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Cancer Research 68, 8499, October 15, 2008. doi: 10.1158/0008-5472.CAN-08-0907
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

MicroRNA-Mediated Inhibition of Prostate-Derived Ets Factor Messenger RNA Translation Affects Prostate-Derived Ets Factor Regulatory Networks in Human Breast Cancer

Victoria J. Findlay, David P. Turner, Omar Moussa and Dennis K. Watson

Department of Pathology and Laboratory Medicine, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina

Requests for reprints: Dennis K. Watson, Hollings Cancer Center, Medical University of South Carolina, 86 Jonathan Lucas Street, Charleston, SC 29425. Phone: 843-792-3962; Fax: 843-792-3940; E-mail: watsondk{at}musc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Prostate-derived Ets factor (PDEF) is an ETS transcription factor expressed in normal tissues with high epithelial cell content and noninvasive breast cancer cells. A putative tumor suppressor PDEF protein expression is often lost during progression to a more invasive phenotype. Interestingly, PDEF mRNA has been found to be retained or even overexpressed in the absence of protein; however, the mechanisms for this remain to be elucidated. This study identifies two microRNAs (miRNA) that directly act on and repress PDEF mRNA translation, leading to the loss of PDEF protein expression and the gain of phenotypes associated with invasive cells. In addition, we show that these miRNAs are elevated in human breast tumor samples. Together, these data describe a mechanism of regulation that explains, for the first time, the lack of correlation between PDEF mRNA and protein levels, providing insight into the underexplored role of posttranscriptional regulation and how this contributes to dysregulated protein expression in cancer. These observations have critical implications for therapeutically targeting miRNAs that contribute to cancer progression. [Cancer Res 2008;68(20):8499–506]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Cancer death is due in large part to metastases. One of the more interesting challenges is to understand the cellular changes that occur during progression toward invasive cancer. ETS proteins are a large family of transcription factors with diverse functions and activities that activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, apoptosis, differentiation, and transformation (1, 2). The ETS family gene prostate derived Ets factor (PDEF) is expressed in normal epithelial tissues, including prostate, breast, and colon (3). In normal tissue and noninvasive cancers, mRNA and PDEF protein are easily detectable by Northern and Western blot. However, PDEF protein loss is correlated with prostate, breast, and colon cancer progression to an invasive phenotype both in vitro and in vivo (3–5). Interestingly, this loss of protein does not always correlate well with PDEF mRNA levels (5). Indeed, some invasive cancers retain or have elevated levels of PDEF mRNA in the absence of protein (3, 6, 7). PDEF reexpression in multiple invasive prostate, breast, and colon cancer cells results in reduced cell growth, migration, and invasion (2, 3).1 Reciprocal small interfering RNA–mediated knockdown experiments in PDEF-expressing noninvasive cells result in increased migration and invasion together with an altered morphology consistent with a more invasive phenotype (2). Together, these and other data support the model that PDEF target genes control several aspects of the multistep metastatic process and, specifically, loss of PDEF regulatory networks is a key event in the development of invasive cancer (8). The processes involved in the loss of PDEF protein during cancer progression have not been elucidated. The goal of this study was to identify pathways involved in the posttranscriptional regulation of PDEF that ultimately results in protein loss, providing novel mechanistic insight into the cellular events leading to a more aggressive phenotype.

