International Journal of Urology (2015) 22, 867--877

doi: 10.1111/iju.12829

Original Article: Laboratory Investigation

MicroRNA-205 inhibits cancer cell migration and invasion via modulation of centromere protein F regulating pathways in prostate cancer Rika Nishikawa,1,2 Yusuke Goto,1,2 Akira Kurozumi,1,2 Ryosuke Matsushita,3 Hideki Enokida,3 Satoko Kojima,4 Yukio Naya,4 Masayuki Nakagawa,3 Tomohiko Ichikawa2 and Naohiko Seki1 1

Department of Functional Genomics, 2Department of Urology, Chiba University Graduate School of Medicine, Chiba, 3Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, and 4Department of Urology, Teikyo University Chiba Medical Center, Chiba, Japan

Abbreviations & Acronyms CENPF = centromere protein F GAPDH = glyceraldehyde-3phosphate dehydrogenase GEO = gene expression omnibus KEGG = Kyoto encyclopedia of gene and genomes MCM = minichromosome maintenance protein miRNA = microRNA miR-205 = microRNA-205 PCa = prostate cancer PIN = prostatic intraepithelial neoplasias PSA = prostate-specific antigen RT–PCR = reverse transcription polymerase chain reaction UTR = untranslated region Correspondence: Naohiko Seki Ph.D., Department of Functional Genomics, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. Email: [email protected] Received 23 December 2014; accepted 29 April 2015. Online publication 7 June 2015

© 2015 The Japanese Urological Association

Objectives: To investigate the functional roles of microRNA-205 in the modulation of novel cancer pathways in prostate cancer cells. Methods: Functional studies of microRNA-205 were carried out to investigate cell proliferation, migration and invasion in prostate cancer cell lines (PC3 and DU145) by restoration of mature microRNA. In silico database and genome-wide gene expression analyses were carried out to identify molecular targets and pathways mediated by microRNA-205. Loss-of-function studies were applied to microRNA-205 target genes. Results: Restoration of microRNA-205 in cancer cell lines significantly inhibited cancer cell migration and invasion. Our data showed that the centromere protein F gene was overexpressed in prostate cancer clinical specimens and was a direct target of microRNA-205 regulation. Silencing of centromere protein F significantly inhibited cancer cell migration and invasion. Furthermore, MCM7, an oncogenic gene functioning downstream of centromere protein F, was identified by si-centromere protein F transfectants in prostate cancer cells. Conclusions: Loss of tumor-suppressive microRNA-205 seems to enhance cancer cell migration and invasion in prostate cancer through direct regulation of centromere protein F. Our data describing pathways regulated by tumor-suppressive microRNA-205 provide new insights into the potential mechanisms of prostate cancer oncogenesis and metastasis.

Key words: centromere protein F, microRNA, microRNA-205, prostate cancer, tumor suppressor.

Introduction MiRNA are endogenous small ncRNA molecules (19–22 bases in length) that regulate protein-coding gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner.1 At present, a substantial amount of evidence has suggested that miRNA are aberrantly expressed in many human cancers, and play significant roles in human oncogenesis and metastasis.2–4 It is believed that normal regulatory mechanisms can be disrupted by the aberrant expression of tumor-suppressive or oncogenic miRNA in cancer cells. Therefore, identification of aberrantly expressed miRNA is an important first step toward elucidating the details of miRNA-mediated oncogenic pathways. Based on this background, we have previously evaluated miRNA expression signatures using PCa clinical specimens.5 According to these miRNA signatures, we have focused on miRNA that are downregulated in PCa, and have identified tumor-suppressive miRNA that mediate novel PCa pathways.5–11 Elucidation of aberrantly expressed miRNA in PCa specimens, and identification of novel cancer pathways regulated by tumor-suppressive miRNA will provide new insights into the potential mechanisms of PCa oncogenesis and metastasis. PCa is the most frequently diagnosed cancer and the second leading cause of cancer death among men in developed countries.12 Most patients are initially responsive to androgen-deprivation therapy, but their cancers eventually become resistant to androgen-depriva867

R NISHIKAWA ET AL.

tion therapy and progress to castration-resistant prostate cancer. Castration-resistant prostate cancer is difficult to treat, and most clinical trials for advanced PCa have shown limited benefits, with disease progression and metastasis to the skeleton or other sites.13,14 Therefore, understanding the molecular mechanisms of castration-resistant prostate cancer and the metastatic pathways underlying PCa using current genomic approaches, including ncRNA networks, would facilitate the development of novel therapies for and prevention of PCa. Elucidation of miRNA expression signatures, including our PCa signature, has shown that miR-205 is frequently downregulated in PCa tissues, suggesting that miR-205 could act as a tumor suppressor.15 The aim of the present study was to investigate the functional significance of miR-205, and to identify the molecular targets and pathways mediated by miR-205 in PCa cells. Our data showed that restoration of mature miR-205 inhibited cancer cell migration and invasion. Furthermore, gene expression data and in silico database analysis showed that CENPF was a direct target of miR-205

regulation. Silencing of CENPF significantly inhibited the migration and invasion of cancer cells. Furthermore, we investigated the downstream genes regulated by CENPF in PCa cells. The discovery of pathways mediated by tumorsuppressive miR-205 provides important insights into the potential mechanisms of PCa oncogenesis, and suggests novel therapeutic strategies for the treatment of PCa.

Methods Clinical prostate specimens Specimens were obtained from patients with PCa who underwent radical prostatectomy at Chiba University Hospital (Chiba, Japan) from 2009 to 2013. A total of 17 paired samples of PCa and corresponding normal tissues were used for the present study (Table 1). The samples considered normal were free of cancer cells as determined by pathological examination, as described previously.10,11 Clinical prostate specimens obtained by needle biopsy were collected from patients admitted to Teikyo University Chiba

Table 1 Patients’ characteristics (radical prostatectomy specimens) TNM classification No.

