SNCG promotes the progression and metastasis of high-grade serous ovarian cancer via targeting the PI3K/AKT signaling pathway

The 312 EOC tissues were obtained from the Department of Pathology, Chongqing Medical University, between January 2006 and September 2016. All patients underwent primary surgery and followed by cisplatin-based chemotherapy. No patients received neoadjuvant chemotherapy or radiation therapy. The borderline tumor tissues (n = 21), benign tumor tissues (n = 24), normal ovaries (n = 26), and fallopian tubes (n = 28, resected for non-ovarian diseases) were collected from patients who received gynecological surgeries in the First Affiliated Hospital of Chongqing Medical University between April 2014 and December 2016. All specimens were reevaluated for histological type and grade by three senior pathologists. The study protocols have been approved by the local Medical Ethics Committee, and the written informed consent was obtained from the patients.

Human ovarian tumor cell lines SKOV3, OVCAR3, OVCAR5, ES-2 were purchased from Keygen Biotech (Jiang Su, China). HO-8910, HO-8910 PM cell lines were purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China). The HEY cell line was purchased from Shanghai Genechem (Shanghai, China). All cell lines were authenticated by profiling of short tandem repeat analysis. Cells were cultured in RPMI1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum, penicillin (100 U/ml, Gibco) and streptomycin (100 μg/ml, Gibco). Cells were incubated in a humidified atmosphere containing 5% CO₂ at 37 °C and checked regularly for mycoplasma infection.

Antibodies against SNCG (ab55424), Phalloidin (ab176753), Ki-67 (ab15580), F-actin (ab112124) were obtained from Abcam (Cambridge, UK). Antibodies against mTOR (sc-517,464), p-mTOR (Sec2448, sc-293,133), p70S6 kinase (sc-8418), GAPDH (sc-47,724), β-Actin (sc-47,778), Cyclin D1 (sc-8396), MMP9 (sc-393,859, sc-21,733), Vimentin (sc-6260) were purchased from Santa Cruz (CA, USA). Antibodies against p-p70S6 kinase (Thr389, #97596), p-Akt (Sec473, #4060), Akt (#4961), MMP2 (#40994), E-cadherin (#14472), N-cadherin (#13116) were purchased from Cell Signaling Technology. Secondary antibodies, including HRP-labeled goat anti-mouse IgG (A0216), HRP-labeled goat anti-rabbit IgG (A0208), Cy3-labeled goat anti-rabbit IgG (P0183), were purchased from Beyotime (Shanghai, China). LY294002 (ab120243) was obtained from Abcam (Cambridge, UK), Puromycin (sc-205,821) was purchased from Santa Cruz (CA, USA). insulin-like growth factor 1 (IGF-1) (P5502) was purchased from Beyotime (Shanghai, China). DMSO (PYG0040) and DAPI (AR1177) were purchased from Bosterbio (Hubei, China).

The paraffin-embedded blocks were cut at 4 μm and mounted on slides. Following the deparaffinization, rehydration, blocking endogenous peroxidase, and antigen retrieval, the tissues were incubated with antibodies overnight at 4 °C. After washing in PBS, the tissues were incubated with the secondary antibodies for 30 min at 37 °C. Finally, sections were stained with a DAB staining solution and counterstained with hematoxylin. Negative control slides were incubated with normal goat serum. The immunostains were scored based on the intensity and the percentage of stained cells as described previously. Scores of 0 to 4 were defined as low expression, whereas scores of 5 to 9 were defined as high expression [16].

Cells were seeded on sterile glass coverslips at 37 °C overnight and then fixed with 4% paraformaldehyde for 10 min, followed by blocking with 10% normal goat serum at 37 °C for 30 min. Subsequently, cells were incubated with the SNCG, MMP9, Vimentin, and F-actin primary antibodies at 4 °C overnight. After washing, cells were incubated with the secondary antibodies for 1 h. The F-actin cytoskeleton was visualized by staining with Phalloidin for 30 min following DAPI nuclear staining for 5 min. All images were taken with a laser scanning confocal microscope.

