Apelin Promotes Prostate Cancer Metastasis by Downregulating TIMP2 via Increases in miR-106a-5p Expression

Prostate cancer commonly affects the urinary tract of men and metastatic prostate cancer has a very low survival rate. Apelin belongs to the family of adipokines and is associated with cancer development and metastasis. However, the effects of apelin in prostate cancer metastasis is undetermined. Analysis of the database revealed a positive correlation between apelin level with the progression and metastasis of prostate cancer patients. Apelin treatment facilitates cell migration and invasion through inhibiting tissue inhibitor of metalloproteinase 2 (TIMP2) expression. The increasing miR-106a-5p synthesis via c-Src/PI3K/Akt signaling pathway is controlled in apelin-regulated TIMP2 production and cell motility. Importantly, apelin blockade inhibits prostate cancer metastasis in the orthotopic mouse model. Thus, apelin is a promising therapeutic target for curing metastatic prostate cancer.


Introduction
Prostate cancer commonly affects the urinary tract of men, whose susceptibility for this cancer increases with age [1]. Around 77% of newly diagnosed prostate cancer cases present with localized disease; 11% and 5% of cases present with disease that has metastasized to the regional lymph nodes and distant organs, respectively [2]. Five-year survival is around 30% in patients with metastatic prostate cancer, whereas almost 100% of patients with local or regional disease remain alive at 5 years [3]. A clear understanding of the mechanism of malignant progression and metastasis in prostate cancer would enable the development of early prevention and intervention strategies.
Tumor metastasis involves various processes, such as reorganizing and degrading the extracellular matrix (ECM) barrier to allow cancer cell growth, invasion and migration. Matrix metalloproteases (MMPs) are zinc-dependent proteases [4,5] that are key mediators

Study Datasets
Levels of apelin, TIMP2 and miRNA in healthy and tumor tissue samples collected from The Cancer Genome Atlas (TCGA) were analyzed by the Gene Expression Profiling Interactive Analysis (GEPIA), and the Oncomine and UALCAN websites [34].

Transwell Assay
Cell migratory and invasive activity was evaluated through Transwell inserts in 24-well dishes (Costar, New York, NY, USA) mentioned in our previous research [35]. Migratory and invasive cells were imaged under ×200 magnification using an Eclipse Ti2 microscope (Nikon, Tokyo, Japan).
Luciferase activity was assayed using the method described in our previous publications [36]. Briefly, after 24 h transfection, the lysis buffer (Promega, Madison, WI, USA) was added, and 100 µL was added to each well. Cell lysate were collected and an equal volume of luciferase substrate was added into cell lysates, relative luciferase activity was determined by Dual-Luciferase ® Reporter Assay System (Promega, Madison, WI, USA).

Metastatic Prostate Cancer Model
All procedures in the animal studies were approved by the Institutional Animal Care and Use Committee, and performed according to the Guidelines of Animal Experimentation issued by China Medical University. Six-week old male nude mice, bought from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan), were injected using a 22-gauge needle in the anterior prostate with 5 × 10 5 PC3 or PC3/sh-APLN cells suspended in 50 µL Matrigel. Tumor development and distal metastasis was monitored using the IVIS Spectrum scanner (Xenogen, Tucson, AZ, USA). Mice were humanely sacrificed after 7 weeks and distant organs (liver and lungs, as well as leg bone), were harvested. The percentages of lung metastases (No. of positive signal lungs/No. of total lungs) and other metastases were conducted using the IVIS Imaging System. Hematoxylin and eosin (H&E) staining was used to investigate distal metastasis in bone, liver and lung specimens.

Immunohistochemistry (IHC)
Human and mouse tissues were stained with anti-apelin or TIMP2 antibody and the procedure as according our previous works [37,38]. The sum of the intensity and percentage scores were analyzed [34].

Statistics
All values are given as the mean ± standard deviation (S.D.). The Student's t-test assessed the differences between the groups. A p value of <0.05 was considered to be statistically significant.

