Targeting the SPOCK1-snail/slug axis-mediated epithelial-to-mesenchymal transition by apigenin contributes to repression of prostate cancer metastasis
Prostate cancer (PCa) is considered one of the most prevalent malignancy globally, and metastasis is a major cause of death. Apigenin (API) is a dietary flavonoid which exerts an antimetastatic effect in various cancer types. Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1) is a crucial modulator of tumor growth and metastasis in cancers. However, the role and underlying regulatory mechanisms of SPOCK1 in the API-mediated antimetastatic effects of PCa remain unclear.
MTS, colony formation, wound-healing, and transwell assays were conducted to evaluate the effects of API on PCa cell proliferative, migratory, and invasive potentials. In vivo orthotopic bioluminescent xenograft model were employed to determine antitumor activity of API. PCa cells were transfected with either Snail-, Slug-, SPOCK1-overexpressing vector, or small hairpin (sh)SPOCK1 to determine the invasive abilities and expression levels of SPOCK1 and epithelial-to-mesenchymal transition (EMT) biomarkers in response to API treatment. Immunohistochemical (IHC) assays were carried out to evaluate the expression level of SPOCK1 in PCa xenografts and a PCa tissue array. Associations of SPOCK1 expression with clinicopathological features and prognoses of patients with PCa were analyzed by GEO or TCGA RNA-sequencing data.
API significantly suppressed in vitro PCa cell proliferation, migration, and invasion and inhibited in vivo PCa tumor growth and metastasis. Moreover, survival times of animals were also prolonged after API treatment. Mechanistic studies revealed that API treatment resulted in downregulation of SPOCK1, which was accompanied by reduced expressions of mesenchymal markers and subsequent attenuation of invasive abilities of PCa cells. Overexpression of SPOCK1 in PCa xenografts resulted in significant promotion of tumor progression and relieved the anticancer activities induced by API, whereas knockdown of SPOCK1 had opposite effects. In clinical, SPOCK1 levels were higher in tumor tissues compared to non-tumor tissues, which was also significantly correlated with shorter disease-free survival in PCa patients.
Levels of SPOCK1 increase with the progression of human PCa which suggests that SPOCK1 may act as a prognostic marker or therapeutic target for patients with PCa. Suppression of SPOCK1-mediated EMT signaling contributes to the antiproliferative and antimetastatic activities of API in vitro and in vivo.
KeywordsProstate cancer SPOCK1 Metastasis Snail Slug Epithelial-to-mesenchymal transition Apigenin
Androgen deprivation therapy
Castration resistant prostate cancer
Epithelial to mesenchymal transition
Gene Expression Omnibus
Glycogen synthase kinase 3β
Luteinizing hormone-releasing hormone
Phosphatase and tensin homolog
Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1
The Cancer Genome Atlas
Prostate cancer (PCa) is the most commonly diagnosed cancer in males and the second leading cause of cancer-associated deaths worldwide . Androgen deprivation therapy (ADT) remains the primary clinical treatment for patients in the early stage of PCa. However, despite androgen ablation, nearly all patients with advanced stages of PCa experience recurrence as the disease develops into castration-resistant PCa (CRPC), which is characterized by aggressive growth and distal organ metastasis, and is incurable . Therefore, early diagnosis and treatment before the tumor metastasizes are critical for improving survival of patients with PCa. Despite advances in detection and treatment strategies, there are currently no effective therapeutics to treat metastatic PCa. Hence, the means to prevent PCa progression and conduct necessary interventions before the cancer has spread to other organs remain major clinical challenges.
The epithelial-to-mesenchymal transition (EMT) is a biological process, and activation of the EMT program contributes to cell invasion and metastasis in multiple cancers . Clinical evidence suggests that aberrant activation of the EMT is correlated with therapeutic resistance and tumor aggressiveness and negatively impacts survival of patients with PCa . One oncogene, Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1), was observed to affect the EMT process by facilitating metastasis in various cancers [5, 6]. SPOCK1, also known as testican-1, is a proteoglycan that belongs to a Ca2+-binding proteoglycan family that was implicated in cell proliferation, DNA replication, apoptosis, and the migration and invasion of cancer cells . More importantly, clinicopathologic analyses revealed that SPOCK1 is frequently overexpressed in PCa tissues , and is involved in cancer recurrence, drug resistance, and metastasis [8, 9]. However, in light of our current knowledge, the function of SPOCK1 in PCa metastasis is not entirely understood, and even less is known about the underlying mechanism responsible for the SPOCK1-mediated EMT process in PCa development and progression.
