Exosome-derived microRNAs contribute to prostate cancer chemoresistance

  • Authors:
    • Jing Li
    • Xin Yang
    • Hao Guan
    • Atsushi Mizokami
    • Evan T. Keller
    • Xiaozhen Xu
    • Xia Liu
    • Jiyong Tan
    • Longyuan Hu
    • Yi Lu
    • Jian Zhang
  • View Affiliations

  • Published online on: June 3, 2016     https://doi.org/10.3892/ijo.2016.3560
  • Pages: 838-846
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Certain microRNAs (miRNAs) play a key role in cancer cell chemoresistance. However, the pleiotropic functions of exosome-derived miRNAs on developing chemoresistance remain unknown. In the present study, we aimed to construct potential networks of miRNAs, which derived from the exosome of chemoresistant prostate cancer (PCa) cells, with their known target genes using miRNA expression profiling and bioinformatic tools. Global miRNA expression profiles were measured by microarray. Twelve miRNAs were initially selected and validated by qRT-PCR. Known targets of deregulated miRNAs were utilized using DIANA-TarBase database v6.0. The incorporation of deregulated miRNAs and target genes into KEGG pathways were utilized using DIANA-mirPath software. To construct potential miRNA regulatory networks, the overlapping parts of miRNAs and their targer genes from the selected KEGG pathway ‘PCa progression (hsa05215)’ were visualized by Cytoscape software. We identified 29 deregulated miRNAs, including 19 upregulated and 10 downregulated, in exosome samples derived from two kinds of paclitaxel resistance PCa cells (PC3-TXR and DU145-TXR) compared with their parental cells (PC3 and DU145). The enrichment results of deregulated miRNAs and known target genes showed that a few pathways were correlated with several critical cell signaling pathways. We found that hub hsa-miR3176, -141-3p, -5004-5p, -16-5p, -3915, -488‑3p, -23c, -3673 and -3654 were potential targets to hub gene androgen receptor (AR) and phosphatase and tensin homolog (PTEN). Hub gene T-cell factors/lymphoid enhancer-binding factors 4 (TCF4) target genes were mainly regulated by hub hsa-miR-32-5, -141-3p, -606, -381 and -429. These results may provide a linkage between PCa chemoresistance and exosome regulatory networks and thus lead us to propose that AR, PTEN and TCF4 genes may be the important genes which are regulated by exosome miRNAs in chemoresistance cancer cells.

Introduction

Prostate cancer (PCa) is the most frequently diagnosed malignancy and the second leading cause of death for male cancer patients (≥50 years old) in western countries (1). Currently, anti-androgen therapy is the first line of treatment for patients diagnosed with PCa. A majority of these patients, however, eventually develop androgen-independent PCa which is highly metastatic and has poor prognosis (2). Taxanes are effective in treating patients diagnosed with androgen-independent PCa. Although clinical trials have proved the initial efficacy of paclitaxel (PTX) in increasing survival in PCa patients (3), about half of patients develop drug resistance. There are a few effective approaches availabe for treating chemoresistant PCa. In addition, the pleotropic functions of exosome-drived miRNAs on developing chemoresistance remain unknown.

Cancer cells release abundant soluble or membranous factors that facilitate their growth and survival. Evidence suggests that small microvesicles with exosomes play a pivotal role in this process. Exosomes are membrane vesicles with a size of 40–100 nm. They contain proteins, mRNAs, microRNAs (miRNAs) and signaling molecules, that reflect the physiological state of their cells of origin and consequently provide a rich source of potential biomarker molecules (4). Exosomes can be taken up by other cells, it is possible that these membrane vesicles could serve as a novel way of intercellular communication and signaling (5). Over the past few years, researchers have revealed that exosomes crosstalk and/or influence major signal pathways in tumor progression, such as hypoxia-driven epithelial-to-mesenchymal transition, angiogenesis and metastasis involving many cell types within the tumor microenvironment (68).

miRNAs are small non-coding RNAs with diverse functions. They can regulate their target genes in a cooperative, combinatorial fashion, where a single miRNA can target multiple mRNA transcripts and distinct miRNAs can target the same mRNA, ensuring control over a large number of cellular functions. Notably, recent studies found that the secretion of miRNAs is also partially mediated through vesicular/exosome-mediated mehanisms (9). Hence, exosomes play an important role in miRNA regulation. Therefore, we cannot ignore them while studying miRNA targets or designing miRNA-targeted therapeutic strategies.

miRNA profiling through microarrays is an invaluable technique to determine a miRNA signature, which is necessary to figure out the general and specific expression alterations in difference cells or tissue. In the present study, we aimed to explore the exosome derived miRNA contributing to taxanes-resistance (TXR) PCa cells compared with their parental cells for elaborating potential effect of exosomal miRNA of drug resistance of PCa cells.

Materials and methods

Cell culture and preparation of culture medium

Human metastatic PCa cell lines DU145 and PC3 and their PTX resistant versions DU145-TXR and PC3-TXR used in this study were given by Dr Atsushi Mizokami (Kanazawa University, Kanazawa, Japan). All cell lines were maintained in RPMI culture media supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS; Gibco-Life Technologies, Carlsbad, CA, USA) in a humidified incubator containing 5% CO2 at 37°C as previously described (10).

