Background
Prostate cancer (PCa) is the second common diagnosed cancer in men worldwide and the fifth leading cause of cancer-related deaths [
1]. The 5-year relative survival rate of primary PCa patients is > 99%, while that of patients with distant metastasis sites is no more than 30% [
2]. Bone is among the most preferential metastatic site of PCa [
3]. Once tumor cell metastasis to bone, it will cause several bone- associated complications, including hypercalcemia, intractable pain, fracture, or nerve compression syndrome, contributing to the poor survival in PCa patients [
4]. A major challenge for treatment of advanced metastatic disease is due to incomplete understanding of the molecular mechanisms underlying the high avidity of PCa to bone. Therefore, it’s significantly necessary to unveil the molecular mechanism underlying the high bone metastatic propensity of PCa.
Since its initial discovery as a proto-oncogene signaling, the critical roles of the phosphoinositide 3-kinase (PI3K)/Akt Signaling in diverse cellular processes, including cell growth, proliferation and survival, has seized considerable attention [
5,
6]. The PI3K family is activated in response to multiple extracellular stimuli, including EGF [
7], IGF-1 [
8], insulin [
9] and CaM [
10]. Then, the activated PI3K phosphorylates phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatidylinositol − 3,4,5-trisphosphate [PI(3,4,5)P3], at the 3′-hydroxyl group of the inositol ring of phosphatidylinositol, which further recruits Akt and phosphoinositide-dependent kinases to the plasma membrane, leading to activation of Akt kinase by two phosphorylation sites at Thr 308 and Ser 473 [
11,
12]. The activated Akt further phosphorylates multiple downstream effectors, which promotes cells unlimited proliferation and growth [
13‐
15]. Constitutive activation of the PI3K/Akt pathway has been reported to contribute to the pathogenesis of many types of cancer [
16]. Furthermore, increasing evidence is accumulating that activity of PI3K/Akt signaling plays a crucial role in the metastasis of cancer. A study by Xue and colleagues has shown that Akt-mediated Twist1 phosphorylation promoted breast cancer lung metastasis [
17]. Ectopic expression of cyclin G1 promoted epithelial-mesenchymal transition (EMT) and metastasis of hepatocellular carcinoma cells via enhancing Akt activation-mediated the stabilization of Snail, a critical EMT mediator [
18]. Importantly, Ni et al. have reported that PI3K/Akt signaling -mediated stabilization of histone methyltransferase WHSC1 profoundly increased bone metastasis and osteolytic bone lesions in PCa [
19]. However, the underlying mechanism responsible for activation of PI3K/Akt signaling in bone metastasis of PCa remains largely unknown.
microRNAs (miRNAs) play important roles in cellular differentiation, proliferation, and embryo development [
20], and have been involved in the development, progression and metastasis of cancer [
21‐
23]. An accumulating body of studies has determined the pivotal roles of miRNAs in bone metastasis of PCa [
24‐
28]. miR-133a-3p was one of the frequently deregulated miRNA in cancer [
29‐
31], and low expression of miR-133a-3p has been associated with recurrence and metastasis of PCa [
32‐
36]. However, the clinical significance of miR-133a-3p in the progression and bone metastasis of PCa, and the biological role of miR-133a-3p and its molecular mechanisms underlying bone metastasis of PCa have not been reported. Here we reported that miR-133a-3p was decreased in PCa tissues and further downregulated in bone metastatic PCa tissues, which was positively associated with poor clinicopathological characteristics and bone metastasis-free survival in PCa patients. Moreover, upregulating miR-133a-3p dramatically inhibited cancer stem cells characteristics and anoikis resistance in vitro, and tumorigenesis and bone metastasis in vivo in PCa cells. Our results further demonstrated that miR-133a-3p repressed activity of PI3K/AKT signaling by simultaneously targeting EGFR, FGFR1, IGF1R and MET, which further suppressed bone metastasis of PCa. Therefore, our results clarify the underlying mechanism to determinate the anti-bone metastatic role of miR-133a-3p in PCa.