Natural products and their derivatives have been further developed as anticancer agents and provide potential sources for novel drugs to treat cancer . Flavonoids are naturally occurring polyphenolic metabolites, which are present in a wide variety of edible plant sources, such as fruits, vegetables, grains, nuts, seeds, tea, and traditional medicinal herbs . Epidemiological studies and systematic analyses have shown an inverse association between the dietary intake of flavonoids and the risk of cancer . Apigenin (API), 4′,5,7-trihydroxyflavone, is one of the most common flavonoids and is found in significant quantities in a variety of vegetables and fruits. Accumulating evidence has revealed that the anticancer properties of API are due to its ability to cause cell cycle arrest, trigger apoptosis, induce autophagy, inhibit migration/invasion, attenuate drug resistance, and stimulate immune responses in various cancer types in vitro and in vivo [13, 14]. API was recently shown to effectively suppress migration and invasion through modulation of the EMT process in PCa . Recently, we also showed that API suppresses CD26/DPPIV expression, and the interplay between p-Akt and Snail/Slug contributed to inhibition of the EMT-mediated invasive ability and subsequently blocked tumor metastasis in a human A549 xenograft model .
Although API was shown to be a promising molecule for use in PCa prevention and therapy , the precise mechanism underlying its modulation of cell motility and the antimetastatic effects of API so far remain underexplored. In the present study, we found that SPOCK1, which is upregulated in PCa, is involved in the invasion and metastasis of cancer cells and was correlated with poor prognoses. More importantly, we demonstrate for the first time that API suppressed SPOCK1 expression leading to attenuation of PCa metastasis by targeting the Snail/Slug-mediated EMT process.
API (A3145) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS), antibiotics, molecular weight standards, trypsin-EDTA, and all medium additives were obtained from Life Technologies (Gaithersburg, MD). The CellTiter 96 AQueous One Solution Proliferation Assay System was purchased from Promega (Madison, WI). An enhanced chemiluminescence (ECL) kit was purchased from Amersham (Arlington Heights, IL). Antibodies specific for fibronectin (ab2413) and SPOCK1 (ab229935) were obtained from Abcam (Cambridge, MA). Antibodies specific for cleaved-PARP (#9541), phospho-Ak (ser473, #9271), Akt (#4691), Snail (#3895), Slug (#9585), Twist (#46702), and E-cadherin (#3195) were obtained from Cell Signaling Technology (Danvers, MA). Antibodies specific for vimentin (550513) and N-cadherin (610920) were purchased from BD Biosciences (San Jose, CA). Antibodies specific for cyclin D1 (sc-8396), cyclin E (sc-377,100), β-actin (sc-47,778), and goat anti-rabbit (sc-2004) and anti-mouse (sc-2005) immunoglobulin G (IgG) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyvinylidene fluoride (PVDF) membranes for Western blotting were purchased from Bio-Rad (Hercules, CA). Unless otherwise specified, other chemicals used in this study were purchased from Sigma Chemical (St. Louis, MO).
Cell lines and cell culture
The human transformed benign prostate cell line PNT2 and human PCa cell lines including metastatic hormone-sensitive cell line (LNCap) and metastatic CRPC cell lines (DU145, PC-3, and PC-3 M) were all purchased from American Type Culture Collection (ATCC, Manassas, VA). PNT2 cells were cultured in RPMI 1640 medium (Gibco BRL); PC-3 and PC-3 M cells were cultured in minimal essential medium (MEM; Gibco BRL, Grand Island, NY); DU145 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL); and LNCap cells were maintained in T-medium (Gibco BRL) supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin-glutamine (Invitrogen). Cells were regular passaged at 70%~ 80% confluence via trypsinization using 1 × trypsin and 0.05% EDTA followed by resuspension in complete medium. All cells were incubated at 37 °C in a 5% CO2 and 95% air atmosphere.
