MiR-148a Attenuates Paclitaxel Resistance of Hormone-refractory, Drug-resistant Prostate Cancer PC3 Cells by Regulating MSK1 Expression

Abstract

MicroRNAs are involved in cancer pathogenesis and act as tumor suppressors or oncogenes. It has been recently reported that miR-148a expression is down-regulated in several types of cancer. The functional roles and target genes of miR-148a in prostate cancer, however, remain unknown. In this report, we showed that miR-148a expression levels were lower in PC3 and DU145 hormone-refractory prostate cancer cells in comparison to PrEC normal human prostate epithelial cells and LNCaP hormone-sensitive prostate cancer cells. Transfection with miR-148a precursor inhibited cell growth, and cell migration and invasion, and increased the sensitivity to anti-cancer drug paclitaxel in PC3 cells. Computer-aided algorithms predicted mitogen- and stress-activated protein kinase, MSK1, as a potential target of miR-148a. Indeed, miR-148a overexpression decreased expression of MSK1. Using luciferase reporter assays, we identified MSK1 as a direct target of miR-148a. Suppression of MSK1 expression by siRNA, however, showed little or no effects on malignant phenotypes of PC3 cells. In PC3PR cells, a paclitaxel-resistant cell line established from PC3 cells, miR-148a inhibited cell growth, and cell migration and invasion, and also attenuated the resistance to paclitaxel. MiR-148a reduced MSK1 expression by directly targeting its 3′-UTR in PC3PR cells. Furthermore, MSK1 knockdown reduced paclitaxel-resistance of PC3PR cells, indicating that miR-148a attenuates paclitaxel-resistance of hormone-refractory, drug-resistant PC3PR cells in part by regulating MSK1 expression. Our findings suggest that miR-148a plays multiple roles as a tumor suppressor and can be a promising therapeutic target for hormone-refractory prostate cancer especially for drug-resistant prostate cancer.

Introduction

MicroRNAs (miRNAs)² are small non-coding RNAs composed of about 22–24 nucleotides and control protein expression through translational inhibition or mRNA degradation by binding to the 3′-untranslated region (3′-UTR) of target mRNAs (1). miRNAs regulate a number of biological processes such as development, proliferation, differentiation, and apoptosis. Aberrant expression of miRNA has been reported in a variety of cancers, some of which have been shown to act as tumor suppressors or oncogenes (2).

MiR-148a expression is down-regulated in human breast cancer and undifferentiated gastric cancer (3, 4). DNA methylation-associated silencing of miR-148 expression is identified in human cancer cell lines established from lymph node metastasis of colon, melanoma, and head and neck cancer, suggesting its role for the development of metastasis (5). Direct targets of miR-148a so far reported include transcription growth factor-β-induced factor 2 (TGIF2), DNA (cytosine-5-)-methyltransferase 3β (DNMT3b) and pregnane X receptor (PXR) (5–7). However, the functional roles and target genes of miR-148a in prostate cancer have not yet been documented.

Mitogen- and stress-activated kinase 1 (MSK1), also known as ribosomal protein S6 kinase, 90kDa, polypeptide 5 (RPS6KA5), is a serine/threonine kinase that serves as a downstream target of extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein kinase in response to various stimuli including epidermal growth factor (EGF), phorbol ester (TPA), UV-irradiation, and anisomycin (8–11). Activated MSK1 phosphorylates chromatin-related proteins such as histone H3 and HMG-14 and transcription factors such as CREB, ATF1, NF-κB, and ER81 (8, 11–15). MSK1 is also involved in transcriptional activation of genes including immediate early genes c-fos and c-jun and inflammatory gene IL-6 (16–23). Relevant to cancer is the report that MSK1 is required for tumor promoter-induced cell transformation of JB6 Cl41 mouse epidermal skin cells (24). The detailed mechanisms underlying regulation of MSK1 expression and its functional roles in cancer are poorly understood.

Most patients with prostate cancer initially respond to androgen ablation, but eventually the tumor becomes refractory to the treatment. Although patients with hormone-refractory prostate cancer are usually treated with taxane anti-cancer drugs such as docetaxel and paclitaxel, the clinical outcome is not satisfactory. There is an urgent need to develop novel treatments to enhance the chemosensitivity in patients with hormone-refractory, drug-resistant prostate cancer.