Ultracentrifugation exosome isolation

Exosomes were prepared from the supernatant of cancer cells using differential centrifugations as previously described (11). In brief, supernatant were harvested, centrifuged at 300 x g for 10 min to eliminate cells and at 16,500 x g for 20 min to remove cell debris and particles. Exosomes were pelleted by ultracentrifugation at 120,000 x g for 70 min (all steps were performed at 4°C). The exosome pellet was dissolved in nuclease free water and subsequently split and transferred to different RNase free tubes for RNA isolation. Each exosomal sample was harvested from 400 ml cell suspension with 1–4×106 cells/ml. Exosomes were then immediately lysed in respective lysing solution and continued for RNA purification.

Electron microscopy (EM)

EM imaging of exosome preparations were performed as follows. Briefly, the pellet from ultracentrifugation was suspended in 50 μl of PBS and vortexed briefly and then 5 μl was adsorbed to a carbon-coated grid that had been made hydrophilic by a 30-sec exposure to a glow discharge. Excess liquid was removed with filter paper, and the samples were stained with 0.75% uranylformate for 30 sec. After removing the excess uranylformate, the grid was examined in a Hitachi electron transmission microscope (H-7650).

Western blot analysis

Exosome samples were lysed in RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) with 1 mM PMSF and protease inhibitor cocktail on ice for 30 min, and then sonicated and quantified using the BCA protein assay kit (Beyotime Institute of Biotechnology, Nanjing, China). Protein fractions were separated by 10% SDS-PAGE and then were transferred to polyvinylidenedifluoride membranes (0.22 μm; Millipore, Billerica, MA, USA). In addition, the membranes blocked with 5% (w/v) skim milk powder in Tris-buffered saline with 0.05% (v/v) Tween-20 (TTBS) and incubate with mouse anti-TSG101 (1:1,000; Cell Signaling Technology) and mouse anti-Alix (1:1,000; Cell Signaling Technology) in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20) with 5% non-fat dried milk overnight at 4°C. In addition, they were incubated with horseradish peroxidase secondary antibody for 1 h at room temperature. Immunodetection was visualized using a chemiluminescent ECL reagent (Beyotime Institute of Biotechnology).

microRNA microarray chip analysis

Total RNA was isolated from exosomal samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. miRNAs of exosome releasing by two pairs of PCa cell lines were detected by microRNA microarrays chip analysis. Microarray analysis was performed on 5 μg of total RNA, miRNA expression profiling microarray was completed by using Agilent miRNA microarrays version 2.3 (Agilent Technologies, Santa Clara, CA, USA) and Agilent's miRNA Complete Labeling and Hyb kit (p/n 5190-0456) generates fluorescently labeled miRNAs with a sample input of 100 ng of total RNA. For identification of upregulated and downregulated miRNA, we calculated the miRNA expression ratio of PC3-TXR to PC3 or DU145-TXR to DU145 separately, and then we found the upregulated or downregulated miRNAs in the two pairs of cancer cell lines.

miRNA validation by quantitative real-time PCR (qRT-PCR)

To verify mature miRNA expression levels, qRT-PCR was performed using a High-Specificity qRT-PCR detection kit in conjunction with an ABI 7500 thermal cycler, according to the manufacturer's recommendations. qRT-PCR primer sequences are shown in Table I. We used U6 small nuclear RNA (U6 snRNA) as an endogenous control for normalization. The qRT-PCR results were expressed relative to miRNA expression levels at the threshold cycle (Ct) and were converted to fold changes (2−ΔΔCt).

Table I

qRT-PCR primer sequences of 12 miRNAs.

Table I

qRT-PCR primer sequences of 12 miRNAs.

miRNAAccessionmiRNA mature sequencesPrimer sequence
miR-32-5pMIMAT0000090 UAUUGCACAUUACUAAGUUGCA GTATTGCACATTACTAAGTTGC
miR-23cMIMAT0000418 AUCACAUUGCCAGUGAUUACCC GCATCACATTGCCAGTGATTAC
miR-3915MIMAT0018189 UUGAGGAAAAGAUGGUCUUAUU ACGTTGAGGAAAAGATGGTCT
miR-451aMIMAT0001631 AAACCGUUACCAUUACUGAGUU GCAAACCGTTACCATTACTGAG
miR-1204MIMAT0005868 UCGUGGCCUGGUCUCCAUUAU TCGTGGCCTGGTCTCCA
miR-3607-3pMIMAT0017985 ACUGUAAACGCUUUCUGAUG ACGACTGTAAACGCTTTCTG
miR-99bMIMAT0000689 CACCCGTAGAACCGACCTTGCG TCACCCGTAGAACCGACCT
miR-16-5pMIMAT0000069 UAGCAGCACGUAAAUAUUGGCG AGTAGCAGCACGTAAATATTG
miR-192-3pMIMAT0004543 CUGCCAAUUCCAUAGGUCACAG ACTGCCAATTCCATAGGTC
miR-429MIMAT0001536 UAAUACUGUCUGGUAAAACCGU CGTAATACTGTCTGGTAAAACCG
miR-141-3pMIMAT0000432 TAACACTGTCTGGTAAAGATGG ACTAACACTGTCTGGTAAAGATG
miR-3176MIMAT0015053 ACTGGCCTGGGACTACCGG CGACTGGCCTGGGACTAC
Bioinformatic analysis

Target genes of deregulated miRNAs were listed using DIANA-TarBase database v6.0, which include experimentally validated miRNA targets in the literature. To explore the potential function of the whole miRNAs and target genes signature, DIANA-mirPath, a web-based DNA Intelligent Analysis (DIANA)-miRPath v2.0 was used (http://www.microrna.gr/miRPathv2) to perform enrichment analysis of differentially expressed miRNA gene targets in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (12). The pathways were obtained with a P-value <0.05 and gene count. ‘PCa progression (hsa05215)’ in enriched KEGG pathways and related miRNAs were visualized by Cytoscape software (13).

