagnosed, and 342 000 deaths occurred worldwide due to this malignancy [2]. The failure of cervical cancer therapy and poor outcomes are mostly due to the development of local invasion and distant metastasis [3]. A large amount of evidence has demonstrated that epithelial-mesenchymal transition (EMT) plays a key role in the progression and metastasis of cervical cancer (4). Dysregulation of EMT-associated genes, such as N-cadherin, E-cadherin and Twist1, induces the dysregulation of EMT and contributes to the invasion and metastasis of cervical carcinoma [4–6]. N-cadherin, as a marker of ongoing EMT, is widely recognized as a pivotal factor involved in cellular adhesion and tumor metastasis [7]. Overexpression of N-cadherin is associated with high aggressiveness and motility of tumour cells by participating in EMT, across a range of tumours such as cervical cancer, melanoma and non-small cell lung cancer [8–10]. Histone acetylation is highlighted as one of the most important regulators of cancer EMT progression. However, the mechanism of specific histone acetyltransferases in regulating the progression of EMT in cervical cancer is poorly understood.

Histone acetylation has effects on transcriptionally active euchromatin and is regulated by the coordination between histone acetyltransferases (HATs) and histone deacetylases (HDACs) [11]. A growing amount of evidence suggests that histone acetylation has a key role in the dysregulation of EMT-associated genes and represents an essential mechanism of cancer progression and metastasis [12–14]. Wang et al reported that Tip60 histone acetyltransferase mediates the acetylation of SPZ1 and Twist1, promoting EMT progression in liver cancer [15]. Hou et al demonstrated that HDACs inhibit the acetylation and transcriptional activity of p65 and negatively regulate the EMT process in gastric cancer [16]. Histone acetylation also plays an essential role in the development of cervical cancer. There is research reporting that the expression levels of histone H3 acetyl K9 and histone H3 trimethyl K4 are strongly correlated with the prognosis of cervical cancer patients [17]. In addition, a previous study demonstrated that HAT GCN5 contributes to cell cycle proliferation in HPV-16 E7-expressing cells and suggests its role in cervical cancer [18]. However, to what extent histone acetylation plays a role in cervical cancer metastasis by regulating the EMT process remains to be explored.

In this study, we comprehensively analysed the expression patterns of histone acetylation genes in 128 cervical tissue specimens. We discovered seven histone acetylation genes can potentially regulation the EMT pathway, including CSRP2BP, LPCAT1, NAT14, CREBBP, MSL3, ATF2 and GNPNAT1. In particular, CSRP2BP (also known as lysine (K) acetyltransferase 14, KAT14) was markedly overexpressed in cervical cancer tissues and cervical cancer cell lines and significantly associated with a poor prognosis and metastasis in cervical cancer patients. Overexpression of CSRP2BP significantly promoted cervical cancer cell proliferation, migration, invasion and resistant to cisplatin chemotherapy. We further identified that HAT CSRP2BP mediated EMT signalling and cervical cancer metastasis by interacting with SMAD4 to form a complex to activate N-cadherin transcription. Notably, CSRP2BP overexpression increased the acetylation level of H4 at the N-cadherin promoter. Our study suggests that HAT CSRP2BP is a key regulator of cervical cancer EMT and metastasis and may be a potential therapeutic target for cervical cancer.

Gene expression profiles of cervical cancer

Gene expression profiles of cervical cancer were obtained from Gene Expression Omnibus (GEO) under the accession number GSE63514 [19]. In total, transcriptomes of 128 frozen cervical samples spanning normalcy, increasingly severe cervical intraepithelial neoplasia (CINI-CINIII), and cervical cancer were analyzed in this study. Gene expression profiles were measured by Affymetrix U133 Plus 2.0 microarray platform. The processed data were downloaded for further analysis.

Collection of histone acetylation-related genes

The histone acetylation-related genes were obtained from GO [20]. The genes in GO term ‘ACETYLTRANSFERASE_ACTIVITY’ were downloaded from MSigDB database [20]. In total, 88 genes were expressed in the transcriptomes of cervical cancers.

EMT scores

To calculate the EMT score for each sample, we first obtained the EMT-related genes from MSigDB [21]. Next, we performed the single sample gene set enrichment analysis (ssGSEA) to calculate EMT enrichment score for each patient [22].

Prioritization of genes in cervical cancer

To identify the histone acetylation-related genes that potentially correlated with EMT, we performed a two-step method to prioritize the genes. First, differential expression analysis was performed for patients with different states. Wilcoxon’s rank sum test was used to analyze the differences between the two states of patients in cervical cancer. Genes with p value ≤ 0.05 was considered as differentially expressed in cervical cancer. For all histone acetylation-related genes, we calculated the rank score R based on the combination of fold-change and p-value:

\({R}_{i}=-log10\) (\({\text{p}}_{i}\))\(*log2\left({FC}_{i}\right)\)

where \({\text{p}}_{i}\) is p-value for gene i and \({FC}_{i}\) is the fold-change comparison between cancer and normal samples.

Next, we identified the genes of which expressions were correlated with EMT scores in cervical cancer. Spearman correlation coefficients were calculated between the expressions of histone acetylation-related genes and EMT scores. Genes with p value ≤ 0.05 and absolute SCC ≥ 0.3 were identified. Finally, genes with top ranked R scores and correlated with EMT scores were overlapped.

Gene set enrichment analysis

For identifying the pathways potentially correlated with gene of interest, we first calculated the SCC between the expressions of candidate gene and all other genes. All protein coding genes were ranked based on the SCC and subjected into gene set enrichment analysis (GSEA) [22]. The EMT pathway was used as the pathway for analysis.

Tissue specimens

A total of 208 paraffin-embedded human cervical cancer tissues collected from January 2010 to December 2016 were obtained from the Southern Medical University Nanfang Hospital, Hainan Provincial People's Hospital and the First Affiliated Hospital of Hainan Medical University. Nineteen additional matched pairs of fresh cervical cancer tissue specimens (T) and adjacent noncancerous tissue (ANT) samples were obtained from the Department of Gynecological of Hainan Provincial People's Hospital from June to July 2019. All patients involved in this study were selected from the database and histologically confirmed as having cervical cancer, and none of the patients were treated with radiotherapy, immunotherapy or chemotherapy before surgery. For total protein isolation, 19 matched pairs of fresh cervical cancer tissue and ANT samples were obtained from patients immediately after surgery and snap-frozen at −80°C until use. The percentages of tumour purity in these tissues used for protein analyses were established by routine histopathological analyses [23]. The study was approved by the Research Ethics Committee of the Southern Medical University Nanfang Hospital, Hainan Provincial People's Hospital and First Affiliated Hospital of Hainan Medical University IRB (HYLL-2020-060). Informed consent was obtained from each participant.

Immunohistochemistry (IHC)

IHC was performed on paraffin-embedded human cervical tissue, ANT and tumour xenograft sections as previously described [23]. Primary antibodies, including anti-CSRP2BP (1:400; LS-B11653; LSBio), anti-N-cadherin (1:300; 13116S; Cell Signaling Technology), anti-E-cadherin (1:300; 3195; Cell Signaling Technology), and anti-Ki67 (1:400; sc-15402; Santa Cruz Biotechnology), were used to detect protein expression. For the negative controls, the procedures were performed without primary antibody. CSRP2BP staining was scored by two different pathologists who acted independently with regard to the evaluation of the intensity of staining and the proportion of positive staining. The staining index for CSRP2BP expression in cervical cancer was calculated by multiplying the two scores of the staining intensity and the proportion of positive cells. The median of all scores was used as a cut-off value for CSRP2BP. The optimal cut-off value was used as follows: a score of ≥ 6 was used to define tumours with high CSRP2BP expression, and a score of ≤ 4 indicated low CSRP2BP expression.

