SPAG9 promotes prostate cancer growth and metastasis

: Sperm-associated antigen 9 (SPAG9) expression is increased in prostate tissues of prostate cancer patients. This experimental study aimed to investigate the role of SPAG9 in bone metastasis of prostate cancer. Immunohistochemical analysis showed that SPAG9 staining was positive in 81.67% of 240 cases of prostatic carcinoma but only in 6.67% of 120 cases of benign prostate hyperplasia. Strong PAG9 staining was positively correlated with Gleason score and bone metastasis in 240 prostate cancer patients ( p < 0.05), but not with the age or serum prostate-specific antigen level ( p > 0.05). PC-3 cells were transfected with shRNA against SPAG9, and CCK-8 assay in triplicate showed that PC-3 cell viability was inhibited by SPAG9 knockdown. In addition, transwell assay in triplicate showed that PC-3 cell invasion was inhibited by SPAG9 knockdown. Furthermore, total 2 × 106 PC-3 cells were injected subcutaneously into the right flank of nude mice which were randomly divided into three groups (N = 8) and treated by intratumoral injection of SPAG9 shRNA, control shRNA or PBS, respectively. SPAG9 shRNA inhibited the growth, invasion and angiogenesis while promoted apoptosis of xenografted PC-3 cells. SPAG9 knockdown led to the upregulation of E-cadherin and the downregulation of MMP2 and vimentin in xenografted tumors. In conclusion, this is the first study to provide evidence that SPAG9 promotes bone metastasis of prostate cancer, and SPAG9 is a promising target to prevent or treat bone metastasis of prostate cancer.


Introduction
Despite numerous diagnostic and therapeutic advances in recent years, prostate adenocarcinoma still has a high incidence in the Western population (Siegel et al., 2017). Prostate cancer is prone to bone metastasis (Battafarano et al., 2018). The incidence of bone metastasis is high in prostate cancer patients and their survival is only 12-53 months (Macedo et al., 2017;Liu et al., 2017).
Androgen deprivation therapy (ADT) is often used to decrease circulating testosterone levels by surgical or chemical castration (Dai et al., 2017;Yap et al., 2016). However, the response is transient and most patients will develop resistance to ADT and ultimately progress to castration-resistant prostate cancer (CRPC) (Wadosky et al., 2016). For CRPC with bone metastasis, additional therapies have been approved recently, including targeted alpha therapy with radium-223 dichloride (Sumanasuriya et al., 2017). However, the disease will ultimately relapse, indicating that *Address Correspondence to: Ye Tian, yangchunhua820203@126.com effective therapy is needed to further improve the outcome.
Sperm-associated antigen 9 (SPAG9) emerges as a novel regulator of mitogen-activated protein kinases (MAPKs) pathway to modulate cancer growth and metastasis (Pan et al., 2018). SPAG9 expression was increased in prostate cancer tissues (Chen et al., 2014). However, the role of SPAG9 in bone metastasis of prostate cancer is unknown.
In this study, we hypothesized that SPAG9 promotes bone metastasis of prostate cancer and aimed to analyze the correlation of SPAG9 expression to bone metastasis and clinical pathological features of prostate cancer. Furthermore, we used highly metastatic PC-3 prostate cancer cell line to understand the mechanism by which SPAG9 promotes prostate cancer growth, angiogenesis, and invasion.

Immunohistochemistry
The protocols were approved by Institute Ethics Committee and all subjects provided informed consent. A total of 240 patients with prostate cancer (120 cases with bone metastasis and 120 cases without bone metastasis) were enrolled who underwent surgical resection at Second Affiliated Hospital of Soochow University between 2010 and 2017. 120 patients with benign prostate hyperplasia were enrolled as control. Clinical data were collected from their medical records. Only patients with confirmed diagnosis of prostate cancer or benign prostate hyperplasia by experienced urologists met the eligibility criteria and were included in the study groups, respectively. Immunohistochemical analysis of SPAG9 expression in clinical samples was performed as described previously (Chen et al., 2014). Briefly, tissue samples were fixed in paraformaldehyde, embedded and cut into 4 μm sections. next, endogenous peroxidases were quenched and the sections were washed with 0.01 M phosphate buffered saline (PBS). The sections were blocked with 2% goat serum at room temperature for 1 h, then incubated with rabbit polyclonal anti-SPAG9 antibody (1:500 dilution, nB110-17780, novus Biologicals, Littleton, CO, USA) at 4°C overnight, washed and then incubated with diaminobenzidine (Zhongshan Biotech, Beijing, China). For negative control, the sections were incubated with rabbit IgG serum. The stained sections were observed under microscope, and SPAG9 staining was scored by three pathologists independently in a blind manner based on the intensity and percentage of cells as follows: −, negative staining of almost all cells; +, weak staining of almost all cells; ++, moderate staining of more than 50% cells or strong staining of more than 5% cells; +++ strong staining of more than 50% cells. The correlation of SPAG9 staining with histopathological factors was analyzed by Pearson's test.
Cell culture PC-3 prostate cancer cells were obtained from Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China), and cultured in RPMI1640 medium (Gibco, USA) supplemented with 10% fetal calf serum (Gibco, USA), penicillin and streptomycin at 37°C in a humid incubator with 5% CO 2 . Cells were passaged to maintain exponential growth, and only cells at exponential growth phase were used for further experiments. SPAG9 specific shRnA was constructed by GenePharma (Shanghai, China) and transfected into PC-3 cells using lipofectamin 2000 (Invitrogen, USA) according to the manufacturer's instructions. For negative control, cells were transfected with control shRnA.