Statistical analysis

Statistical testing was conducted with the assistance of the SPSS 13.0 software. All data are expressed as means ± standard deviation (SD). All miRNAs in exosome derived from drug-resistant PCa cells and their parental cells were compared using a t-test to define differentially expressed miRNAs. Results were considered significant when P-values were <0.05.

Results

Confirmation of exosomal vesicles isolated from PCa cell culture supernatant

The enrichment of typical exosome specific marker proteins such as Alix and TSG101 were assessed using western blot analysis (Fig. 1A). In addition, morphological analysis of the exosome using EM revealed a heterogeneous population of vesicles comprising round-shaped 40–100 nm diameter vesicles, consistent with exosome (Fig. 1B and C).

Altered miRNA expression between two pairs of cancer cells

To identify differentially expressed miRNA between two pairs of cancer cells, we used GenomeStudio software to select probe-expressed miRNAs (probe signals have significant differences in the background) and chose the miRNAs with the expression ratio (PC3-TXR to PC3 or DU145-TXR to DU145) >2.0 (as upregulated miRNA) or <0.5 (as downregulated miRNA). From the differentially expressed miRNAs, we selected 19 upregulated miRNAs (Table II) and 10 down-regulated miRNAs (Table III) which may contribute to PCa chemoresistance.

Table II

miRNAs upregulated in drug resistant cells compared to parent cells.

Table II

miRNAs upregulated in drug resistant cells compared to parent cells.

miRNA symbolPCT vs. PC (FC)Log FCDUT vs. DU (FC)Log FC
hsa-miR-16-5p2.056961.0405142.876451.524289
hsa-miR-203a4.694482.2309653.443011.78367
hsa-miR-32-5p2.269271.1822283.638241.863241
hsa-miR-515-3p2.138541.0966264.001832.00066
hsa-miR-99b-5p3.089521.6273833.040171.604152
hsa-miR-590-5p2.562551.357582.002991.002155
hsa-miR-451a2.94021.5559142.565171.359054
hsa-miR-12044.297632.1035412.365631.242224
hsa-miR-42914.469562.1601332.433541.283056
hsa-miR-36732.01151.0082726.231882.639667
hsa-miR-23c4.932212.3022342.417991.273808
hsa-miR-36545.799252.5358662.864581.518324
hsa-miR-3607-3p2.299861.20154684.68736.404074
hsa-miR-391513.09083.7104812.506031.325404
hsa-miR-4716-3p2.633671.3970752.780281.47523
hsa-miR-4722-5p2.079091.0559522.060611.043071
hsa-miR-488-3p4.929812.3015327.659262.937205
hsa-miR-46692.076311.0540222.580331.367556
hsa-miR-5004-5p2.110741.0777494.033212.011929

Table III

miRNAs downregulated in drug resistant cells compared to parent cells.

Table III

miRNAs downregulated in drug resistant cells compared to parent cells.

miRNA symbolPCT vs. PC (FC)Log FCDUT vs. DU (FC)Log FC
1hsa-miR-141-3p−2.881124063−1.526632−2.133284129−1.093076
2hsa-miR-429−17.98522167−4.16874−3.115885179−1.639642
3hsa-miR-192-5p−2.65166288−1.406897−2.436187999−1.284625
4hsa-miR-192-3p−5.965686275−2.576688−2.431876456−1.28207
5hsa-miR-606−2.791088936−1.480828−2.155900291−1.10829
6hsa-miR-3176−4.066972836−2.023955−2.471960338−1.305656
7 hsa-miR-1224-3p−2.073545435−1.0521−2.843015695−1.507422
8hsa-miR-381-3p−2.563924725−1.358354−2.769265562−1.469503
9hsa-miR-933−4.804709053−2.264449−2.477625641−1.308958
10hsa-miR-34b-3p−3.828528334−1.93679−2.636757694−1.398765
miRNA microarray results were validated by qRT-PCR

For validation using qRT-PCR, we chose 8 miRNAs that were upregulated and 4 miRNAs that were downregulated by at least 3 standard deviations in exosome derived from drug-resistant PCa cells compared to those of their parental cells. After normalization with U6 snRNA as the endogenous control, the qRT-PCR data confirmed significant expression (P<0.05) of all miRNAs in all samples except for miRNA-16 expression in the ratio of PC3-TXR to PC3 cells (Fig. 2).

KEGG pathway analysis

From the differentially expressed miRNAs, we selected 19 upregulated miRNAs (Table II) and 10 downregulated miRNAs (Table III) to predicte target genes using by DIANA-Tarbase v6.0 databases for target analysis (Table IV). Following the addition of upregulated miRNAs, the DIANA-miRPath v2.0 identified top 10 important signaling pathways (Table V) as significantly enriched (P<0.05). In addition to PCa and ErbB signaling pathway, insulin signaling pathway and PI3K-Akt signaling pathway were among the top 5 associated pathways (Table V). Similarly to the down-regulated miRNAs, significant enrichment was seen in top 10 important signaling pathways that were mainly influenced by 10 miRNAs (Table VI). Among these, the top 10 important pathways included those involved in focal adhesion, regulation of actin cytoskeleton, TGF-β signaling pathway, ubiquitin mediated proteolysis, ErbB signaling pathway (Table VI).

Table IV

Number of known targets of deregulated miRNAs from DIANA microT-CDS.

Table IV

Number of known targets of deregulated miRNAs from DIANA microT-CDS.

