Potential Role of SWI/SNF Complex Subunit Actin-Like Protein 6A in Cervical Cancer

SWI/SNF complex subunit Actin-like protein 6A (ACTL6A) has been regarded as an oncogene, regulating the proliferation, migration and invasion of cancer cells. However, the expression pattern and biological role of ACTL6A in cervical cancer have not been reported. In this study, the mRNA expression and protein level of ACTL6A in cervical cancer samples were determined by public database and immunohistochemical (IHC) analysis. The effects of ACTL6A on cervical cancer cells were investigated via MTT, colony-formation assay, tumor xenografts and flow cytometry. Gene set enrichment analysis (GSEA) was used to explore the potential mechanism of ACTL6A in regulating tumorigenesis of cervical cancer. The results revealed that ACTL6A was markedly upregulated in cervical cancer tissues. Silencing ACTL6A expression resulted in decreased cervical cancer cell proliferation, colony formation and tumorigenesis in vitro and in vivo. Furthermore, we demonstrated that knockdown of ACTL6A induced cell cycle arrest at G1 phase, ACTL6A-mediated proliferation and cell cycle progression were c-Myc dependent. Our study provides the role of ACTL6A in cervical oncogenesis and reveals a potential target for therapeutic intervention in this cancer type.


INTRODUCTION
Cervical cancer is the fourth most common malignant tumor in women worldwide, with an estimated 0.604 million new cases and 0.342 million cancer-related deaths (1). It is also the second leading cause of cancer death in women aged 20 to 39 years (2). Although recent global cancer assessments have shown that early and high-quality screening programs have reduced the mortality rate of cervical cancer, premature cervical cancer deaths have risen rapidly in most low-and middleincome countries (3). Current treatment strategies for advanced cervical cancer is a combination of radical surgery, radiotherapy, chemotherapy and immunotherapy, with limited efficacy and potential adverse reactions. The identification of more valuable therapeutic targets can improve the survival of patients with cervical cancer.
Actin-like protein 6A (ACTL6A, also known as BAF53A) is a subunit of the chromatin remodeling complex SWI/SNF that plays a role in the development of neuronal progenitor cells, stem cells and epidermal cells (4,5). It has been paid more and more attention in the development and progression of various malignant tumors. We reported that ACTL6A is amplification in ovarian cancer and silencing ACTL6A attenuated folliclestimulating hormone (FSH)-driven glycolysis by downregulation of PGK1 (6). ACTL6A is highly expressed in hepatocellular carcinoma (HCC) tissues and associated with poor prognosis. ACTL6A binds to SOX2 promoter, up-regulates SOX2 expression, and induces epithelial-mesenchymal transition (EMT) of HCC cells through Notch1 pathway (7). ACTL6A also interacts with p63 in head and neck squamous cell carcinoma (HNSCC), and activates the Hippo-YAP pathway to control the transcriptional regulation of proliferation and inhibition of differentiation (8). One recent study reported that ACTL6A could induce resistance to cisplatin treatment (9). These studies suggest that ACTL6A may be a new potential therapeutic target for inhibiting tumor progression. However, it's biological role in cervical cancer has not been reported.
In this study, we focused on the role of ACTL6A in cervical cancer. We found that the protein level of ACTL6A in cervical cancer tissues was higher than that in adjacent normal tissues. Functional assays confirmed that ACTL6A is an oncogene that promotes the proliferation and cell cycle progression of cervical cancer cells in a c-Myc-dependent manner. Our results reveal the role of ACTL6A in promoting the proliferation of cervical cancer cells and indicate that ACTL6A may be a promising anticancer therapeutic target.

Cell Lines and Tissue Specimens
Cervical cancer cell lines Hela and C33A were obtained from the American Type Culture Collection (ATCC, USA). HEK293T was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured with DMEM (high glucose, Invitrogen, USA) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and 1% streptomycin/ penicillin at 37°C in a humidified atmosphere incubator containing 5% CO 2 .
A total of 124 patients with cervical cancer underwent radical hysterectomy and lymphadenectomy in Shanghai Tenth People's Hospital from January 2006 to December 2014. All enrolled patients were in the IB1-IIA2 stage. Pathological diagnoses of the samples were performed by two experienced pathologists (Yingze Wei and Jiangtao Xu) basing on the World Health Organization classification in a double-blinded manner. Clinical and pathological characteristics of these samples are shown in Supplementary Table S1. This study was approved by the institutional Ethics Committee of Shanghai Tenth People's Hospital. All patients provided written informed consent.