Methods
Cell lines and cell culture
The human PCa cell lines 22RV1, PC-3, VCaP, DU145, LNCaP and normal prostate epithelial cells RWPE-1 were obtained from Shanghai Chinese Academy of Sciences cell bank (China). RWPE-1 cells were grown in defined keratinocyte-SFM (1×) (Invitrogen). PC-3, LNCaP and 22Rv1 cells were cultured in RPMI-1640 medium (Life Technologies, Carlsbad, CA, US) supplemented with penicillin G (100 U/ml), streptomycin (100 mg/ml) and 10% fetal bovine serum (FBS, Life Technologies). DU145 and VCaP cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% FBS. The C4-2B cell line was purchased from the MD Anderson Cancer Center and maintained in T-medium (Invitrogen) supplemented with 10% FBS. All cell lines were grown under a humidified atmosphere of 5% CO2 at 37 °C.
Plasmids, transfection and generation of stable cell lines
The human MIR133A gene was PCR-amplified from genomic DNA and cloned into a pMSCV-puro retroviral vector (Clontech, Japan). The 3’UTR of EGFR, FGFR1, IGF1R and MET were PCR-amplified from genomic DNA and cloned into pmirGLO vectors (Promega, USA), and the list of primers used in cloning reactions is shown in Additional file
1: Table S1. AgomiR-133a-3p was synthesized and purified by RiboBio. Cells were treated with MK-2206 (Selleck Chemicals, Houston, TX, USA) at the concentrations (1 μM). Transfection of miRNA, siRNAs, and plasmids was performed using Lipofectamine 3000 (Life Technologies, USA) according to the manufacturer’s instructions.
Total RNA from tissues or cells was extracted using the RNA Isolation Kit (Qiagen, USA) according to the manufacturer’s instructions. Messenger RNA (mRNA) were reverse transcribed from total mRNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA). The random primer was used for reverse transcription of mRNA and the primer for reverse transcription of miRNA were synthesized and purified by RiboBio (Guangzhou, China). Complementary DNA (cDNA) was amplified and quantified on the CFX96 system (BIO-RAD, USA) using iQ SYBR Green (BIO-RAD, USA). The primers are provided in Additional file
2: Table S2. Real-time PCR was performed according to a standard method, as described previously [
37]. Primers for U6 and miR-133a-3p were synthesized and purified by RiboBio (Guangzhou, China). U6 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous controls. Relative fold expressions were calculated with the comparative threshold cycle (2
-ΔΔCt) method.
Patients and tumor tissues
A total of 225 individual and 20 paired PCa tissues, and 48 benign prostate lesions tissues were obtained during surgery or needle biopsy at The Clinical Biobank of Collaborative Innovation Center for Medical Molecular Diagnostics of Guangdong Province, The Affiliated Jiangmen Hospital of Sun Yat-sen University (Guangdong, China), and the Second Affiliated Hospital of Guangzhou Medical University (Guangdong, China) between January 2008 and December 2016. Patients were diagnosed based on clinical and pathological evidence, and the specimens were immediately snap-frozen and stored in liquid nitrogen tanks. For the use of these clinical materials for research purposes, prior patient’ consents and approval from the Institutional Research Ethics Committee were obtained. The clinicopathological features of the patients are summarized in Additional file
3: Table S3, Additional file
4: Table S4, Additional file
5: Table S5. The median of miR-133a-3p expression in PCa tissues was used to stratify high and low expression of miR-133a-3p.