Upregulated miRNADownregulated miRNA


miRNA symbolNumber of target genesmiRNA symbolNumber of target genes
1hsa-miR-16-5p419hsa-miR-141-3p308
2hsa-miR-203a355hsa-mi-429148
3hsa-miR-32-5p289hsa-miR-192-5p39
4hsa-miR-515-3p58hsa-miR-192-3p43
5hsa-miR-99b-5p10hsa-miR-60676
6hsa-miR-590-5p115hsa-miR-317624
7hsa-miR-451a9 hsa-miR-1224-3p46
8hsa-miR-12042hsa-miR-381-3p268
9hsa-miR-4291121hsa-miR-9333
10hsa-miR-3673225hsa-miR-34b-3p118
11hsa-miR-23c192
12hsa-miR-365439
13 hsa-miR-3607-3p304
14hsa-miR-3915101
15 hsa-miR-4716-3p11
16 hsa-miR-4722-5p73
17hsa-miR-488-3p117
18hsa-miR-46692
19 hsa-miR-5004-5p43

Table V

Top 10 important pathways of upregulated miRNA in drug resistant cells from DIANA miRPath v.2.0.

Table V

Top 10 important pathways of upregulated miRNA in drug resistant cells from DIANA miRPath v.2.0.

KEGG pathwayP-valueGenesmiRNAs
1ErbB signaling pathway (hsa04012)3.40E-234518
2Long-term potentiation (hsa04720)2.63E-223516
3Insulin signaling pathway (hsa04910)4.36E-205917
4Prostate cancer (hsa05215)1.52E-194115
5PI3K-Akt signaling pathway (hsa04151)1.93E-1912117
6Wnt signaling pathway (hsa04310)1.86E-186616
7mTOR signaling pathway (hsa04150)5.12E-183215
8Focal adhesion (hsa04510)2.89E-177817
9Regulation of actin cytoskeleton (hsa04810)2.73E-157917
10Neurotrophin signaling pathway (hsa04722)1.23E-135017

Table VI

TOP 10 important pathways of downregulated miRNA in drug resistant cell from DIANA miRPath v.2.0.

Table VI

TOP 10 important pathways of downregulated miRNA in drug resistant cell from DIANA miRPath v.2.0.

KEGG pathwayP-valueGenesmiRNAs
1Focal adhesion (hsa04510)1.91E-16618
2Regulation of actin cytoskeleton (hsa04810)2.71E-15629
3TGF-β signaling pathway (hsa04350)1.76E-14307
4Ubiquitin mediated proteolysis (hsa04120)1.76E-14449
5ErbB signaling pathway (hsa04012)1.65E-13318
6Axon guidance (hsa04360)1.30E-10398
7Gap junction (hsa04540)2.05E-10319
8Prostate cancer (hsa05215)2.50E-10288
9Pathways in cancer (hsa05200)2.50E-10869
10PI3K-Akt signaling pathway (hsa04151)4.73E-09789
miRNA regulated gene networks associated with PCa chemoresistance

The pathway of ‘PCa progression (hsa05215)’ category from enriched KEGG pathways was selected for further investigation of the miRNA regulatory networks in PCa (data not shown). Based on these data, the overlapping parts of tow pathways were visualized by Cytoscape software (Fig. 3). We determined hub AR and PTEN target genes mainly regulated by upregulated miRNAs (hsa-miR-16-5p, -23c, -32-5p, -3915, -5004-5p, -488-3p, -3673 and -3654) and downregulated miRNAs (hsa-miR-3176 and -141-3p). Additionally, hub TCF4 target genes mainly regulated by upregulated miRNAs (hsa-miR-32-5p) and downregulated miRNAs (hsa-miR-141-3p, -606, -381 and -429).

Discussion

miRNAs are a kind of regulatory RNAs that function primarily by targeting specific mRNAs for degradation or inhibition of translation and, thus, decrease the expression of the target protein, and their role in tumor development would be through the regulation of their target protein genes (14). Many studies have shown that miRNAs tend to be expressed abnormally in tumor tissues and cells (15,16). miRNAs function as tumor inhibiting or cancerogenic factors in the development of tumors and also have extensive application value for diagnosing and predicting the prognosis of tumors.

Exosomes are topology identical to that of a cell and contain a broad array of biologically active material including proteins, nucleotides, deoxynucleotides and non-coding miRNAs (17). Emerging evidence indicates that exosomes play a key role in tumor-host crosstalk, and exosome secretion, composition, and functional capacity are altered as tumors progress to an aggressive phenotype. In addition to transmitting signals to other cancer cells, the exosomes released by cancer cells can also impact tumor cell growth, metastasis and angiogenesis and generating the cancer microenvironment (18,19).

In this study, we used the microarray technology to search for miRNA with abnormal expression in exosomes released by PTX resistant PCa cells and their parental cells. We identified and selected 29 miRNAs differentially expressed in two kinds of cells, and this expression profiling might provide a useful clue for indepth research of PCa. In the present study, miR-203 was highly expressed in exosome derived from chemoresistence PCa cells. Recently, a study found that miR-203 is upregulated in three oxaliplatin (L-OHP)-resistant metastatic colorectal cancer cell (CRC) lines and induces L-OHP resistance in CRCs by negatively regulating ataxia telangiectasia mutated kinase (20). Furthermore, miR-203 promote cisplatin resistance through suppression of the suppressor of cytokine signaling 3 (21). miR-451 in this study following qPCR validation demonstrated a significant change in exosome derived from chemoresistance PCa cells as compared to their parental cells. In contrast, miR-451 was downregulated in docetaxel-resistant lung adenocarcinoma cells (22). However, miR-451 was upregulated in multidrug-resistant (MDR) human ovarian cancer cell line (A2780DX5) and human cervix carcinoma cell line (KB-3-1), compared with their parental lines. Inversely, treatment of A2780DX5 cells with the antagomirs of miR-451 decreased the expression of P-glycoprotein and MDR1 mRNA (23). Additionally, miR-23a was also upregulated in this study. Inhibition of miR-23a expression increases the sensitivity of drug-resistant ovarian cancer cells to cisplatin possibly by miR-23a targeting genes that causes inhibition of P-gp protein expression (24).