Reverse transcription polymerase chain reaction (RT-PCR)
Total RnA was extracted from the cells using TRIzol reagent (Invitrogen, USA) following the manufacturer's protocols. cDnA was synthesized by reverse transcription using Access RT-PCR System (Promega, Madison, WI, USA). Realtime PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) on ABI Prism 7700 Sequence Detection System (Applied Biosystems) with the following primers: SPAG9 5'-AGAGGAAGGCTTGGGAGTA-3' and 5'-GGGCAGCAGAGCTATGTG-3' . The reaction conditions were 40 cycles at 94°C for 1 min, 50°C for 1 min and 68°C for 1 min. PCR was performed three times in triplicate, and relative mRnA levels of SPAG9 were calculated by 2 -ΔΔCt method.

Western blot analysis
Cells were collected by centrifugation and lysed in RIPA lysis buffer (Santa Cruz Biotechnology, USA). Equal amounts of proteins in cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with SPAG9 or β-actin antibody (Santa Cruz Biotechnology, USA) at 4°C overnight, and then with secondary antibodies (Santa Cruz Biotechnology, USA) at room temperature for 1 h. The membranes were developed by using ECL kit (Pierce, USA) and the bands were quantitated by an image analyzer (LabWorks, Upland, CA, USA) with β-actin as loading control.

Cell proliferation assay
The proliferation of PC-3 cells was evaluated by CCK-8 method as described previously (Zhong et al., 2018). Briefly, the cells were seeded into 96-well plates. After culture at 37°C in a humid incubator with 5% CO 2 for 48 h, 10 µL CCK-8 solution (Sigma, USA) was added into each well and after incubation for 1 h, the OD at 450 nm was measured by a microplate reader.

In vitro invasion assay
The invasion of PC-3 cells was evaluated by transwell chamber as described previously (Tian et al., 2017). Briefly, 10 5 cells in serum-free medium were loaded into the upper chamber while 10% FBS were loaded into the lower chamber. After incubation at 37°C in a humid incubator with 5% CO 2 for 20 h, the cells on the top of the filters were removed by cotton swab. The remaining cells were fixed in 4% paraformaldehyde, stained with Crystal Violet and stained cells were counted in five randomly selected fields. At least three chambers from three different experiments were analyzed.

Xenograft tumor model in nude mice
Animal study protocols were approved by Institutional Animal Care and Use Committee. BALB/c nude mice (4-5 weeks old) were obtained from Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). Total 2 × 10 6 PC-3 cells were injected subcutaneously into the right flank of mice. When tumors grew to about 100 mm 3 , the mice were randomly divided into three groups (n = 8) and were treated by intratumoral injection of SPAG9 shRnA, control shRnA vectors (all constructed by GenePharma, Shanghai, China) or PBS, respectively. Tumor volume was calculated as: V (mm 3 ) = length of tumor x width of tumor 2 /2. At the end, tumor tissues were dissected and apoptotic cells were detected by TUnEL assay using in situ apoptosis detection kit (Roche, Indianapolis, In, USA) following the manufacturer's protocols.

Statistical analysis
Statistical analysis was performed using Student's t test for the comparison between two groups or analysis of variance (AnOVA) test followed by Tukey's test for the comparison among multiple groups. p < 0.05 was considered to be significant. was only 6.67% in 120 cases of benign prostate hyperplasia. SPAG9 staining was dominant in the nucleus (Fig. 1). Strong PAG9 staining was positively correlated with Gleason score and bone metastasis (P < 0.05), but not with the age or serum prostate-specific antigen level (P > 0.05) (Tab. 1).

Discussion
To our knowledge, this is the first study to report that SPAG9 expression in prostate cancer showed a positive correlation with Glean score and bone metastasis, suggesting that SPAG9 may play a role in bone metastasis of prostate cancer and could be a therapy target for prostate cancer. However, due to limited data, potential bias of correlation analysis may be overlooked and need to be further addressed in future clinical studies.
In present study, we silenced SPAG9 expression with shRnA and found that SPAG9 knockdown inhibited the viability and invasion of PC-3 cells. Our results are in agreement with previous findings that SPAG9 knockdown and SPAG9 overexpression decreased and increased cancer cell proliferation, respectively (Li et al., 2014).
To further elucidate the mechanism by which SPAG9 promotes prostatic carcinoma progression and metastasis in vivo, we focused on EMT markers such as E-cadherin and vimentin (Tanaka et al., 2013). We found that SPAG9 knockdown led to decreased expression of vimentin and MMP2 and increased expression of E-cadherin. These data indicate that SPAG9 enhances EMT to promote prostate cancer metastasis.
Angiogenesis is essential to tumor metastasis (Wu et al., 2018). SPAG9 could regulate JnK pathway and JnK induces the activation of VEGF (Garg et al., 2008;Guma et al., 2009). Thus, SPAG9 may promote cancer angiogenesis via JnK signaling. In our study, we found that the well-known marker of angiogenesis CD31 was downregulated in pGPU6/GFP/ neo-SPAG9 group. Thus, SPAG9 knockdown inhibited the formation of blood vessels. Moreover, SPAG9 knockdown induced apoptosis in prostate cancer cells xenografted into nude mice. Taken together, SPAG9 expression is high in prostate cancer tissues and is positively correlated with Glean score and bone metastasis. Silencing SPAG9 could inhibit the growth, invasion and angiogenesis while promote apoptosis of xenografted PC-3 cells. These findings suggest that SPAG9 promotes the occurrence and bone metastasis of prostate cancer, and further studies are needed to confirm that SPAG9 is a promising target to prevent or treat bone metastasis of prostate cancer.