Cell culture

The human cervical cancer cell lines Hela (HPV18 +), SiHa (HPV -) and C-33A (HPV16 +) were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai China), primary human cervical cancer cells (T1, T2, T3) were established based on previously reported methods [24]. All cell lines were subjected to high glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, CA, USA) supplemented with 10% foetal bovine serum (FBS, Gibco, CA, USA) and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin; both from Gibco, CA, USA) and maintained in 5% CO2 and 37°C atmosphere.

Plasmids construction and transfection

The coding sequence of the human CSRP2BP gene (NM_001392073, Origene, USA) was amplified and subcloned into the Xhol and BamHI sites of the pLVX-AcGFP-N1 lentiviral vector (PT3994-5, Clontech, USA) to generate CSRP2BP expression plasmids. The Agel and EcoRI sites of the GV248-EGFP-puromycin lentiviral vector (GIDE77111, GENE, CN) were used to generate CSRP2BP shRNA constructs. The human CDH2 gene (NM_001308176, Origene, USA) was subcloned into the EcoRl and BamHl sites of the pSin-EF1α-puro lentiviral vector (SBI, USA). CSRP2BP mutants lacking the HAT domain (711-714 aa deleted) and N-cadherin-Luc reporter mutants lacking the SEB2 were generated using the KOD Plus Mutagenesis kit (Cade No. SMK-101, TOYOBO, Japan) according to the manufacturer’s instructions.

The vectors pMD2.G and psPAX2 were packaged in 293T cells using calcium phosphate transfection. Then, transduced cells were selected for 7 days with puromycin (P7255-25MG, Sigma, USA). The surviving cells were amplified by monoclonal culture. Hela and C-33A stable cell lines expressing CSRP2BP and shCSRP2BP were established (Hela/C-33A-CSRP2BP, Hela/C-33A-Vector, Hela/C-33A-shCSRP2BP and Hela/C-33A-shcon). Protein and mRNA of transfected cells were taken for real time-PCR and Western blotting analyses. The primers used in this study are listed in supplementary Table S1.

Western blotting

Western blotting was performed as described previously [25] by using anti-CSRP2BP (LifeSpan BioSciences, USA), anti-H4, anti-acetylated H4 on lysine 5 (H415Kac), anti-acetylated H4 on lysine 12 (H412Kac) (Abcam, USA), and anti-N-cadherin (Cell Signaling Technology, MA) antibodies. Next, the membranes were incubated with secondary antibody for 1 hour at RT, and then incubated with chemiluminescent substrate kit reagents (Millipore, USA); images were captured on a Tanon 4600SF instrument (Shanghai, China). The antibodies used in this study are listed in supplementary Table S2.

RNA isolation and quantitative real-time PCR

Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, USA) based on standard procedures. Complementary DNA (cDNA) was prepared with the Prime Script® RT reagent kit (Takara, Japan) according to the manufacturer’s instructions. The primers used in this study are listed in supplementary Table S3. The expression level of gene mRNA was quantified using a SYBR® Premix E2x Taq TM II kit (Takara, Japan). The relative expression level was determined by normalizing the expression level of each target to GAPDH, and the relative mRNA fold change was determined using the 2 (-∆∆Ct) method. Samples were run in triplicate. Three independent experiments were performed.

Cell proliferation examination

For the cell growth curve, 1×10⁴ cells were allowed to grow in 6-well plates and cultured for 6 days. The cells were counted every day to draw the cell growth curve. The 5-ethynyl-2’-deoxyuridine (EdU) assay was performed with the Cell-Light EdU Apollo 567 kit (RiboBio, China) according to the manufacturer’s instructions in vitro. All images were photographed with a fluorescence microscope (Olympus, DP72). For colony formation, cells were harvested and seeded into 6-well plates with a mount of 1 × 10³ cells/well. On day 10, the cell colonies were stained with Giemsa stain for 15 min after fixation with 4% paraformaldehyde (Biosharp, China) for 30 min. Colonies with > 50 cells were counted. Three independent experiments were performed.

Wound healing assay

Wound healing assays were established by using Ibidi Culture Insert chambers (Ibidi, Germany) following the manufacturer’s protocol. A total of 45 × 10⁴ cells were added to each well of the chamber. After 24 h, wound closure was monitored, and images were captured at different time points using an Olympus microscope (Olympus). Data were analyzed by using previously published methods .

Transwell migration assays

Cell invasion assays were performed using 12-well tissue culture plate inserts (8.0 μm pores, BIOFIL, China) precoated with Matrigel (BD Biosciences). First, cells suspended in 200 μL of serum-free medium were plated in the upper chambers, whereas 600 μL of medium supplemented with 10% FBS was placed in the lower chambers. After 24 h of incubation, the cells on the lower surface of the membrane filter were fixed and stained and then counted with an inverted microscope (Olympus, IX71).

Immunofluorescence

Cells grown on cover slides were fixed with methanol and acetone (1:1) for 20 min at -20°C, permeabilized with 0.5% Triton X-100 for 20 min, blocked with 5% BSA (Beyotime, China) in phosphate buffered saline (PBS) containing 0.1% Tween-20 for 1 h, and then incubated with a primary antibody overnight at 4°C. Subsequently, the slides were incubated with Alexa Flour® 488 IgG anti-mouse (Abcam, US) or Alexa Flour® 594 IgG anti-rabbit (Abcam, US) at room temperature for 1 hour. DAPI was used to stain the nuclei for 5 min. Fluorescence images were taken using confocal microscopy (Olympus Fluoview FV3000).

Flow cytometry and chemoresistance model in vitro

Flow cytometry was performed using a BD FACS Aria II cell sorter (Becton Dickinson, San Jose, CA) to analyse the cell cycle through propidium iodide (Sigma, China) staining. Modfit LT 3.1 trial cell cycle analysis software was used to analyse the cell cycle. The cells were plated in 6-well plates (1 × 10⁵ cells/well) and cultured until they reached 90% confluency. Through flow cytometry, the annexin V+/PI cells were analysed after the indicated cells were treated with cisplatin (20μg/ml, 40μg/ml, 80 μg/ml) for a 24 h culture.

Coimmunoprecipitation (Co-IP) assay

Plasmid vectors expressing Flag-HA-tagged SMAD3 and Flag-HA-tagged SMAD4 were cotransfected into Hela cells with HA-tagged CSRP2BP by using Lipo2000TM Transfection Reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. Hela cells were lysed and incubated with RAPI (Biosharp, China) lysis fluid containing 1× PMSF (Cell Signaling Technology, MA, USA) and 1× protease inhibitor cocktail (Cell Signaling Technology, MA, USA). After centrifugation, the protein supernatant was collected and incubated with the corresponding antibody at 4°C for 2 hours. The antigen-antibody complex was precipitated with protein A/G (Invitrogen, CA, USA), and Western blotting was performed to examine the target proteins.

siRNA transfection

siRNA duplexes against N-cadherin and SMAD4 were transfected into Hela-CSRP2BP, Hela-Vector cells by using Lipofectamine^TMRNA iMAX Transfection Reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. The siRNA duplex sense sequences were as follows: si-N-cadherin: 5’-CCAGUGACUCUUAAGAGAA-3’, si-SMAD4: 5’-CCACCAAGUAAUCGUGCAU-3’.

Luciferase reporter assays

Regions of the CDH2 promoter (~2 kb) were amplified using the primers and subcloned into the PLG3-basic luciferase reporter plasmid to synthesize the pGL-3-N-cadherin promoter plasmid. The coding sequences of the human CSRP2BP gene (NM_001392073, Origene, USA) and SMAD4 gene (NM_000018.10, Origene, USA) were amplified and subcloned into the Xhol and BamHI sites of the PCGN vector (PT3994-5, Clontech, USA). Luciferase activity was measured as described previously [28].