MicroRNAs (miRNA) are endogenous 19-25 nucleotide noncoding RNAs that have recently emerged as a novel class of small, evolutionarily conserved important gene regulatory molecules involved in many critical developmental and cellular functions (9). Through specific base pairing with target mRNA sequences in the 3' untranslated region (UTR), miRNAs induce mRNA degradation, translational repression, or both (10). Individual miRNAs can target numerous mRNAs, often in combination with other miRNAs, thereby providing a mechanism for controlling complex regulatory networks. It is estimated that there are over 600 miRNAs in mammalian cells and that ~30% of all genes are regulated by miRNAs (11, 12). Over 3,000 identified mature miRNAs exist in species ranging from plants to humans. Their existence and conservation throughout species support the concept that they perform critical functions in gene regulation (13). Indeed, the conserved evolution of both miRNAs and transcription factors highlights their importance in and the complexity of gene regulation (14). miRNAs have been implicated in the control of many fundamental cellular and physiologic processes, including tissue development, cellular differentiation, and proliferation, metabolic and signaling pathways, apoptosis, and stem cell maintenance (15–17). Mounting evidence indicates that miRNAs may also play a significant role in cellular transformation and carcinogenesis acting either as oncogenes or tumor suppressors (18, 19). Hence, there are few cellular processes that are not affected by miRNAs. In addition, specific miRNA signatures have been identified for both solid cancers and hematologic malignancies (20–23), and mounting evidences suggest that the power of miRNAs lies in the ability to distinguish specific cancer subtypes based on their miRNA profile, including, as well as of direct relevance to the studies described herein, breast cancer (20, 24). Nonetheless, the identification and validation of specific targets have been limited. We report here PDEF as a novel target for miR-204 and miR-510 and describe for the first time a mechanism for the loss of PDEF protein expression during breast cancer progression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell culture. Human breast cancer cell lines (MCF7, BT474, CAMA-1, HBL100, HCC202, Hs578t, MDA-MB-157, MDA-MB-175 VII, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-436, and MDA-MB-453) were cultured according to American Type Culture Collection (ATCC) website. The breast cancer cell lines CAMA-1, HBL100, HCC202, MDA-MB-415, and MDA-MB-436 were a kind gift of R. Neve (University of California). All other lines were obtained from ATCC. For the generation of stable MCF7 cells overexpressing miR-204, pSuppressor-neo vector (Imgenex) expressing miR-204 was transfected into MCF7 cells and stable cells were selected in medium containing G418 (Invitrogen). This vector system was also used for transient expression of miR-204 and miR-510 into MCF7 cells.

Tumor samples. Matched tumor and nontumor breast samples were obtained from the Hollings Cancer Center tumor bank at Medical University of South Carolina (MUSC). Before surgery at the center, all patients provided written informed consent to allow any excess tissue to be used for research studies. Samples were snap frozen in OCT and stored at –80°C until use. The pathologic status of the specimens was confirmed by histologic examination of 10 µm sections taken at the start, middle, and end of the 5 x 20 µm sections taken for RNA analysis. Each tumor section contained between 65% and 80% malignant epithelial cells and 0 to 5% nonmalignant epithelial cells. Nontumor sections contained 100% benign epithelial cells. To determine PDEF expression, human breast cancer paraffin blocks of tissues available from the same patients were obtained from the HCC Tumor Bank (MUSC).

Immunohistochemistry. Antigen retrieval was done by heating in a microwave oven for 2 x 5 min on half power in 10 mmol/L citrate (pH 6.0). Sections were washed and treated with 1% H2O2 for 15 min, and nonspecific binding was blocked with 2.5% horse serum (ImmPRESS Vector staining kit; Vector Laboratories) for 1 h and then incubated overnight at 4°C with PDEF primary antibody at a 1:100 dilution in 2% bovine serum albumin (BSA) in PBS. Overnight incubation at 4°C was followed by 3 x 10 min washes in PBS, Immpress antirabbit secondary antibody was incubated (Vector Laboratories) for 2 h at room temperature. After washing with H2O, 3,3'-diaminobenzidine substrate (Sigma) was added for 2 min followed by washing in H2O. Slides were counterstained with hematoxylin.

Quantitative reverse transcription–PCR. Total RNA from cancer cell lines was extracted using the RNeasy Plus Mini kit (Qiagen). Total RNA from breast tumor and nontumor samples was extracted using Trizol as per the manufacturer's instructions (Invitrogen). One microgram total RNA was reverse transcribed in a 20-µL reaction using Superscript III reverse transcriptase (Invitrogen) for miRNA analyses and iScript (Bio-Rad) for all other studies. Real-time PCR was performed with 1 µL of a 1:10 dilution of reverse-transcribed cDNA using the Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) in a LightCycler (Roche). The cycling conditions for all genes were preincubation at 50°C for 2 min, 95°C for 2 min, followed by 30 to 50 cycles of denaturation at 94°C for 10 s, annealing at 1° below the lowest Tm for each gene-specific primer pair (Supplementary Table S1) for 10 s and extension for 30 s at 72°C, with a single data acquisition at the end of each extension. All ramping was done at 20°C/s. Triplicate reactions were run for each cDNA sample. The relative expression of each gene was quantified on the basis of Ct value measured against an internal standard curve for each specific set of primers (Supplementary Table S1) using the software provided by the instrument manufacturer (Roche). These data were normalized to S26, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or U6 (see individual experiments). The size and purity of the PCR products were also analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining.