PCa or non-PCa

Age

PSA (ng/mL)

Gleason score

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

PCa PCa PCa PCa PCa PCa PCa PCa PCa PCa PCa PCa PCa PCa PCa PCa PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa

64 68 70 69 64 61 68 66 70 60 70 72 56 65 65 65 65

5.4 12.8 16.1 25.8 29.9 7.9 8.8 6.1 11.8 22.1 8.9 4.5 7.1 13.1 9.5 5.8 4.6

3 3 4 4 4 3 4 4 4 3 3 3 3 4 4 4 5

868

+ + + + + + + + + + + + + + + + +

4 5 5 5 3 4 5 3 4 4 4 4 4 3 4 3 4

Stage

T

N

M

C C C B B C B B C B B B C B B B B

3a 3a 3b 2a 2b 3a 2b 2b 3b 2b 2a 2b 3a 2b 2b 2a 2b

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

© 2015 The Japanese Urological Association

MiR-205 targeting CENPF in PCa

Table 2 Patients’ characteristics (needle biopsy specimens)

No.

PCa or non-PCa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

PCa PCa PCa PCa PCa PCa PCa PCa PCa PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa Non-PCa

Age

PSA (ng/mL)

Gleason score

78 69 78 73 72 65 81 78 79 58 60 56 61 62 73 57 64 60 63 65

989 26 19.3 478 102 212 11.4 121 633 482 5.6 8.4 8.6 35.5 6.0 5.2 4.4 5.7 11.4 13.2

4 3 4 4 4 4 4 4 4 4

Medical Center Hospital from 2008 to 2013. A total of 20 needle biopsy specimens were used in this study (Table 2). A pair of needle biopsy specimens was collected from the same region; one specimen was subjected to pathological analysis, whereas the other was used for molecular studies, as described previously.8,9 The patients’ backgrounds and clinicopathological characteristics are summarized in Tables 1 and 2. The protocol was approved by the institutional review board of Chiba University and Teikyo University.

Cell culture and RNA extraction PC3 and DU145 cells, human PCa cells obtained from the American Type Culture Collection (Manassas, VA, USA), were maintained and used for this study, as described previously.7–11 Total RNA from clinical specimens and formalin-fixed paraffin-embedded samples was extracted according to previously described methods.7–11

Quantitative real-time RT–PCR The procedure for PCR quantification was described previously.7–11 TaqMan probes and primers for CENPF (P/N: Hs01118845_m1; Applied Biosystems, Foster City, CA, USA) and GUSB (the internal control; P/N: Hs00939627_m1; Applied Biosystems) were assay-ondemand gene expression products. The expression levels of miR-205 (Assay ID: 000509; Applied Biosystems) were analyzed by TaqMan quantitative real-time PCR (TaqMan MicroRNA Assay; Applied Biosystems) and normalized to the expression of RNU48 (Assay ID: 001006; Applied Biosystems). All reactions were carried out in duplicate, and © 2015 The Japanese Urological Association

+ + + + + + + + + +

4 4 4 3 4 4 4 5 5 4

TNM classification T

N

M

4 3b 3a 3b 3a 4 2 3b 3b 3b

1 0 0 0 0 1 0 1 1 1

1 0 0 1 0 1 0 0 0 0

each assay included negative control reactions that lacked cDNA.

Transfections with mature miRNA and small interfering RNA Transfection procedures and transfection efficiencies of miRNA in PC3 and DU145 cells were described previously.7–11 The following mature miRNA species were used in the present study: mature miRNA and Pre-miR miRNA Precursor (hsa-miR-205-5p; Product ID: PM11015, Applied Biosystems). The following siRNA were used: Stealth Select RNAi siRNA, si-CENPF (cat no. HSS101791, HSS101793; Invitrogen, Carlsbad, CA, USA) and negative control miRNA/ siRNA (P/N: AM17111; Applied Biosystems).

Cell proliferation, migration, and invasion assays PC3 and DU145 cells were transfected with 10 nmol/L miRNA or siRNA by reverse transfection. Cells were transfected with 10 nmol/L miRNA or si-RNA by reverse transfection and plated in 96-well plates at 3 9 103 cells per well. After 72 h, cell proliferation was determined with the XTT assay using a Cell Proliferation Kit II (Roche Molecular Biochemicals, Mannheim, Germany), as previously reported.7–11 Cell migration assays were carried out using BD Falcon Cell Culture Inserts (BD Biosciences, Franklin Lakes, NJ, USA) that contained uncoated Transwell polycarbonate membrane filters with 8-lm pores in 24-well tissue culture plates. Cells were transfected with 10 nm miRNA or siRNA by reverse transfection and plated in 10 cm dishes at 8 9 105 cells. After 48 h, the cells were collected and 2 9 105 cells 869

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were added to the upper chamber of each migration well and were allowed to migrate for 48h. After gentle removal of the nonmigratory cells from the filter surface of the upper chamber, the cells that migrated to the lower side were fixed and stained with Diff-Quick (Sysmex Corporation, Kobe, Japan). The number of cells that migrated to the lower surface was determined microscopically by counting four areas of constant size per well.7–11 Cell invasion assays were carried out using modified Boyden chambers containing Transwell membrane filter inserts precoated with Matrigel with 8-lm pores in 24-well tissue culture plates (BD Biosciences), as described previously.7–11 All experiments were carried out in triplicate.