Cell viability assay
LNCap, DU145, PC-3, and PC-3 M human PCa cells (5 × 103) were seeded onto 96-well plates in 100 μL of culture medium. After 24 h of incubation to allow cells to adhere, cells were treated with various concentrations of API (0~80 μM) for 24, 48, and 72 h and then subjected to a cell-viability assay (MTS assay; Promega, Madison WI). The absorbance was measured at 490 nm using a microplate reader. Values are the mean ± standard deviation (SD) of triplicate wells and were normalized to that of the control group to determine the percent viability. Values of the half maximal inhibitory concentration (IC50) were determined through the dose-response curves by using GraphPad Prism 6.0 (GraphPad software, La Jolla, CA).
Colony formation assay
Human PCa cells were diluted and seeded at 1000 cells/well in 6-well culture plates. After 24 h, cells were treated with various concentrations of API (0~80 μM) or the vehicle for 48 h and then cultured under standard conditions for 10~14 days. The medium was replaced every 3 days. Finally, colonies were stained with 0.1% crystal violet, and colonies with more than 50 cells were counted.
Wound healing assays
PC-3 M and DU145 cells were grown to full confluence in 6-well plates and a small area was then disrupted by scratching the monolayer with a 200-μl plastic pipette tip. Cells were washed twice with phosphate-buffered saline (PBS) and replaced with complete medium containing various concentrations of API, and wound closure was observed after 48 h. Images were immediately captured under a phase-contrast microscope at 100x magnification, while at 48 h, cells were washed with PBS and then fixed with 4% paraformaldehyde followed by staining with 0.1% crystal violet.
Transwell migration and invasion assays
Cell motility was analyzed with the aid of a transwell. To analyze cell migration, 3 × 104 cells in 0.2 ml of serum-free medium were seeded in an uncoated top chamber (24-well insert; pore size, 8 μm; Corning Costar, Corning, NY), and medium supplemented with 10% FBS in the lower chamber was used as a chemoattractant. An invasion assay was conducted following the same procedure, with the exception that 5 × 104 cells were plated in a Matrigel (BD Biosciences, Bedford, MA)-coated top chamber. After 48 h of incubation, cells that had migrated or invaded to the bottom surface of the insert were fixed in 100% methanol for 5 min, stained in 0.1% crystal violet for 30 min, and rinsed in PBS, and cells on the top surface of the insert were removed by wiping with a cotton swab. The number of cells migrating or invading through the membrane was visualized and counted under a light microscope (200×, three random fields per well).
Western blot analysis
Cells were washed with PBS plus zinc ions (1 mM), and lysed with lysis buffer (10 mmol/l Tris-hydrochloride, 0.25 mol/l sucrose, 5 mmol/l EDTA, 50 mmol/l NaCl, 30 mmol/l sodium pyrophosphate, 50 mmol/l NaF, 1 mmol/l Na3VO4, 1 mmol/l PMSF, and 2% protease inhibitor cocktail; at pH 7.5). The protein concentration in the resulting lysate was determined with a bicinchoninic acid protein assay (Merck, Darmstadt, Germany). Appropriate quantities of protein (30~50 μg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) or 4–20% gradient SDS-PAGE (TOOLS, New Taipei City, Taiwan) and transferred onto nitrocellulose membranes. The membranes were inhibited and incubated overnight with the appropriate primary antibody at dilutions according to the manufacturer’s instructions. The membranes were subsequently washed and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody at a 1:1000 dilution in Tris-buffered saline-Tween 20 (10 mM Tris-HCl at pH 7.4, 150 mM NaCl and 0.1% Tween-20). The bound secondary antibody was then detected using an ECL system (Pierce Biotechnology, Rockford, IL).
Transient transfection of DNA
To overexpress Snail, or Slug , semiconfluent cultures of PCa cells in a 6-mm2 Petri dish were transfected with 3 μg of an empty or expression vector using the Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA) for 6 h according to the manufacturer’s instructions. At 24 h after transfection, cells were analyzed for invasion/migration and Snail and Slug expressions.
Lentiviral production and infection
The short hairpin (sh)RNA oligonucleotide sequence specifically targeting human SPOCK1 gene messenger (m)RNA was purchased from the National RNAi Core Facility at Academic Sinica (Taipei, Taiwan). The target sequences of SPOCK1 shRNA were 5′-CTGCTGGATGACCTAGAATAT-3 and 5′-GCTTTCGAGACGATGATTATT-3. The shRNA lentivirus was produced as previously described .