In the present study, we demonstrated that miR-148a is down-regulated in hormone-refractory prostate cancer cells compared with normal and hormone-sensitive cancer cells. Then, we investigated effects of ectopic expression of miR-148a on malignant phenotypes of hormone-refractory prostate cancer PC3 cells and found that miR-148a plays multiple tumor suppressive roles. We also identified MSK1 as a direct target of miR-148a, which, however, did not mediate miR-148a effects in PC3 cells. In paclitaxel-resistant PC3 cells, miR-148a inhibited malignant phenotypes including paclitaxel -resistance and reduced MSK1 expression via acting on its 3′-UTR. Finally, we proved that miR-148 attenuates paclitaxel-resistance by regulating MSK1 expression. We thus provided evidence that miR-148a has potential as a novel therapeutic target for treatment of hormone-refractory prostate cancer especially for drug-resistant prostate cancer.

EXPERIMENTAL PROCEDURES

Cell Culture

PrEC normal human prostate epithelial cells were obtained from Clonetics and cultured as recommended by the supplier. LNCaP, PC3, and DU145 human prostate cancer cells were purchased from ATCC and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Paclitaxel-resistant cancer cells, PC3PR, were established from parental PC3 cells by stepwise increases of paclitaxel in the culture medium. First, PC3 cells were incubated with 5 nm of paclitaxel for 2 days. Then, the culture medium was changed to fresh medium without paclitaxel. After several passages with 5 nm paclitaxel, cells were exposed to 10 nm paclitaxel. The same procedure was repeated until resistant cells viable at 500 nm paclitaxel were obtained. HEK293 human embryonic kidney cells were obtained from ATCC and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.

RNA Extraction and Quantitative Reverse Transcriptase-PCR

Total RNA containing miRNA was extracted using the miRNeasy Mini kit (Qiagen, Hilden, Germany). Target miRNA was reverse transcribed to cDNA by a gene-specific RT primer using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). TaqMan MicroRNA Assay was performed with Premix Ex Taq (Takara, Shiga, Japan) using the Thermal Cycler Dice Real Time System (Takara). The relative quantification value of the target, normalized to a control, was calculated by the comparative Ct methods. 18 S rRNA was used as an internal control gene. To determine the MDR1 mRNA levels, semi-quantitative RT-PCR was performed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) and Takara Taq (Takara).

Western Blot Analysis

Whole cell lysates were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking with 5% skimmed milk for 1 h, membranes were incubated overnight at 4 °C with a primary antibody in TBS-T and then reacted with a horseradish peroxidase-conjugated secondary antibody for 1 h. Immunoreactive proteins were detected with the ECL Plus Western blotting Detection System (GE Healthcare, Waukesha, WI) and visualized using the LAS-4000 Lumino-Image Analyzer (Fujifilm, Tokyo, Japan). Antibodies against MSK1 and MDR1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-actin was obtained from Sigma-Aldrich.

Transfection

Cells seeded onto 6-well plates were transfected with hsa-miR-148a precursor (Ambion, Austin, TX) or siGENOME SMARTpool Human MSK1 (Thermo Scientific Dharmacon, Waltham, MA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and cultured in Opti-MEM I reduced serum medium for 8 h. Then, culture medium was changed to normal medium. As a negative control, Pre-miR miRNA precursor negative control 1 and 2 (Ambion) or Stealth RNAi Negative Control Medium GC Duplex (Invitrogen) was used.

Cell Growth Assay

Transfected cells were re-seeded onto 12-well plates at 5 × 10⁴ cells/well and cultured for 5 days. The number of viable cells was determined using the trypan blue dye exclusion assay.

Colony Formation Assay

Transfected cells were re-seeded onto 6-well plates at 400 cells/well. Two weeks later, colonies were fixed with 100% methanol for 15 min and stained with crystal violet for 20 min. After taking photographs, the number of colonies with diameter more than 1.5 mm was counted.

Cell Migration Assay

Transfected cells in 6-well plates were cultured until cells reached confluency. Cell layers were wounded using a 1-ml tip and cultured for 24 h. Photographs were taken at time 0 and 24 h, and the wound area was determined using Photoshop software. The area of cell migration was determined by subtracting the area free of cells at 24 h from that at 0 h.

Cell Invasion Assay

Transfected cells were re-seeded onto Matrigel Invasion Chamber (Becton Dickinson, Franklin Lakes, NJ) at 1 × 10⁵ cells/well. Twenty-four hours later, cells invaded through the Matrigel membrane were fixed with 100% methanol and stained with crystal violet. After taking photographs, the number of invaded cells was counted.