miRNA immunoprecipitation
Cells were co-transfected with HA-Ago2, followed by HA-Ago2 immunoprecipitation using anti-HA-antibody. Real-time PCR analysis of the IP material was performed to test the association of the mRNA of EGFR, FGFR1, IGF1R and MET with the RISC complex. The specific processes were performed as previously described [
38]. Briefly, Cells (5 × 10
5) were plated in 60-mm cell culture dishes, proliferating to 60–80% confluence after 24 h of culture, and the pIRESneo-FLAG/HA-Ago2 plasmas were cotransfected into cells using Lipofectamine 3000. After 48-h transfection, cells were washed and lysed in radioimmunoprecipitation buffer (Sigma-Aldrich) containing 10% proteinase inhibitor cocktail (Sigma-Aldrich) and 1 mMphenylmethylsulfonyl fluoride (Sigma-Aldrich). A fraction of the whole cell lysate was used for RNA isolation, and the remaining lysate was subjected to immunoprecipitation (IP) using an antibody against Ago2 (Abcam) or immunoglobulin G (IgG) (Abcam). RNA from whole cell lysates and RNA IP (RIP) fractions was extracted with TRIzol (Life Technologies) according to the manufacturer’s instructions. The relative levels of mRNA were determined using real-time RT-PCR as described above. The relative mRNA enrichment in the RIP fractions was computed based on the ratio of relative mRNA levels in the RIP fractions and the relative mRNA levels in the whole cell lysates.
Western blot
Western blot was performed according to a standard method, as previously described [
39]. Antibodies against Bcl2, Bcl-xl, Surviva and Mcl-1 were purchased from Abcam (Cambridge, USA), and EGFR, FGFR1, IGF1R, MET, p-AKT (S473), p-AKT (T308) and AKT were purchased from Cell Signaling Technology. As a loading control, membranes were stripped and reprobed with an anti-α-tubulin antibody (Sigma-Aldrich, USA).
Luciferase reporter assay
Cells (4 × 10
4) were seeded in triplicate in 24-well plates and cultured for 24 h and performed as previously described [
40]. Luciferase and Renilla signals were measured 36 h after transfection using a Dual Luciferase Reporter Assay Kit (Promega). The mutations can be designed at any site, but the best one should be around the seed nucleotides in miRNA target tracks. You can design the two pairs of PCR primers, one pair for uspstream fragment, in which the primer for the bottom strand has the nucleotides in the seed region mutated; the other pair for the downstream fragment, in which the 5′ end of primer for the top strand should be overlapping at least 6 nucleotides with the 5′ end of the bottom strand primer for the upstream fragment. Two rounds of PCR are to be run, first round is run to amplify the upstream and downstream fragments separately. After PCR, check on the gel and make purifications for the two PCR fragments. In second round the top strand primer for the upstream fragment and the bottom strand primer for the downstream fragment should be used together with the two purified first-round PCR fragments. The extension reaction is started to join the overlapping region, followed by the regular PCR to amplify the joined upstream and downstream fragments containing mutated nucleotides at the seed motif of miRNA target tracks. Check on the gel after PCR, and make subclone first and sequencing to confirm the mutations. Then insert the mutated 3’-UTR fragment into your testing vector.
Akt activity assay
To measure Akt kinase activities of in cells or tumor tissues, Akt activity assay was performed as previous described [
41]. The immune complexes were then incubated with a biotinylated peptide substrate that became phosphorylated in the presence of activated Akt. The phosphorylated substrates, which reflected the activity of Akt kinase in the extract, was then quantified with the K-LISA Akt Activity Kit (Calbiochem, Darmstadt, Germany) that comprises a primary antibody recognizing the phosphorylated substrate peptides.