Apelin Is Highly Expressed in Patients with Progressing Prostate Cancer or Metastatic Disease
Apelin promotes cancer progression and metastasis [22,23]. Our analysis of apelin mRNA expressions in samples from the TCGA database revealed higher apelin expression in prostate cancer samples than in normal healthy tissue ( Figure 1A) and apelin levels were positively correlated with T and N tumor classification, as well as Gleason grade in the prostate cancer samples ( Figure 1B). Consistently, analyses of the Oncomine database and GEPIA tool, as well as IHC data from the tissue array, confirmed significantly upregulated apelin expression in prostate cancer samples ( Figure 1C-E) and higher levels of apelin were negatively associated with survival ( Figure 1F). Interestingly, apelin expression was higher  Figure 1G). Our results indicate that high levels of apelin are positively related with increasing tumor stage, poor survival and metastasis in prostate cancer. sion in prostate cancer samples than in normal healthy tissue ( Figure 1A) and apelin levels were positively correlated with T and N tumor classification, as well as Gleason grade in the prostate cancer samples ( Figure 1B). Consistently, analyses of the Oncomine database and GEPIA tool, as well as IHC data from the tissue array, confirmed significantly upregulated apelin expression in prostate cancer samples ( Figure 1C-E) and higher levels of apelin were negatively associated with survival ( Figure 1F). Interestingly, apelin expression was higher in metastatic prostate cancer (androgen receptor [AR] positive) compared to nonmetastatic disease ( Figure 1G). Our results indicate that high levels of apelin are positively related with increasing tumor stage, poor survival and metastasis in prostate cancer. (G) Metastatic prostate cancer exhibited higher levels of apelin expression compared to primary prostate cancer (exposed by AR amplification). Scale bar for all images is 100 μm. NS, no significant difference. (G) Metastatic prostate cancer exhibited higher levels of apelin expression compared to primary prostate cancer (exposed by AR amplification). Scale bar for all images is 100 µm. NS, no significant difference.

Apelin Facilitates Prostate Cancer Cell Motility by Reducing TIMP2 Expression
To examine the effects of apelin in prostate cancer cell motility, the PC3 and DU145 cell lines were stimulated with apelin. The results showed that apelin concentration dependently enhances cell migration and invasion (Figure 2A,B) but did not influence viability of PC3 and DU145 cells ( Figure S1). A previous report has indicated higher migratory ability of PC3 cells compared to DU145 and LNCaP cells [39]. Our data indicated higher levels of apelin expression in PC3 cells than in DU145 and LNCaP cells ( Figure 2C), implying that apelin is associated with migratory capacity in prostate cancer lines. Overexpression in LNCaP cells (lowest expressed apelin) or knockdown of apelin in PC3 cells (highest expressed apelin) promoted and reduced prostate cancer cell motility, respectively ( Figure 2D-I), suggesting that apelin facilitates prostate cancer migration and invasion.
higher levels of apelin expression in PC3 cells than in DU145 and LNCaP cells ( Figure 2C), implying that apelin is associated with migratory capacity in prostate cancer lines. Overexpression in LNCaP cells (lowest expressed apelin) or knockdown of apelin in PC3 cells (highest expressed apelin) promoted and reduced prostate cancer cell motility, respectively ( Figure 2D-I), suggesting that apelin facilitates prostate cancer migration and invasion. TIMPs inhibit MMPs synthesis and suppress tumor metastasis in different cancer types [40]. Stimulation of PC3 cells with apelin significantly inhibited TIMP2 synthesis by a greater extent than TIMP1 and TIMP3 synthesis ( Figure 3A). Apelin also concentrationdependently decreased mRNA and protein synthesis of TIMP2 ( Figure 3B,C). Furthermore, knockdown apelin increases the TIMP2 expression ( Figure S2). Overexpression of TIMP2 antagonized apelin-induced promotion of cell motility ( Figure 3D-F). The analysis of TCGA and GEPIA records as well as IHC data from the tissue array also revealed lower levels of TIMP2 in prostate cancer tissue than in normal healthy specimens ( Figure 3G-I). Thus, apelin facilitates prostate cancer disease by inhibiting TIMP2 expression. a greater extent than TIMP1 and TIMP3 synthesis ( Figure 3A). Apelin also concentrationdependently decreased mRNA and protein synthesis of TIMP2 ( Figure 3B,C). Furthermore, knockdown apelin increases the TIMP2 expression ( Figure S2). Overexpression of TIMP2 antagonized apelin-induced promotion of cell motility ( Figure 3D-F). The analysis of TCGA and GEPIA records as well as IHC data from the tissue array also revealed lower levels of TIMP2 in prostate cancer tissue than in normal healthy specimens ( Figure 3G-I). Thus, apelin facilitates prostate cancer disease by inhibiting TIMP2 expression.