Luciferase Reporter Assay

The 3′-UTR of human MSK1 gene was amplified by PCR using the following primers: forward; 5′-GATCGAGCTCGCATGGTAGGAGTGTATCAG-3′, reverse; 5′-GATCACGCGTGAAATGTTTTCATCACATAG-3′. SacI and Mlu I sites (underlined) were introduced to the primers for subcloning. PCR products were digested with these restriction enzymes and then cloned into the multi-cloning sites of the pMIR-REPORT miRNA expression reporter plasmid (Ambion), obtaining the MSK1 3′-UTR WT firefly luciferase reporter gene. To introduce mutations into the seed sequences of all three predicted miR-148a target sites within the MSK1 3′-UTR and generate the MSK1 3′-UTR MT reporter gene, overlap extension PCR was performed using above-mentioned primers and the following primers: forward; 5′-TGTTTTAAGTAATATTGCACTTTATAAAAAAGTATGAATAAAGCAAACTATTTTATAAAGAATCGGGTTTAAAGCATTAATCGGGTATTTTTGCC-3′, reverse; 5′-TGCAATATTACTTAAAACAGAGAGACATGTACCTTAATATTCTGCAATAACCATTAAAACAAATTGCCAGGAAAGAGCCCGATTAAATTAAAAAC. The mutated DNA sequences were underlined. Similarly, the MSK1 3′-UTR MT1, MT2, and MT3 reporter genes with mutations at each of three potential binding sites (at positions 651–673, 776–798, and 794–816, respectively) were constructed by overlap extension PCR. The primers used were as follows: MT1 forward; 5′-CTAAGCTATGTGTATGTTTTTAATTTAATCGGGCTCTTTCCTGGC-3′, MT1 reverse; 5′-GCCAGGAAAGAGCCCGATTAAATTAAAAACATACACATAGCTTAG-3′, MT2 forward; 5′-AAGCAAACTATTTTATAAAGAATCGGGTTTAAAGCATTTGCACTG-3′, MT2 reverse; 5′-CAGTGCAAATGCTTTAAACCCGATTCTTTATAAAATAGTTTGC-3′, MT3 forward; 5′-TTTTATAAAGTGCACTGTTTAAAGCATTAATCGGGTATTTTTGCC-3′, MT3 reverse; 5′-AAAATAAATGGCAAAAATACCCGATTAATGCTTTAAACAGTGCAC-3′. After construction of the reporter genes, DNA sequences of the 3′-UTR were verified. Cells seeded onto 24-well plates were co-transfected with firefly reporter constructs containing the wild-type or mutant MSK1 3′-UTR, Renilla-expressing plasmid, phRL-TK, and miR-148a or negative control precursor using Lipofectamine 2000. Firefly luciferase activity and Renilla luciferase activity were measured 24 h after the initiation of transfection by the Pikkagene Dual Luciferase Assay systems (TOYO B-Net, Tokyo, Japan). Firefly luciferase activity was normalized to Renilla luciferase activity.

Statistical Analysis

Statistical significance was determined by the Student's t test.

RESULTS

MiR-148a Was Down-regulated in PC3 and DU145 Cells

Previous works (3, 4) showed that miR-148a expression is down-regulated in several types of cancer. We investigated expression levels of miR-148a in PrEC normal prostate epithelial cell line, LNCaP hormone-sensitive prostate cancer cell line and PC3 and DU145 hormone-refractory prostate cancer cell lines. MiR-148a expression was significantly decreased in PC3 and DU145 cells compared with PrEC and LNCaP cells (Fig. 1A). We previously reported that miR-34a expression is down-regulated in PC3 and DU145 cells because of functional defects of tumor suppressor gene p53 (25). Because the expression pattern of miR-148a among these four cell lines was similar to that of miR-34a, we tested if p53 could regulate the expression of miR-148a. In PC3 cells, over-expression of p53 resulted in up-regulation of miR-34a but not miR-148a, indicating that miR-148a expression is likely to be regulated by p53-independent mechanisms (Fig. 1B).

FIGURE 1.

MiR-148a expression levels in prostate cancer cell lines. A, total RNA containing miRNA was extracted from PrEC, LNCaP, PC3, and DU145 cells and subjected to quantitative RT-PCR for miR-148a. Data are expressed as mean ± S.D. (n = 3). B, empty vector or p53 expression vector (2 μg) was transiently transfected into PC3 cells. Forty-eight hours later, total RNA was extracted and subjected to quantitative RT-PCR for miR-34a and miR-148a. Data are expressed as mean ± S.D. (n = 3).