In the present study, we observed that expression of 10 miRNAs decreased significantly in the exosome derived from chemoresistance prostate cancer cells compared to their parental cells. Downregulated expression of miR-141 plays a role in selective resistance to L-OHP and epithelial-mesenchymal transition (EMT) in CRCs during repeated treatments with L-OHP (25). miR-429 were downregulated in drug- resistant epithelial ovarian cancer tissues (26). Interestingly, overexpression of miR-429 in ovarian cancer cells (OCI-984) induced morphological, functional and molecular changes consistent with EMT and a concomitant significant increase in the sensitivity of the converted cells to cisplatin (27). In addition, miR-429 upregulation induced apoptosis and suppressed invasion by targeting Bcl-2 and SP-1 in esophageal carcinoma (28). Hence, miR-429 may be a potential cancer therapeutic target. miR-381 were strongly downregulated in MDR cells (K562/ADM) by targeting the 3′-UTR of the MDR1 gene and restoring expression of miR-381 in K562/ADM cells was correlated with reduced expression of the MDR1 gene and its protein product, P-gp and increased drug uptake by the cells (29). Additionally, the ubiquitin-specific protease 2a (USP2a) induced drug resistance in PCa cells, and inhibition of miR-34b made USP2aWT cells trigger drug resistance via miR-34b-driven c-Myc regulation (30).

Several pathways appeared to be enriched by the upregulated miRNAs. Among 10 important pathways, the pathway which associated with ErbB signaling pathway was found to be influenced by 18 miRNAs, which was predicted to target 45 genes. Homo- or heterodimerization of ErbB receptors activate multiple downstream signaling pathways, which are critically involved in multiple biological consequences and thereby promote tumor initiation and progression. Genetic and/or epigenetic alterations of ErbB pathway genes were detected in 80% of adenocarcinomas (31). Such as the AKT/ERK signaling pathway, which is the pathway downstream of ErbB, was predicted to be active in taxanes-resistant gastric cancer cell lines (32). In addition, the activation of ErbB3 was mainly through PI-3K/Akt signaling playing a vital role in the progression of castration-resistant PCa into docetaxel-resistance (33).

Similarly, top 10 important signaling pathways of the downregulated miRNAs were noted. Focal adhesion and the regulation of actin cytoskeleton were among the top 5 associated pathways. Notably, these pathways have been implicated in PCa as suggested by other authors. It has been reported that focal adhesion kinase (FAK) could promote the growth, survival, migration, metastasis and androgen-independence of prostate tumors in vitro and in vivo through the activation of major oncogenic pathways (34). More importantly, it was reported that a potential clinical niche for FAK tyrosine kinase inhibitor (TKI) may be used in patients with PCa to overcome chemoresistance because cotreatment with FAK TKI and docetaxel resulted in an additive attenuation of FAK and Akt phosphorylation and overcame the chemoresistant phenotype in PCa PC3 and DU-145 cells (35). Interestingly, FAK inhibition with VS-6063 overcame YB-1-mediated PTX resistance by an AKT-dependent pathway (36) in ovarian cancer. Additionally, the acquisition of drug resistance in ovarian cancer cells induced an extensive reorganization of the actin cytoskeleton, which governed the cellular mechanical properties, motility, and possibly intracellular drug transportation (37).

PCa development is driven by aberrant androgen signaling via the androgen receptor (AR) activity for growth promotion and apoptosis inhibition. Evidence established that taxane stabilization of microtubules inhibits the AR translocation into the nucleus, thus, preventing the transcriptional activity of AR (38). Additionally, taxanes lead to an increase in forkhead box O (FOXO)1, a transcriptional repressor of AR, consequently resulting in inhibition of ligand-dependent and ligand-independent transcription (39). In this study, several miRNA was overexpressed in exosome of taxanes-resistant prostate cancer cells which might be induced by taxanes and target the AR gene to inhibit its activity. However, PC3 and DU145 are of androgen-independent cell lines, known to possess low AR levels (40). Therefore, our findings may indicate the existence of an AR-independent pathway that may be associated with castration PCa. Most importantly, phosphatase and tensin homolog (PTEN) and T-cell factors/lymphoid enhancer-binding factors (TCFs) were regulated by several miRNAs in the present study. PTEN is a tumor-suppressor gene. Deletion of PTEN frequently resulted in tumorigenesis, including primary glioblastomas, breast and lung cancer (41,42). It was shown that chemoresistance is associated with Beclin-1 and PTEN expression in epithelial ovarian cancers. The status of the functional PTEN/FOXO pathway and the drug bioavailability may be the two key determinants for taxol chemoresistance of CRPC in the clinic (43). In the present study, at least 5 upregulated miRNAs and 2 downregulated miRNAs were regulated by the PTEN gene. Our hypothesis is that PTEN is one of the important genes regulated by several upregulated miRNAs via exosomes secreted by PTX-resistant PCa cells in tumor microenvironment. TCF4 are a major class of transcription factors that control the nuclear response to Wnt/β-catenin signaling. Some studies indicated that TCF4 functions to promote cellular proliferation (44,45). TCF4 silencing sensitizes the CRC line to L-OHP as a common chemotherapeutic drug (46). Hereby, TCF4 was related to hsa-miR-606, -381 and -429. In addition, several downregulated miRNAs could promote TCF4 expression. We proposed that TCF4 is one of the important factors regulated by exosomes in tumor micro-environment. However, the exact regulatory networks remain elusive, and it is hard to estimate the actual false-positive rate of bioinformatics tools. Therefore, further experimental studies should be carried out in order to confirm our results.