Xenograft models

BALB/c-nu mice (n=20, 5-6 weeks of age, Gem Pharmatech. Co. LTD) were kept in aseptic conditions under constant temperature and humidity. The mice were randomly divided into four groups, and each mouse received a groin subcutaneous injection of a 100 μL suspension of 1×106 cells. Tumour growth was detected with callipers every 2 days, and tumour volume was calculated using the following formula: Volume = (length×width2)/2. Magnetic resonance imaging (MRI) was used to evaluate the tumours in the mice. To further evaluate the metastatic potential of CSRP2BP, mice (n=5) were transplanted with 1x106 cells suspended in 100 μL PBS by tail intravenous injection. Six weeks after transplantation, all mice were sacrificed, and the tumours were harvested and imaged by a chemiluminescent imaging system (Sacecreation).

Chromatin immunoprecipitation (ChIP)

Cells were fixed with 37% formaldehyde for 10 min, treated with 0.125 M glycine for 5 min and centrifuged at 3000 × g at 4 °C for 5 min to collect a crude nuclear fraction. The nuclear pellet was incubated with 1% SDS lysis buffer and sonicated to shear genomic DNA into 100~400 bp fragments. Genomic DNA fragments were transferred to slide-A-LyzerTM G2 (Invitrogen Life Technologies, CA, USA) at 4 °C for 4 h. Immune complexes were precipitated with protein A/G beads (Invitrogen Life Technologies, USA), and soluble chromatin complexes were immunoprecipitated with human IgG antibody (Proteintech), H4ac antibody (Active Motif) or CSRP2BP antibody (Life Span Biotechnology) in ChIP dilution buffer overnight at 4 °C. The beads were sequentially washed with a low salt buffer, a high salt buffer, LiCl wash buffer, and TE buffer. The immunoprecipitated chromatin complexes were eluted in ChIP direct elution buffer at 65 °C for 30 min and incubated overnight at 65 °C to cross-link the chromatin complexes. DNAs were isolated using a QIAGEN DNA kit (28106, Germany). The extracted DNA was analysed by real-time PCR. Primers to detect N-cadherin promoter occupancy were listed in supplementary Table S4.

RNA-seq

Total RNA from cells for RNA sequencing was isolated using TRIzol reagent (Invitrogen Life Technologies, USA) based on standard procedures. Subsequently, an Agilent 2100 bioanalyzer and a NanoDrop 2000 were used to examine quality. Libraries were constructed using the standard Illumina library construction process. Each library was sequenced on an Illumina NovaSeq 6000 in 150 PE mode by Beijing Berry Genomics Co., Ltd. (Beijing, China).

Statistical analyses

All statistical analyses were performed using the SPSS software package (version 19.0, SPSS, Inc.) and Prism 5.0 software (GraphPad, La Jolla, CA, USA). The data are presented as the mean ± SD of at least three independent experiments. The independent sample t test was used for comparing groups to identify significant differences. The Kaplan‒Meier method was used for PFS and OS analysis, and significance was determined by the log-rank test. Multivariate logistic regression was performed to identify the independent risk factors related to the prognosis of cervical cancer. The relationships between CSRP2BP expression level and clinicopathological features were tested by the χ2 test or Fisher’s exact test. The results were considered statistically significant at P < 0.05.

Integrative analyses identify CSRP2BP correlated strongly with cervical cancer progression and EMT

Histone acetylation has demonstrated as one of the most important regulators of EMT pathway. To systematically identify the histone acetylation-related genes that are potentially correlated with EMT, we first obtained 88 genes with histone acetyltransferase activity from Gene ontology (GO). We next analysed the expression patterns of histone acetyltransferase genes in 128 cervical tissue specimens. In total, 28 genes (e.g., ATF2, BRCA2, CSRP2BP and EP300) were up-regulated and 16 genes (e.g., CDY1, CHAT and CRAT) were down-regulated in cervical cancer (Fig. 1A). We ranked the genes based on R scores (see methods in detail) and identified the top 30% genes in cervical cancer (Fig. 1B). BRCA2 was ranked top one and emerging evidence have demonstrated that BRCA2 play important roles in cervical cancer [26].

Next, we performed the spearman correlation coefficient (SCC) analysis and identified genes of which expressions were correlated with EMT scores in cervical cancer. In total, 19 genes and 26 genes that were negatively and positively correlated with EMT scores were identified (Fig. 1C). In particular, there were seven genes showed higher expression in cervical cancer and positively correlated with EMT scores, including CSRP2BP, LPCAT1, NAT14, CREBBP, MSL3, ATF2 and GNPNAT1 (Fig. 1D). We found that histone acetyltransferase CSRP2BP is a component of the ATAC chromatin remodelling complex containing HAT activity [27]. CSRP2BP is a co-activator for CRP2 to drive smooth muscle gene expression. Knockdown of CSRP2BP expression suppressed the migration of both A7r5 rat smooth muscle cells and primary human vascular smooth muscle cells (hVSMCs) [28]. However, the function of CSRP2BP in cervical cancer progression and metastasis is still unknown. Thus, we next analysed the expressions of CSRP2BP in detail. We illustrated CSRP2BP was highly expressed in cervical cancer compared to normal samples (Fig. 1E, p=0.022). In addition, CSRP2BP exhibited increased expression with increasingly severe cervical intraepithelial neoplasia (CIN) and invasive cancer (Fig. 1F). Scatter plot showed that the expressions of CSRP2BP were significantly correlated with EMT scores in cervical cancer (Fig. 1G, R=0.34 and p=7.5E-5). Moreover, the gene set enrichment analysis (GSEA) found that genes correlated with CSRP2BP were significantly enriched in EMT pathway (Fig. 1H, ES=0.377 and p=1.11E-8). Together, these results suggested that CSRP2BP plays oncogenic roles in cervical cancer by potentially regulating EMT pathway.

CSRP2BP was overexpressed and correlated with a poor prognosis in cervical cancer

Previous studies have found that CSRP2BP is involved in cell differentiation, cell apoptosis and embryonic development [27, 29]. However, its functional role in tumorigenesis remains unclear. To investigate the role of CSRP2BP in cervical cancer, we performed Western blotting and immunohistochemical staining (IHC) for the expression of CSRP2BP on human cervical tissues. We found that CSRP2BP was mainly expressed in carcinoma nests and was expressed at significantly higher levels in cervical cancer tissues (T) than in adjacent noncancerous tissues (ANTs) (Fig. 2A-C). Cervical cancer primary cells (T1, T2, T3) were isolated from three cervical cancer tissues, and we found CSRP2BP expression was increased in primary cervical cancer cells compared with ANTs (Fig. 2D, E). These data indicated that the expression of CSRP2BP was upregulated in cervical cancer.

To further validate the association of CSRP2BP expression with the prognosis of human cervical cancer, specimens from a total of 208 patients who received surgery were analysed by IHC. According to the immunostaining scores of CSRP2BP, these patients were divided into high (score ≥ 6) or low/undetected (score ≤ 4) expression groups (Fig. S1). As shown in supplementary Table S5, high expression of CSRP2BP was associated with International Federation of Gynecology and Obstetrics (FIGO) stage (P<0.001), type of tumour growth (P<0.001), tumour size (P<0.001), stromal invasion (P<0.001), lymphovascular space invasion (LVSI) (P<0.026) and HPV16/18 infection (P<0.001). To find out the relationship between HPV16/18 infection and CSRP2BP, we tested the expression of E6/E7 in overexpressed/knockdown CSRP2BP cervical cancer cells. The results revealed that overexpression of CSRP2BP increased the E6/E7 expression and knockdown CSRP2BP suppressed the E6/E7 expression (Fig. 2F, G). Consistent with our results, we found that CSRP2BP expression was higher in CINIII and cancer compared with normal tissues (Fig. 1F). The HPV 16/18 infection and development of the cervical lesion were therefore associated with an increased expression of CSRP2BP.