Animal study
All mouse experiments were approved by The Institutional Animal Care and Use Committee of Sun Yat-sen University and the approval-No. was L102012016110D. The 6-week-old BALB/c-nu mice were randomly divided into four groups (n = 6 per group). The PC-3 cells (1 × 106, 1 × 105, 1 × 104 and 1 × 103) were inoculated subcutaneously together with Matrigel (final concentration of 25%) into the inguinal folds of the nude mice respectively. In the experiment testing, animals were injected with 100 μl agomir-133a-3p or agomir scramble through the lateral tail vein every four days for 4 weeks. The mice were sacrificed 35 days after inoculation and the tumors were excised and weighted. For the bone metastasis study, BALB/c-nu mice (5–6 weeks old) were anaesthetized and inoculated into the left cardiac ventricle with 1 × 105 PC-3 cells in 100 μl of PBS. Agomir-133a-3p was injected through tail vein 2 days after inoculation of PC-3 cells. Osteolytic lesions were identified on radiographs as radiolucent lesions in the bone. The area of the osteolytic lesions was measured using the Metamorph image analysis system and software (Universal Imaging Corporation), and the total extent of bone destruction per animal was expressed in square millimeters. Each bone metastasis was scored based on the following criteria: 0, no metastasis; 1, bone lesion covering < 1/4 of the bone width; 2, bone lesion involving 1/4~ 1/2 of the bone width; 3, bone lesion across 1/2~ 3/4 of the bone width; and 4, bone lesion > 3/4 of the bone width. The bone metastasis score for each mouse was the sum of the scores of all bone lesions from four limbs.
Flow cytometric analysis
Flow cytometric analyzed of apoptosis were used the FITC Annexin V Apoptosis Detection Kit I (BD, USA), and was presented as protocol described. Briefly, cells were dissociated with trypsin and resuspended at 1 × 106 cells/mL in binding buffer with 50 μl/ml FITC Annexin V and 50 ul/ml PI. The cells were subsequently incubated for 15 min at room temperature, and then were analyzed by Gallios flow cytometer (Beckman Coulter, USA). The cell’s inner mitochondrial membrane potential (Δψm) was detected by flow cytometric using MitoScreen JC-1 staining kit (BD), and was presented as protocol described. Briefly, cells were dissociated with trypsin and resuspended at 1 × 106 cells/mL in Assay Buffer, and then incubated at 37 °C for 15 min with 10 μl/ml JC-1. Before analyzed by flow cytometer, cells were washed twice by Assay Buffer. Flow cytometry data were analyzed using FlowJo 7.6 software (TreeStar Inc., USA).
Caspase-9 or − 3 activity assays
Activity of caspase-9 or − 3 was analysis by spectrophotometry using Caspase-9 Colorimetric Assay Kit or Caspase-3 Colorimetric Assay Kit (Keygen, China), and was presented as protocol described. Briefly, 5 × 106 cells or 100 mg fresh tumor tissues were washed with cold PBS and resuspended in Lysis Buffer and incubated on ice for 30 min. Mixed the 50 μl cell suspension, 50 μl Reaction Buffer, and 5 μl Caspase-3/− 9 substrate, and then incubated at 37 °C for 4 h. The absorbance was measured at 405 nm, and BCA protein quantitative analysis was used as the reference to normal each experiment groups.
Side population analysis
The cell suspensions were labeled with Hoechst 33,342 (Molecular probes – #H-3570) dye for side population analysis as per standard protocol [
42]. Briefly, cells were resuspended at 1× pre-warmed OptiMEM (Gibco, USA) containing 2% FBS (Gibco, USA) at a density of 106/mL. Hoechst 33,342 dye was added at a final concentration of 5 lg/mL in the presence or absence of verapamil (50 lmol/L; Sigma) and the cells were incubated at 37 °C for 90 min with intermittent shaking. At the end of the incubation, the cells were washed with OptiMem containing 2% FBS and centrifuged down at 4 °C, and resuspended in ice-cold OptiMem containing 2% FBS and 10 mmol/L HEPES. Propidium iodide (Sigma, USA) at a final concentration of 2 lg/mL was added to the cells to gate viable cells. The cells were filtered through a 40-lm cell strainer to obtain single cell suspension before sorting. Analysis and sorting was done on a FACS AriaI (Becton Dickinson). The Hoechst 33,342 dye was excited at 355 nm and its dual-wavelength emission at blue and red region was plotted to get the SP scatter.