In conclusion, the present study provided novel information that might contribute to a better understanding of molecular mechanisms as well as biological pathways implicated in progressive PCa chemoresistance. This study showed 29 differentially expressed miRNA that bear the potential molecular marker of insensitive PCa cells through targeting known genes to regulate the pathogenesis of PCa. Particularly, hub genes and miRNAs of our constructed network might be central actors of molecular alterations in PCa. Further bench works are needed to confirm their exact roles.

Acknowledgements

The present study was supported by the Natural Science Foundation of China (NSFC) Key Project (81130046); the NSFC (81171993; 81272415; 81560505); the Guangxi Projects of China (2013GXNSFEA053004; 2012GXNSFCB053004; 2013GX NSF BA019177; 13550 0 4 -5; 201201Z D0 0 4; GZPT13-35; 14122008-22; 11-031-05-K2; KY2015YB057; 14-045-12-K2). The authors thank Drs Jiejun Fu and Chunlin Zou for helpful discussions and Ms. Xin Huang for editing.

References

1 

Fendler A, Jung M, Stephan C, Honey RJ, Stewart RJ, Pace KT, Erbersdobler A, Samaan S, Jung K and Yousef GM: miRNAs can predict prostate cancer biochemical relapse and are involved in tumor progression. Int J Oncol. 39:1183–1192. 2011.PubMed/NCBI

2 

van Brussel JP and Mickisch GH: Multidrug resistance in prostate cancer. Onkologie. 26:175–181. 2003.PubMed/NCBI

3 

Tannock IF, de Wit R, Berry WR, Horti J, Pluzanska A, Chi KN, Oudard S, Théodore C, James ND, Turesson I, et al; TAX 327 Investigators. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. N Engl J Med. 351:1502–1512. 2004. View Article : Google Scholar : PubMed/NCBI

4 

Simpson RJ, Lim JW, Moritz RL and Mathivanan S: Exosomes: Proteomic insights and diagnostic potential. Expert Rev Proteomics. 6:267–283. 2009. View Article : Google Scholar : PubMed/NCBI

5 

Camussi G, Deregibus MC, Bruno S, Cantaluppi V and Biancone L: Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney Int. 78:838–848. 2010. View Article : Google Scholar : PubMed/NCBI

6 

An T, Qin S, Xu Y, Tang Y, Huang Y, Situ B, Inal JM and Zheng L: Exosomes serve as tumour markers for personalized diagnostics owing to their important role in cancer metastasis. J Extracell Vesicles. 4:275222015. View Article : Google Scholar : PubMed/NCBI

7 

Zhang J, Li S, Li L, Li M, Guo C, Yao J and Mi S: Exosome and exosomal microRNA: Trafficking, sorting, and function. Genomics Proteomics Bioinformatics. 13:17–24. 2015. View Article : Google Scholar : PubMed/NCBI

8 

Hannafon BN, Carpenter KJ, Berry WL, Janknecht R, Dooley WC and Ding WQ: Exosome-mediated microRNA signaling from breast cancer cells is altered by the anti-angiogenesis agent docosahexaenoic acid (DHA). Mol Cancer. 14:1332015. View Article : Google Scholar : PubMed/NCBI

9 

Yang M, Chen J, Su F, Yu B, Su F, Lin L, Liu Y, Huang JD and Song E: Microvesicles secreted by macrophages shuttle invasion-potentiating microRNAs into breast cancer cells. Mol Cancer. 10:1172011. View Article : Google Scholar : PubMed/NCBI

10 

Li F, Danquah M, Singh S, Wu H and Mahato RI: Paclitaxel- and lapatinib-loaded lipopolymer micelles overcome multidrug resistance in prostate cancer. Drug Deliv Transl Res. 1:420–428. 2011. View Article : Google Scholar : PubMed/NCBI

11 

Lässer C, Eldh M and Lötvall J: Isolation and characterization of RNA-containing exosomes. J Vis Exp. 9:e30372012.

12 

Vlachos IS, Kostoulas N, Vergoulis T, Georgakilas G, Reczko M, Maragkakis M, Paraskevopoulou MD, Prionidis K, Dalamagas T and Hatzigeorgiou AG: DIANA miRPath v.2.0: Investigating the combinatorial effect of microRNAs in pathways. Nucleic Acids Res. 40(W1): W498–504. 2012. View Article : Google Scholar : PubMed/NCBI

13 

Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B and Ideker T: Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res. 13:2498–2504. 2003. View Article : Google Scholar : PubMed/NCBI

14 

Bian Z, Li LM, Tang R, Hou DX, Chen X, Zhang CY and Zen K: Identification of mouse liver mitochondria-associated miRNAs and their potential biological functions. Cell Res. 20:1076–1078. 2010. View Article : Google Scholar : PubMed/NCBI

