SPAG5 interacts with CEP55 and exerts oncogenic activities via PI3K/AKT pathway in hepatocellular carcinoma

Twenty-seven fresh HCC specimens were collected for determination of mRNA and protein levels of SPAG5 from Sun Yat-sen University Cancer Center (SYSUCC). A cohort of 298 paraffin-embedded HCC cases diagnosed between Jan 2012 to Dec 2013 at Dongguan Third People’s Hospital and SYSUCC was recruited. Another 93 HCC samples with venous metastases were obtained from SYSUCC. None of the patients had received radiotherapy or chemotherapy before surgery. All samples were anonymous. This project was approved by Institute Research Ethics Committee of the above two hospitals. The clinical implication of SPAG5 was further determined in The Cancer Genome Atlas (TCGA) dataset (http://​www.​cbioportal.​org) and the Oncomine dataset (https://​www.​oncomine.​org).

HCC cell lines PLC8024, Huh7 and QGY-7703 were purchased from the Cell Resource Center, Chinese Academy of Science Committee (Shanghai, China). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) in a humidified incubator at 37 °C and 5% CO₂. The cells were transfected with SPAG5 overexpression vector or shRNAs by Lipofectamine 2000, according to the instruction, and then selected by G418 for 4 weeks to establish stable cells.

qRT-PCR was performed according to our previous study. The sequences of the PCR primers are as followings: SPAG5, forward: 5′- CATCTCACAGTGGGATAACTAATAAAC-3′ and reverse: 5′- CAGGGATAGGTGAAGCAAGGATA-3′; CEP55, forward: 5’-TGAAGAGAAAGACGTATTGAAACAA-3′ and reverse: 5′- ACTGTGGCTCCAAACTGCTT-3′; β-actin, forward: 5’-TGGCACCCAGCACAATGAA-3′ and reverse: 5’-CTAAGTCATAGTCCGCCTAGAAGCA-3′. The relative expression of SPAG5 and CEP55 was presented as –ΔCT value.

Equal amounts of protein (30 μg) were resolved by SDS-PAGE and then electrophoretically transferred onto PVDF membranes (Millipore, Bedford, MA). After blocked in 5% non-fat milk 1 h at room temperature, the membranes were incubated with appropriately diluted primary antibodies overnight at 4 °C. After washed twice with TBST, the blotted membranes were incubated with HRP-conjugated secondary antibody at 1:20000 dilutions for 1 h at room temperature. The membranes were visualized by the enhanced Phototope TM-HRP Detection Kit and exposed to Kodak medical X-ray processor (Carestream Health, USA). The primary antibodies are as followings: SPAG5, 1:500 (Sigma-Aldrich), CEP55 (1:1000, #81693, Cell Signaling Technology), AKT (1:1000, #2920, Cell Signaling Technology), p-AKT at Ser473 (1:1000, #4046, Cell Signaling Technology), ERK1/2 (1:1000, #4695, Cell Signaling Technology), p-ERK1/2 at Thr202/Tyr204 (1:1000, #4370, Cell Signaling Technology), E-Cadherin, β-Catenin, N-Cadherin, Vimentin, Fibronectin and MMP-2 (Epitomics, Burlingame, CA) and β-actin (1:1000, #3700, Cell Signaling Technology).

IHC staining was performed on a HCC tissue microarray (TMA). The expression levels were scored as proportion of immunopositive staining area (0%, 0; 1–25%, 1; 26–50%, 2; 51–75%, 3; 76–100%, 4) multiplied by intensity of staining (0, negative; 1, weak; 2, moderate; 3, intense). The scores were independently rendered by two pathologists (Dr. Yang YF and Dr. Zhang MF). The median IHC score (4.0) was chosen as the cut-off value to define high and low expression.

Stable cells were constructed. Cells were collected and seeded in 6-well plates at a density of 1.0 × 10³ per well, and then incubated for 14 days. Colonies were fixed with methanol, stained with 0.1% crystal violet and counted.

A total of 3.0 × 10⁴ cells were re-suspended in 200 μl of serum-free medium and placed in the upper compartment of a Transwell chamber (Corning; 24-well insert, pore size: 8 mm). The lower chamber was filled with 15% fetal bovine serum as a chemo attractant and incubated for 48 h for the migration assay. After 48 h, the cells on the upper surface of the membrane were removed, and the cells on the lower surface were fixed and stained with 0.1% crystal violet. Five visual fields of each insert were randomly chosen and counted under a light microscope.

PLC8024 cells were co-transfected with miR-363-3p mimics or the negative control and psiCHECK-2-SPAG5–3’-UTR reporter. Cells were collected 36 h after transfection and analyzed with the Dual-Luciferase Reporter Assay System (Promega, CA, USA).

