Synaptopodin 2 represses cervical cancer cell growth and enhances the sensitivity of cervical cancer cells to cisplatin via Hippo pathway

Purpose: To explore the functions and regulatory mechanisms of synaptopodin-2 (SYNPO2) in cervical cancer progression. Methods: Normal cervical cell lines (Ect1/E6E7) and cervical cancer cell lines (HeLa, SiHa, C-33A, and CaSki) were cultured. The pcDNA3.1 vector overexpressing SYNPO2, a negative control (vector), and blank control (control) were transfected into HeLa and SiHa cells. Protein expression from normal and cervical cancer cell lines was examined by western blot. Cell viability and proliferation were evaluated in HeLa and SiHa cells using Cell Counting Kit-8 and colony formation assays, while cell migration and invasion were assessed by wound healing and Transwell assays, respectively. Cell apoptosis was determined by flow cytometry. Results: SYNPO2 expression was decreased in cervical cancer based on the Gene Expression Profiling Interactive Analysis website (p < 0.05). Additionally, Kaplan–Meier Plotter website showed that cervical cancer patients with low SYNPO2 expression showed worse prognoses than patients with high SYNPO2 expression (p < 0.05). Subsequent investigations revealed that SYNPO2 overexpression repressed cell proliferation, migration, and invasion in cervical cancer (p < 0.01). Furthermore, SYNPO2 overexpression enhanced cervical cancer cell apoptosis (p < 0.001) and increased the sensitivity of cervical cancer cells to cisplatin (p < 0.01). The regulatory function of SYNPO2 on Hippo pathway in cervical cancer indicate that SYNPO2 inactivated Hippo pathway (p < 0.05). Conclusion: Synaptopodin 2 represses cervical cancer cell growth and enhances the sensitivity of cervical cancer cells to cisplatin via Hippo pathway, thus indicating its potentials for development for the treatment of cervical cancer.


INTRODUCTION
Cervical cancer is a common pernicious tumor, and the fourth-leading cause of cancer-related death in women [1]. Human papillomavirus (HPV) infection is a key factor for cervical cancer, but other factors play a role as well [2]. Treatments for cervical cancer include surgery, chemotherapy, and radiotherapy. However, treatments for this disease are difficult, and treatments have only limited effectiveness. Treatment outcomes for cervical cancer patients remain poor due to the high recurrence rate. The underlying pathogenesis of cervical cancer is also vague. With the evolution of molecular biology, useful molecular targets have been identified, thus enabling breakthroughs in cancer treatment [3]. It is, therefore, crucially important to examine these novel molecular targets for cervical cancer treatment.
Synaptopodin-2 (SYNPO2) was the second member of the podin family to be identified, and its encoding gene is located in chromosome 4q26. Previous reports have revealed that SYNPO2 is expressed in diverse cancers, and it acts as a tumor repressor. For instance, SYNPO2 targets the yes-associated protein 1 (YAP)-Krueppel-like factor 5 (KLF5) axis in colorectal cancer, and reduces hypoxiastimulated cell proliferation and migration [4]. Moreover, in glioma, vitamin C enhances SYNPO2 expression, which then inhibits cell proliferation and migration [5]. Additionally, SYNPO2 affects the PI3K/Akt/mTOR pathway and suppresses breast cancer progression [6]. In triple-negative breast cancer, vitamin C modulates YAP1 and SYNPO2 so as to inhibit metastasis [7]. However, the functions and regulatory mechanisms of SYNPO2 in cervical cancer remain unclear.
The Hippo pathway was shown to be involved in cell apoptosis and proliferation. More importantly, it has been shown that SYNPO2 reduces YAP/TAZ activity in order to inhibit triple-negative breast cancer metastasis [8]. Nevertheless, it is unclear whether SYNPO2 affects the Hippo pathway in cervical cancer.

EXPERIMENTAL Cell lines and cultures
Normal cervical cell line (Ect1/E6E7) and cervical cancer cell lines (HeLa, SiHa, C-33A, and CaSki) were obtained from the American Tissue Culture Collection (ATCC, USA). Cell cultivation was performed in Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA) and 10 % fetal bovine serum (FBS, Gibco) in a drippy incubator at 37 °C containing 5 % CO2.

Cell Counting Kit-8 (CCK-8) assay
The transfected cervical cancer cells were seeded in 96-well plates. The CCK-8 solution (10 μL; Dojindo Laboratories, Kumamoto, Japan) was then added to the plates at 0, 1, 2, and 3 days. The optical density (OD) value was measured with a microplate reader (BioTek Instruments, USA) at 450 nm. For IC50 value determination, Cisplatin (DDP) was added in 96well plates, and then CCK-8 solution was added.

Colony formation assay
Cervical cancer cells (1,000 cells/well) were cultured for 14 days in 6-well plates. The cells were then fixed with 4 % paraformaldehyde) and stained with 0.1 % crystal violet so as to assess colony formations. The images of the colonies were obtained with a microscope.

Wound-healing assay
Cervical cancer cells were grown to 80 -90 % confluency in the 6-well plates. A scratch-wound was made with a sterile pipette tip (10 µL). Cell migration distance was measured at the beginning and after 24 h under a microscope.

Transwell assay
Cell invasion assays were performed using Transwell Matrigel inserts (BD Biosciences, MD, USA). Serum-free DMEM medium was added into the upper chamber. The DMEM medium with FBS (20 %) was added into the lower chamber. Crystal violet was used to stain the invading cells, and they were observed with a microscope.

Flow cytometry
The Annexin V-FITC Apoptosis Determination Kit (Beyotime) and a BD FACS Calibur (BD Bioscience, USA) were utilized for flow cytometry. After washing, the cells were stained with Annexin V-FITC and propidium iodide in the dark. Cell apoptosis was evaluated by flow cytometry.