15 

Zhang C, Wang C, Chen X, Yang C, Li K, Wang J, Dai J, Hu Z, Zhou X, Chen L, et al: Expression profile of microRNAs in serum: A fingerprint for esophageal squamous cell carcinoma. Clin Chem. 56:1871–1879. 2010. View Article : Google Scholar : PubMed/NCBI

16 

Iorio MV and Croce CM: microRNA involvement in human cancer. Carcinogenesis. 33:1126–1133. 2012. View Article : Google Scholar : PubMed/NCBI

17 

Makino DL, Halbach F and Conti E: The RNA exosome and proteasome: Common principles of degradation control. Nat Rev Mol Cell Biol. 14:654–660. 2013. View Article : Google Scholar : PubMed/NCBI

18 

Janowska-Wieczorek A, Wysoczynski M, Kijowski J, Marquez-Curtis L, Machalinski B, Ratajczak J and Ratajczak MZ: Microvesicles derived from activated platelets induce metastasis and angiogenesis in lung cancer. Int J Cancer. 113:752–760. 2005. View Article : Google Scholar

19 

Kruger S, Abd Elmageed ZY, Hawke DH, Wörner PM, Jansen DA, Abdel-Mageed AB, Alt EU and Izadpanah R: Molecular characterization of exosome-like vesicles from breast cancer cells. BMC Cancer. 14:442014. View Article : Google Scholar : PubMed/NCBI

20 

Zhou Y, Wan G, Spizzo R, Ivan C, Mathur R, Hu X, Ye X, Lu J, Fan F, Xia L, et al: miR-203 induces oxaliplatin resistance in colorectal cancer cells by negatively regulating ATM kinase. Mol Oncol. 8:83–92. 2014. View Article : Google Scholar :

21 

Ru P, Steele R, Hsueh EC and Ray RB: Anti-miR-203 upregulates SOCS3 expression in breast cancer cells and enhances cisplatin chemosensitivity. Genes Cancer. 2:720–727. 2011. View Article : Google Scholar : PubMed/NCBI

22 

Wang R, Chen DQ, Huang JY, Zhang K, Feng B, Pan BZ, Chen J, De W and Chen LB: Acquisition of radioresistance in docetaxel-resistant human lung adenocarcinoma cells is linked with dysregulation of miR-451/c-Myc-survivin/rad-51 signaling. Oncotarget. 5:6113–6129. 2014. View Article : Google Scholar : PubMed/NCBI

23 

Zhu H, Wu H, Liu X, Evans BR, Medina DJ, Liu CG and Yang JM: Role of MicroRNA miR-27a and miR-451 in the regulation of MDR1/P-glycoprotein expression in human cancer cells. Biochem Pharmacol. 76:582–588. 2008. View Article : Google Scholar : PubMed/NCBI

24 

Jin AH, Zhou XP and Zhou FZ: Inhibition of microRNA-23a increases cisplatin sensitivity of ovarian cancer cells: The possible molecular mechanisms. Nan Fang Yi Ke Da Xue Xue Bao. 35:125–128. 2015.(In Chinese). PubMed/NCBI

25 

Tanaka S, Hosokawa M, Yonezawa T, Hayashi W, Ueda K and Iwakawa S: Induction of epithelial-mesenchymal transition and down-regulation of miR-200c and miR-141 in oxaliplatin-resistant colorectal cancer cells. Biol Pharm Bull. 38:435–440. 2015. View Article : Google Scholar : PubMed/NCBI

26 

Liu L, Zou J, Wang Q, Yin FQ, Zhang W and Li L: Novel microRNAs expression of patients with chemotherapy drug-resistant and chemotherapy-sensitive epithelial ovarian cancer. Tumour Biol. 35:7713–7717. 2014. View Article : Google Scholar : PubMed/NCBI

27 

Wang L, Mezencev R, Švajdler M, Benigno BB and McDonald JF: Ectopic over-expression of miR-429 induces mesenchymal-to-epithelial transition (MET) and increased drug sensitivity in metastasizing ovarian cancer cells. Gynecol Oncol. 134:96–103. 2014. View Article : Google Scholar : PubMed/NCBI

28 

Liu D, Xia P, Diao D, Cheng Y, Zhang H, Yuan D, Huang C and Dang C: MiRNA-429 suppresses the growth of gastric cancer cells in vitro. J Biomed Res. 26:389–393. 2012. View Article : Google Scholar

29 

Xu Y, Ohms SJ, Li Z, Wang Q, Gong G, Hu Y, Mao Z, Shannon MF and Fan JY: Changes in the expression of miR-381 and miR-495 are inversely associated with the expression of the MDR1 gene and development of multi-drug resistance. PLoS One. 8:e820622013. View Article : Google Scholar : PubMed/NCBI

30 

Benassi B, Marani M, Loda M and Blandino G: USP2a alters chemotherapeutic response by modulating redox. Cell Death Dis. 4:e8122013. View Article : Google Scholar : PubMed/NCBI

31 

Hoque MO, Brait M, Rosenbaum E, Poeta ML, Pal P, Begum S, Dasgupta S, Carvalho AL, Ahrendt SA, Westra WH, et al: Genetic and epigenetic analysis of erbB signaling pathway genes in lung cancer. J Thorac Oncol. 5:1887–1893. 2010. View Article : Google Scholar : PubMed/NCBI

32 

Wu G, Qin XQ, Guo JJ, Li TY and Chen JH: AKT/ERK activation is associated with gastric cancer cell resistance to paclitaxel. Int J Clin Exp Pathol. 7:1449–1458. 2014.PubMed/NCBI