Ectopic Expression of miR-148a Inhibited Cell Growth in PC3 Cells

To study whether decreased expression of miR-148a contributes to malignant phenotypes of prostate cancer cells, we first evaluated effects of ectopic expression of miR-148a on cell growth in PC3 cells. Cell growth assay revealed that miR-148a significantly inhibited cell growth at late phase (days 4 and 5 after transfection) (Fig. 2A). Phase contrast microscopic analysis showed that intercellular contacts appeared to be inhibited at late phase in PC3 cells transfected with miR-148a precursor (Fig. 2B). To further confirm the ability of miR-148a to inhibit cell growth, we performed colony formation assay and found that the number of colonies formed was reduced in PC3 cells transfected with miR-148a compared with those transfected with a control precursor (Fig. 2C). These results suggest that miR-148a inhibits cell growth of PC3 cells.

FIGURE 2.

Effects of ectopic expression of miR-148a on cell growth in PC3 cells. A, PC3 cells transfected with a negative control or miR-148a precursor (10 nm) were re-seeded onto 12-well plates. Viable cell number was counted each day for 5 days after transfection using the trypan blue dye exclusion assay. Data are expressed as mean ± S.D. (n = 3). **, p < 0.01, compared with cells transfected with a negative control precursor. B, phase contrast photographs of PC3 cells at 5 days after transfection with a negative control or miR-148a precursor were shown. C, PC3 cells transfected with a negative control or miR-148a precursor were re-seeded onto 6-well plates and cultured for 14 days. After fixation and staining, colonies were counted. Data are expressed as mean ± S.D. (n = 4). *, p < 0.05, compared with cells transfected with a negative control precursor.

Ectopic Expression of miR-148a Attenuated Cell Migration and Invasion in PC3 Cells

It has been recently reported that miR-148a inhibits cell motility of cell lines established from lymph node metastasis of colon, melanoma, and head and neck cancer (5). Here we examined whether ectopic expression of miR-148a could inhibit cell migration of PC3 cells using cell migration assay. The area of migration during 24 h was lower in PC3 cells expressing miR-148a than in those expressing a negative control precursor (Fig. 3A). To evaluate miR-148a effects on the invasive ability, we performed the invasion assay using the Matrigel Invasion Chamber. The number of invaded cells was significantly decreased in PC3 cells transfected with miR-148a precursor compared with those transfected with a negative control precursor (Fig. 3B). These results suggest that ectopic expression of miR-148a attenuates cell migration and invasion of PC3 cells.

FIGURE 3.

Effects of ectopic expression of miR-148a on cell migration and invasion, and the sensitivity to paclitaxel in PC3 cells. A, PC3 cells transfected with a negative control or miR-148a precursor (10 nm) were cultured until confluency. Cell layers were wounded and cultured for 24 h. Photographs were taken at time 0 and 24 h. The area of cell migration was determined by subtracting the area free of cells at 24 h from that at 0 h. Data are expressed as mean ± S.D. (n = 4). B, PC3 cells transfected with a negative control or miR-148a precursor were re-seeded onto the Matrigel invasion chambers. Twenty-four hours later, invaded cells were fixed, stained, and counted. Data are expressed as mean ± S.D. (n = 3). C, PC3 cells transfected with a negative control or miR-148a precursor were re-seeded onto 6-well plates at 1.5 × 10⁵ cells/well and incubated for 24 h. Then, cells were treated with (right panel) or without (left panel) 20 nm of paclitaxel for 48 h. The number of viable cells at time 0 and 48 h was counted. The time at which paclitaxel treatment was initiated was set as 0 h. Data are expressed as mean ± S.D. (n = 3). **, p < 0.01, compared with cells transfected with a negative control precursor.

Ectopic Expression of miR-148a Increased the Sensitivity to Paclitaxel of PC3 Cells

To our knowledge, little is known about the effects of miR-148a on the sensitivity to anti-cancer drugs. We studied whether the sensitivity to paclitaxel could be altered by miR-148a in PC3 cells. In the absence of paclitaxel, miR-148a showed no effect on proliferation of PC3 cells at the time points examined (Fig. 3C, left panel), which was consistent with the data shown in Fig. 2A. In the presence of 20 nm paclitaxel at which concentration no cell growth was observed, miR-148a decreased cell viability of PC3 cells (Fig. 3C, right panel), which was associated with increased apoptosis as determined by Hoechst 33342 staining (data not shown). These results show that miR-148a can increase the sensitivity to paclitaxel of hormone-refractory PC3 cells.