Real-Time Quantitative PCR (RT-qPCR)
Total RNA was isolated from the cells using RNAiso Plus (9108, Takara) and reverse transcription was performed using PrimeScript ™ Reverse Transcriptase (2690S, Takara). All qPCR analyses were performed using SYBR qPCR Master Mix (TOYOBO). Primer sequences are shown in Supplementary  Table S2.

Tumor Xenografts
The animal experiment was conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) and the protocol was approved by the ethics committee of Shanghai Tenth People's Hospital. 5×10 6 Hela cells infected with lentiviral control shRNA and shACTL6A were subcutaneously injected into the dorsal flank of six-week-old female nude mice (Shanghai SLAC laboratory Animal Co., Ltd). Tumor size was measured every 4 days after 12 days, and the average tumor volume was calculated according to the standard formula: volume = (length × width 2 )/2. On 36th day, the mice were euthanized, tumors were excised and weighed.

Western Blotting
The transfected cells were extracted using RIPA lysis buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.4, 1 mM EDTA, 1% NP40, 0.1% SDS, 1% sodium deoxycholate, proteinase inhibitor cocktail and phosphatase inhibitor cocktail) for 30 minutes at 4°C and then centrifuged at 12,000 rpm 4°C for 15 minutes, the supernatant was extracted as total protein. The protein sample was mixed with 4×loading buffer, boiled at 100°C for 15 minutes, and the SDS-PAGE gel was used for separation. After that, the proteins on the SDS-PAGE gel were transferred to a nitrocellulose membrane and incubated with the primary antibody at 4°C overnight. Next day, the membrane was incubated with 5% skimmed milk for 45 minutes, and then incubated with secondary antibody for 60 minutes. The immunoblots were detected by using the Odyssey system (LI-COR Biosciences, Lincoln, USA) or the electrochemiluminescence (ECL) imaging system (Tanon, Shanghai, China).

MTT and Colony-Formation Assay
SiRNA-transfected Hela and C33A cells were seeded into 96-well plates with 1000 cells per well. At different time points (1, 2, 3, 4 or 5 days after plating), 10 ml MTT solution (5 mg/ml final concentration, C0009, Beyotime, China) was added to each well. After incubation at 37°C for 4 hours, 200 ml dimethyl sulfoxide (DMSO) was added, and the color formation was quantified at 490 nm wavelength. In each individual experiment, proliferation under each condition was studied in sextuplicate. For the colony formation experiment, shRNA transfected Hela or C33A cells were seeded into a 12-well plate at a ratio of 1000 cells per well. After 10 days, the cells were fixed with methanol and stained with 0.4% crystal violet. The number of colonies was counted for analysis.

Cell Cycle Analysis
The cell cycle analysis kit (C1052, Beyotime, China) was used according to the manufacturer's protocol. At 72 hours after transfection, Hela and C33A cells were harvested by trypsin method and fixed in 70% cold ethanol at -20°C overnight. Then propidium iodide (PI) was stained at 37°C for 30 minutes and the cell cycle distribution was analyzed by flow cytometry with a FACScan flow cytometer (BD Biosciences, USA). The percentage of cells in the G0/G1, S and G2/M phases was analyzed by using FlowJo (version 7.6.2, Tree Star, USA).

Luciferase Reporter Analysis
HEK293T were inoculated at 5×10 4 cells per well in 24-well plates. At the same time, 10 ng of Renilla luciferase reporter plasmid and 50 ng of E2F1 promoter luciferase reporter plasmid were transfected. Twenty-four hours after transfection, luciferase activity was measured by using the Dual Luciferase Reporter Gene Kit (Promega, CA, USA), following the manufacturer's protocol.