33 

Jathal MK, Chen L, Mudryj M and Ghosh PM: Targeting ErbB3: the new RTK(id) on the prostate cancer block. Immunol Endocr Metab Agents Med Chem. 11:131–149. 2011. View Article : Google Scholar : PubMed/NCBI

34 

Figel S and Gelman IH: Focal adhesion kinase controls prostate cancer progression via intrinsic kinase and scaffolding functions. Anticancer Agents Med Chem. 11:607–616. 2011. View Article : Google Scholar : PubMed/NCBI

35 

Lee BY, Hochgräfe F, Lin HM, Castillo L, Wu J, Raftery MJ, Martin Shreeve S, Horvath LG and Daly RJ: Phosphoproteomic profiling identifies focal adhesion kinase as a mediator of docetaxel resistance in castrate-resistant prostate cancer. Mol Cancer Ther. 13:190–201. 2014. View Article : Google Scholar

36 

Kang Y, Hu W, Ivan C, Dalton HJ, Miyake T, Pecot CV, Zand B, Liu T, Huang J, Jennings NB, et al: Role of focal adhesion kinase in regulating YB-1-mediated paclitaxel resistance in ovarian cancer. J Natl Cancer Inst. 105:1485–1495. 2013. View Article : Google Scholar : PubMed/NCBI

37 

Seo YH, Jo YN, Oh YJ and Park S: Nano-mechanical reinforcement in drug-resistant ovarian cancer cells. Biol Pharm Bull. 38:389–395. 2015. View Article : Google Scholar : PubMed/NCBI

38 

Darshan MS, Loftus MS, Thadani-Mulero M, Levy BP, Escuin D, Zhou XK, Gjyrezi A, Chanel-Vos C, Shen R, Tagawa ST, et al: Taxane-induced blockade to nuclear accumulation of the androgen receptor predicts clinical responses in metastatic prostate cancer. Cancer Res. 71:6019–6029. 2011. View Article : Google Scholar : PubMed/NCBI

39 

Gan L, Chen S, Wang Y, Watahiki A, Bohrer L, Sun Z, Wang Y and Huang H: Inhibition of the androgen receptor as a novel mechanism of taxol chemotherapy in prostate cancer. Cancer Res. 69:8386–8394. 2009. View Article : Google Scholar : PubMed/NCBI

40 

Alimirah F, Chen J, Basrawala Z, Xin H and Choubey D: DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: Implications for the androgen receptor functions and regulation. FEBS Lett. 580:2294–2300. 2006. View Article : Google Scholar : PubMed/NCBI

41 

Mizoguchi M, Nutt CL, Mohapatra G and Louis DN: Genetic alterations of phosphoinositide 3-kinase subunit genes in human glioblastomas. Brain Pathol. 14:372–377. 2004. View Article : Google Scholar : PubMed/NCBI

42 

Kohno T, Takahashi M, Manda R and Yokota J: Inactivation of the PTEN/MMAC1/TEP1 gene in human lung cancers. Genes Chromosomes Cancer. 22:152–156. 1998. View Article : Google Scholar : PubMed/NCBI

43 

Jiang J and Huang H: Targeting the Androgen Receptor by taxol in castration-resistant prostate cancer. Mol Cell Pharmacol. 2:1–5. 2010.PubMed/NCBI

44 

van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I, Hurlstone A, van der Horn K, Batlle E, Coudreuse D, Haramis AP, et al: The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell. 111:241–250. 2002. View Article : Google Scholar : PubMed/NCBI

45 

Shin HW, Choi H, So D, Kim YI, Cho K4, Chung HJ5, Lee KH6, Chun YS7, Cho CH1, Kang GH, et al: ITF2 prevents activation of the beta-catenin-TCF4 complex in colon cancer cells and levels decrease with tumor progression. Gastroenterology. 147:430–442.e438. 2014. View Article : Google Scholar

46 

Gheidari F, Bakhshandeh B, Teimoori-Toolabi L, Mehrtash A, Ghadir M and Zeinali S: TCF4 silencing sensitizes the colon cancer cell line to oxaliplatin as a common chemotherapeutic drug. Anticancer Drugs. 25:908–916. 2014. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

August-2016
Volume 49 Issue 2

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Li J, Yang X, Guan H, Mizokami A, Keller ET, Xu X, Liu X, Tan J, Hu L, Lu Y, Lu Y, et al: Exosome-derived microRNAs contribute to prostate cancer chemoresistance. Int J Oncol 49: 838-846, 2016
APA
Li, J., Yang, X., Guan, H., Mizokami, A., Keller, E.T., Xu, X. ... Zhang, J. (2016). Exosome-derived microRNAs contribute to prostate cancer chemoresistance. International Journal of Oncology, 49, 838-846. https://doi.org/10.3892/ijo.2016.3560
MLA
Li, J., Yang, X., Guan, H., Mizokami, A., Keller, E. T., Xu, X., Liu, X., Tan, J., Hu, L., Lu, Y., Zhang, J."Exosome-derived microRNAs contribute to prostate cancer chemoresistance". International Journal of Oncology 49.2 (2016): 838-846.
Chicago
Li, J., Yang, X., Guan, H., Mizokami, A., Keller, E. T., Xu, X., Liu, X., Tan, J., Hu, L., Lu, Y., Zhang, J."Exosome-derived microRNAs contribute to prostate cancer chemoresistance". International Journal of Oncology 49, no. 2 (2016): 838-846. https://doi.org/10.3892/ijo.2016.3560