PF-transfected PC3 cells. An oligo-microarray (Agilent Technologies) was used for gene expression studies. The strategy behind this analysis procedure was described previously.7–11

Identification of putative target genes regulated by miR-205 in PCa cells

Statistical analysis

To identify miR-205 target genes, we used in silico analysis and genome-wide gene expression analysis. First, we screened genes using TargetScan Release 6.2 (http://www.targetscan.org/). Next, we attempted to identify miR-205 target genes using miR-205-transfected PC3 cells. A SurePrint G3 Human GE 60K Microarray (Agilent Technologies, Santa Clara, CA, USA) was used for expression profiling of miR205 transfectants in comparison with miRNA-negative control transfectants. Genes containing putative miR-205 target sites and showing downregulation in miR-205 transfectants were listed. Furthermore, to identify upregulated genes in clinical PCa specimens, we analyzed a publicly available gene expression data set in the GEO database (accession number: GSE29079). We merged these datasets and selected putative miR-205 target genes in the present study.

Western blotting Cells were harvested 96 h after transfection, and lysates were prepared. Protein lysates (50 lg) were separated on MiniPROTEAN TGX gels (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes. Immunoblotting was carried out with rabbit anti-CENPF antibodies (1:1500; ab5; Abcam, Cambridge, UK); anti-GAPDH antibodies (1:1000; ab8245; Abcam) were used as an internal loading control.

Plasmid construction and dual-luciferase reporter assay Partial wild-type sequences of the CENPF 30 -UTR or those with a deleted miR-205 target site (position 461–468) were inserted between the XhoI–PmeI restriction sites in the 30 UTR of the hRluc gene in the psiCHECK-2 vector (C8021; Promega, Madison, WI, USA). The procedure for the dualluciferase reporter assay was described previously.7–11 All experiments were carried out in triplicate.

Identification of downstream targets regulated by CENPF To identify molecular targets regulated by CENPF in PCa cells, we carried out gene expression analysis using si-CEN870

Immunohistochemistry A tissue microarray containing 60 PCa specimens, 10 PINs and 10 prostatic hyperplastic samples was obtained from Provitro (Berlin, Germany; cat. no. 401 2209, Lot no. 146P160910.10-24, Berlin, BRD). Tissue specimens were immunostained following previously described protocols.6 Primary rabbit polyclonal antibodies against CENPF (Abcam) were diluted 1:400.

The relationships between two groups and the numerical values obtained by real-time RT–PCR were analyzed using paired t-tests. The relationships among three variables and numerical values were analyzed using Bonferroni-adjusted Mann–Whitney U-tests. All analyses were carried out using Expert StatView Software (version 4; SAS Institute, Cary, NC, USA).

Results Expression of miR-205 in PCa clinical specimens and cell lines To validate our previous expression signature of PCa, we evaluated miR-205 expression in clinical PCa specimens. The expression levels of miR-205 were significantly lower in tumor tissues and cell lines (PC3 and DU145) than in corresponding non-cancerous prostate tissues (Fig. 1a, left and right).

Effects of restoring miR-205 on cell proliferation, migration, and invasion activities in PCa cell lines To investigate the functional effects of miR-205, we carried out gain-of-function studies using miRNA transfection of PC3 and DU145 cell lines. XTT assays showed that cell proliferation was not inhibited in miR-205 transfectants in comparison with mock or miR-control transfectants (Fig. 1b). Matrigel invasion assays showed that cell invasion activity was significantly inhibited in miR-205 transfectants in comparison with mock or miR-control transfectants (Fig. 1c). Finally, migration assays showed that cell migration activity was significantly inhibited in miR-205 transfectants in comparison with mock or miR-control transfectants (Fig. 1d).

Identification of putative targets of miR-205 regulation in PCa cells To gain further insights into the genes affected by miR-205, we used a combination of in silico analysis and gene expression analysis using miR-205 transfectants. First, we screened miR-205-targeted genes using the TargetScan database and © 2015 The Japanese Urological Association

MiR-205 targeting CENPF in PCa

P = 0.0022

50 40 30 20 10 Non PCa cancerous

120 100 80 60 40 20 0

3.0 2.5 2.0 1.5 1.0 0.5 0

Cell lines (c)

(%) 140

Non PCa cancerous (d)

(%) 140 120 100 80

** **

60 40

PC3

CENPF was a direct target of miR-205 in PCa cells We carried out quantitative real-time RT–PCR and western blotting in PC3 and DU145 cells to investigate whether CENPF expression was reduced by restoration of miR-205. Expression of CENPF mRNA was significantly repressed in miR-205 transfectants in comparison with mock or miR-control transfectants (Fig. 3a). The protein expression levels of CENPF were also repressed in miR-205 transfectants (Fig. 3b). We also carried out luciferase reporter assays in PC3 cells to determine whether CENPF had an authentic binding site for miR-205. The TargetScan database predicted that one putative miR-205-binding site existed in the 30 -UTR of CENPF (position 461–468, Fig. 3c). We used vectors encoding either a partial wild-type sequence (including the predicted miR-205 target site) or deletion of the seed sequence of the 30 -UTR of CENPF mRNA. We found that the luminescence intensity was significantly reduced by cotransfection with miR-205 and the vector carrying the wild-type 30 -UTR of CENPF (Fig. 3c).

0

DU145 mock

(%) 140 120 100

**

80 60

**

40 20

20

identified 3175 putative candidate genes. Next, we paired down the 3175 genes based on two types of gene expression data as follows: (i) upregulated genes determined by the gene expression dataset of PCa clinical specimens in GEO (accession number: GSE29079); and (ii) downregulated genes (log2 ratio less than 1.0) after transfection of PC3 cells with miR205. The procedure for selection of candidate miR-205 targets is shown in the flowchart in Figure 2. From this selection, 12 candidate genes were identified as putative miR-205 targets (Table 3). Among these candidates, we focused on the CENPF gene in further analyses.