Cells (500 cells/well) were seeded into 6-well Ultra Low Cluster plates (Corning) and cultured in suspension in serum-free DMEM-F12 (BioWhittaker), supplemented with B27 (1:50, Invitrogen), 20 ng/ml endothelial growth factor (EGF; BD Biosciences), 0.4% bovine serum albumin (Sigma), and 4 mg/ml insulin (Sigma). After 10–12 days, the number of cell spheroids (tight, spherical, non-adherent masses > 50 μm in diameter) were counted, and images of the spheroids were scored under an inverse microscope (spheroids formation efficiency = colonies/input cells× 100%).
High throughput data processing and visualization
The miRNAs expression levels and clinical profile of PCa dataset were downloaded from The Cancer Genome Atlas (TCGA:
https://tcga-data.nci.nih.gov/tcga/). The log2 values of miRNAs in each sample were analyzed using Excel 2010 and GraphPad 5, as well as statistically analyze the miRNAs expression level of all PCa tissues using paired t-test or unpaired t-test. The expression levels of miRNAs in each sample were analyzed as previously described [
43].
Statistical analysis
All values are presented as the mean ± standard deviation (SD). Significant differences were determined using the GraphPad 5.0 software (USA). One-way ANOVA was used to determine statistical differences between multiple testing and the post hoc test after ANOVA is Tukey. Unpaired or paired t-test was used to determine statistical differences between two groups. The chi-square test was used to analyze the relationship between miR-133a-3p expression and clinicopathological characteristics. Survival curves were plotted using the Kaplan Meier method and compared by log-rank test. P < 0.05 was considered statistical significant. All experiments were repeated three times.
Discussion
Numerous studies have demonstrated that copy number variation, chromosomal rearrangements and genetic mutations are implicated in the progression and metastasis of PCa [
48‐
50]. In metastatic PCa tumor tissues, phosphatase and tensin homolog (PTEN) loss-of-function mutations or genomic alterations in components of the PI3K/AKT signaling even reaches up to 70% [
48,
51], supporting the critical roles of PI3K/AKT signaling in metastatic PCa. Furthermore, epigenetic regulations are emerging as important contributing factors for the unrestrained activation of AKT signaling [
52]. Among these factors, aberrant expression of miRNAs constitutes a compelling component in epigenome. miR-27b has been reported to decreased in diffuse large B-cell lymphoma (DLBCL). Forced expression of miR-27b suppressed DLBCL cell proliferation and tumor growth via repressing PI3K/AKT pathway by targeting MET [
53]. In addition, miR-508 directly targeted multiple phosphatases, including INPP4A, INPP5J and PTEN, resulting in constitutive activation of PI3K/Akt signaling, which promoted the aggressive phenotype of oesophageal squamous cell carcinoma [
41]. In PCa, several miRNAs, including miR-16, miR-106b, miR-148a, miR-4534 and miR-195, have been reported to be implication in the activation of the PI3K/Akt signaling pathway [
54‐
56]. In the current study, our results demonstrated that several cytokine receptors of Akt signaling, including EGFR, FGFR1, IGF1R and MET, were direct targets of miR-133a-3p in PCa cells. Downregulation of miR-133a-3p dramatically augmented the activity of PI3K/Akt signaling in PCa cells. Therefore, our results uncover a novel mechanism for the constitutive activation of PI3K/Akt signaling in bone metastasis of PCa.
The PI3K/Akt signaling cascade can be activated by several stimuli, including integrins, receptor tyrosine kinases, cytokine receptors and G-protein-coupled receptors, all of which can induce production of phospha- tidylinositol (3,4,5) trisphosphates (PIP3) by phosphoinositide 3-kinase (PI3K) [
5,
6]. Among these, cytokine activation of PI3K/Akt pathway has been regarded as a primary manner mediating PI3K/Akt signaling cascade. In this manner, activation of PI3K/Akt signaling starts with the binding of cytokine to the corresponding receptor, such as EGF [
7], IGF-1 [
8] and insulin [
9]. Excessive secretion of cytokines in an autocrine or paracrine manner, or increased expression of the corresponding receptors has been reported to contribute to constitutive activation of PI3K/AKT signaling [
57‐
60]. However, how these cytokines and the receptors are simultaneously disrupted in cancers, resulting in the activation of PI3K/AKT signaling, remains unclear. In this study, through analyzing publicly available algorithms, we found that several cytokine receptors, including EGFR, ERBB4, FGFR1, IGF1R, IFG2R, IN2R, MET and NGFR, may be potential target of miR-133a-3p, in which only EGFR, FGFR1, IGF1R and MET were targeted by miR-133a-3p in PCa cells. Importantly, autocrine levels of the corresponding cytokines were not affected by upregulating or silencing miR-133a-3p in PCa cells. Taken together, our results suggest that miR-133a-3p represses AKT signaling activity via inhibiting cytokine receptors, but has no any effect on cytokines secretion in PCa cells.