MSK1 Was a Direct Target of miR-148a in PC3 Cells

To identify a direct target of miR-148a, we searched for potential targets of miR-148a using the TargetScan software. MSK1 was one of the predicted targets with the highest score, which contained three putative miR-148a binding sites within its 3′-UTR (Fig. 4A). Other computer-aided algorithms, PicTar and miRanda also predicted MSK1 as a putative target of miR-148a. Furthermore, DNA sequences of three potential miR-148a binding sites predicted within the human MSK1 3′-UTR were highly conserved among species including chimpanzee and rhesus (Fig. 4B). These in silico data suggested that three putative binding sites could be involved in regulation of MSK1 expression by miR-148a. Western blot analysis showed that the expression levels of miR-148a in LNCaP, PC3, DU145 cells were higher than that in PrEC cells (Fig. 4C). Combined with the data shown in Fig. 1A, there was an inverse correlation between the expression of MSK1 and miR-148a in PrEC, PC3, and DU145 cells except for LNCaP cells. In LNCaP cells, MSK1 expression may be also regulated by mechanisms other than those involving miR-148a. As shown in Fig. 4D, miR-148a precursor significantly decreased the MSK1 protein level in PC3 cells on days 2, 3, 4, and 5 after transfection, suggesting that MSK1 could be a direct target of miR-148a. To prove this possibility, we constructed a firefly luciferase reporter gene containing the MSK1 3′-UTR with three potential target sites (MSK1 3′-UTR WT) and a mutant version of the reporter gene in which all these sites were mutated (MSK1 3′-UTR MT). Introduction of miR-148a precursor into PC3 cells decreased relative luciferase activity of the reporter gene containing the MSK1 3′-UTR WT compared with a control precursor, while that of the reporter gene containing the MSK1 3′-UTR MT was not affected by miR-148a (Fig. 5A). Similar results were obtained in other cell line, HEK293 human embryonic kidney cells (Fig. 5B). To determine contribution of each of three putative miR-148a binding sites (site 1, 2, or 3) to the MSK1 3′-UTR activity, we generated MSK1 3′-UTR firefly luciferase reporter genes in which each site was mutated (MSK1 3′-UTR MT1, MT2, or MT3, respectively). Suppression of luciferase activity by miR-148a was seen with the MSK1 3′-UTR MT2 reporter gene, which was almost similar to that with the WT reporter gene (Fig. 5C). However, miR-148 showed little or no effects on luciferase activity with the MT1 and MT3 reporter genes. In all, these results suggest that miR-148a may directly bind to the sites 1 and 3 of the MSKI 3′-UTR and inhibit its protein expression.

FIGURE 4.

Effects of ectopic expression of miR-148a on MSK1 expression in PC3 cells. A, putative miR-148a binding sites within the human MSK1 3′-UTR are shown (sites 1, 2, and 3). The position of the binding sites was numbered relative to the first nucleotide of the 3′-UTR. Mutations were introduced into the seed regions of all three binding sites as indicated by underline. B, interspecies conservation of putative miR-148a binding sites within the MSK1 3′-UTR is shown. Open box denotes perfectly conserved sequences between humans and other species. C, cell lysates were extracted from PrEC, LNCaP, PC3, and DU145 cells and subjected to Western blot analysis for MSK1. D, PC3 cells were transfected with a negative control or miR-148a precursor (10 nm) and cultured for 2, 3, 4, and 5 days. Cell lysates was harvested and subjected to Western blot analysis for MSK1 and β-actin.

FIGURE 5.

Effects of ectopic expression of miR-148a on the MSK1 3′-UTR activity in PC3 cells. A and B, MSK1 3′-UTR WT or MSK1 3′-UTR MT firefly luciferase reporter gene (20 ng) was transfected into PC3 cells (A) or HEK293 cells (B) along with the Renilla luciferase expressing plasmid (20 ng) and either a negative control or miR-148a precursor (30 nm). Luciferase assay was performed 24 h after transfection. Relative luciferase activity was expressed as a ratio of firefly/Renilla luciferase activity. Data are expressed as mean ± S.D. from three independent experiments, each performed in triplicate. C, MSK1 3′-UTR WT or MSK1 3′-UTR MT1, MT2 or MT3 firefly luciferase reporter gene (20 ng) was transfected into PC3 cells along with the Renilla luciferase expressing plasmid (20 ng) and either a negative control or miR-148a precursor (30 nm). Luciferase assay was performed as described above.