© 2015 The Japanese Urological Association

DU145

Cell invasion (relative to mock)

(b)

PC3

3.5

Cell migration (relative to mock)

60

0

Cell proliferation (relative to mock)

Fig. 1 The expression levels of miR-205 in clinical specimens and PCa cell lines, and the functional significance of miR-205 in PCa cell lines. (a) Real-time PCR showed that the expression levels of miR-205 were significantly lower in PCa tissues and cell lines than in non-cancerous prostate tissues. RNU48 was used as an internal control. (Left) Formalin-fixed paraffin-embedded samples and PCa cell lines. (Right) Needle biopsy samples. (b) Cell proliferation was determined with XTT assays 72 h after transfection with 10 nmol/L miR-205, miR-control or mock transfection. (c) Cell invasion activity was determined using Matrigel invasion assays. (d) Cell migration activity was determined using wound healing assays. The error bars show standard deviation. **P < 0.0001.

P = 0.0023 microRNA-205 expression (normalized to RNU48)

microRNA-205 expression (normalized to RNU48)

(a)

PC3

miR-control

DU145

0

PC3

DU145

miR-205

Effects of silencing CENPF on cell proliferation, migration and invasion in PCa cell lines To investigate the functional role of CENPF in PCa cells, we carried out loss-of-function studies using si-CENPF transfectants. First, we evaluated the knockdown efficiency of si-CENPF transfection in PC3 and DU145 cells. In the present study, we used two types of si-CENPF and used as silencing effect for PCa cells. Quantitative real-time RT– PCR and western blotting showed that the both siRNA effectively downregulated CENPF expression in both cell lines (Fig. 4a,b). The XTT assay showed that cell proliferation was inhibited in si-CENPF transfectants in comparison with the mock or si-control transfectant cells (Fig. 4c). The Matrigel invasion assay showed that cell invasion activity was significantly inhibited in si-CENPF transfectants in comparison with the mock or si-control transfectant cells (Fig. 4d). The migration assay showed that cell migration activity was significantly inhibited in si-CENPF transfectants (Fig. 4e).

Expression of CENPF in PCa clinical specimens The expression of CENPF was significantly upregulated in PCa clinical specimens, and Spearman’s rank test showed a significant negative correlation between CENPF expression and miR-205 expression (Fig. 5a,b). We compared the expression levels in PCa, intraepithelial neoplasm (PIN) and non-cancerous prostatic tissue. The CENPF was strongly expressed in several cancer lesions, 871

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and various tested clinicopathological parameters (Gleason score and tumor stage; data not shown).

TargetScan Database search 3175 genes

Identification of downstream pathways regulated by CENPF in PCa cells Finally, we investigated the molecular pathways regulated by CENPF in PCa cells using genome-wide gene expression analysis in si-CENPF transfectants. A total of 408 genes were downregulated (log2 ratio less than 0.5) in si-CENPF-transfected PC3 cells. We also assigned the downregulated genes to KEGG pathways using the GeneCodis program (http://genecodis.cnb.csic.es), as described previously.7 A total of 22 pathways were identified as significantly enriched (Table 4). Genes related to the “DNA replication pathway” are listed in Table 5, and genes related to the “cell cycle pathway” are listed in Table 6.

GEO expression data (GSE29079) Upregulated in PCa

Expression data downregulated in miR-205-transfected PC3 cells (log2 ratio < –1.0)

Discussion

12 genes as putative targets of miR -205

Fig. 2 Strategy for identification of putative candidate genes regulated by miR-205 in PCa cells. Study flow for identification of miR-205 target genes by in silico analysis of TargetScan database and genome-wide gene expression analysis of miR-205 transfectants and PCa clinical specimens.

whereas no or low expression was observed in PIN and the non-cancerous tissues. Expression patterns of CENPF in non-cancerous tissue, PIN and PCa tissues are shown in Figure S1. In Figure 5c, CENPF was strongly expressed in cancerous lesions, whereas low expression was observed in non-cancerous tissues. There was no significant correlation between CENPF expression

Based on the miRNA signature of PCa, we identified tumorsuppressive miRNA (miR-1, miR-29s, miR-133a, miR-145, miR-218 and miR-224), the molecular pathways targeted by these miRNA, and the targets of these miRNA in PCa cells.5–11 In the present study, we focused on miR-205, because miR-205 has been shown to be downregulated in several PCa signatures.15 Our present data showed that miR205 was significantly reduced in PCa specimens, and restoration of the miRNA inhibited cancer cell migration and invasion, providing insights into the functional roles of miR-205 as a tumor suppressor in PCa cells. Several studies of miR205 have reported that this miRNA has multiple functions (as an oncogenic or tumor-suppressive miRNA) and targets several types of genes in cancer cells.16,17 Several studies in PCa cells have reported the tumor-suppressive functions of miR205 and its target genes.18–20 Interestingly, the p63 gene, which is a homolog of tumor-suppressive p53 gene, regulates expression of miR-205 in PCa cells.21 Loss of p63 and miR205 enhanced PCa metastasis.21 Transcriptional regulation of the p63–miR-205 axis plays an important role in PCa onco-

Table 3 Candidate of putative miR-205 target genes Entrez gene ID

Gene symbol

1063 23097 983 54902 5583 54927

CENPF CDK19 CDK1 TTC19 PRKCH CHCHD3

10618 6596 7171 8935 23265

TGOLN2 HLTF TPM4 SKAP2 EXOC7

872

Gene name

Location

Expression in miR-205 transfectant PC3 (log2 ratio)

GEO expression data fold change (tumor/normal)

Conserved cites

Poorly conserved cites

Centromere protein F, 350/400 kDa Cyclin-dependent kinase 19 Cyclin-dependent kinase 1 Tetratricopeptide repeat domain 19 Protein kinase C, eta Coiled-coil-helix-coiled-coil-helix domain containing 3 Trans-golgi network protein 2 Helicase-like transcription factor Tropomyosin 4 Src kinase associated phosphoprotein 2 Exocyst complex component 7