Plasmid construction and transfection
SPOCK1 Gateway donor complementary (c)DNA was purchased from DNasu Plasmid Repository and then recombined into the plenti6.3-DEST (Invitrogen) vector by Clonase LR (Invitrogen). The Plenti-6.3-SPOCK1, pMD.G, and pCMVDR8.91 plasmids were transfected into 293 T cells for packing the lentivirus. Target cells were incubated with viral supernatants for 48 h.
Intracardiac experimental metastasis model
PC-3 M-Luc cells were cultured in MEM supplemented with 10% FBS, and API’s curative effects on the progression of established metastases were evaluated as follows. For intracardiac experimental metastasis assays, male NOD-scid IL2Rγnull (NSG) mice (6~7 weeks old) were intraperitoneally (IP) injected with API (3 mg/kg of body weight (BW)) or 10% DMSO 3 days prior to an intracardiac injection and then approximately 106 PC-3 M-Luc cells were inoculated into the left ventricle of the heart by nonsurgical means. Bioluminescence imaging was done 30 min after the intracardiac injection to detect the distribution of PCa cells. Then each treated mouse was administered an IP injection of 3 mg/kg of API 6 days/week for 5 weeks. The injection volume was 100 μL (10 μL of a stock solution and 90 μL of PBS) each day. The control group received 100 μL of vehicle (10 μL of DMSO and 90 μL of PBS). Mice that showed whole-body bioluminescence signals were further monitored with weekly bioluminescence imaging (BLI). Images were acquired and analyzed with an In Vivo Imaging System (IVIS) Spectrum Imaging System (Xenogen, Alameda, CA). Ex vivo images of tumor-bearing tissues excised from the mice at necropsy were also obtained. All experiments were conducted in accordance with guidelines and regulations approved by the Institutional Animal Care and Use Committee of Taipei Medical University.
Orthotopic xenograft mouse model
For SPOCK1 overexpression and knockdown experiments in an orthotopic xenograft mouse model, 5-week-old male NSG mice were anesthetized with pentobarbital; then the PC-3-mock-luciferase, PC-3-SPOCK1-luciferase, PC-3 M-mock-luciferase, or PC-3 M-sh-SPOCK1-luciferase stable cell lines (5 × 105) were resuspended in a 1:1 mixture of PBS and GFR-Matrigel and inoculated into the anterior prostate using a 30-gauge needle, which was inserted through a lower abdominal incision. The incision was closed using a 4–0 Vicryl filament. After 7 days, the mice were randomly assigned to the experimental and control groups according to the Xenogen IVIS spectrum BLI results, such that treatment was initiated at a similar mean tumor size in each group. Then each treated mouse received daily IP injections of 3 mg/kg of API or the vehicle (10% DMSO in PBS) 6 days/week. The day after API treatment, mice were injected with D-luciferin and imaged for 1~2 min using this live imaging device to monitor the tumor size and location in real time. After 35 days, mice were sacrificed, and ex vivo images of tumor-bearing tissues excised from the mice at necropsy were further determined using the IVIS-Spectrum system. Tumors were also weighed and fixed, sectioned, and stained with hematoxylin and eosin (H&E) for IHC. All experiments were conducted in accordance with guidelines and regulations approved by the Institutional Animal Care and Use Committee of Taipei Medical University.
Mice anesthetized by exposure to 1%~ 3% isoflurane were placed in the IVIS Imaging System (Xenogen) and imaged from ventral views approximately 2 min after an IP injection of 100 μl of d-luciferin (Caliper Life Sciences) at 30 mg/ml per mouse. Established metastasis was assessed by imaging during the treatment period. The acquisition time was 2 min at the beginning of the time course and was progressively reduced afterward in accordance with the signal strength to avoid saturation. The analysis was performed using Living Image software (Xenogen) by measuring the photon flux (photons/s/cm2) with a region of interest drawn around the whole-animal or organ bioluminescence signal.
Tissue sections were routinely de-waxed and rehydrated. Sections were stained in hematoxylin for 5 min, and washed in running tap water for 5 min. Then, sections were stained in eosin for 30 s, dehydrated, and mounted by routine methods. Representative fields were chosen to be presented in the figures.