Knockdown of MSK1 Showed Little or No Effect on Malignant Phenotypes of PC3 Cells

To investigate whether effects of miR-148a on various malignant phenotypes of PC3 cells could be mediated by MSK1, we performed its knockdown experiments. As shown in Fig. 6A, MSK1 siRNA almost completely diminished its protein expression in PC3 cells on days 2, 3, 4, and 5 after transfection. Cell growth assay showed that introduction of MSK1 siRNA inhibited cell growth on days 2 (p < 0.01), 3 (p < 0.01), and 4 (p < 0.05) after transfection (Fig. 6B), but the inhibitory effect was rather marginal. Also, MSK1 knockdown had little or no effects on colony formation, cell migration and invasion, and the sensitivity to paclitaxel of PC3 cells (supplemental Fig. S1, A–D, respectively). Taken together, it is suggested that MSK1 does not play an obvious role in mediating the miR-148a effects in PC3 cells.

FIGURE 6.

Effects of MSK1 knockdown on cell growth in PC3 cells. A, PC3 cells were transfected with a negative control or MSK1 siRNA (5 nm) and cultured for 2, 3, 4, and 5 days. Cell lysates were harvested and subjected to Western blot analysis for MSK1. B, PC3 cells transfected with a negative control or MSK1 siRNA were re-seeded onto 12-well plates. Viable cell number was counted in triplicate each day for 5 days after transfection. Data are expressed as mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01, compared with cells transfected with a negative control precursor.

Establishment of Paclitaxel-resistant Cells from PC3 Cells

To study the roles of miR-148a on the resistance to anti-cancer drug, we established a PC3PR paclitaxel-resistant cell line from PC3 cells. The IC₅₀ of PC3PR cells was 2577.3 nm, whereas that of parental PC3 cells was 8.6 nm (Fig. 7A), indicating that the resistance to paclitaxel was markedly increased in PC3PR cells. The expressions of MDR1 protein and mRNA, which is involved in the export of drugs and drug resistance, were significantly increased in PC3PR cells compared with parental PC3 cells (Fig. 7B).

FIGURE 7.

Characterization of PC3PR cells. A, PC3 cells and PC3PR paclitaxel-resistant cells were seeded onto 6-well plates. After 24 h, cells were cultured for additional 48 h in medium containing paclitaxel at indicated concentrations. Then, viable cell number was counted in triplicate. Data are expressed as mean ± S.D. B, cell lysates and total RNA extracted from PC3 cells and PC3PR cells were subjected to Western blot analysis for MDR1 and β-actin (upper panel) and semi-quantitative RT-PCR for MDR1 and 18 S rRNA (lower panel), respectively.

Ectopic Expression of miR-148a Attenuated Malignant Phenotypes of PC3PR Cells Including Paclitaxel Resistance

Here we tested if miR-148a could affect malignant phenotypes of PC3PR cells. Introduction of miR-148a precursor inhibited cell growth, colony formation, and cell migration and invasion of PC3PR cells (Fig. 8, A–D, respectively). In the absence of paclitaxel, miR-148a inhibited proliferation of PC3PR cells (Fig. 8E, left panel), which was consistent with the data shown in Fig. 8A. In the presence of 2 μm paclitaxel, miR-148a decreased cell viability of PC3PR cells (Fig. 8E, right panel), which was accompanied by increased apoptosis (data not shown). These results show that miR-148a can attenuate malignant phenotypes of PC3PR cells including paclitaxel resistance.

FIGURE 8.

Effects of miR-148a overexpression on malignant phenotypes of PC3PR cells. A, PC3PR cells transfected with a negative control or miR-148a precursor (10 nm) were re-seeded onto 12-well plates. Viable cell number was counted each day for 5 days after transfection. Data are expressed as mean ± S.D. (n = 3). B, PC3PR cells transfected with a negative control or miR-148a precursor were re-seeded onto 6-well plates and cultured for 14 days. After fixation and staining, colonies were counted. Data are expressed as mean ± S.D. (n = 4). C, PC3PR cells transfected with a negative control or miR-148a precursor (10 nm) were cultured until confluency. Cell layers were wounded and cultured for 24 h. Photographs were taken at time 0 and 24 h. The area of cell migration was determined by subtracting the area free of cells at 24 h from that at 0 h. Data are expressed as mean ± S.D. (n = 4). D, PC3PR cells transfected with a negative control or miR-148a precursor were re-seeded onto the Matrigel invasion chambers. Twenty-four hours later, invaded cells were fixed, stained, and counted. Data are expressed as mean ± S.D. (n = 3). E, PC3PR cells transfected with a negative control or miR-148a precursor (10 nm) were re-seeded onto 6-well plates. After 24 h, cells were treated with (right panel) or without (left panel) 2 μm of paclitaxel for additional 48 h. The number of viable cells at time 0 and 48 h was counted. The time at which paclitaxel treatment was initiated was set as 0 h. Data are expressed as mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 compared with cells transfected with a negative control precursor.