Male BALB/c nude mice aged 3–4 weeks were randomly divided into two groups. Four million cells were implanted subcutaneously under the right armpits into the flanks of the mice. Each group included 6 mice. Tumor size and body weight were measured once every 3 days. Four weeks later, the mice were sacrificed, and tumor weight and size were measured again. Volumes were calculated using the following formula: Volume (mm³) = [width² (mm²) × length (mm)]/2. For metastasis observations, five-week-old male nude BALB/c mice were injected with 5 × 10⁵ cells via the tail vein. Six weeks later, the mice were killed. The lungs of the mice were fixed and stained with hematoxylin and eosin. Lung metastasis was quantified by counting the number of tumor nodule in 10 randomly selected high-power fields. All animal studies were conducted with the approval of the Medical Experimental Animal Care Commission of SYSUCC.

The expression of SPAG5 was firstly examined in fresh HCC tissues, using qRT-PCR, western blot and IHC. Results showed that no gene amplification was found for SPAG5 in HCC (Additional file 1: Figure S1). The mRNA level of SPAG5 in 27 paired HCC tissues was frequently up-regulated, compared to the corresponding nontumorous tissues (Fig. 1a). This was validated by other liver studies in Oncomine dataset (Fig. 1b). Consistently, the protein expression of SPAG5 in fresh HCC specimens was elevated by 2.65 folds on average (Fig. 1c). The protein expression of SPAG5 in HCC cell lines was much higher than that in immortalized liver cell line (L-02) (Additional file 2: Figure S2). Results of TMA-based IHC in SYSUCC cohort containing 298 patients with HCC demonstrated that more SPAG5 was expressed in HCC tissues (Fig. 1d). The patients were divided into two groups according to the median IHC score (4.0): high SPAG5 and low SPAG5. High expression of SPAG5 was associated with larger tumor size, poor tumor differentiation, advanced TNM stage, more lymph node metastasis and tumor vascular invasion (Table 1). Furthermore, a cohort consisting of 93 patients with tumor embolus was recruited to determine the expression of SPAG5 in metastatic tumor tissues. Increased expression of SPAG5 was found in tumor metastasis, compared with the primary tumor (Fig. 1d).

The prognostic implication of SPAG5 in HCC was next explored. In SYSUCC cohort, patients with high SPAG5 expression were likely to survive shorter and experience tumor relapse in a shorter time, compared with those with low SPAG5 expression (Fig. 1e). The median overall survivals in high SPAG5 and low SPAG5 groups were 16.0 and 27.1 months, respectively. Multivariate analyses using Cox regression model revealed SPAG5 as an independent prognostic factor for overall survival in HCC (Table 2). The data from TCGA database confirmed the prognostic value of SPAG5 in HCC (Fig. 1f). The 5-year survival in high SPAG5 and low SPAG5 groups were 41% and 52% respectively. These data suggest that overexpression of SPAG5 serves as a promising factor for the prognosis of patients with HCC.

The biological function of SPAG5 in HCC was next investigated. PLC8024 and Huh7 cells were stably transfected with SPAG5 overexpression vector, and QGY-7703 cells were treated with SPAG5 shRNAs. The mRNA and protein expression of SPAG5 in stable cell lines were examined by qRT-PCR and western blot (Fig.2a and b). In HCC cells, overexpression of SPAG5 markedly enhanced the cell proliferation. Foci formation and soft agar assays demonstrated that SPAG5 significantly increased the frequency of colony formation on solid plates (Fig. 2c) and in soft agar (Fig. 2d). In contrast, the silence of SPAG5 resulted in reduced colonies in QGY-7703 cells. These findings indicated that SPAG5 promoted cell growth in both anchorage-dependent and -independent manners.

In vivo tumor formation assay was used to examine the effect of SPAG5 on tumor growth. Tumors were found in 6/6 and 4/6 of mice injected with SPAG5-transfected and empty vector-transfected PLC8024 cells, respectively. The related numbers in Huh7 cells were 6/6 and 5/6. Tumors formed by PLC8024 and Huh7 cells with SPAG5 overexpression grew much faster than those in control groups. However, the tumors derived from QGY-7703 cells transfected with SPAG5 shRNAs were much smaller and lighter than those in control group. Tumor formation was observed only in 3/6 of mice injected with SPAG5 shRNAs-transfected QGY-7703 cells, but in 5/6 of mice in control group (Fig. 2e). The cell proliferation in tumors was evaluated by Ki67 staining. Results showed that cell proliferation was enhanced by SPAG5 overexpression but attenuated by SPAG5 knockdown (Fig. 2f).