1q41 6q21 10q21.1 17p12 14q23.1 7q33

2.14 2.05 2.02 1.65 1.46 1.43

1.32 1.66 1.30 1.14 1.24 1.11

1 1 0 1 0 0

0 4 1 1 2 1

2p11.2 3q25.1–q26.1 19p13.1 7p15.2 17q25.1

1.31 1.24 1.22 1.21 1.07

1.32 1.19 1.14 1.19 1.22

0 0 0 0 0

2 1 1 1 1

© 2015 The Japanese Urological Association

MiR-205 targeting CENPF in PCa

miR-205

miR-cont

mock

miR-cont

(b)

miR-205

miR-cont

mock

PC3

PC3 (%) 120 100

(%) 120 100

80 60

80 60

40 20

40 20

**

**

0

** **

genesis. These findings are consistent with our present results, showing that miR-205 functions as a tumor suppressor and strongly contributes to PCa oncogenesis. A single miRNA might regulate multiple protein-coding genes; indeed, bioinformatics studies have shown that

**

60 40 20 PC3

DU145

100

DU145

*

80 60

**

**

40

**

20 0

(e)

Cell migration (relative to mock)

PC3

80

** * **

(d) (%) 120 Cell invasion (relative to mock)

GAPDH

si-CENPF-2

si-CENPF-1

miR-cont

si-CENPF-1

miR-cont

si-CENPF-2

si-CENPF-1

mock

miR-cont

100

0

0

mock

miR-205

(c) (%) 120

DU145

si-CENPF-2

mRNA expression of CENPF (relative to mock)

0.2

position 461–468 of CENPF 3’ UTR

DU145

CENPF

© 2015 The Japanese Urological Association

**

0.6 0.4

5' ..UCAGAGCUGAGUAAAAUGAAGGA..3' Wild 3' ...GUCUGAGGCCACCUUACUUCCU..5' miR-205 5' ..UCAGAGCUGAGUAAA-------A..3' Deletion

(b)

Fig. 4 Effects of si-CENPF transfection on PCa cell lines. (a) CENPF mRNA expression levels were measured by RT–PCR 72 h after transfection with 10 nmol/L si-CENPF. GUSB was used for normalization. (b) CENPF protein expression 96 h after transfection with si-CENPF. GAPDH was used as a loading control. (c) Cell proliferation was determined by XTT assay 72 h after transfection with 10 nmol/L si-CENPF. (d) Cell invasion was determined by Matrigel invasion assay 48 h after transfection with 10 nmol/L si-CENPF. (e) Cell migration was determined by migration assay 48 h after transfection with 10 nmol/L si-CENPF. The error bars show standard deviation. *P < 0.01; **P < 0.0001.

1.0 0.8

miR-cont

GAPDH

mock

Deletion

0

CENPF

(a)

Luminescence (relative to control)

0

Wild

1.2

miR-205

40 20

700 bP

miR-205 binding site

*

80 60 40 20 0

*

Human CENPF 3' UTR 500 300 100

(c)

DU145 (%) 120 100

mock

mRNA expression of CENPF (relative to mock)

Fig. 3 CENPF expression was directly regulated by miR-205 in PCa cell lines. (a) CENPF mRNA expression 72 h after transfection with miR-205. GUSB expression was used for normalization. (b) CENPF protein expression 96 h after transfection with miR-205. GAPDH was used as a loading control. (c) The miR-205 binding site in the 30 -UTR of CENPF mRNA. Luciferase reporter assays were carried out using vectors that included (wild) or lacked (deletion) the wild-type sequences of the putative miR-205 target site. Renilla luciferase assays were normalized to firefly luciferase values. The error bars show standard deviation. *P < 0.01; **P < 0.0001.

PC3 (%) 120 100 80 60

Cell proliferation (relative to mock)

(a)

PC3

DU145

(%) 120 100

**

80 60

**

40

** **

20 0

PC3

DU145

miRNA regulate more than 30–60% of the protein-coding genes in the human genome.22 Reduced expression of tumorsuppressive miRNA might cause overexpression of oncogenic genes in cancer cells. To better understand PCa oncogenesis and metastasis, we identified miR-205 target genes using a 873

R NISHIKAWA ET AL.

P = 0.0002

(b)

1.4

1.4

1.2

1.2 CENPF expression

mRNA expression of CENPF (relative to GUSB)

(a)

1.0 0.8 0.6 0.4 0.2

P = 0.0010 r = –0.758

0.6 0.4 0.2 0

–0.2 –0.5

0 Non Cancerous (c)

1.0 0.8

PCa

0

0.5

1

1.5

2

2.5

3

3.5

miR-205 expression

Gleason 3+5, pT2bN0

Gleason 3+3, pT3aN0

(x50)

(x200)

(x50)

(x200)

(x200)

(x200)

Fig. 5 Expression levels of CENPF in PCa clinical specimens. (a) Expression levels of CENPF in PCa clinical specimens. GUSB was used for normalization. (b) Correlation between CENPF expression and miR-205. (c) Immunohistochemical staining of CENPF in PCa tissues. Overexpression of CENPF was observed in cancer lesions (left: Gleason 3 + 5, pT2bN0; right: Gleason 3 + 3, pT3aN0). In contrast, normal prostate glands and stromal tissues did not express CENPF.