Knockdown of MSK1 Attenuated Paclitaxel Resistance of PC3PR Cells

Finally, we investigated if miR-148a effects might be mediated by MSK1. As shown in Fig. 9A, MSK1 expression in PC3PR cells was higher than that in PC3 cells (left panel), but miR-148a expression was not decreased in PC3PR cells compared with PC3PR cells (right panel). These results suggest that up-regulation of MSK1 expression in PC3PR cells is not caused by miR-148a down-regulation, but by other unidentified mechanisms. However, ectopic expression of miR-148a decreased MSK1 protein levels (Fig. 9B) via acting on its 3′-UTR (Fig. 9C). MSK1 knockdown exhibited no effects on cell growth, colony formation, and cell migration and invasion of PC3PR cells (supplemental Fig. S2, A–D, respectively). In the absence of paclitaxel, MSK1 knockdown showed no effect on proliferation of PC3PR cells (Fig. 9D, left panel). In the presence of 2 μm paclitaxel, miR-148a decreased cell viability of PC3PR cells (Fig. 9D, right panel). Collectively, these results indicate that miR-148a attenuates paclitaxel resistance of PC3PR cells through regulating MSK1 expression.

FIGURE 9.

Effects of MSK1 knockdown on paclitaxel-resistance of PC3PR cells. A, cell lysates and total RNA harvested from PC3 and PC3PR cells were subjected to Western blot analysis for MSK1 (left panel) and quantitative RT-PCR for miR-148a (right panel), respectively. RT-PCR data are expressed as mean ± S.D. (n = 4). B, PC3PR cells were transfected with a negative control or miR-148a precursor (10 nm) and cultured for 2 days. Cell lysates were harvested and subjected to Western blot analysis for MSK1. C, MSK1 3′-UTR WT or MSK1 3′-UTR MT firefly luciferase reporter gene (20 ng) was transfected into PC3PR cells along with the Renilla luciferase expressing plasmid (20 ng) and either a negative control or miR-148a precursor (30 nm). Luciferase assay was performed 24 h after transfection. Relative luciferase activity was expressed as a ratio of firefly/Renilla luciferase activity. Data are expressed as mean ± S.D. (n = 3). D, PC3PR cells transfected with a negative control or MSK1 siRNA (5 nm) were re-seeded onto 6-well plates. After 24 h, cells were treated with (right panel) or without (left panel) 2 μm of paclitaxel for additional 48 h. The number of viable cells at time 0 and 48 h was counted. The time at which paclitaxel treatment was initiated was set as 0 h. Data are expressed as mean ± S.D. (n = 3). **, p < 0.01, compared with the control.

DISCUSSION

MiRNAs act as tumor suppressors or oncogenes in a variety of cancers including prostate cancer (26–32). However, the functional roles of miRNAs in hormone-refractory prostate cancer are largely unknown. MiRNA expression profiling of prostate cancer tissues and cell lines has been recently documented (33–36). Microarray data reported by Mattie et al. (33) revealed that miR-148a expression was lower in advanced prostatic tumor (Gleason score 8), prostatic lymph node metastasis, and PC3 cells than in pooled normal adjacent to tumor sample, transitional cell metaplasia and LNCaP cells. Porkka et al. (34) identified differentially expressed miRNAs between prostate carcinoma samples and benign prostatic hyperplasia (BPH), which showed that miR-148a was down-regulated in hormone-refractory carcinomas compared with BPH. Our microarray data also demonstrated that the levels of miR-148a in hormone-refractory PC3 and DU-145 cells were lower than those in PrEC normal prostate epithelial cells and hormone-sensitive LNCaP cells.³ In an attempt to identify miRNA that behaves as a tumor suppressor and possesses potential as a therapeutic target for hormone-refractory prostate cancer, we focused on miR-148a in the present study. Using quantitative RT-PCR, we demonstrated that miR-148a expression was down-regulated in hormone-refractory PC3 and DU145 cells as compared with PrEC normal prostate epithelial cells and hormone-sensitive LNCaP cells. As revealed by transfection studies, the mechanism underlying the suppression of miR-148a expression did not involve defects of p53 in PC3 cells. In primary breast cancer specimens and cancer cell lines derived from lymph node metastasis, aberrant hypermethylation of the CpG islands has been found upstream of the miR-148a gene (3, 5). It is thus possible that hypermethylation of the miR-148a promoter contributes to decreased expression of miR-148a in PC3 cells, which remains to be studied.