Downexpression of miR-133a-3p has been extensively reported in a various types of cancer and predicted a poor prognosis [
61‐
67]. However, several lines of evidence have shown that miR-133a-3p was upregulated in hepatocellular carcinoma [
29], multiple myeloma [
31], breast cancer [
68] and osteosarcoma [
69], suggesting that miR-133a-3p plays an oncogenic or tumor-suppressive miRNA depending on tumor types. Furthermore, low expression of miR-133a-3p has been correlated with the recurrence and distant metastasis of PCa [
32‐
36]. However, the clinical significance of miR-133a-3p in the progression and bone metastasis of PCa, as well as the biological role of miR-133a-3p and its molecular mechanisms underlying bone metastasis of PCa have not been elucidated. In this study, our results demonstrated that miR-133a-3p was downregulated in PCa tissues and further reduced in bone metastatic PCa tissues. Low expression of miR-133a-3p closely correlated with advanced clinicopathological characteristics and poor bone metastasis-free survival in PCa patients. Furthermore, our results clarified that miR-133a-3p repressed bone metastasis of PCa via inactivating PI3K/AKT signaling by directly targeting EGFR, FGFR1, IGF1R and MET. Therefore, our results indicate that miR-133a-3p functions as a tumor-suppressive miRNA via disrupting PI3K/AKT signaling in bone metastasis of PCa.
Strikingly, our results revealed that the low miR-133-3p expression did not affect overall survival in PCa patients, although the low miR-133-3p levels positively and significantly correlated with advanced clinicopathological characteristics of PCa patients, including serum PSA levels, Gleason score and TNM status. In fact, these pathological parameters predict poor overall survival in PCa patients [
70‐
74]. The possibility that low levels of miR-133a-3p were significantly associated with serum PSA levels, Gleason score and TNM status in PCa patients, but had no effect on overall survival in PCa patients is that the majority of our PCa samples were obtained from 2011 to 2016, and PCa patients have a relatively high rate of 5-year overall survival. Therefore, the solid conclusion about the prognostic prediction of miR-133a-3p expression levels in overall survival in PCa patients is well warranted to follow up in the following couples of years.
Circulating microRNAs have drawn a great deal of interest as promising novel non-invasive biomarkers for the early diagnosis of cancer. Recently, the involvement of circulating miR-133a-3p as a potential biomarker for diagnosis and prognosis of cancer are becoming increasingly appreciated. In breast cancer, higher miR-133a-3p levels in the plasma or serum of breast cancer patients provided considerable discrimination compared with the healthy controls [
68,
75,
76]. Conversely, low expression level of miR-133a-3p expression was observer in the serum of astrocytomas and colorectal cancer patients with a high sensitivity and specificity [
77,
78]. However, little is known about the potential applicable values of circulating miR-133a-3p in PCa and its bone metastatic phenotypes. In this study, our results demonstrated that low expression of miR-133a-3p strongly correlated with bone metastasis-free survival in PCa patients, suggesting that miR-133a-3p may serve as a potential bone metastasis diagnostic marker in PCa patients. However, whether miR-133a-3p expression in the serum or plasma samples of PCa patients may be used as a potential non-invasive marker to predict bone metastasis of PCa requires further investigation.