Plasmid construction. To overexpress miR-204 and miR-510, the genomic region surrounding the pri-miRNA sequence of miR-204 and miR-510 were amplified with ThermalAce (Invitrogen). The cycling conditions were preincubation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 65°C for 1 min, and extension for 1 min at 72°C. The PCR products (~500 bp) were directionally cloned into the pSuppressor-neo vector (Imgenex) using XhoI and XbaI. The 5' UTR, open reading frame (ORF), 3' UTR, and full-length sequences of PDEF were amplified from human genomic DNA of MCF7 cells and directionally cloned into pcDNA3 (Invitrogen). The wild-type 3' UTR of PDEF was cloned into the XbaI site of the pGL3-promoter vector (Promega). The sequences complementary to the seed of the miR-204 and miR-510 were deleted using a QuikChange site-directed mutagenesis kit (Stratagene). For primer sequences and Tas, see Supplementary Table S2. All constructs were validated by sequencing at the MUSC sequencing facility.

Oligonucleotide transfection. The miRNA inhibitors (Ambion) are single-stranded, chemically enhanced oligoribonucleotides designed to inhibit the endogenous miRNAs. Cells were transfected with 100 nmol/L of the indicated oligoribonucleotide using the Oligofectamine reagent, as per the manufacturer's instructions (Invitrogen). At 48 h after transfection, cells were harvested for protein or RNA extraction.

Luciferase assays. Cells were plated at 200,000 cells per well in a six-well plate. The pGL3 reporter constructs (0.5 µg, firefly luciferase) were cotransfected with pRL-TK (0.05 µg, Renilla luciferase) using Lipofectamine 2000 as per the manufacturer's instructions (Invitrogen). The media was changed the next day, and luciferase activity measured after 48 h using the dual luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each transfected well.

Western blot analysis. Cell lysate preparation and Western blot analysis using enhanced chemiluminescence were performed, as described previously (2). Experimental antibodies include human PDEF (prepared as described previously; ref. 3) and E-cadherin (BD BioSciences). GAPDH and β-actin (Abcam) were used as loading controls.

Transwell migration and invasion assay. Stably transfected MCF7 cells were seeded into the upper chamber of a Transwell insert precoated with 5 µg/mL fibronectin for migration or a BD Matrigel invasion chamber for invasion in serum-free medium at a density of 50,000 cells per well (24-well insert; pore size, 8 µmol/L; BD Biosciences). Medium containing 10% serum was placed in the lower chamber to act as a chemoattractant, and cells were further incubated for 24 h. Nonmigratory cells were removed from the upper chamber by scraping with a cotton bud. The cells remaining on the lower surface of the insert were stained using Diff-Quick (Dade Behring, Inc.). Cells were quantified as the number of cells found in 10 random microscope fields in two independent inserts. Error bars represent the SD from three separate experiments.

Immunofluorescence. Cells were seeded onto sterile cover slides (18-mm diameter) coated with 5 µg/mL fibronectin and allowed to attach overnight. Cells were then fixed with 2% formaldehyde, permeabilized with 0.1% Triton X-100, and blocked in 2% BSA for 1 h at room temperature. E-cadherin expression was examined using the antibody detailed above and visualized using Alexa Fluor secondary antibody (Invitrogen). Immunofluorescence was examined using an Olympus IX70 confocal microscope.

Colony formation assay. Wild-type and miR-204 stably transformed MCF7 cells were seeded at a cell density of ~4 cells/mm2 in normal growth media. Cells were incubated as normal, and colonies were counted after 7 to 10 d.

Soft agar assay. Two milliliters of 0.6% agarose in 2x DMEM were plated in each well of a six-well plate and left to set for 20 min. This layer was overlaid with 1.0 x 104 wild-type and miR-204 stably transfected MCF7 cells in 3 mL of 0.4% agarose diluted in 2x DMEM. Cells were incubated as normal for ~14 d, and the colonies were counted.