Apelin Inhibits TIMP2 Expression and Subsequently increases Prostate Cancer Cell Motility by Promoting miR-106a-5p Synthesis via the c-Src/PI3K/Akt Signaling
c-Src is involved in several key signaling pathways and plays an important role in tumorigenesis and metastasis [42]. Treatment of prostate cancer cells with the c-Src  Figure 5G). The c-Src inhibitor and siRNA both antagonized apelin-induced increases in miR-106a-5p synthesis ( Figure 5H,I). c-Src activation of PI3K/Akt pathway is crucial for cancer metastasis [43]. Treating prostate cancer cells with PI3K and Akt inhibitors or transfecting them with PI3K and Akt siRNAs reversed apelin-mediated effects upon TIMP2 synthesis, cell motility and miR-106a-5p synthesis ( Figure 6A-H). Apelin also promoted the phosphorylation of PI3K and Akt, while the c-Src and PI3K inhibitors diminished apelin-induced promotion of PI3K and Akt phosphorylation ( Figure 6I-K). Knockdown apelin decreases the c-Src, PI3K and Akt phosphorylation ( Figure S2). Thus, apelin induces TIMP2-dependent prostate cancer motility by increasing miR-106a-5p expression via the c-Src/PI3K/Akt pathway.

Apelin Blockade Inhibits Prostate Cancer Metastasis in the Orthotopic Model
Next, we addressed whether apelin blockade helps to prevent prostate cancer metastasis in tumor xenograft mouse models. First, we implanted PC-3 cells stably expressing

Apelin Blockade Inhibits Prostate Cancer Metastasis in the Orthotopic Model
Next, we addressed whether apelin blockade helps to prevent prostate cancer metastasis in tumor xenograft mouse models. First, we implanted PC-3 cells stably expressing pLenti CMV V5-Luc into nude mouse anterior prostates, then sacrificed the mice 7 weeks later ( Figure 7A). Apelin blockade significantly suppressed tumor growth, according to IVIS imaging and manual tumor weight measurements ( Figure 7A-C). Importantly, apelin blockade also prevented distant metastases in the lung, liver and bone ( Figure 7D,E). IHC staining demonstrated that apelin blockade significantly suppressed levels of apelin expression and increased levels of TIMP2 ( Figure 7E). These results support the targeting of apelin for reducing the development of metastatic prostate cancer. Figure 8 represents the overall study results. pLenti CMV V5-Luc into nude mouse anterior prostates, then sacrificed the mice 7 weeks later ( Figure 7A). Apelin blockade significantly suppressed tumor growth, according to IVIS imaging and manual tumor weight measurements ( Figure 7A-C). Importantly, apelin blockade also prevented distant metastases in the lung, liver and bone ( Figure 7D,E). IHC staining demonstrated that apelin blockade significantly suppressed levels of apelin expression and increased levels of TIMP2 ( Figure 7E). These results support the targeting of apelin for reducing the development of metastatic prostate cancer. Figure 8 represents the overall study results.

Discussion
It is well established that advanced prostate cancer is related with aggressive growth and a high metastatic likelihood [44]. The dysfunction of apelin, a cytokine secreted from adipose tissue, plays a role in tumor metastasis by enhancing cell proliferation, migration and cell survival [45]. This study reports high levels of apelin expression in human metastatic prostate cancer samples. Upregulation of apelin expression predicts a poor prognosis with lower survival in patients with prostate cancer. Apelin facilitates TIMP2-dependent migration and invasion of prostate cancer cells. Elevating miR-106a-5p expression via the c-Src/PI3K/Akt signaling cascades was mediated in apelin-induced promotion of prostate cancer motility. Importantly, apelin blockade inhibited prostate cancer metastasis in the orthotopic mouse model. The evidence indicates that apelin is a promising therapeutic object in metastatic prostate cancer.
Secretion of androgen by testicular Leydig cells plays a pivotal role in prostate epithelial cell growth and differentiation [46], as well as prostate cancer development and metastasis [47]. The requirement for androgen in prostate cancer progression led to the facilitation of androgen deprivation therapy [48]. In this study, apelin overexpression clearly enhanced the motility of androgen-dependent prostate cancer cells (the LNCaP cell line). Exogenous apelin significantly enhanced the migration and invasion of two androgen-independent cell lines (PC3 and DU145), while shRNA knockdown of apelin autocrine inhibited c-Src/PI3K/Akt signaling pathway ( Figure S2) and motility of PC3 and