Cells were harvested and lysed with the lysis buffer (Beyotime, Jiangsu, China) and centrifuged for at 12,000 g for 10 min at 4 °C. Following extraction, the total proteins were determined with BSA protein assay kits (Beyotime, Jiangsu, China). Next, equal amounts of protein (40 μg) were loaded onto 6–12% sodium dodecyl sulfate-polyacrylamide gels and electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, MA, USA). Membranes were blocked with 5% non-fat dry milk in TBS with 0.1% Tween-20 for 1 h at 37 °C and then incubated with primary antibodies overnight at 4 °C, followed by exposure to secondary antibodies for 1 h at 37 °C. The GAPDH and β-actin were used as the internal control. Protein bands were detected by enhanced chemiluminescence plus detection reagents (Beyotime, Jiangsu, China).

Total RNA was extracted using Trizol reagent (Takara, Japan), and a cDNA reverse transcription kit (Takara, Japan) was used to synthesize the first-strand DNA following the manufacturer’s instructions. Briefly, each well contained a reaction volume of 25 μl. And the reaction was carried out using SYBR Green Kit and a CFX96™ real-time PCR Detection System (BioRad, USA). The primers sequences are listed in Table S1. The 2^−△△Ct method was used to determine the mRNA expression levels, with GAPDH as an internal control.

To suppress SNCG expression, three small hairpin RNA (shRNA) sequences targeting SNCG were synthesized by Sangon Biotech, Shanghai, China (sh1: 5′-GAAGCAGCTGAGAAGACCAAG-3′, sh2:5′-TCATGTATGTGGGAGCCAA GA-3′, and sh3: 5′-CCAAGGAGAATGTTGTACAGA-3′), and a scrambled shRNA was synthesized for the negative control (Scr: 5′-TTCTCCGAACGTGTCA CGTAA-3′). The SNCG cDNA was amplified and subcloned into pcDNA3.1 to construct SNCG overexpression vectors (Ove), and an empty vector was used as the negative control (Ctrl). Sequences are listed in Table S2. Subsequently, stable cell lines silencing or overexpressing SNCG were generated using the lentivirus vector following the manufacturer’s protocols. Infected cells were treated with 2 μg/ml puromycin, and the puromycin resistant clones were isolated. The transfection efficiency was confirmed by Western blotting and qRT-PCR.

For cell viability assays, cells (500 per well) were seeded in 96-well plates over 24 h to 96 h. Briefly, 20 μl of MTT solution was added to each well. After incubation for 4 h at 37 °C, 150 μl of DMSO was added to each well. Finally, A450 was measured using a microplate reader. To analyze the colony formation, cells (200 per well) were seeded in 24-well plate for 2 weeks, and then colonies were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. The colony formation was also assessed using a two-layer soft agar assay. Cells (1× 10³ per well), mixed in with a 1.5 ml of 0.3% top agar solution in RPMI1640 medium containing 10% FBS, were plated over a solidified base agar layer (1.5 ml of 0.6% agar solution) in 6-well plates. After 3 weeks, colonies containing over 50 cells were counted.

Transwell assays were used to evaluate cell migration and invasion. The 8-μm pores (BD Bioscience, CA, USA) coated with a 1:5 dilution of Matrigel (BD Bioscience, CA, USA) were used in the invasion assay. Meanwhile, membranes without Matrigel coating in the chambers were used in the migration assay. The protocols were the same for both assays. Cells (1 × 10⁴ per well), suspended in the medium containing 5% FBS, were seeded in the upper chambers. Next, the medium containing 10% FBS was added to the lower chambers as a chemoattractant. After a 24 h incubation at 37 °C, cells on the lower side were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, photographed and counted under a microscope. Besides, a wound-healing assay was used to assess cell migration ability. Cells were seeded into 6-wells (1 × 10⁶ per well) and cultured until 90% confluence. Subsequently, cells were scratched with a 200-μL sterile tip and washed with PBS twice to remove the detached cells. Cells were allowed to grow for 24 h in a serum-free medium. The wound margins were observed and photographed under a microscope.