Statistical analysis. For statistical testing, two-sided paired Student's t tests were done using Excel spreadsheet. P values are given for each individual experiment, but in general, P < 0.05 was considered statistically significant. Error bars represent SDs of three independent experiments, unless indicated otherwise.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
PDEF is posttranscriptionally regulated during breast cancer progression. The discordance between PDEF mRNA and protein levels supported the model that PDEF may be posttranscriptionally regulated during breast cancer progression. To identify appropriate cell line systems to examine this model, we evaluated PDEF mRNA and protein levels in multiple breast cancer cell lines by real-time PCR and Western analyses, respectively. MCF7, MDA-MB-361, and BT474 cell lines expressed detectable levels of both PDEF mRNA and protein, and MDA-MB-175 VII, MDA-MB-415, MDA-MB-453, CAMA-1, and HCC202 cell lines had PDEF mRNA, but little or no detectable levels of protein (Fig. 1A and B ). These cell lines are derived from luminal cells, appear more differentiated, form tight cell-cell junctions, and are noninvasive or weakly invasive (25). In contrast, MDA-MB-157, MDA-MB-436, MDA-MB-231, HBL100, and Hs578t cells had neither PDEF mRNA or protein (Fig. 1A and B) and are derived from basal B cells, appear less differentiated, have a more mesenchymal-like appearance, and are highly invasive (25). Therefore, cells lose PDEF protein during progression to a more invasive cancer.


Figure 1
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  		Figure 1. PDEF is regulated posttranscriptionally through sequences present in the 3' UTR. A, quantitative real-time PCR of PDEF mRNA normalized to S26. B, Western blot analysis of breast cancer cell lines probed with primary antibodies for PDEF and GAPDH. Bottom, semiquantitative analysis. C, schematic representation of full-length, ORF alone, ORF + 5' UTR (5' UTR), and ORF + 3' UTR (3' UTR) constructs and Western blot (WB) and RT-PCR analysis of PDEF expression in MDA-MB-157 cells transiently transfected with the constructs illustrated above. D, luciferase activity of MDA-MB-157 cells transfected with a reporter luciferase gene (pGL3) and pGL3 fused to the PDEF 3'UTR (3' UTR) normalized to Renilla luciferase activity. Columns, mean for three experiments conducted in triplicate; bars, SD.

 
Our previous gain-of-function studies used a construct expressing only the ORF of PDEF to overexpress the protein in multiple breast cancer cell lines (2, 3). These observations supported the notion that PDEF may be regulated posttranscriptionally through sequences in its UTRs. To study UTR-dependent translational control of PDEF transcripts, the level of PDEF mRNA and protein expression after transfection of MDA-MB-157 cells with constructs lacking 5' and/or 3' UTR sequences were compared with that, allowing expression of the full-length PDEF transcript (Fig. 1C). Reverse transcription–PCR (RT-PCR) analysis showed that PDEF mRNA is expressed after transfection of each construct (Fig. 1C). In contrast, PDEF protein was not detectable in cells transfected with PDEF constructs containing both or either UTR (Fig. 1C), supporting the model that elements present within the 3' UTR of PDEF negatively regulate its mRNA translation in breast cancer. One mechanism of posttranscriptional regulation is through miRNA-mediated repression of elements present in the 3' UTR of genes. To investigate whether PDEF mRNA translation was regulated by factors binding to its 3' UTR, we fused the 3' UTR of PDEF mRNA to a luciferase reporter. The presence of the 3' UTR of PDEF resulted in a significant reduction in luciferase activity compared with the unmodified control luciferase reporter (Fig. 1D).