Bioinformatics Analysis
Gene expression data from Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (CESC) available at the TCGA database (TCGA, Provisional) was assessed by using the cBioPortal (http://cbioportal.org) (11,12). The Oncomine platform (https://www.oncomine.org/) and Gene Expression Omnibus (GEO) datasets were used to compare the expression of ACTL6A in normal and tumor tissues. GSE63514 compared the expression of ACTL6A in 24 normal, 14 cervical intraepithelial neoplasia (CIN) I, 22 CIN II, 40 CIN III and 28 cervical cancers specimens. GSE7803 was used to analyze the expression of ACTL6A between 10 normal squamous cervical epithelia samples and 21 invasive squamous cell carcinomas of the cervix. GSE6791 showed the expression of ACTL6A in 8 cervix uteri samples and 20 cervical cancer samples. GSE7410 evaluated the expression of ACTL6A between 5 non-cervical carcinoma and 19 patients with cervical cancer. GSE9750 compared the expression of ACTL6A in 24 normal cervical epithelium and 33 cervical cancer samples. GSE56303 assessed the expression of ACTL6A in 55 cervical squamous cell carcinomas and 5 cervical adenocarcinomas.
Biological states or processes related to the activities of ACTL6A were evaluated by GSEA v3.0 software (13). Using different gene sets deposited in the GSEA Molecular Signatures Database v6.2 (MSigDB) resource, we identified the differential pathways between the high-and low-ACTL6A expression specimens.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism (Version 6.01, GraphPad Software, Inc., USA). Data were shown as the mean ± SD from at least three independent experiments. The relationship between ACTL6A protein level and clinicopathological features was analyzed by Fisher's Exact Test and Pearson's chi-square test. Correlations between the expressions of ACTL6A and relatedgenes were determined using Spearman's correlation analysis. Immunoreactivity scores were compared using the Mann-Whitney U test. Student t test was used to calculate the significance of difference between control groups and experimental groups. All tests were two-tailed and p-value of less than 0.05 was considered statistically significant.

Expression Pattern of ACTL6A in Cervical Cancer
We first studied the ACTL6A genome and gene expression profile in 310 CESE samples from the TCGA database by using cBioPortal. The mRNA upregulation of ACTL6A and BRD9 was most common in CESC, while changes in other SWI/SNF components were relatively rare ( Figure 1A). In addition, ACTL6A co-expressed several important genes in the cervical cancer 3q amplicon, including PIK3CA, SOX2 and TP63 (Supplementary Figure 1A). Then we analyzed the expression data from the Oncomine and GEO databases. As shown in Figure 1B, ACTL6A expression was significantly upregulated in various malignant tumor tissues, including cervical cancer, bladder cancer, breast cancer and esophageal cancer. Compared with the normal cervix, the expression of ACTL6A was higher in CIN and cervical cancer tissues, and the expression of ACTL6A in CIN increased with the rise of the grade (GSE63514, Figure 1C). Moreover, ACTL6A expression in multiple data sets of cervical cancer was significantly higher than that of normal cervix (GES7803, GSE6791, GSE7410, GSE9750, Figures 1D-F, Supplementary Figure 1B). However, there was no difference in ACTL6A between cervical squamous cell carcinoma and adenocarcinoma (GSE56303, Supplementary Figure 1C). Then we examined the protein level of ACTL6A in cervical cancer tissue. The cytoplasmic and nuclear immunoreactivity of ACL6A in cervical cancer tissues was significantly higher than that in adjacent nontumor tissues (p < 0.01, Figures 2A, B). The relationship between ACTL6A and clinicopathological features was also analyzed. According to IRS scores, 124 patients were classified into two groups: low or no rating group (n = 30) and high rating group (n = 94). As shown in Table 1, high levels of ACTL6A protein were significantly associated with FIGO stage (p < 0.01) and differentiation grades (p < 0.01), but not with tumor size (p = 0.452), deep stromal invasion (p = 0.146) and patient age (p = 0.061). These data suggest that ACTL6A amplification may be associated with the progression of cervical cancer.

Correlation Between ACTL6A and Differentiation Genes
One previous study demonstrates that ACTL6A is required to repress differentiation (4). Here, we investigated the correlation between ACTL6A and differentiation genes in cervical cancer samples from TCGA. As shown in Figure 3A, the expression of ACLT6A in cervical cancer cohort was negatively correlated with differentiated genes (S100P, S100A4, KRT7 and TGM2). Furthermore, the expressions of S100P, S100A4, KRT7 and TGM2 were also induced by silencing of ACTL6A in cervical cancer cells ( Figure 3B).

Role of ACTL6A in Cervical Cancer Cell Proliferation
Next, we examined the role of ACTL6A in cervical cancer cell proliferation by using the MTT and colony formation assay. As demonstrated in Figures 4A, B, knockdown of ACTL6A reduced the proliferation and colony formation of Hela and C33A cell lines compared with the control group. We also found that the subcutaneous tumors formed by Hela cells transfected with lentiviral shACTL6A were smaller, in both size and weight, than those with shRNA control cells (Figures 4C-E).