Table 4 Significantly enriched KEGG pathways regulated by si-CENPF in PC3 cells

Number of genes

Hypergeometric P-value

Corrected hypergeometric P-value

Annotations

14 genes 16 genes 7 genes 9 genes 7 genes 8 genes 8 genes 7 genes 5 genes 11 genes 5 genes 4 genes 8 genes 3 genes 7 genes 5 genes 5 genes 10 genes 6 genes 3 genes 4 genes 2 genes

2.11E-19 1.88E-13 2.4E-09 2.13E-07 5.78E-06 5.41E-06 1.87E-05 3.03E-05 6.12E-05 8.43E-05 0.000299 0.000352 0.000713 0.00078 0.001282 0.001668 0.002159 0.002048 0.002775 0.004652 0.005025 0.007599

3.04E-17 1.35E-11 1.15E-07 7.67E-06 0.000139 0.000156 0.000385 0.000545 0.000979 0.001214 0.003912 0.00423 0.007895 0.008018 0.012307 0.015015 0.017276 0.017345 0.021028 0.033496 0.03446 0.049739

(KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG) (KEGG)

874

03030: 04110: 03430: 05323: 04115: 00240: 04114: 04914: 03420: 04060: 04621: 03410: 04062: 00670: 00230: 04640: 05322: 05200: 04120: 05143: 05120: 03450:

DNA replication Cell cycle Mismatch repair Rheumatoid arthritis p53 signaling pathway Pyrimidine metabolism Oocyte meiosis Progesterone-mediated oocyte maturation Nucleotide excision repair Cytokine-cytokine receptor interaction NOD-like receptor signaling pathway Base excision repair Chemokine signaling pathway One carbon pool by folate Purine metabolism Hematopoietic cell lineage Systemic lupus erythematosus Pathways in cancer Ubiquitin mediated proteolysis African trypanosomiasis Epithelial cell signaling in Helicobacter pylori infection Non-homologous end-joining

© 2015 The Japanese Urological Association

MiR-205 targeting CENPF in PCa

Table 5 Downregulated genes involved in DNA replication pathway by si-CENPF transfectant Expression in si-CENPF transfectant (log2 ratio) Entrez gene ID

Gene symbol

Gene name

si-CENPF_1 (HSS101791)

si-CENPF_3 (HSS101793)

10535 4173 4176 3978 5422 5982 5981 5985 4174 4175 23649 5557 2237 5111

RNASEH2A MCM4 MCM7 LIG1 POLA1 RFC2 RFC1 RFC5 MCM5 MCM6 POLA2 PRIM1 FEN1 PCNA

Ribonuclease H2, subunit A Minichromosome maintenance complex component 4 Minichromosome maintenance complex component 7 Ligase I, DNA, ATP-dependent Polymerase (DNA directed), alpha 1, catalytic subunit Replication factor C (activator 1) 2, 40 kDa Replication factor C (activator 1) 1, 145 kDa Replication factor C (activator 1) 5, 36.5 kDa Minichromosome maintenance complex component 5 Minichromosome maintenance complex component 6 Polymerase (DNA directed), alpha 2 (70 kD subunit) Primase, DNA, polypeptide 1 (49 kDa) Flap structure-specific endonuclease 1 Proliferating cell nuclear antigen

0.69 0.70 0.78 0.74 0.65 0.71 0.52 0.83 0.61 0.64 0.72 0.99 0.60 0.56

0.90 1.23 0.78 0.78 0.56 0.99 0.65 1.02 0.63 0.93 1.19 1.88 0.82 0.83

Table 6 Downregulated genes involved in cell cycle pathway by si-CENPF transfectant Expression in si-CENPF transfectant (log2 ratio) Entrez gene ID

Gene symbol

Gene name

si-CENPF_1 (HSS101791)

si-CENPF_3 (HSS101793)

4173 9133 991 890 4176 1111 891 4174 4175 701 8318 8454 993 699 5111 983

MCM4 CCNB2 CDC20 CCNA2 MCM7 CHEK1 CCNB1 MCM5 MCM6 BUB1B CDC45 CUL1 CDC25A BUB1 PCNA CDK1

Minichromosome maintenance complex component 4 Cyclin B2 Cell division cycle 20 homolog (S. cerevisiae) Cyclin A2 Minichromosome maintenance complex component 7 CHK1 checkpoint homolog (S. pombe) Cyclin B1 Minichromosome maintenance complex component 5 Minichromosome maintenance complex component 6 Budding uninhibited by benzimidazoles 1 homolog beta (yeast) Cell division cycle 45 homolog (S. cerevisiae) Cullin 1 Cell division cycle 25 homolog A (S. pombe) Budding uninhibited by benzimidazoles 1 homolog (yeast) Proliferating cell nuclear antigen Cyclin-dependent kinase 1

0.70 0.55 0.87 1.03 0.74 0.68 0.95 0.61 0.64 0.61 0.60 0.75 0.84 0.74 0.60 0.54

1.23 0.80 1.32 1.05 0.78 1.63 1.43 0.63 0.93 1.08 0.92 0.65 0.67 1.24 1.07 1.65

combination of gene expression analysis and in silico database analysis. In the present study, we identified 12 putative candidate genes (CENPF, HMGB3, CDK19, CDK1, TTC19, PRKCH, CHCHD3, TGOLN2, HLTF, TPM4, SKAP2 and EXOC7) regulated by miR-205 in PCa cells. Among them, we confirmed the direct binding of miR-205 to the 30 -UTR of CENPF using luciferase reporter assays. To show the effectiveness on the list of putative candidate targets of miR-205 regulation, we investigated the functional significance of the HMGB3 in PCa cells. Our data showed that HBGB3 was a direct target of miR-205, and silencing of the gene inhibited cancer cell migration and invasion, like the CENPF phenotype (data not shown). These data show that tumor-suppressive miR-205 targeting multiple oncogenic © 2015 The Japanese Urological Association

genes contributed to cancer cell migration and invasion in PCa cells. It is considered that the continuous study of miR205 regulation in PCa cells is important to the elucidation of PCa oncogenesis and metastasis. CENPF is a 367-kDa coiled-coil protein whose expression is increased during the early G2 phase.23 Previous studies showed that CENPF is a member of the centromere protein family, and centromere protein complex acts as a critical chromosomal segregation process for kinetochore assembly and spindle checkpoint signaling during mitosis.24,25 Aberrant expression of CENPF has been observed in several types of cancers, showing that overexpression of this gene could influence oncogenesis.26,27 Interestingly, a recent study showed that FOXM1, encoding forkhead box M1, and 875