Since clinical data revealed that SPAG5 expression was correlated with metastatic features in HCC, the effect of SPAG5 on HCC cell migration was next determined. Transwell assays were performed to show that SPAG5 overexpression increased the migrated cells in PLC8024 and Huh7 cells, whereas SPAG5 depletion inhibited the cell movement in QGY-7703 cells (Fig. 3A). Invasion assays demonstrated that SPAG5 enhanced the ability of cell invasion in HCC cells (Fig. 3b). These data indicated SPAG5 might be involved in tumor metastasis. Western blot showed that the expression of Fibronectin, Vimentin, N-cadherin and MMP2 was induced by SPAG5, but reduced by SPAG5 knockdown in HCC cells. In contrast, the expression of E-cadherin and β-catenin was down-regulated by SPAG5 overexpression (Fig. 3c). In vivo tumor metastasis models demonstrated that more lung nodules were depicted in mice injected with PLC8024 and Huh7 cells with SPAG5 overexpression. shRNAs against SPAG5 significantly reduced the tumor metastasis in mice (Fig. 3d). These data suggested SPAG5 triggered the epithelial-mesenchymal transition (EMT) process in HCC cells to facilitate cell migration.

The mechanism responsible for SPAG5 upregulation in HCC was next explored. Seven microRNAs were predicted by two bioinformatic algorithms (Targetscan and miRanda) to be the potential upstream regulator of SPAG5, including two tumor suppressors (miR-539-5p and miR-363-3p) (Additional file 3: Figure S3A). Since miR-539-5p has been reported to modulate the expression of SPAG5 in HCC cells, miR-363-3p, which was down-regulated in HCC cells (Additional file 4: Figure S4), was chosen for further studies. A putative site for the binding of miR-363-3p and SPAG5 3’UTR was identified (Additional file 3: Figure S3B). Re-introduction of miR-363-3p into PLC8024 and QGY-7703 resulted in a significant decrease of SPAG5 mRNA (Fig. 4a), whereas suppression of miR-363-3p by its specific inhibitor led to the increase of SPAG5 mRNA (Fig. 4b). Consistently, the protein level of SPAG5 was downregulated by miR-363-3p mimics but upregulated by its inhibitor in both HCC cell lines (Fig. 4c). Dual luciferase reporter assays showed that overexpression of miR-363-3p markedly inhibited the activity of wild type SPAG5 3’UTR but not the mutant 3’UTR (Fig. 4d). In clinical samples, the expression of miR-363-3p was reversely associated with SPAG5 mRNA (Fig. 4e). These data suggest that miR-363-3p is capable of modulating the expression of SPAG5 in HCC cells.

Previous literatures reported that miR-363-3p functioned as a tumor suppressor in HCC, which was validated by our studies (Fig. 4f-h). The effect of SPAG5 on miR-363-3p-mediated phenotypes was determined. Overexpression of SPAG5 increased the colony formation and cell migration in HCC cells expressing miR-363-3p (Fig. 4F and G). Furthermore, the G1 phase arrest caused by miR-363-3p was relieved by the ectopic expression of SPAG5 (Fig. 4h). Collectively, these data suggest that SPAG5 contributes to the tumor suppressive activity of miR-363-3p.

The underlying mechanism via which SPAG5 exerted oncogenic functions towards HCC was investigated. According to the expression of SPAG5 mRNA in TCGA datasets, patients were divided into two groups. Genes co-expressing with SPAG5 was determined and shown by heatmap (Additional file 5: Figure S5A and Additional file 6: Table S1). Gene Set Enrichment Analysis (GSEA) indicated that pathway involved in akapcentrosome was activated in cases with high SPAG5 expression (Additional file 5: Figure S5B). SPAG5 expression was positively correlated with the expression of CEP55 that contributes to the centrosome modulation, in both TCGA and SYSUCC samples (Fig. 5a-b). HCC patients with high SPAG5 were frequently accompanied with high expression of CEP55 (Fig. 5c). In HCC cells, ectopic SPAG5 expression did not alter the expression of CEP55 at both mRNA and protein levels (Fig. 5d-e), which implies that SPAG5 can not modulate the expression of CEP55. Instead, CEP55 was detectable in the precipitant mediated by the specific antibody of SPAG5 in both PLC8024 and Huh7 cells (Fig. 5f). Confocal assays demonstrated the co-localization of SPAG5 and CEP55 in the cytoplasm of the two cell lines (Fig. 5g). Functionally, the knockdown of CEP55 significantly attenuated SPAG5-promoted cell proliferation and migration (Fig. 5h and i). These data indicate that SPAG5 exhibits pro-HCC activities through interacting with CEP55.

Current studies have shown that CEP55 promotes tumor progression via PI3K/AKT/mTOR pathway. The effect of SPAG5 on the activation of PI3K/AKT signaling was next determined. Western blot showed that the phosphorylation of AKT at Ser473, but not of ERK1/2 at Thr202/Tyr204, was increased in HCC cells with SPAG5 overexpression, and decreased in cells with SPAG5 silence (Fig. 6a). Upon the treatment of wortmannin (an inhibitor for PI3K/AKT pathway), the SPAG5-mediated cell growth and migration in HCC cells were dramatically suppressed (Fig. 6b and c). Furthermore, the activation of AKT by SPAG5 was partly blocked by the incubation of CEP55 siRNA (Fig. 6d). These data suggest that SPAG5 triggers the PI3K/AKT pathway in HCC via the interaction with CEP55.