Identification of Biological Processes and Signaling Pathways
To gain insight into the potential biological pathways of ACTL6A implicated in the tumorigenesis of cervical cancer, GESA was performed by using Linked Omics platform. The results showed that ACTL6A may be involved in DNA replication, chromosome segregation, mitotic cell cycle phase transitions, cell cycle G2/M and G1/S phase transitions, protein localization of chromosomes, etc. (Supplementary Figure 2A). Chromatin DNA binding, nucleosome binding, histone binding, ubiquitin-like protein binding, doublestranded RNA binding and translation initiation factor binding were also potential molecular functions (Supplementary Figure  2B). In addition, high expression of ACTL6A was positively correlated with various cancer-related processes of cervical cancer mapping in KEGG pathway ( Figure 5A). For example, DNA replication, cell cycle, mismatch repair, Wnt signaling pathway and Hippo signaling pathway were closely associated with the expression of ACTL6A in cervical cancer. Moreover, the other four gene sets of cell cycle including REACTOME CELL CYCLE (NES = 2.293, p < 0.01), WHITFIELD CELL CYCLE LITERATURE (NES = 2.115, p < 0.01), REGULATION OF CELL CYCLE (NES = 1.645, p < 0.01) and MITOTIC CELL CYCLE (NES = 1.956, p < 0.01) were also proved to be significantly correlated with high expression of ACTL6A ( Figure 5B).

A B
FIGURE 3 | Correlation between ACTL6A and differentiation genes. (A) Correlation of ACTL6A mRNA expression with differentiation genes (S100P, S100A4, KRT7 and TGM2) based on TCGA database by using LinkedOmics. p value and correlation coefficient were shown. (B) The expressions of differentiation genes (S100P, S100A4, KRT7 and TGM2) were detected with RT-qPCR. Each bar represents the mean ± SD of triplicate experiments, *p < 0.01.

Silencing ACTL6A Induces Cell Cycle Arrest in Cervical Cancer Cell
As ACTL6A expression was closely associated with cell cycle according to GSEA, we then investigated whether ACTL6A affects the cell cycle of cervical cancer cells. Cell cycle analysis by flow cytometry showed that silencing of ACTL6A significantly increased the percentage of cells in the G1 phase and decreased the proportion of cells in the S phase compared to the control group ( Figure 6A). Moreover, we evaluated the expression of cell cyclerelated genes (CCNA2, MCM2 and SKP2) in cervical cancer cells transfected with siNC or siACTL6A. The mRNA expressions and protein levels of these genes/proteins were decreased in both Hela and C33A cells after knockdown of ACTL6A ( Figures 6B, C). Correlation analysis also revealed that ACTL6A was positively correlated with these cell cycle regulation genes in clinical samples of cervical cancer from TCGA ( Figure 6D).

ACTL6A Induces Cell Cycle Progression via c-Myc
To further investigate the molecular mechanism of ACTL6Amediated cell cycle, GSEA showed that the gene set HALLMARK_MYC_TARGETS was positively correlated with the high expression of ACTL6A (Supplementary Figure 3A). In addition, the online analysis tool of the string database of the protein-protein interaction (PPI) network revealed that ACTL6A may form a complex with c-Myc and its dimerization partner Max (Supplementary Figure 3B). Then we evaluated the effect of ACTL6A on c-Myc dependent transfection of the well-defined c-Myc target gene E2F1 (15). HEK293T cells transfected with either ACTL6A or c-Myc plasmid and E2F1 promoter. We observed that both c-Myc and ACTL6A activated the E2F1 promoter, while overexpression of c-Myc and ACTL6A further stimulated E2F1 promoter activity ( Figure 7A). In addition, silencing of either c-Myc or ACTL6A decreased E2F1 expression ( Figure 7B), while overexpression of both c-Myc and ACTL6A resulted in increased E2F1 mRNA expression ( Figure 7C). Importantly, c-Myc knockdown blocked ACTL6A-induced E2F1 ( Figure 7D). Moreover, we demonstrated that the expression of ACTL6A was significantly correlated with c-Myc target genes, such as HDAC2, CDC45, CDK4, PSMD7 and MCM4 (Supplementary Figure 3E).
To further verify the cell growth-promoting function of ACTL6A is mediated by c-Myc, we showed that overexpression of ACTL6A promoted the proliferation of Hela and C33A cells, while knockdown of c-Myc significantly inhibited ACTL6Aincreasd proliferation ( Figure 7E). Flow cytometry analysis also demonstrated that ACTL6A-induced cell cycle progression was abolished by knockdown of c-Myc ( Figure 7F). These results indicated that ACTL6A regulates the proliferation and cell cycle progression of cervical cancer cells through c-Myc.