Discussion
It is well established that advanced prostate cancer is related with aggressive growth and a high metastatic likelihood [44]. The dysfunction of apelin, a cytokine secreted from adipose tissue, plays a role in tumor metastasis by enhancing cell proliferation, migration and cell survival [45]. This study reports high levels of apelin expression in human metastatic prostate cancer samples. Upregulation of apelin expression predicts a poor prognosis with lower survival in patients with prostate cancer. Apelin facilitates TIMP2-dependent migration and invasion of prostate cancer cells. Elevating miR-106a-5p expression via the c-Src/PI3K/Akt signaling cascades was mediated in apelin-induced promotion of prostate cancer motility. Importantly, apelin blockade inhibited prostate cancer metastasis in the orthotopic mouse model. The evidence indicates that apelin is a promising therapeutic object in metastatic prostate cancer.
Secretion of androgen by testicular Leydig cells plays a pivotal role in prostate epithelial cell growth and differentiation [46], as well as prostate cancer development and metastasis [47]. The requirement for androgen in prostate cancer progression led to the facilitation of androgen deprivation therapy [48]. In this study, apelin overexpression clearly enhanced the motility of androgen-dependent prostate cancer cells (the LNCaP cell line). Exogenous apelin significantly enhanced the migration and invasion of two androgen-independent cell lines (PC3 and DU145), while shRNA knockdown of apelin autocrine inhibited c-Src/PI3K/Akt signaling pathway ( Figure S2) and motility of PC3 and DU145 cells. Epithelial-mesenchymal transition (EMT) involves changes in epithelial cells to mesenchymal cells [49] and associated with tumor cell invasion and cancer metastasis [50,51]. However, our results ( Figure S3) show that apelin did not significantly change the EMT markers expression. These results indicate that apelin facilitates the migration and invasion but not EMT function in both androgen-dependent and -independent prostate cancer cells.
TIMPs are well recognized for their inhibition of MMPs, which promote tumor metastasis [16]. In this study, stimulation of prostate cancer cells with apelin abolished TIMP2 expression by a greater extent than TIMP1 or TIMP3, suggesting that TIMP2 is more important than other TIMPs in apelin-mediated motility of prostate cancer cells. TIMP2 regulates metastasis in several cancers, including cervical [52], lung [53] and ovarian cancer [54]. Our study analyses indicate that compared with normal controls, prostate cancer patients have lower levels of TIMP2 expression. We observed that apelin promoted prostate cancer cell migration and invasion that was antagonized when the cells were transfected with TIMP2 overexpression. These results suggest that apelin facilitates TIMP2-dependent prostate cancer cell motility.
The APJ receptor is a major receptor of apelin. The apelin/APJ system is a critical regulator of various physiological functions, such as glycometabolism, liver disease and macrophage activation [55][56][57]. Here we also found that the APJ expression is consistence with aplein levels, which is highest expressed in PC3 cells and lowest expressed in LNCaP cells ( Figure S4), associating with apelin facilitates the migration and invasion ability. c-Src pathway is critical for controlling different cellular functions [58], such as the facilitation of cell invasion and metastasis [59,60]. In current study found that apelin increases c-Src phosphorylation, while the c-Src inhibitor or siRNA reversed apelin-regulated TIMP2 synthesis and the motility of prostate cancer cells. PI3K/Akt signaling is crucial downstream of c-Src for regulating the migratory ability of human chondrosarcoma cells during metastasis [61]. Our data showed that the PI3K and Akt inhibitor or siRNA antagonized apelin-mediated TIMP2-dependent prostate cancer motility. Our results also reveal that apelin enhances PI3K and Akt phosphorylation, which was antagonized by the c-Src inhibitor, suggesting that c-Src-dependent PI3K/Akt activation mediates apelin-controlled TIMP2 synthesis and the metastasis of human prostate cancer cells.
The distinguishing feature of miRNAs is their ability to target several genes and affect multiple biological functions, including cancer [62,63]. Numerous reports indicated that pharmacotherapy capable of modulating miRNA expression would antagonize cancer cell migration and thus be an novel therapeutic approach for tumor metastasis [64,65]. In current study, our investigation of miRNA software databases predicted that miR-106a-5p interrupts with TIMP2 transcription. This was confirmed by the results revealing higher miR-106a-5p level in prostate cancer tissue than in normal control tissue. In addition, apelin stimulation increases miR-106a-5p expression, while treatment of prostate cancer cells with the miR-106a-5p inhibitor reversed apelin-induced inhibition of TIMP2 synthesis and promotion of cell migration and invasion. c-Src, PI3K and Akt inhibitors, as well as their respective siRNAs, inhibited apelin-facilitated promotion of miR-106a-5p synthesis, suggesting that apelin inhibits TIMP2 synthesis and facilitates prostate cancer migration by increasing miR-106a-5p production through the c-Src/PI3K/Akt pathway. Our in vivo studies also showed that knockdown apelin inhibited miR-106a-5p expression ( Figure S5) and the nodular metastasis in the lives and bones of nude mice versus negative control groups, which is consistence with our in vitro experiments ( Figure S2B) and previous report [66].

Conclusions
Apelin suppresses TIMP2 production and subsequently facilitates the metastatic potential of human prostate cancer cells by increasing miR-106a-5p synthesis via the c-Src, PI3K and Akt signaling cascades. We now have a better understanding about how apelin-mediated miRNA and TIMP2 synthesis contributes to prostate cancer cell motility, which may help investigators design more effective treatment for metastatic disease.