IHC was performed as previously reported . Mouse prostate tissues were fixed in 10% (v/v) formaldehyde in PBS, embedded in paraffin, and cut into 3-μm sections. Prostate sections were deparaffinized in a xylene solution and rehydrated using gradient ethanol concentrations. Deparaffinized sections were boiled in a microwave in 0.1 M citric acid buffer (pH 6.0) for antigen retrieval and then blocked by hydrogen peroxide and blocking serum, followed by incubation overnight with corresponding primary antibody for SPOCK1 (1:200, ab229935) at 4 °C. A secondary antibody was then incubated with the slides at room temperature for 1 h. Sections were observed after diaminobenzidine (DAB) kit (Boster, Wuhan, China) incubation and scored under a light microscope.
The stained SPOCK1 were evaluated with an Olympus BX50 light microscope. For semi-quantitative evaluation of the slides, a multi-score of staining frequency and intensity was applied. The frequency score ranged from 0~5 (negative = 0, < 10% positive cells = 1+, 10%~ 25% = 2+, 25%~ 50% = 3+, 50%~ 75% = 4+, and 75%~ 100% = 5+). The intensity score ranged from 0~3 (weak = 1, moderate = 2, strong = 3). The two results for staining intensity and frequency were multiplied, so that a ‘Multi-Score’ reflected both. Result are scored by multiplying the frequency score by the intensity.
Data are presented as the mean ± standard deviation (SD), and statistical comparisons between groups were made using Student’s t-test (two-tailed). Animal survival time was determined utilizing a Kaplan-Meier survival analysis and log-rank test. A p value of < 0.05 was considered a statistically significant difference.
API treatment results in reduced cell viability and motility of human PCa cells
Comparison of cell viability under API treatment in four Different PCa cell lines
API reverses changes in EMT biomarkers which contributed to suppression of the invasive property of human PCa cell lines
API suppresses PCa metastasis and prolongs survival in an intracardiac injection model
API inhibits PCa cells invasion through modulation of the SPOCK1-mediated EMT process
Targeting SPOCK1 with API suppresses orthotopic prostate tumor growth and spontaneous metastasis
Suppression of SPOCK1 protein expression by API contributes to inhibition of EMT activation in vivo
SPOCK1 was overexpressed in PCa and correlated with poor survival
PCa is one of the leading causes of cancer-related deaths in men worldwide, and metastasis is the primary factor in PCa mortality. Until recently, the mainstay of therapy for patients with metastatic PCa principally focused on targeting the androgen receptor (AR) [22, 23]. Thus, ADT is universally accepted as the initial treatment for men with locally advanced and metastatic PCa, which reduces levels of androgen hormones and is accomplished by luteinizing hormone-releasing hormone (LHRH) agonist drugs or surgical castration . Unfortunately, as demonstrated in nearly all patients receiving ADT, it ultimately results in relapse and development of clinically androgen-independent PCa (AIPC), and tumor progression inevitably occurs . This suggests that the high rate of mortality from PCa is linked to the development of AIPC and the current lack of effective therapies. Therefore, developing novel therapeutic approaches to target AIPC has considerable potential for improving the quality of life and survival of patients with metastatic PCa. Our present results showed that API significantly inhibited the cell viability of both androgen-independent DU145, PC3, and PC-3 M cells and the androgen-dependent LNCaP human PCa cell lines. Moreover, our results showed that API suppressed the invasive ability of androgen-independent DU145, PC3, and PC-3 M cells, which were respectively derived from brain, bone, and liver metastasis, and possess high tumorigenic and metastatic capacities. Our studies also identified that SPOCK1 plays an important role in regulating the invasive abilities of several AIPC cell lines (PC3 and PC-3 M) and can be downregulated by API treatment in those cell lines. Importantly, for the first time we demonstrated that the antimetastatic effect of API is due to suppression of SPOCK1-mediated EMT activation in a human PCa xenograft model. These results suggest that suppression of SPOCK1 might be a general phenomenon in API-regulated cell motility of PCa cells.