To study the effect of SNCG on tumor growth in vivo, 2 × 10⁶ cells per mice, trypsinized and resuspended in PBS, were injected intraperitoneally into 6-week-old athymic female nude mice (n = 5/group). After five weeks, the mice were sacrificed and autopsied. Visible metastatic implants were harvested and weighed. Excised tumor tissues were formalin-fixed and paraffin-embedded for IHC subsequently. All animal experiments were approved by the Institution Animal Care Committee at Chongqing Medical University, Chongqing, China.

The human phospho-kinase array kit (ARY003B) was purchased from R&D Systems and performed based on the manufacturer’s protocol. Briefly, 1.0 mL of array buffer was firstly added to each well for 1 h at room temperature. After washing, SKOV3 (Scr/sh1) cell lysates (400 μg per well) was added to each well overnight in array membranes at 4 °C. And then membranes were washed and incubated with detection antibodies for 2 h at room temperature. Next, membranes were exposed to streptavidin-HRP for 30 min on a rocking platform. After washing, protein bands were detected by enhanced chemiluminescence for 1 min and exposed to film. The experiment was performed three times.

In our cohort of patients, the mean age was 51.2 years (range, 15–86 years). 239 (76.6%) patients were diagnosed with advanced-stage (III/IV), while 73 (23.4%) patients had early-stage (I/II). Of all patients, 87.5% underwent primary surgery with optimal debulking. The majority of patients were HGSOC (69.2%), and others had low-grade serous (7.7%), endometroid (10.2%), mucinous (5.8%), and clear cell (7.1%) types. The clinicopathologic features are summarized in Table 1.

The high level of SNCG expression was observed in 68.3% of EOC tissues by IHC staining. The expression of SNCG was significantly increased in the EOC tissues compared with the borderline tumor, benign tumor, normal ovary, and fallopian tube (Fig. 1a-b). Moreover, representative staining images of SNCG expressed in different pathological types were also shown in Fig. 1b. The association between the SNCG expression and clinicopathologic parameters of EOC was subsequently analyzed. Results showed that up-regulated SNCG positively correlated with the high CA125 values (p = 0.003), advanced stage (p < 0.001), high-grade (p = 0.018), HGSOC (p < 0.001), and suboptimal debulking tumors (p = 0.015), but not associated with age, tumor histology, or ascites (Table 1). Furthermore, we explored the relationship between SNCG expression and the survival time of EOC patients. The median progression-free survival (PFS) and overall survival (OS) were 19 months and 38 months, respectively. Kaplan-Meier analysis indicated a significantly worse PFS of patients in the SNCG-high expression group than those in the SNCG-low expression group (p = 0.007). However, the data failed to show a statistically significant correlation between SNCG expression and OS (p = 0.058, Fig. 1c). Patients harboring HGSOC mostly diagnose at advanced stages and have shorter progression-free survival. Thus, we hypothesized that overexpression of SNCG might be related to the survival of HGSOC. As expected, a significant correlation was identified between SNCG up-regulation and clinical outcome (PFS and OS) in patients with HGSOC. Moreover, multivariate analysis showed that SNCG was an independent prognostic factor for HGSOC patients (Fig. 1d).

To determine the contribution of SNCG to the malignant behaviors of ovarian cancer cells, we first used Western blot to assess the endogenous expression of SNCG in different cell lines. The results showed that SNCG was highly expressed in SKOV3 and HO-8910 PM cells, whereas a low level of SNCG was detected in OVCAR3 (Fig. 2a). We chose these three kinds of HGSOC cell lines for further study. Next, we lentivirally introduced specific SNCG shRNA into SKOV3 and HO-8910 PM cells and successfully established stable cell lines (SKOV3-sh1 and HO-8910 PM-sh3, Fig. 2b). The MTT and colony formation assays showed that knockdown of SNCG suppressed the viability and colony formation in SKOV3-sh1 and HO-8910 PM-sh3 cells (Fig. 2c-d). Using transwell assays, we observed that the migratory and invasive capacities of SKOV3-sh1 and HO-8910 PM-sh3 cells were noticeably inhibited (Fig. 2e). Besides, we overexpressed SNCG in OVCAR3 cells (OVCAR3-Ove). As expected, the ectopic expression of SNCG significantly enhanced EOC cell proliferation and clonogenicity (Fig. 2c-d). Meanwhile, the transwell assay showed a substantial increase in cell migration and invasion of OVCAR3-Ove cells (Fig. 2e). The effect of SNCG on cancer cell morphology was then evaluated by F-actin cytoskeleton staining combined with FN exposure. We observed that the cell polarization and lamellipodia formation were limited in SNCG-knockdown cells compared with control cells. Furthermore, knockdown of SNCG reduced the expression levels of MMP9 and Vimentin (Fig. 2f and g). Thus, SNCG silencing inhibited the ovarian cancer cell migratory capacity, which might contribute to its metastatic inhibition in ovarian cancer.