miRNAs regulate PDEF expression through its 3' UTR. To determine whether miRNAs played a role in the posttranscriptional regulation of PDEF expression, we conducted a bioinformatics analyses and identified 12 potential miRNA recognition sequences within the 3' UTR of the PDEF transcript. Primers were designed for the top 10 scoring miRNAs. Real-time PCR analysis on a series of breast cancer cell lines revealed that, compared with eight other miRNAs, the expression of two miRNAs, miRNA 204 (miR-204) and miRNA 510 (miR-510), were most correlated with breast cancer cell lines having high PDEF RNA and low PDEF protein levels (Fig. 2A and data not shown). Specifically, miR-204 and miR-510 are expressed at elevated levels relative to MCF7 in four of five and five of five of the breast cancer cell lines with high PDEF RNA and low PDEF protein (MDA-MB-175 VII, MDA-MB-415, MDA-MB-453, CAMA-1, and HCC202), whereas miR-28 is elevated in only two of five lines (MDA-MB-175 VII, MDA-MB-415). Although some elevated expression of miR-204 and miR-510 was also observed in other breast cancer cell lines when compared with MCF7, we chose these two miRNAs as the best candidates of the 10 initially characterized for further investigation in the negative regulation of PDEF expression. The predicted sites for miRNAs within a 3' UTR can overlap or even be identical; however, miR-204 and miR-510 bind to separate sites within the 3' UTR of PDEF (Fig. 2A). To examine whether PDEF expression is repressed by miR-204 and/or miR-510 through these elements, the luciferase reporter construct containing the 3' UTR of PDEF was cotransfected into HeLa cells together with a plasmid construct designed to overexpress either miR-204 or miR-510. Cotransfection with either miR-204 or miR-510 further reduced the luciferase activity to 55% and 35%, respectively (Fig. 2B). The most important criteria for target recognition are the 5' five to eight nucleotide core sequence of a miRNA, known as the "seed sequence." To further validate that miR-204 and miR-510 are direct repressors of PDEF via binding to the identified sites within the 3' UTR, we mutated the seed sequences of miR-204 and miR-510, respectively, in the luciferase reporter construct. Transfection of HeLa cells with these mutated luciferase reporters resulted in ~25% and 40% increase in luciferase activity, respectively (Fig. 2C). Furthermore, miR-204 and miR-510 had no significant repressive effect on luciferase activity when overexpressed in cells transfected with luciferase-UTR constructs containing the respective mutated seed sequences (Fig. 2D).


Figure 2
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  		Figure 2. miR-204 and miR-510 regulate PDEF through its 3' UTR. A, top, quantitative real-time PCR of miR-28 (black columns), miR-204 (white columns), and miR-510 (gray columns) normalized relative to the amount of U6 target ({Delta}Ct). The relative levels of miRNA expression were measured by determining the {Delta}{Delta}Ct values of the indicated cell lines versus MCF7. Bottom, schematic representation of the predicted target sites of miR-204 and miR-510 in the 3' UTR of PDEF mRNA. The numbers (1–469) represent base pairs in the 3' UTR of PDEF and the numbers in parentheses (1425–1894) represent base pairs in the full-length PDEF gene (Genbank accession number AF071538; ref. 42). Complementary base sequences are highlighted in upper case letters. B-D, luciferase activity of HeLa cells transfected with (B) pGL3 PDEF 3' UTR reporter construct (3' UTR) and pSuppressor vector alone (pSupp), pSupp/miR-204 (miR204), or pSupp/miR-510 (miR510); (C) pGL3 reporter construct (pGL3), 3' UTR, 3' UTR reporter construct mutated in the miR-204 seed sequence binding site (mut204), or 3' UTR reporter construct mutated in the miR-510 seed sequence binding site (mut510); or (D) cotransfected with 3'UTR, mut204 or mut510 and pSupp (gray columns), miR204 (white columns), or miR510 (black columns). All luciferase assays were normalized to Renilla luciferase activity. The data are expressed as the mean ± SD for three experiments conducted in triplicate.

 
Regulation by miRNAs of protein translation can be due to translational repression and/or mRNA degradation. To assess whether miR-204 and/or miR-510 have a functional role in the down-regulation of endogenous PDEF expression by either of these mechanisms, two cell lines were selected as model systems: CAMA-1 (elevated PDEF mRNA, low PDEF protein) and MCF7 (PDEF mRNA and protein). Transfection of antisense oligoribonucleotides (ASO) targeted against miR-204 or miR-510 in CAMA-1 cells resulted in an increase in PDEF protein levels, whereas the PDEF mRNA levels remained unchanged (Fig. 3A ). Reciprocal overexpression of either miR-204 or miR-510 in MCF7 cells results in a loss of PDEF protein, without significantly affecting the levels of PDEF mRNA (Fig. 3B and C). PDEF protein is present at very low (miR-204) or undetectable levels (miR-510) in cells transfected with 1 µg plasmid. These results show that miR-204 and miR-510 posttranslationally regulate endogenous PDEF mRNA, most likely through a mechanism of translation inhibition.