We next performed a Kaplan-Meier analysis to investigate the relationship between the expression of CSRP2BP and the survival of patients with cervical cancer. The median overall survival (OS) of patients with a high and low/no expression of CSRP2BP was 55 months (range, 8–88 months) and 62 months (range, 19–129 months) respectively (log-rank test χ2=8.014, P=0.005). The median progression-free survival (PFS) of patients with a high and low/no expression of CSRP2BP was 54 months (range, 1–88 months) and 60 months (range, 11–129 months) respectively (log-rank test χ2=8.319, P=0.004). These results revealed a clear negative correlation between the expression of CSRP2BP and PFS/OS of patients with cervical cancer (Fig. 2H). To analyse the effect of CSRP2BP on the OS of different FIGO stage cervical cancer patients, survival analysis was performed in FIGO I stage and II stage subgroups. Survival analysis revealed that CSRP2BP overexpression was associated with poor OS (P=0.014) in 138 patients with stage I, but not with OS (P=0.85) in 70 patients with stage II (Fig. 2I). To further determine whether CSRP2BP served as an independent prognostic factor, PFS and OS were assessed using multivariate analysis. High CSRP2BP expression, LVSI and advanced FIGO stage were associated with both poor PFS and OS (supplementary Table S6). Taken together, these data revealed a clear association between CSRP2BP expression and poor clinical outcome in cervical cancer patients.

CSRP2BP enhanced cervical cancer cell proliferation and growth in vitro and in vivo

To evaluate the functions of CSRP2BP, Western blotting was used to evaluate the expression of CSRP2BP in cervical cancer cell lines (Hela, SiHa and C-33A) and ANTs. We found that the protein levels of CSRP2BP were prominently increased in cervical cancer cell lines (Fig. 3A), suggesting its potential tumorigenic role in cervical cancer. To test whether CSRP2BP was involved in cervical cancer cell growth and proliferation, we assessed the proliferation potential of CSRP2BP-overexpressing Hela/C-33A cells (Hela-CSRP2BP, C-33A-CSRP2BP) as well as CSRP2BP-knockdown Hela/C-33A cells (Hela-shCSRP2BP, C-33A-shCSRP2BP) (Fig. 3B, C). Cell growth curve and colony formation assays showed that CSRP2BP overexpression significantly increased the proliferation of Hela/C-33A cells, whereas knockdown of CSRP2BP suppressed the proliferation of Hela/C-33A cells (Fig. 3D, E). Furthermore, EdU and flow cytometry assays demonstrated that overexpression of CSRP2BP had a great impact on increasing the proportion of S phase cells, whereas knockdown of CSRP2BP got the opposite result (Fig. 3F, G). Taken together, our data suggested that CSRP2BP accelerates the proliferation of cervical cancer cells in vitro.

To further confirm the functions of CSRP2BP, we subcutaneously examined the effect of CSRP2BP on the tumorigenicity of cervical cancer in vivo by using a subcutaneous xenograft model. As shown in Fig. 3H and 3I, MRI experiments and isolated tumor specimens revealed that CSRP2BP overexpression increased tumour growth and weight, while knockdown of CSRP2BP got the opposite results. Meantime, CSRP2BP promotes Hela cell growth in vivo (Fig. 3J, K). IHC results further illustrated that Ki-67 was increased in CSRP2BP-overexpressing tumors, while Ki-67 was decreased in CSRP2BP-knockdown tumors (Fig. 3L). These results demonstrated that CSRP2BP induced the growth and proliferation of cervical cancer both in vitro and vivo.

Cisplatin is one of the most commonly used chemotherapeutic drugs in the treatment of cervical cancer [30]. We assessed the chemotherapy sensitivity of cervical cancer cells. Hela-vector and Hela-CSRP2BP cells were stained with annexin V+ and PI. The results showed that the overexpression of CSRP2BP induced apoptosis resistance in cisplatin-treated cervical cancer cells (Fig. 3M, N). Our findings revealed that the cell survival rate increased and the sensitivity of cervical cancer cells to cisplatin decreased in a dose-dependent manner with the overexpression of CSRP2BP.

CSRP2BP drove cervical cancer invasion and metastasis

Cancer metastasis is typically characterized by tumour cell properties of increased migration and invasion [31]. We next investigated whether CSRP2BP impacts cell invasion and migration in cervical cancer. As shown in Fig. 4A and 4B, CSRP2BP overexpression greatly increased the migration and invasion of Hela/C-33A cells, while CSRP2BP knockdown cells showed a reduced metastatic capacity in vitro. To elucidate whether CSRP2BP promoted cervical cancer metastasis in vivo, we injected CSRP2BP-overexpressing and CSRP2BP-knockdown Hela cells through the tail vein to establish a pulmonary metastasis model in BALB/c nude mice. Six weeks later, the lungs were collected, and micro-metastatic foci were counted. In line with our in vitro results, CSRP2BP overexpression showed more lung metastatic foci and a higher metastasis incidence, as determined by fluorescence and H&E staining (Fig. 4C, D). The tumor weight and number induced by the injection of Hela-CSRP2BP cells and Hela-shCSRP2BP was respectively signifcantly larger and smaller than Hela-vector control cells (Fig. 4E, F). Taken together, these results indicated that CSRP2BP induced cervical cancer cell invasion and metastasis both in vitro and in vivo.

CSRP2BP activated EMT signals and increased metastasis in cervical cancer cells

Since histone acetylation is a ubiquitous hallmark of transcriptional activity [32], we conducted RNA-Seq in CSRP2BP-overexpressing and control Hela cells for a full transcriptome analysis to monitor potential downstream targets of CSRP2BP in cervical cancer metastasis. Compared with the control groups, 591 genes were upregulated (≥ 2-fold) in Hela-CSRP2BP cells (Fig. S2A). GO analysis showed that most of these genes were involved in cell proliferation and adhesion, consistent with our study (Fig. S2B). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the most significantly activated pathway was the PI3K/AKT signalling pathway (Fig. S2C, D). Intriguingly, N-cadherin (also known as CDH2) was found among the genes that were the most upregulated in each of the Hela-CSRP2BP cells, and this gene is associated with EMT signalling and cancer metastasis (Fig. 5A and Fig. S2E). The GSEA analysis revealed that CSRP2BP expression levels were positively correlated with EMT (Fig. 5B, ES = 0.64, P = 2.97e-10). These results were consistent with our finding that CSRP2BP showed higher expression in cervical cancer and positively correlated with EMT (Fig. 1C, D). Therefore, we hypothesized that N-cadherin might be an important downstream transcription target involved in CSRP2BP-mediated cervical cancer EMT and metastasis.