Introduction of miR-148a into head and neck cancer SIHN-011B cells induces inhibition of cell motility in vitro and also induces reduction of tumor growth and inhibition of metastasis formation in xenograft models (5). In the present study, we demonstrated that ectopic expression of miR-148a resulted in inhibition of cell growth, and cell migration and invasion in PC3 cells, suggesting that dysregulation of miR-148a expression may contribute to metastatic potential of PC3 cells.

DNA methyltransferases play an important role in mediating aberrant methylation patterns in human cancer cells, which is implicated in tumor initiation and progression (37). It has been recently reported that miR-148a directly targets and down-regulates DNA (cytosine-5-)-methyltransferase 3β, DNMT3b (6). In PC3 cells, DNMT3b knockdown results in inhibition of cell growth, increase of apoptotic cells, and reduction of cell migration, whereas the invasive ability is not affected (38). These previous reports suggest that effects of miR-148a on cell growth and cell migration but not cell invasion may be mediated in part by down-regulation of DNMT3b in PC3 cells.

In addition to cell growth, and cell migration and invasion, we investigated effects of miR-148a on the sensitivity to anti-cancer drug paclitaxel. Introduction of miR-148a increased the chemosensitivity of PC3 cells, indicating that miR-148a can augment the sensitivity to paclitaxel in hormone-refractory PC3 cells that are sensitive to the anti-cancer drug. In clinical practice, it is of great importance to ameliorate the resistance of drug-resistant prostate cancer. To test the possibility that miR-148a could attenuate such a drug resistance, we established PC3PR paclitaxel-resistant cells from PC3 cells. PC3PR cells could survive in the presence of 500 nm paclitaxel and the IC₅₀ was higher by 300-fold in PC3PR cells than in parental PC3 cells. The expression level of MDR1 was markedly increased in PC3PR cells compared with PC3 cells. Transfection with miR-148a precursor attenuated paclitaxel resistance of PC3PR cells. Furthermore, miR-148a inhibited cell growth, and cell migration and invasion in PC3PR cells as observed in PC3 cells. Taken all together, our findings suggest that miR-148a can inhibit malignant phenotypes of hormone-refractory, drug-sensitive, and -resistant prostate cancer cells including paclitaxel resistance of drug-resistant cells.

MSK1 is a member of the p90 ribosomal protein S6 kinase family and is activated by Ras-MAPK and p38 stress kinase pathways (8–11). MSK1 is implicated in TPA- and EGF-induced oncogenesis (24). In PC3 cells, RSK2, another member of the p90 ribosomal protein S6 kinase family, is up-regulated and involved in cell growth (39). We demonstrated that MSK1 is a direct target of miR-148a in both PC3 and PC3PR cells and hypothesized that some of the tumor suppressive effects of miR-148a might be mediated through down-regulation of MSK1 expression. In PC3 cells, suppression of MSK1 expression by siRNA showed little or no effects on cell growth, cell migration, and invasion and the sensitivity to paclitaxel. In PC3PR cells, MSK1 expression was elevated compared with PC3 cells, which, however, was not associated with miR-148a down-regulation. Intriguingly, despite the lack of effects on paclitaxel-sensitivity of PC3 cells, MSK1 knockdown decreased paclitaxel-resistance of PC3PR cells. These results imply that up-regulation of MSK1 expression and activation of the MSK1 signaling pathway may be involved in the development of paclitaxel-resistance in PC3PR cells. Nevertheless, additional direct target genes that mediate tumor suppressive effects of miR-148a as well as molecular mechanisms underlying attenuation of paclitaxel-resistance by MSK1 knockdown remain to be elucidated.

In conclusion, we demonstrated that miR-148a is down-regulated in PC3 and DU145 hormone-refractory prostate cancer cells and that miR-148a inhibits cell growth and cell migration and invasion in both PC3 and PC3PR cells. MiR-148a also augmented the chemosensitivity of PC3 cells and attenuated the chemoresistance of PC3PR cells. Thus, miR-148a plays multiple tumor suppressive roles in hormone-refractory, drug-sensitive and -resistant prostate cancer cells. Furthermore, we proved that MSK1 is a novel target gene of miR-148a in both PC3 and PC3PR cells and that miR-148 attenuates paclitaxel-resistance of PC3PR cells by modulating MSK1 expression. Our findings suggest that miR-148a is a promising therapeutic target for hormone-refractory prostate cancer especially for drug-resistant prostate cancer.