Figure 3
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  		Figure 3. miR-204 and miR-510 regulate endogenous PDEF protein expression. A, Western blot analysis of endogenous PDEF expression in CAMA-1 cells transfected with scrambled ASO (control) or ASO against miR-204 (204) or miR-510 (510). MCF7 and MDA-MB-231 cells are control cells positive and negative for PDEF protein expression, respectively. B and C, Western blot analysis of MCF7 cells transfected with increasing concentrations of (B) miR-204 or (C) miR-510. Quantitative real-time PCR analysis of PDEF mRNA levels from the treatments in A, B, and C normalized to GAPDH are shown to the right of the Western blots.

 
Functional consequences of the regulation of PDEF by miR-204. Studies in our laboratory have shown that the overexpression of PDEF in an invasive breast cancer cell line leads to an altered cell morphology (2, 3). MCF7 cells typically have an epithelial morphology described as cobblestone in appearance. However, MCF7 cells that were stably transfected with miR-204 and showed concomitant lower levels of PDEF protein expression (Fig. 4A ) had an altered cell morphology (Fig. 4C). These cells also had a more spindle-like morphology and seemed more mesenchymal, a change similar to that observed in cells that have undergone an epithelial-to-mesenchymal transition (EMT; ref. 26). PDEF is a transcription factor involved in the negative regulation of genes involved in metastatic progression. Studies in our laboratory have identified urokinase-type plasminogen activator (uPA) and slug as negative PDEF response genes, as their mRNA expression levels decrease when PDEF is overexpressed (3),2 and as direct targets, as shown by chromatin immunoprecipitation.3 uPA and slug are involved in metastatic progression, and slug up-regulation plays distinct roles during EMT (27, 28), and therefore, these genes were selected for examination in our miR-204 stable overexpressing cells. Consistent with negative regulation by PDEF, miRNA-mediated reduction in PDEF protein expression resulted in an increase in the levels of both uPA and slug by quantitative real-time PCR analysis (Fig. 4B). The adhesion protein E-cadherin plays critical roles during epithelial morphogenesis (29). Expression of this protein is down-regulated during the acquisition of invasive and metastatic phenotypes at late stages of epithelial tumor progression. Slug is established as a transcriptional repressor of E-cadherin gene expression in this process (28). Therefore, we performed quantitative real-time PCR analysis and confirmed that E-cadherin mRNA levels were reduced in miR-204 overexpressing cells (Fig. 4B). Concurrently, we observed a decrease in total and surface staining of E-cadherin by immunofluorescence (Fig. 4D) and total protein levels by Western blot analysis (Fig. 4A).


Figure 4
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  		Figure 4. Functional consequences of miR-204 overexpression in MCF7 cells. A, Western blot analysis of PDEF and E-cadherin (E-Cad) expression in MCF7 control cells (MCF7) and a miR-204 overexpressing stable clone (miR-204). B, quantitative real-time PCR of E-cadherin, uPA, and SLUG mRNA expression in MCF7 control (black columns) or miR-204 overexpressing cells (gray columns) normalized to GAPDH. Bright field microscopy (C) and E-cadherin immunofluorescence (D) of MCF7 control cells (MCF7) and a miR-204 overexpressing MCF7 stable clone (miR-204).

 
Cells that have undergone an EMT have higher migratory and invasive properties. To assess whether migration and/or invasion are altered in miR-204 overexpressing cells, we performed transwell migration assays across a chemokine gradient and invasion assays through Matrigel. The number of cells found to migrate or invade in miR-204 overexpressing cells was significantly increased compared with the parental control (Fig. 5A ). To further explore the possible role of miR-204 in cancer progression, we investigated the ability of the stably transfected MCF7 cells to proliferate when seeded at low density in vitro using a clonogenic assay and observed an increase in the total number of colonies formed when compared with the parental control (Fig. 5B). A hallmark of transformed cells is their ability to grow independent of anchorage; therefore, to determine whether the overexpression of miR-204 resulted in enhanced transformation, we performed soft agar assays and observed an increase in the total number of colonies that were able to form in the cells overexpressing miR-204 (Fig. 5B). Each of these molecular and cellular phenotypes were confirmed with multiple miR-204 stable clones (data not shown). Similar findings were obtained for cells after transient expression of miR-510 in MCF7 cells (Supplementary Fig. S1).