To test this hypothesis, we examined the expression of N-cadherin and E-cadherin by western blotting and RT‒PCR. As shown in Fig. 5C and Fig. S2F, a significant increase of N-cadherin protein expression and a reduction of E-cadherin protein expression were observed in Hela-CSRP2BP cell extracts. Subsequent IHC also revealed that the expression of N-cadherin was significantly increased in Hela-CSRP2BP xenograft tumours, while the expression of E-cadherin was decreased (Fig. 5D). This led us to further examine a variety of EMT and invasion markers. Of note, MMP-2 and MMP-9 showed increased mRNA and protein levels in CSRP2BP-overexpressing cells, whereas CSRP2BP-knockdown cells displayed the opposite results (Fig. S2G, H). To verify that N-cadherin was, at least in part, under the control of CSRP2BP during EMT, we used small interfering RNA (siRNA) to knockdown N-cadherin in Hela-CSRP2BP cells (Hela-CSRP2BP-siN-cadherin, Fig. 5E). Depletion of N-cadherin reduced cell metastasis and invasion in vitro (Fig. 5F, G). Further study demonstrated that N-cadherin overexpression was able to increase the metastasis and invasion capacity of Hela-shCSRP2BP cells (Hela-shCSRP2BP-N-cadherin, Fig. 5H-J). These data strongly demonstrated a critical role of N-cadherin in CSRP2BP-induced EMT signalling in the metastasis process.

Identification of SMAD4 as a CSRP2BP binding partner that induced N-cadherin transcription

As a transcriptional coactivator, CSRP2BP does not bind DNA itself. It must be recruited to chromatin via protein‒protein interactions with transcription factors to regulate gene expression [28]. Since studies reported that the transcription factors SMAD3 and 4 could bind to the promoter region of N-cadherin to stimulate its transcription and participate in the EMT process [33, 34], we hypothesized that CSRP2BP might associate with SMAD4 or SMAD3 to promote N-cadherin transcription. We cotransfected plasmids expressing Flag-HA-tagged SMAD3 or Flag-HA-tagged SMAD4 in Hela cells with HA-tagged CSRP2BP, and the Co-IP results indicated that CSRP2BP interacted with SMAD4 but not with SMAD3 (Fig. 6A and Fig. S3). Next, we determined the subcellular localization of CSRP2BP and SMAD4 by performing immunofluorescence. As shown in Fig. 6B, endogenous CSRP2BP and SMAD4 were expressed in both the nucleus and cytoplasm of cervical cancer cells. To further explore the function of the CSRP2BP/SMAD4 complex, we conducted a luciferase reporter gene assay using a pGL-3-N-cadherin promoter plasmid with CSRP2BP and SMAD4 expression plasmids. CSRP2BP significantly increased the luciferase activity in the pGL-3-N-cadherin promoter when SMAD4 was present (Fig. 6C).

Next, we explored whether CSRP2BP was recruited to the N-cadherin promoter by SMAD4 in Hela cells. The N-cadherin promoter contains two SMAD binding elements (SBEs), -117 to -235 and -1321 to -1213, in the ~2 Kb promoter region [35-37]. We then conducted ChIP-QPCR with anti-CSRP2BP or control IgG antibodies to test whether these two sites are involved in the binding of CSRP2BP and SMAD4 to the N-cadherin promoter. We also tested the nearby E-box site (-1411 to -1291) as a control. ChIP-QPCR results showed that CSRP2BP levels were high surrounding the SBE2 site, while no obvious binding around SBE1 and the E-box site was observed in Hela cells (Fig. 6D-F). Deletion of SEB2 in N-cadherin-Luc (SEBM-Luc) reporter significantly relieved SMAD4/CSRP2BP complex-increased N-cadherin promoter activity, supporting the idea that SMAD4 recruits CSRP2BP specifically to the SBE site of the N-cadherin promoter in cervical cancer cells (Fig. 6G, H).

To probe whether SMAD4 was the key factor by which CSRP2BP activated N-cadherin expression in cervical cancer cells, we used siRNA to knockdown SMAD4 in Hela-CSRP2BP cells and detected changes in SMAD4 and N-cadherin expression. As expected, SMAD4 knockdown decreased the expression of N-cadherin (Fig. 6I). Furthermore, Transwell and cell migration assays revealed that knockdown of SMAD4 significantly inhibited the invasion and metastasis abilities of CSRP2BP-overexpressing cells (Fig. 6J, K). In summary, CSRP2BP stimulated N-cadherin transcription by cooperating with SMAD4 to bind to the N-cadherin promoter.

CSRP2BP promoted N-cadherin expression partly by acetylating H4

Histone acetylation makes a great contribution to EMT, and CSRP2BP is one of the important HATs [38, 39]. We previously reported that CSRP2BP acetylated H3 and H4 [28]. In addition, Drosophila CSRP2BP was shown to exhibit HAT activity specific for H4 [26]. Thus, to verify whether CSRP2BP promoted N-cadherin expression via histone acetylation in cervical cancer cells, we subsequently checked the levels of histone H3 acetylation (H3Ac) and histone H4 acetylation (H4Ac) in CSRP2BP-overexpressing or CSRP2BP-silenced Hela/C-33A cells. As shown in Fig. 7A-C, CSRP2BP overexpression significantly increased the histone H4 acetylation level at lysine sites 5 and 12 in Hela and C-33A cells. No other checked sites of histone H4 (lysine sites 8 and 16) or histone H3 acetylation were changed.

Next, to verify whether CSRP2BP regulates N-cadherin transcription by histone acetylation, we examined H4 acetylation levels on the N-cadherin promoter by ChIP assay and found that CSRP2BP overexpression led to an increased level of H4Ac on the N-cadherin promoter in Hela cells (Fig. 7D, E). Our previous study showed that the HAT domains of CSRP2BP in mice are located at amino acids 708-710 [21]. Since the HAT domains are highly conserved, we finally generated a human CSRP2BP HAT mutant by deleting four amino acids within the HAT domain (711-714 aa deleted (CSRP2BP MUT)) and assayed the effects of CSRP2BP HAT domain mutation on co-activation of the N-cadherin promoter by SMAD4. CSRP2BP HAT domain mutants marginally reduced the transcription of N-cadherin compared to that of wild-type CSRP2BP (Fig. 7F). All these results indicated that CSRP2BP activates N-cadherin transcription primarily through its histone acetylation activity.

The metastasis of cervical cancer is closely correlated with a poor prognosis and low 5-year survival rates in cervical cancer patients [40]. Therefore, it is an urgent issue to unravel the precise mechanisms regulating the metastasis of cervical cancer. Histone acetylation has demonstrated as one of the most important regulators of cervical cancer metastasis. Our analysis of cervical tissue gene expression data identify the histone acetylation-related genes that potentially correlated with EMT. We found that CSRP2BP, a histone acetyltransferase mediating histone acetylation, was higher expression in cervical cancer and positively correlated with EMT scores. In addition, CSRP2BP exhibited increased expression with increasingly CINIII and invasive cancer. The development of the cervical lesion was therefore associated with an increased expression of CSRP2BP. For in-depth research, CSRP2BP promoted cervical cancer EMT and metastasis by upregulating N-cadherin. Mechanistically, CSRP2BP positively regulates N-cadherin expression by acetylating H4K5 and H4K12 in the promotor region of N-cadherin through cooperation with Smad4 (Fig. 7G). These results provided strong evidence that CSRP2BP was an important histone acetyltransferase and oncogene factor in the EMT and metastasis of cervical cancer.

Acetylation is one of the posttranscriptional modifications (PTMs) influencing various aspects of protein biology [41]. Recently, a large amount of research has demonstrated that aberrant histone acetylation contributes to the occurrence of many diseases, such as cancer, chronic inflammation and diabetes [42, 43]. Histone acetylation is mediated by HATs and HDACs. HATs catalyse acetylation associated with gene transcription, and a growing number of studies have highlighted the role of HAT dysfunction in the initiation and development of various cancers, such as breast cancer and liver cancer [44]. However, the precise mechanisms underlying how HATs regulate the progression and metastasis of cervical cancer are still elusive.