Statistical analysis
Data are reported as mean ± standard deviation (SD). Each experiment was carried out three times. SPSS 22.0 statistical software (IBM Corp, USA) was used for statistical analysis. Comparisons were performed with Student's ttest (for two groups) or one-way analysis of variance (ANOVA) for multiple groups. Statistical significance was set at p < 0.05.

SYNPO2 was associated with a lower expression in cervical cancer
Using the Gene Expression Profiling Interactive Analysis (GEPIA) website, the expression of SYNPO2 was confirmed to be decreased in cervical cancer cells (Figure 1 A). Moreover, using the Kaplan-Meier Plotter website, a worse prognosis was given for patients with low SYNPO2 expression than those with high SYNPO2 expression (Figure 1 B). Compared with normal cervical cell line (Ect1/E6E7), the cervical cancer cell lines (HeLa, SiHa, C-33A, and CaSki) had lower SYNPO2 protein expression (Figure 1 C). These results revealed that SYNPO2 displayed a lower expression in cervical cancer.

SYNPO2 suppressed cell proliferation
The overexpression efficiency of SYNPO2 was shown in Figure 2 A. Moreover, it was found that the OD value was reduced after overexpressing SYNPO2 (Figure 2 B), and the number of colony-forming cells was decreased after SYNPO2 overexpression (Figure 2 C). In general, the data illustrated that SYNPO2 suppressed cell proliferation in cervical cancer.

SYNPO2 inhibited cell migration and invasion
It was demonstrated that cell migration ability was weakened after SYNPO2 up-regulation (Figure 3 A). Additionally, cell invasion was reduced after SYNPO2 overexpression (Figure 3  B). These findings suggest that SYNPO2 inhibited cell migration and invasion in cervical cancer.

SYNPO2 enhanced cell apoptosis
Cell apoptosis was increased after SYNPO2 overexpression (Figure 4 A). Furthermore, Bax protein expression increased, while Bcl-2 protein expression decreased after SYNPO2 overexpression (Figure 4 B). Thus, these results show that SYNPO2 enhanced cell apoptosis in cervical cancer.

SYNPO2 inactivated the Hippo pathway in cervical cancer
It was found that the p-LAST1/LAST1 and p-YAP/YAP levels were elevated after SYNPO2 overexpression (Figure 6 A). In addition, YAP and TAZ levels in the nucleus reduced after SYNPO2 overexpression (Figure 6 B). Thus, in summary, the data suggested that SYNPO2 retarded the Hippo pathway and reduced cervical cancer progression.

DISCUSSION
SYNPO2 has been shown to play a repressive role in many types of cancers [4][5][6][7][8]. However, the detailed impact of SYNPO2 in cervical cancer has not been clarified. In this study, through use of the GEPIA website, it was demonstrated that the expression of SYNPO2 decreased in cervical cancer. Additionally, using the Kaplan-Meier Plotter website, it was found that cervical cancer patients with low SYNPO2 expression had worse prognoses than patients with high SYNPO2 expression. Results from western blot analyses revealed lower levels of SYNPO2 protein expression in cervical cancer cells compared with control cells. Further experiments showed that SYNPO2 overexpression repressed cervical cancer cell proliferation, migration and invasion, and enhanced cervical cancer cell apoptosis. Cisplatin affects cellular processes to regulate cervical cancer progression [9]. In this study, it was observed that SYNPO2 increased the sensitivity of cervical cancer cells to cisplatin.
The Hippo pathway is known to be involved in cell apoptosis and proliferation. The core portion of the Hippo pathway is comprised of coactivators, scaffold proteins, and a kinase cascade [10]. In addition, YAP and TAZ are two downstream members of the Hippo pathway that act as coactivators of TEA domain (TEAD) transcription factors, thereby stimulating cell proliferation and tumorigenesis [11]. Upon Hippo pathway activation, phosphorylated YAP/TAZ is targeted for proteasomal degradation and maintained in the cytoplasm. Conversely, with the inactivation of the Hippo pathway, YAP/TAZ undergoes nuclear translocation and combines with TEAD to form the YAP/TAZ-TEAD complex. This complex modulates pro-survival genes to strengthen cell survival and growth [12].
The Hippo pathway has been observed to participate in multiple cancers. For example, fatty acid-binding protein 5 (FABP5) regulates the Hippo pathway so as to promote gastric cancer cell proliferation and attenuate apoptosis [13]. Additionally, S100A14 modulates the Hippo pathway to aid the suppression of prostate cancer growth and metastasis [14]. Furthermore, NIMA-regulated kinase 2 (NEK2) cooperated with STRIPAK to inactivate the Hippo pathway, thus accelerating the progression of cervical cancer [15]. In addition, ubiquitin-specific peptidase 21 (USP21) de-ubiquitinates forkhead box protein M1 (FOXM1), which modulates the Hippo pathway and enhances cervical cancer radio-resistance [16]. Importantly, YAP/TAZ has been shown to be enhanced in cervical cancer tissues [17], and SYNPO2 inhibits the YAP/TAZ activity in triple-negative breast cancer to reduce tumor metastasis [8]. In this study, it was observed that SYNPO2 inhibits the Hippo pathway in cervical cancer.

Limitations of this study
This study also has the limitations of not using human samples and mouse models.

CONCLUSION
To the best of our knowledge, this study is the first to unravel the roles of SYNPO2 in cervical cancer. The findings indicate that SYNPO2 overexpression reduces cell proliferation, migration, and invasion, and enhances cell apoptosis in cervical cancer by modulating Hippo pathway. Therefore, SYNPO2 as a useful biotarget for drug development for cervical cancer therapy.

Ethical approval
None provided.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.