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CENPF act as synergistic drivers of PCa, and can be used as prognostic markers associated with aggressive PCa.28 Our present data showed that CENPF was upregulated in PCa clinical specimens, and silencing of the CENPF gene significantly suppressed cancer cell migration and invasion in PCa cells. This is the first report showing that CENPF promotes cancer cell migration and invasion, and is directly regulated by tumor-suppressive miR-205, suggesting that miR-205-modulated oncogenic pathways and genes are potential targets for novel PCa therapeutics. Furthermore, to elucidate the functional significance of CENPF in PCa cells, we investigated the effects of CENPF knockdown on the expression of downstream genes. Inhibition of cell proliferation, migration and invasion in PCa cells were induced by enough silencing of CENPF expression by using si-CENPF. A recent study showed that overexpression of CENPF was observed in hepatocellular carcinoma and CENPF knockdown blocked cell cycle at G2/M transition.29 Our present data and other studies showed that overexpression of CENPF plays a critical role in pathogenesis of several types of cancers including PCa. Downstream genes modulated by CENPF were categorized by KEGG pathways. Our data showed that “DNA replication,” “cell cycle” and “mismatch repair” pathways were annotated as the CENPF modulating downstream pathways. Gene expression analysis showed that members of the MCM family were regulated downstream of CENPF. Among the MCM genes, amplification and overexpression of MCM7 has been shown to be associated with the clinicopathological features of PCa, such as relapse, local invasion and tumor grade.30 Furthermore, constitutive expression of MCM7 in PCa cells markedly increases DNA synthesis and cell proliferation.31 These findings suggest that MCM7 contributes to the progression and development of PCa. In conclusion, miR-205 was shown to function as a tumor suppressor in PCa. To the best of our knowledge, this is the first report showing that tumor-suppressive miR-205 directly regulates CENPF in PCa cells. Furthermore, CENPF was upregulated in PCa clinical specimens, and contributed to cancer cell migration and invasion, showing that CENPF functioned as an oncogene. The identification of novel molecular pathways and targets modulated by miR-205 might lead to a better understanding of PCa, and could facilitate the development of new therapeutic strategies for the treatment of this disease.

Acknowledgment This study was supported by the KAKENHI, grant numbers (C) 24592590 and (B) 25293333, and by Futaba Electronics Memorial Foundation.

Conflict of interest None declared.

References 1 Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281–97.

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2 Filipowicz W, Bhattacharyya SN, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 2008; 9: 102–14. 3 Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 2009; 19: 92–105. 4 Iorio MV, Croce CM. MicroRNAs in cancer: small molecules with a huge impact. J. Clin. Oncol. 2009; 27: 5848–56. 5 Fuse M, Kojima S, Enokida H et al. Tumor suppressive microRNAs (miR222 and miR-31) regulate molecular pathways based on microRNA expression signature in prostate cancer. J. Hum. Genet. 2012; 57: 691–9. 6 Kojima S, Chiyomaru T, Kawakami K et al. Tumour suppressors miR-1 and miR-133a target the oncogenic function of purine nucleoside phosphorylase (PNP) in prostate cancer. Br. J. Cancer 2012; 106: 405–13. 7 Kojima S, Enokida H, Yoshino H et al. The tumor-suppressive microRNA143/145 cluster inhibits cell migration and invasion by targeting GOLM1 in prostate cancer. J. Hum. Genet. 2013; 59: 78–87. 8 Goto Y, Nishikawa R, Kojima S et al. Tumour-suppressive microRNA-224 inhibits cancer cell migration and invasion via targeting oncogenic TPD52 in prostate cancer. FEBS Lett. 2014; 588: 1973–82. 9 Goto Y, Kojima S, Nishikawa R et al. The microRNA-23b/27b/24-1 cluster is a disease progression marker and tumor suppressor in prostate cancer. Oncotarget 2014; 5: 7748–59. 10 Nishikawa R, Goto Y, Kojima S et al. Tumor-suppressive microRNA-29s inhibit cancer cell migration and invasion via targeting LAMC1 in prostate cancer. Int. J. Oncol. 2014; 45: 401–10. 11 Nishikawa R, Goto Y, Sakamoto S et al. Tumor-suppressive microRNA-218 inhibits cancer cell migration and invasion via targeting of LASP1 in prostate cancer. Cancer Sci. 2014; 105: 802–11. 12 Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J. Clin. 2013; 63: 11–30. 13 Sturge J, Caley MP, Waxman J. Bone metastasis in prostate cancer: emerging therapeutic strategies. Nat. Rev. Clin. Oncol. 2011; 8: 357–68. 14 Chi KN, Bjartell A, Dearnaley D et al. Castration-resistant prostate cancer: from new pathophysiology to new treatment targets. Eur. Urol. 2009; 56: 594–605. 15 Goto Y, Kurozumi A, Enokida H, Ichikawa T, Seki N. The functional significance of aberrant expressed microRNAs in prostate cancer. Int. J. Urol. 2015; 22: 242–52. 16 Qin AY, Zhang XW, Liu L et al. MiR-205 in cancer: an angel or a devil? Eur. J. Cell Biol. 2013; 92: 54–60. 17 Greene SB, Herschkowits JI, Rosen JM. The ups and downs of miR-205: identifying the roles of miR-205 in mammary gland development and breast cancer. RNA Biol. 2010; 7: 300–4. 18 Hulf T, Sibbritt T, Wiklund ED et al. Epigenetic-induced repression of microRNA-205 is associated with MED1 activation and a poorer prognosis in localized prostate cancer. Oncogene 2013; 32: 2891–9. 19 Gandellini P, Profumo V, Casamichele A et al. miR-205 regulates basement membrane deposition in human prostate: implications for cancer development. Cell Death Differ. 2012; 19: 1750–60. 20 Majid S, Dar AA, Saini S et al. MicroRNA-205-directed transcriptional activation of tumor suppressor genes in prostate cancer. Cancer 2010; 116: 5637–49. 21 Tucci P, Agostini M, Grespi F et al. Loss of p63 and its microRNA-205 target results in enhanced cell migration and metastasis in prostate cancer. Proc. Natl Acad. Sci. USA 2012; 109: 15312–7. 22 Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005; 120: 15–20. 23 Liao H, Winkfein RJ, Mack G, Rattner JB, Yen TJ. CENP-F is a protein of the nuclear matrix that assembles onto kinetochores at late G2 and is rapidly degraded after mitosis. J. Cell Biol. 1995; 130: 507–18. 24 Johnson VL, Scott MI, Holt SV, Hussein D, Taylor SS. Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome congression. J. Cell Sci. 2004; 117: 1577–89. 25 Varis A, Salmela AL, Kallio MJ. Cenp-F (mitosin) is more than a mitotic marker. Chromosoma 2006; 115: 288–95. 26 Kim HE, Kim DG, Lee KJ et al. Frequent amplification of CENPF, GMNN and CDK13 genes in hepatocellular carcinomas. PLoS ONE 2012; 7: e43223. 27 Chen WB, Cheng XB, Ding W et al. Centromere protein F and survivin are associated with high risk and a poor prognosis in colorectal gastrointestinal stromal tumours. J. Clin. Pathol. 2011; 64: 751–5.