CSRP2BP, a histone acetyltransferase, is a co-activator for CRP2, and our previous study revealed that CSRP2BP was a driver of smooth muscle gene expression [28]. However, the role of CSRP2BP in the metastasis of cervical cancer is poorly understood. In our study, we found that the expression level of CSRP2BP was significantly increased in clinical cervical cancer tissues and cervical cancer cell lines. Overexpression of CSRP2BP promoted tumour growth and metastasis in vitro and in vivo. In contrast, knockdown of CSRP2BP significantly inhibited tumour growth and metastasis. Clinically, we found that elevated expression of CSRP2BP was strongly correlated with significantly decreased overall survival rates.

It is important to note that a significant association between the expression of CSRP2BP and the clinicopathological characteristics of patients with cervical cancer was observed, including FIGO stage and HPV16/18 infection. And more importantly, overexpression of CSRP2BP could induce E6/E7 expression and knockdown CSRP2BP suppressed E6/E7 expression. It was notable that CSRP2BP maybe participate in the process of HPV infection and the development of the cervical lesion. The underlying mechanisms need further investigation. CSRP2BP overexpression was associated with poor OS in 138 patients with stage I, but not with OS in 70 patients with stage II. Gene expression analysis showed that the expression of CSRP2BP was higher in cervical cancer and CINIII compared with normal tissues, but there was no significantly different between CINII, CINI and normal tissues. These results suggested that CSRP2BP may play a vital role in the carcinogenesis and progression of cervical cancer. Additional studies on the potential role of CSRP2BP in cervical basement membrane discontinuity (breaks or absence) would be of great interest.

We further found that the sensitivity of cervical cancer cells decreased to cisplatin in a dose-dependent manner due to the overexpression of CSRP2BP, suggesting that CSRP2BP was involved in the chemotherapy resistance of cervical cancer. Based on the above research evidence, we speculated that CSRP2BP might serve as a novel biomarker and it also can be a potential therapeutic target for cervical cancer treatment.

EMT is a vital process contributing cervical cancer progression, invasion and metastasis and is characterized by a loss of epithelial markers, such as E-cadherin, and a gain of mesenchymal markers, such as N-cadherin and vimentin [45]. Many studies have revealed that the activation of EMT, especially the upregulation of N-cadherin, makes a great contribution to cancer metastasis, and HATs play a vital regulatory role in EMT [46]. However, whether CSRP2BP participates in the regulation of the EMT process in cervical cancer is still unknown. Here, we reveal that N-cadherin gene expression was significantly increased in CSRP2BP-overexpressing Hela cells by RNA-seq. KEGG analysis showed that EMT-related signalling was activated in CSRP2BP-overexpressing cells. Our experimental data revealed that CSRP2BP overexpression promoted N-cadherin expression in vitro and in vivo and that N-cadherin plays a key role in CSRP2BP-mediated cervical cancer metastasis. These results revealed that CSRP2BP promoted the metastasis of cervical cancer, at least partly by upregulating N-cadherin. Moreover, the RNA-seq results revealed many other genes associated with cell adhesion that was also significantly changed in CSRP2BP-overexpressing Hela cells. Future studies should investigate the potential role of these genes in cervical cancer progression and metastasis.

Our previous in vitro data showed that CSRP2BP could strongly acetylate H3 and H4 [28]. Whether CSRP2BP upregulates N-cadherin expression and promotes carcinogenesis of cervical cancer by acetylating histones is still unknown. Here, we found that overexpression of CSRP2BP significantly induced H4 lysine 5 and 12 acetylation by binding to the SEB2 region of the promotor of N-cadherin. Furthermore, the CSRP2BP HAT domain mutant (711–714 aa deleted), which reduced HAT ability, was incapable of activating N-cadherin expression with SMAD4, as seen for wild-type CSRP2BP, suggesting that CSRP2BP promoted N-cadherin gene transcription primarily by acetylating histones. A previous study demonstrated that HPV oncoprotein E7, playing a central role in cervical carcinogenesis, promoted E7-expressing cell proliferation through acetylating H3K9 in the promoter of E2F1 mediated by the HAT GCN5, suggesting that H3 acetylation also contributes to cervical carcinogenesis [40]. However, our study revealed that CSRP2BP did not affect the acetylation level of H3 in cervical cancer cells. Thus, the precise mechanism by which CSRP2BP affects H4 acetylation needs further investigation. In future studies, it will also be interesting to explore the histone substrates of CSRP2BP in diverse cells and proteins that participate in histone binding, which may affect substrate specificity. Further investigation of the precise coactivators cooperating with CSRP2BP in histone acetylation would be very helpful for better understanding the mechanisms by which histone acetylation controls the progression and metastasis of various cancers and other diseases.

Our previous studies demonstrated that CSRP2BP acetylated histones and activated smooth muscle gene expression by working with SRF and cofactors [28]. Many reports have shown that SMAD4 is one of the most essential transcriptional regulators of EMT-associated genes involved in the expression of the N-cadherin gene and that Smad4 also plays an essential role in the progression of cervical cancer [48–50]. We aimed to further investigate the precise mechanism by which CSRP2BP promotes N-cadherin expression in cervical cancer, and we hypothesized that SMAD4 plays a key role in this process. Consistent with previous findings, we provided evidence showing that CSRP2BP interacted with SMAD4 and that SMAD4 was a key transcription factor in CSRP2BP-mediated regulation of N-cadherin transcription. It has been reported that CSRP2BP was first identified as a binding partner of CRP2 and combined with many other transcription factors to regulate gene expression [28]. Whether the CSRP2BP/SMAD4 complex coordinates with other factors to control specific H4 acetylation and EMT-associated gene expression in cervical cancer progression and metastasis remains elusive.

Our study reveals the critical role of CSRP2BP in the metastasis of cervical cancer. Increased CSRP2BP expression was closely correlated with a poor prognosis in cervical cancer patients. Mechanistically, CSRP2BP promoted the EMT process partly by promoting H4 acetylation via cooperation with SMAD4, resulting in the upregulation of N-cadherin and finally promoting the invasion, migration and chemoresistance of cervical cancer cells in vitro and the metastasis of cervical cancer in vivo. Our results suggest that CSRP2BP can be a clinical prognostic hallmark for cervical cancer patients and maybe a potential therapeutic target for patients with cervical cancer metastasis.

EMT: Epithelial-mesenchymal transition; HATs: Histone acetyltransferases; HDACs: Histone deacetylases ; KAT14: Lysine (K) acetyltransferase 14; KO: Knockout; hVSMCs: Human vascular smooth muscle cells; T: Tissue specimens; ANT: Adjacent noncancerous tissue; cDNA: Complementary DNA; PBS: Phosphate buffered saline; MRI: Magnetic resonance imaging; ChIP: Chromatin immunoprecipitation; Co-IP: Coimmunoprecipitation; IHC: Immunohistochemistry; FIGO: Federation International of Gynecology and Obstetrics; LVSI: Lymphovascular space invasion; OS: Overall survival ;PFS: Progression-free survival; GO: Gene ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; siRNA: Small interfering RNA; H3Ac: Histone H3 acetylation; H4Ac: Histone H4 acetylation; CSRP2BP MUT: CSRP2BP HAT mutant; PTMs: The posttranscriptional modifications.

Author contributions

The contributions each author made to the study are specified as follows: Yanlin Ma, Mingyao Liu,Yongsheng Li and Qi Li initiated and designed the project. Xiaohui Yang, Fei Sun and Mian Liu wrote the manuscript. Xiaohui Yang, Qiuling Jie, Yibing Wang, Mengyongwei Li, Jiaoqi Mei, Linna Ma, Yuechuan Shi and Jingjing Mei performed the experiments and collected the data; Yueying Gao performed the gene set enrichment analysis. Xiaohui Yang, Fei Sun, Manling Chen and Yunjian Wei analyzed the data. Yanlin Ma and Mingyao Liu supervises the designed structure of manuscript, reviews and helps revise this manuscript. All authors read and approved the manuscript.