To determine the potential impact of SNCG in cell growth and metastasis in vivo, we intraperitoneally injected SNCG knockdown (SKOV3-sh1 and HO-8910 PM-sh3) and overexpression (OVCAR3-Ove) cells into female athymic BALB/c nude mice to form peritoneal metastases. Mice were sacrificed after five weeks. Consistent with human disease, the overt metastatic lesions were attached to the mesentery, omentum, and liver. Compared with the control groups, both the number of metastatic nodules and tumor weight were significantly reduced in SNCG-knockdown groups. In contrast, the SNCG-overexpression group significantly increased the metastatic colonization and weight of implants (Fig. 3a). Next, tumor sections were stained to evaluate the proliferation, epithelial-mesenchymal transition (EMT) and metastasis-associated markers by IHC. As shown in Fig. 3b-f, we found that knockdown of SNCG was accompanied by reduced Ki-67, cyclin D1, MMP2, MMP9, N-cadherin, and Vimentin and increased E-cadherin immunostaining in metastatic tissues. Results were also supported in the SNCG overexpression group. Together, the data corroborated the in vitro results and provided evidence that SNCG was critical for the establishment of ovarian cancer metastasis in vivo.

Since loss- and gain-of-function of SNCG could regulate the aggressive behaviors of ovarian cancer cells both in vitro and in vivo, we tried to identify the underlying molecular mechanism. Thus, we performed a human phospho-kinase array, which is closely related to cell proliferation and migration. As shown in Fig. 4a-b, 13 kinases significantly changed after knockdown of SNCG in SKVO3 cells. The phosphorylation of kinases within the PI3K/Akt signaling pathway, including AKT1/2/3 (Sec473) and its downstream kinases p70S6K (Thr398), mTOR (Sec2448), was down-regulated in SKOV3-sh1 cells relative to control cells. These three kinases, members of the PI3K/Akt family, have been reported to be involved in the induction of EMT, which is a critical process in the pathogenesis of metastasis [17, 18]. Since recent studies indicate the role of SNCG on the PI3K/Akt signaling pathway in cancers [19, 20], we chose to further focus on the effect of SNCG on PI3K/Akt signaling pathway in HGSOC. To verify SNCG overexpression promoted EOC progression through regulating the PI3K/Akt pathway, we examined the phosphorylated proteins using western blot analysis. Our results showed that the expression levels of p-Akt, p-p70S6K, and p-mTOR were significantly decreased after knockdown of SNCG in SKOV3 and HO-8910 PM cells, and markedly increased after exogenous overexpression of SNCG in OVCAR3 cells (Fig. 4c). Furthermore, we observed that the IGF-1 (a PI3K agonist) could restore the expressions of p-Akt, p-p70S6K, and p-mTOR downregulated by knockdown of SNCG (Fig. 4d). In contrast, the high levels of these phosphorylated proteins could be decreased by LY294002 (a PI3K inhibitor) in SNCG ectopic-expressed cells (Fig. 4e). Moreover, the colony formation assay and the wound healing assay confirmed that the SNCG regulates cell proliferative and invasive abilities via the PI3K/Akt pathway in vitro (Fig. 4f). Overall, the PI3K/Akt signaling pathway activation is involved in SNCG-mediated promotion of HGSOC proliferation and metastasis.