DISCUSSION
Increasing evidences have suggested that ACTL6A plays important roles in the carcinogenesis processes. However, the pathological role of ACTL6A was remained poorly understood in cervical cancer. Here, we reported a significant increase in ACTL6A protein levels in cervical cancer tissues. Our study suggested that ACTL6A promoted cell proliferation through regulation of cell cycle via c-Myc in cervical cancer. We suggest that ACTL6A plays an oncogenetic role in the development of cervical cancer and has potential clinical values in targeted therapy.
The SWI/SNF complex was originally described as a multisubunit protein complex that controls gene expression and various important signaling pathways (16). Mutations, deletions and translocations involving SWI/SNF complex subunits occur frequently in cancer (17)(18)(19). In contrast, some studies have reported that ACTL6A amplification and overexpression are common events in cancers (6)(7)(8)(9)(20)(21)(22)(23)(24)(25). Consistent with these studies, we found an increase in ACTL6A in cervical cancer by combining the CESC genome and expression profile based on multiple public database and IHC detection in cervical cancer tissues. In addition, by comparing different CIN stages, we found that the expression of ACTL6A increased from low-level CIN to pre-invasive CIN lesions. However, other subunits of the SWI/SNF complex have relatively little genetic alterations in CESC. This supports the idea that ACTL6A is a special oncogenic subunit in the SWI/SNF complex in cervical cancer. In addition, one previous study revealed that depletion of ACTL6A in lysates from HNSCC cells could induce quantitative depletion of other SWI/SNF complex subunits, confirming the binding of ACTL6A with an intact SWI/SNF complex (8). In that case, more evidences for the assembly status of SWI/SNF components upon ACTL6A silencing might help to reveal the role of ACTL6A in more detail.
Previous studies have shown that ACTL6A has carcinogenic activity and acts as an inducer of EMT (7,25). In epidermal squamous cell carcinoma (SCC), ACTL6A has been proven to interact with p53 DNA response elements in the gene promoter of p21 Cip1 and maintain the aggressive phenotype (22). ACTL6A is also co-localized and interacts with Sox2 and p53, and inhibits differentiation in leukemia cells (21). Moreover, ACTL6A enhances malignant behaviors of glioma cells by increasing the stability and abundance of YAP/TAZ protein (20). It also interacts with p63, promoting cell proliferation and inhibiting differentiation of HNSCC through Hippo-YAP pathway (8). Notably, although it has been reported that knockdown of ACTL6A inhibits HPV E6/E7 oncogene expression and restores the p53-related signaling pathway in cervical cancer cells (26), its biological roles in cervical cancer remain largely unknown. In current study, we provide evidence that ACTL6A promotes the proliferation, clone formation and in vivo tumor formation of cervical cancer cells. GSEA analysis showed that the increased expression of ACTL6A was related to biological processes such as cell cycle and DNA replication. In fact, we found that silencing ACTL6A increases the percentage of G1 phase cells and decreased the expression of cell cycle related genes. These results are similar to some previous studies (4,24), which further confirmed that ACTL6A can promote the occurrence of different malignancy through regulating cell cycle progression.
We also provided data on the potential mechanism by which ACTL6A promotes cell cycle progression. C-Myc is an oncogenic transcription factor, which is frequently deregulated in cancers and is associated with malignant progression (27). It is now clear that c-Myc is closely related to the cell cycle process, and the molecular mechanism by which c-Myc stimulates the cell cycle has been well elucidating (28). Previous studies showed that ACTL6A can form a complex with c-Myc, which is critical for the oncogenic activity of c-Myc (29), and ACTL6A also promotes the recruitment of Myc on CDK2 promoters (23). The role of ACTL6A-stimulated c-Myc activity in cervical cancer was demonstrated here. We found that co-expression of ACTL6A enhanced the activity of E2F1 promoter induced by c-Myc, while knockdown of c-Myc reduced the activity and expression of E2F1 stimulated by ACTL6A. The expression of ACTL6A was also significantly correlated with c-Myc target genes. Moreover, the functions of ACTL6A to promote cell growth and induce cell cycle progression is dependent on c-Myc. These findings confirm an important role of ACTL6A in cervical cancer by influencing c-Myc-driven tumorigenesis. Nevertheless, more researches are needed to reveal the mechanism in greater detail.

CONCLUSION
In summary, the present study showed that overexpression of ACTL6A was significantly associated with malignant behavior of cervical cancer. Mechanistic data revealed that ACTL6A promoted tumor growth and cell cycle progress by regulating c-Myc activity. Therefore, inhibition of ACTL6A might be a promising potential therapeutic strategy for cervical cancer.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The study was reviewed and approved by the Ethics Committee of Shanghai Tenth People's Hospital of Tongji University. The patients/participants provided their written informed consent to participate in this study. The study was reviewed and approved by the Ethics Committee of Shanghai Tenth People's Hospital of Tongji University (SHDSYY-2018-2578).