Acknowledgements

We thank Dr. Robert J Schwartz and Dr. Jun Wang for their kind technical support. We thank Dr. Genhai Zhu for his help in collecting clinical samples. We thank all staffs in Department of Reproductive Medicine, the First Affiliated Hospital of Hainan Medical University (especially Dr. Yuanhua Huang) for their help and support.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

All data supporting this study are available within this article and supplementary Information file. The dataset used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

The study was approved by the Research Ethics Committee of the Southern Medical University Nanfang Hospital, Hainan Provincial People's Hospital and First Affiliated Hospital of Hainan Medical University IRB. The Laboratory Animal Care and Use Committees of the hospital approved all experimental procedures. Written informed consents were received from all patients.

Funding

This work was supported by National Natural Science Foundation of China (82072880, 82003144, 81960283), the Major Science and Technology Program of Hainan Province (ZDK2021037, ZDKJ202003), Hainan Province Science and Technology Special Fund (ZDYF2020117), Natural Science Foundation of Guangdong Province, China (2019A1515010019), Tropical Translational Medicine Education Key Laboratory Open Project (2020TTM008), Hainan Province Science and Technology Project (LCYX202102, LCYX202203), project supported by Hainan Province Clinical Medical Center andthe specific research fund of The Innovation Platform for Academicians of Hainan Province, Innovation Project for graduate students in Hainan Province (Hyb2020-57, Hyb2020-356), Students Innovation and Entrepreneurship Training Program of Hainan (S202111810047), China Postdoctoral Science Foundation(2022T150180) and the Postdoctoral Science Foundation of Hainan Province.

Consent for publication

All authors have agreed to publish the final manuscript.

Buskwofie A, David-West G, Clare CA. A Review of Cervical Cancer: Incidence and Disparities. J Natl Med Assoc. 2020; 112(2):229-32.
Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-49.
Liontos M, Kyriazoglou A, Dimitriadis I, Dimopoulos MA, Bamias A. Systemic therapy in cervical cancer: 30 years in review. Crit Rev Oncol Hematol. 2019;137: 9-17.
Pastushenko I, Blanpain C. EMT Transition States during Tumor Progression and Metastasis. Trends Cell Biol. 2019;29(3):212-26.
Li Q, Zhou ZW, Duan W, Qian CY, Wang SN, Deng MS, et al. Inhibiting the redox function of APE1 suppresses cervical cancer metastasis via disengagement of ZEB1 from E-cadherin in EMT. J Exp Clin Cancer Res. 2021;40(1):220.
Sharma A, Kaur H, De R, Srinivasan R, Pal A, Bhattacharyya S. Knockdown of E-cadherin induces cancer stem-cell-like phenotype and drug resistance in cervical cancer cells. Biochem Cell Biol. 2021;99(5):587-95.
Kaszak I, Witkowska-Piłaszewicz O, Niewiadomska Z, Dworecka-Kaszak B, Ngosa Toka F, Jurka P. Role of Cadherins in Cancer-A Review. Int J Mol Sci. 2020;21(20):7624.
Qureshi R, Arora H, Rizvi MA. EMT in cervical cancer: its role in tumour progression and response to therapy. Cancer Lett. 2015;356:321-31.
Tang Y, Durand S, Dalle S, Caramel J. EMT-Inducing Transcription Factors, Drivers of Melanoma Phenotype Switching, and Resistance to Treatment. Cancers (Basel). 2015;12(8):2154.
O'Leary K, Shia A, Schmid P. Epigenetic Regulation of EMT in Non-Small Cell Lung Cancer. Curr Cancer Drug Targets. 2018;18(1):89-96.
Zhang Y, Sun Z, Jia J, Du T, Zhang N, Tang Y, Fang Y, Fang D. Overview of Histone Modification. Adv Exp Med Biol. 2021;1283:1-16.
Chang R, Zhang Y, Zhang P, Zhou Q. Snail acetylation by histone acetyltransferase p300 in lung cancer. Thorac Cancer. 2017;8(3):131-7.
Mobley RJ, Abell AN. Controlling Epithelial to Mesenchymal Transition through Acetylation of Histone H2BK5. J Nat Sci. 2017;3(9):e432.
Li B, Lu W, Qu J, Zhang Y, Wan X. DICER1 regulates endometrial carcinoma invasion via histone acetylation and methylation. J Cancer. 2017;8(6):933-9.
Wang LT, Wang SN, Chiou SS, Liu KY, Chai CY, Chiang CM, et al. TIP60-dependent acetylation of the SPZ1-TWIST complex promotes epithelial-mesenchymal transition and metastasis in liver cancer. Oncogene. 2019;38(4):518-32.
Hou J, Wang T, Xie Q, Deng W, Yang JY, Zhang SQ, et al. N-Myc-interacting protein (NMI) negatively regulates epithelial-mesenchymal transition by inhibiting the acetylation of NF-kappaB/p65. Cancer Lett. 2016;376(1):22-33.
Beyer S, Zhu J, Mayr D, Kuhn C, Schulze S, Hofmann S, et al. Histone H3 Acetyl K9 and Histone H3 Tri Methyl K4 as Prognostic Markers for Patients with Cervical Cancer. Int J Mol Sci. 2017;18(3):477.
Mustachio LM, Roszik J, Farria AT, Guerra K, Dent SY. Repression of GCN5 expression or activity attenuates c-MYC expression in non-small cell lung cancer. Am J Cancer Res. 2019;9(8):1830-45.
den Boon JA, Pyeon D, Wang SS, Horswill M, Schiffman M, Sherman M, et al. Molecular transitions from papillomavirus infection to cervical precancer and cancer: Role of stromal estrogen receptor signaling. Proc Natl Acad Sci U S A. 112(25):E3255-64.
Gene Ontology Consortium. Gene Ontology Consortium: going forward. Nucleic Acids Res 43(Database issue). 2015;43:D1049-56.
Liberzon A, Birger C, Thorvaldsdóttir H, Ghandi M, Mesirov JP, Tamayo P. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst. 2015;1(6):417-425.
Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545-15550.
Sun F, Xiong Y, Zhou XH, Li Q, Xiao L, Long P, et al. Acylglycerol kinase is over-expressed in early-stage cervical squamous cell cancer and predicts poor prognosis. Tumour Biol. 2016;37(5):6729-36.
Qiu F, Zhao X. Study on the sensitivity of primary tumor cells of patients with cervical cancer to chemotherapeutic combinations. J Buon. 2018;23:117-23.
Jie Q, Sun F, Li Q, Zhu J, Wei Y, Yang H, et al. Downregulated ribosomal protein L39 inhibits trophoblast cell migration and invasion by targeting E-cadherin in the placenta of patients with preeclampsia. Faseb J. 2018;35(4):e21322.
Chung SH, Woldenberg N, Roth AR, Masamed R, Conlon W, Cohen JG, et al. BRCA and Beyond: Comprehensive Image-rich Review of Hereditary Breast and Gynecologic Cancer Syndromes. Radiographics. 2020; 40(2):306-325.
Guelman S, Kozuka K, Mao Y, Pham V, Solloway MJ, Wang J, et al. The double-histone-acetyltransferase complex ATAC is essential for mammalian development. Mol Cell Biol. 2009;29(5):1176-88.
Ma Y, Li Q, Li A, Wei Y, Long P, Jiang X, et al. The CSRP2BP histone acetyltransferase drives smooth muscle gene expression. Nucleic Acids Res. 2017;45(6):3046-58.
Suganuma T, Gutiérrez JL, Li B, Florens L, Swanson SK, Washburn MP, et al. ATAC is a double histone acetyltransferase complex that stimulates nucleosome sliding. Nat Struct Mol Biol. 2008;15(4):364-72.
Liang BQ, Zhou SG, Liu JH, Huang YM, Zhu X. Clinicopathologic features and outcome of cervical cancer: implications for treatment. Eur Rev Med Pharmacol Sci. 2021;25(2):696-709.
Terranova VP, Hic S, Diflorio RM, Lyall RM. Tumor cell metastasis. Crit Rev Oncol Hematol. 1986;5(2):87-114.
Narita T, Weinert BT, Choudhary C. Functions and mechanisms of non-histone protein acetylation. Nat Rev Mol Cell Biol. 2019;20(3):156-74.
Yang H, Wang L, Zhao J, Chen Y, Lei Z, Liu X, et al. TGF-beta-activated SMAD3/4 complex transcriptionally upregulates N-cadherin expression in non- small cell lung cancer. Lung Cancer. 2015;87(3):249-57.
Yang L, Yu Y, Xiong Z, Chen H, Tan B, Hu H. Downregulation of SEMA4C Inhibit Epithelial-Mesenchymal Transition (EMT) and the Invasion and Metastasis of Cervical Cancer Cells via Inhibiting Transforming Growth Factor-beta 1 (TGF- β1)-Induced Hela cells p38 Mitogen-Activated Protein Kinase (MAPK) Activation. Med Sci Monit. 2020;26:e918123.
Li F, Lan Y, Wang Y, Wang J, Yang G, Meng F, et al. Endothelial Smad4 maintains cerebrovascular integrity by activating N-cadherin through cooperation with Notch. Dev Cell. 2011;20(3):291-302.
Kang Y, Ling J, Suzuki R, Roife D, Chopin-Laly X, Truty MJ, et al. SMAD4 regulates cell motility through transcription of N-cadherin in human pancreatic ductal epithelium. Plos One. 2014;9(9): e107948.
Alexander NR, Tran NL, Rekapally H, Summers CE, Glackin C, Heimark RL. N-cadherin gene expression in prostate carcinoma is modulated by integrin- dependent nuclear translocation of Twist1. Cancer Res. 2006;66(7):3365-9.
Wei T, Liu H, Zhu H, Chen W, Wu T, Bai Y, et al. Two distinct males absent on the first (MOF)-containing histone acetyltransferases are involved in the epithelial-mesenchymal transition in different ways in human cells. Cell Mol Life Sci. 2020;79(5):238.
Hatanaka H, Mukai A, Ito E, Ueno M, Sotozono C, Kinoshita S, et al. Epigenetic regulation of the epithelial mesenchymal transition induced by synergistic action of TNF-alpha and TGF-beta in retinal pigment epithelial cells. Biochem Biophys Res Commun. 2021;544:31-7.
Cohen PA, Jhingran A, Oaknin A, Denny L. Cervical cancer. Lancet. 2019;393(10167):169-82.
Chen Y, Zhou Y, Yin H. Recent advances in biosensor for histone acetyltransferase detection. Biosens Bioelectron. 2021;175:112880.
Hou X, Gong R, Zhan J, Zhou T, Ma Y, Zhao Y, et al. p300 promotes proliferation, migration, and invasion via inducing epithelial-mesenchymal transition in non-small cell lung cancer cells. Bmc Cancer. 2018;18(1):641.
Menzies KJ, Zhang H, Katsyuba E, Auwerx J, Protein acetylation in metabolism - metabolites and cofactors. Nat Rev Endocrinol. 2016;12(1):43-60.
Lu M, Zhu WW, Wang X, Tang JJ, Zhang KL, Yu GY, et al. ACOT12-Dependent Alteration of Acetyl-CoA Drives Hepatocellular Carcinoma Metastasis by Epigenetic Induction of Epithelial-Mesenchymal Transition. Cell Metab. 2019;29(4):886-900.
Brabletz S, Schuhwerk H, Brabletz T, Stemmler MP. Dynamic EMT: a multi-tool for tumor progression. Embo J. 2021;40(18):e108647.
Li Y, Lv Z, Zhang S, Wang Z, He L, Tang M, et al. Genetic Fate Mapping of Transient Cell Fate Reveals N-Cadherin Activity and Function in Tumor Metastasis. Dev Cell. 2020;54(5):593-607.
Qiao L, Zhang Q, Zhang W, Chen JJ. The lysine acetyltransferase GCN5 contributes to human papillomavirus oncoprotein E7-induced cell proliferation via up-regulating E2F1. J Cell Mol Med. 2018;22(11):5333-45.
Baldus SE, Schwarz E, Lohrey C, Zapatka M, Landsberg S, Hahn SA, et al.Smad4 deficiency in cervical carcinoma cells. Oncogene. 2005;24(5):810-9.
Phuah NH, Azmi MN, Awang K, Nagoor NH. Down-Regulation of MicroRNA-210 Confers Sensitivity towards 1'S-1'-Acetoxychavicol Acetate (ACA) in Cervical Cancer Cells by Targeting SMAD4. Mol Cells. 2007;40(4):291-8.
Wu N, Jiang M, Liu H, Chu Y, Wang D, Cao J, et al. LINC00941 promotes CRC metastasis through preventing SMAD4 protein degradation and activating the TGF-beta/SMAD2/3 signaling pathway. Cell Death Differ. 2021;28(1):219-232.