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MiR-205 targeting CENPF in PCa

28 Aytes A, Mitrofanova A, Lefebvre C et al. Cross-species regulatory network analysis identifies a synergistic interaction between FOXM1 and CENPF that drives prostate cancer malignancy. Cancer Cell 2014; 25: 638–51. 29 Dai Y, Liu L, Zeng T et al. Characterization of the oncogenic function of centromere protein F in hepatocellular carcinoma. Biochem. Biophys. Res. Commun. 2013; 436: 711–8. 30 Ren B, Yu G, Tseng GC et al. MCM7 amplification and overexpression are associated with prostate cancer progression. Oncogene 2006; 25: 1090–8. 31 Padmanabhan V, Callas P, Philips G, Trainer TD, Beatty BG. DNA replication regulation protein Mcm7 as a marker of proliferation in prostate cancer. J. Clin. Pathol. 2004; 57: 1057–62.

Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Fig. S1 Immunohistochemical staining of CENPF in PCa clinical specimens. (a) Negative staining in non-cancerous tissue without malignancy. (b) Weakly stained PIN lesion. (c) Strongly stained cancer lesion (Gleason score 3 + 4, pT3aN0).

Editorial Comment Editorial Comment to MicroRNA-205 inhibits cancer cell migration and invasion via modulation of centromere protein F regulating pathways in prostate cancer In an original article published in this issue of International Journal of Urology, Nishikawa et al. showed that loss of tumor-suppressive microRNA-205 (miR-205) enhanced cancer cell migration and invasion in prostate cancer (PCa) cells.1 The authors should be congratulated for their discovery of centromere protein F (CENPF) as a target of miR205, which mediates migration and invasion of PCa cells. It has been reported that loss of miR-205 expression is associated with cellular and clinical aggressiveness of prostate cancer.2 In the previous study, miR-205, under control of DNp63, suppressed expression of epithelial-mesenchymal transition markers including ZEB1 and vimentin in prostate cancer cells. Furthermore, loss of expression of DNp63 and miR-205 was significantly associated with higher Gleason scores, an increased likelihood of metastatic and infiltration events, and poorer biochemical recurrence-free survival and overall survival. The present study is consistent with the previous study in terms of the functional role of miR-205 in PCa cell migration. There have been several reports on the tumor promoting function of CENPF in PCa and other malignancies. In a previous report, cross-species analyses on genetic regulatory networks established based on gene expression profiles of human and mouse PCa identified CENPF and FOXM1 among 20 master regulators for PCa.3 Functional assays showed that FOXM1 and CENPF synergistically promote PCa growth. The present study also shows higher expression of CENPF in PCa tissue compared with benign prostate epithelia and prostatic intraepithelial neoplasia lesions, although significant correlations were unfortunately not observed between CENPF expression and clinicopathological characteristics of PCa, including Gleason score. In this regard, a recent report has shown higher CENPF expression in PCa tissue in immunohistochemical assay.4 The previous study also showed that

© 2015 The Japanese Urological Association

higher expression of CENPF mRNA is associated with higher Gleason score and disease stage, and poorer treatment outcomes. Thus, there is accumulating evidence for a variety of tumor-promoting functions of CENPF in PCa as well as other malignancies. Although the previous reports mainly focused on the role of CENPF in tumor growth and cell proliferation, it is noteworthy that the present study has reported for the first time a potential role of CENPF expression for cell migration and invasion, which should be further elucidated in future studies. Takashi Kobayashi M.D., Ph.D. Department of Urology, Kyoto University Graduate School of Medicine, Kyoto, Japan [email protected] DOI: 10.1111/iju.12883

Conflict of interest None declared.

References 1 Nishikawa R, Goto Y, Kurozumi A et al. MicroRNA-205 inhibits cancer cell migration and invasion via modulation of centromere protein F regulating pathways in prostate cancer. Int. J. Urol. 2015; 22: 867–77. 2 Tucci P, Agostini M, Grespi F et al. Loss of p63 and its microRNA-205 target results in enhanced cell migration and metastasis in prostate cancer. Proc. Natl. Acad. Sci. USA 2012; 109: 15312–7. 3 Aytes A, Mitrofanova A, Lefebvre C et al. Cross-species regulatory network analysis identifies a synergistic interaction between FOXM1 and CENPF that drives prostate cancer malignancy. Cancer Cell 2014; 25: 638–51. 4 Zhuo YJ, Xi M, Wan YP et al. Enhanced expression of centromere protein F predicts clinical progression and prognosis in patients with prostate cancer. Int. J. Mol. Med. 2015; 35: 966–72.

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