Metastasis is a multistep cellular process, and most PCa-related deaths are due to metastatic disease rather than due to the corresponding primary tumors. Strikingly, the EMT is a crucial step in tumor progression and plays a pivotal role during cancer invasion and metastasis . SPOCK1 encodes a matricellular glycoprotein belonging to the secreted protein, acidic, cysteine-rich (SPARC) family, which consists of SPARC, Hevin, testican-2, testican-3, and follistatin-like protein 1. Members of this protein family act to modulate interactions of extracellular proteins with cell surfaces to direct factors to their suitable extracellular sites . Clinical association studies found that SPOCK1 expression in metastatic tissues was significantly higher than in non-metastatic cancerous tissues . Similar to previous findings, we also observed that SPOCK1 was expressed more strongly in PCa tissues relative to noncancerous tissues, and a high SPOCK1 expression level was significantly correlated with worse disease-free survival compared to PCa patients with lower levels. Additionally, in vivo studies of lung cancer demonstrated that SPOCK1 is not only associated with metastasis but also induces the EMT , suggesting the extensive roles of SPOCK1 in promoting cancer cell invasiveness and metastasis through involvement in the EMT process [5, 29, 30]. Our results showed lower expression of SPOCK1 in PC-3 cells than the isogenic but metastatic variant PC-3 M cells. Moreover, transcription factors of the Snail family (Snail and Slug) were associated with EMT progression during PCa metastasis . Notably, our present study first demonstrated that API treatment attenuated growth and metastasis of PCa through inactivation of the Snail/Slug-dependent EMT process by suppressing SPOCK1 expression.
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endoproteinases, that directly degrade components of the extracellular matrix (ECM), an important proteolytic event in the invasion and metastasis of tumors . Several clinical assays have confirmed the MMP expression is tied to tumor aggressiveness, disease progression, and clinical outcomes in patients with various types of tumors [33, 34]. Of note, MMP-3 and MMP-9 are detected as mesenchymal markers that stimulate the EMT process and contribute to metastasis [35, 36]. On the other hand, transcription factors of the Snail family (Snail and Slug), known to regulate EMT pathways, were also shown to regulate several MMPs [37, 38]. This evidence suggests that cooperation and crosstalk between MMPs and EMT-associated transcription factors may be involved in the process of SPOCK1 inducing the EMT. Indeed, a recent investigation revealed that SPOCK1-promoted tumor growth and metastasis are accompanied by upregulation of MMP-3 and MMP-9 expressions in PCa , suggesting that MMPs may be involved in the SPOCK1-mediated EMT process and subsequent metastasis. The antimetastatic properties of API are that it inhibits cancer cell migration and invasion through attenuation of MMP-9 expression in vitro and in vivo [39, 40]. Our recently published report indicated that API also suppressed CD26 expressions and the EMT-mediated cell invasion in several NSCLC cell lines . In this study, we founded that API treatment of PCa cells caused a decrease in SPOCK1 expression subsequently leading to inhibition of the invasive abilities and EMT-related markers (Snail, Slug and vimentin), while overexpression of SPOCK1 could reverse the API-mediated inhibitory effects, suggesting that SPOCK1 inhibition by API may be the main cause for the API-mediated suppression of Snail family-induced cell motility in PCa.
The PI3K/Akt signaling pathway has been linked to the apoptosis, autophagy, and tumor development, growth, and metastasis. Aberrant expression and activity of the PI3K-Akt pathway was shown to be more frequently observed as PCa progresses toward therapeutic-resistant or metastatic disease , suggesting that this pathway may be implicated in the aggressive phenotype of PCa. Accumulating evidence has demonstrated that activation of the PI3K/Akt pathway can upregulate expressions of Snail and Slug by targeting glycogen synthase kinase (GSK)-3β-mediated degradation, thereby triggering the EMT process . On the other hand, activation of the PI3K/Akt pathway was also reported to play a role in cancer cell invasion and metastasis through increasing MMP production via its multiple downstream target proteins such as mammalian target of rapamycin (mTOR), nuclear factor (NF)-κB, GSK-3, and activator protein (AP)-1 [43, 44, 45]. Recently, it is worth noting that knockdown of SPOCK1 obviously significantly decreased levels of PI3K and Akt phosphorylation in PCa cells . Moreover, SPOCK1 was able to block apoptosis and promote proliferation, invasion, and metastasis in vitro and in vivo through activating PI3K/Akt signaling in various human malignancies [7, 8, 29]. According to recent literature data, we noted that API can directly block the ATP-binding site of PI3K resulting in suppression of PI3K activity and subsequent inhibition of Akt kinase activity . Additionally, API was observed to exhibit chemopreventive and/or anti-carcinogenic properties in many different cancer cell types due to the inhibition of PI3K/Akt, mitogen-activated protein kinase (MAPK), and NF-κB signaling pathways [46, 47]. Actually, our current results showed that treatment of SPOCK1-overexpressed DU145 and PC-3 cells with a PI3K inhibitor, LY294002, or API all can reverse SPOCK1-induced increases of Akt activation and Snail/Slug expression. (Additional file 1: Figure S4a). Moreover, treatment of PC-3 M cells with LY294002 showed the similar inhibitory effect with API treatment or SPOCK1 knockdown on Akt activation and Snail/Slug expression. (Additional file 1: Figure S4b). Taken together, these data suggest that PI3K/Akt pathway participates in the SPOCK1-regulated Snail family expression and EMT progression in PCa cells and SPOCK1-Akt-Snail/Slug signaling pathway could be a critical target of API to suppress metastasis of PCa. In addition, whether the SPOCK1-Akt-MMP pathway might be another target of API to control the invasive ability of PCa should be further evaluated in the future.