FigureS1.tif
Supplementary Fig. S1. Expression of CSRP2BP in cervical cancer. Representative images ofIHC staining for CSRP2BP in paraffin-embedded sections of cervical cancer patients.
FigrueS2.tif
Supplementary Fig. S2. CSRP2BP induces EMT-related signals in Hela cells.A-B Volcano Plots and Heatmap representation of up-regulated and down-regulated genes in Hela- CSRP2BP cells compare with control group. C GO analysis of top 20 biological functions of up-regulated genes in Hela-CSRP2BP cells. D KEGG pathways enriched by the selected up-regulated genes. E The protein expression levels of the PI3K/AKT signaling pathway were detected in Hela-CSRP2BP cells, Hela-shCSRP2BP andthe respective control cells by Western blotting. F The mRNA expression of N-cadherin and E-cadherin in Hela-CSRP2BP and Hela-shCSRP2BP cells were measured by RT-PCR. Statistical analysis was shown as mean SD (n=3). *P＜0.05, **＜0.01. G The protein expression levels of EMT-related markers were measured by Western blotting. H The mRNA expression levels of EMT-related markers were detected by RT-PCR. Statistical analysis is shown as mean ± SD (n=3).*P＜0.05, **＜0.01, ***P＜0,001.
FigureS3.tif
Supplementary Fig. S3. CSRP2BP does not bind to SMAD3 in Hela cells. The interaction between HA-tagged CSRP2BP and HA & Flag-tagged SMAD3 was tested by Co-IP assay with anti-Flag antibody or control normal IgG. Western blotting was stained with anti-HA antibody.
SupplementaryTable1234567.docx

Download PDF

Journal Publication

published 17 Oct, 2023

Read the published version in Journal of Experimental & Clinical Cancer Research →

Editorial decision: Major revision
20 Apr, 2023
Reviewers agreed at journal
22 Feb, 2023
Reviewers invited by journal
31 Jan, 2023
Editor assigned by journal
31 Jan, 2023
First submitted to journal
27 Jan, 2023

You are reading this latest preprint version

Histone acetyltransferase CSRP2BP promotes the epithelial–mesenchymal transition and metastasis of cervical cancer cells by activating N-cadherin

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Materials And Methods

Results

Discussion

Conlusions

Lis Of Abbreviation

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1