Despite ADT remaining the principal treatment for PCa patients with locally advanced and metastatic disease, most patients exhibit ADT failure and progress to CRPC, leading to their death within a few years. Of note, inhibition of the PI3K pathway causes increased androgen receptor (AR) protein levels and AR target gene expression [48, 49]. Conversely, the PI3K/Akt pathway can suppress AR transcriptional activity and thus contributes to castration resistance, suggesting that this pathway is implicated in the progression to castration resistance . Moreover, androgen-dependent LNCaP cells grown in androgen-depleted medium developed androgen-independent growth and high levels of Akt activation. Similarly, Akt activation also increased in LNCaP xenografts grown in castrated mice . Accordingly, the tumor suppressor, phosphatase and tensin homolog (PTEN), is a lipid phosphatase that restrains PI3K/Akt signaling, and loss of PTEN protein expression was associated with Akt hyperactivation. Subsequent experiments in mice with prostate PTEN deletion (Ptenloxp/loxp) clearly demonstrated that castrate-resistant cancer developed in regions of the prostate with simultaneous loss of AR expression . Recently, overexpression of SPOCK1 was observed in CRPC tissues , suggesting that SPOCK1 activates PI3K/Akt signaling [7, 8, 29], which may partially be a potential mechanism for CRPC development. Moreover, in CRPC cells, the EMT is associated with metastatic processes and leads to drug resistance . Despite many recent advances in PCa therapies, the ultimate development of metastatic (m)CRPC with recurrence to lethal disease remains incurable. Our study demonstrated that API inhibits cell invasion and metastasis through suppressing the SPOCK1-mediated EMT process in mCRPC cell lines (PC3, PC3M, and DU145). Collectively, it was interesting to note that cooperation and crosstalk between SPOCK1-induced PI3K/Akt signaling and MMPs may be involved in PCa progression, and the effects of API on these processes in mCRPC should be further evaluated in the future. Finally, our findings reinforced the notion of API as a dietary supplement or potential therapeutic agent for managing mCRPC.
We would like to thank Dr. Tsang-Chih Kuo (National Taiwan University, Taipei, Taiwan) for the gift of the Snail- and Slug-overexpressing plasmids.
W-JL designed this study and drafted the manuscript. M-HC and Y-WL performed the experiments and contributed equally to this work. Y-CW and Y-CY conducted the statistical analysis and analyzed TCGA data. J-LC and H-CH contributed IHC analysis. H-CH performed the animal experiments. W-JL and M-HC review and/or revision of the manuscript. All authors had edit and approved the final manuscript.
This study was supported by a grant (TMU105-AE1-B14) from Taipei Medical University (to W.-J. Lee). This study was supported by a grant (108-wf-eva-04) from Wan Fang Hospital, Taipei Medical University (to W.-J. Lee and Y.-C. Wen). This study was also supported by the TMU Research Center of Cancer Translational Medicine from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan (to M.-H. Chien).
Ethics approval and consent to participate
All animal experiments were carried out in accordance with guidelines of a protocol approved by the Taipei Medical University Animal Ethics Research Board.
Consent for publication
The authors declare that they have no competing interests.
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