Figure 5
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  		Figure 5. miRNA overexpression increases migration, invasion, and transformed phenotypes in vitro. A, transwell migration (left) and Matrigel invasion (right) of MCF7 control (black columns) or miR-204 overexpressing MCF7 (gray columns) cells. B, colony formation (left) and soft agar (right) assays of MCF7 control (black columns) or miR-204 overexpressing MCF7 (gray columns) cells. C, quantitative real-time PCR of uPA (left) and SLUG (right) mRNA expression of miR-204 overexpressing cells transiently transfected with PDEF (dark gray columns) or vector alone (black columns). D, Transwell migration (left) and Matrigel invasion (right) of miR-204 overexpressing cells transiently transfected with PDEF (dark gray columns) or vector alone (black columns).

 
PDEF expression inhibits the miR-204 overexpressing phenotype. miRNAs have multiple targets, and therefore, the effects observed after miR-204 expression may be the result of the increased PDEF protein, as well as non–PDEF-related miR-204 effects. One way to evaluate these possibilities is to examine the phenotypes in cells in which a nontargeted PDEF is expressed. To do this, the ORF of PDEF was transfected into the miR-204 overexpressing cells, and molecular and cellular phenotypic effects were measured. Quantitative real-time PCR analysis showed that exogenous PDEF expression in miR-204 overexpressing cells resulted in a decrease in both uPA and SLUG mRNA levels when compared with the vector alone (Fig. 5C). In addition, these cells were less migratory and less invasive (Fig. 5D). Thus, the miRNA-mediated loss of PDEF was responsible for the observed changes. The collective data suggest that, during breast cancer progression, elevated miR-204 reduces PDEF protein expression, and the resultant change in expression of PDEF-regulated genes and their downstream targets contributes to a more invasive phenotype.

miR-204 and miR-510 levels are elevated in breast tumor samples. Based upon the effect of miR-204 and miR-510 on PDEF protein expression and PDEF-dependent phenotypes, we evaluated the levels of miR-204 and miR-510 in RNA prepared from human breast tumor and matched nontumor samples by quantitative real-time PCR. Relative to that found in nontumor tissue samples, the levels of miR-204 and miR-510 were found to be significantly elevated in all tumor samples (Fig. 6A and B ; Supplementary Table S3). These data suggest that miR-204 and miR-510 function as "oncomiRs" and further support the model that elevated expression of miRNAs contribute to the loss of PDEF protein expression during breast cancer progression. We have previously shown that PDEF protein expression is reduced in invasive breast cancer specimens (3). PDEF expression was evaluated in four of the five samples evaluated for miRNA expression, as they were available as formalin-fixed, paraffin-embedded blocks from the same patients (1335, 1412, 1420, and 1844). As shown in Fig. 6C and D, PDEF protein is found predominantly in the nucleus of nontumor epithelial cells. However, consistent with our previous findings, epithelial cells present in invasive ductal carcinomas show decreased protein expression. Taken together, these data suggest that miR-204 and miR-510 levels are elevated in invasive breast cancer and that these levels are inversely correlated to PDEF protein expression.


Figure 6
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  		Figure 6. miR-204 and miR-510 levels are elevated in human breast cancer. Quantitative real-time PCR analysis of (A) miR-204 and (B) miR-510 levels in human breast tumor (gray columns) and matched nontumor (black columns) samples (for P values, see Supplementary Table S3). Representative immunohistochemical staining of PDEF in human (C) nontumor and (D) tumor breast tissue (case 1335; magnification, 400x).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
The rapidly evolving field of miRNAs has established close correlations between their altered expression and many types of cancer (30–32). However, the identification and validation of specific targets have been limited. This study identifies two miRNAs (miR-204 and miR-510) that are involved in the negative regulation of PDEF mRNA translation and describes, for the first time, a mechanism that contributes to PDEF protein loss during breast cancer progression. Specifically, overexpression of miR-204 and miR-510 in MCF7 cells results in a dose-dependent loss of PDEF protein expression. Although ~60% transfection efficiency was achieved in MCF7 cells, an apparently greater loss of PDEF protein expression was observed, perhaps due to a threshold level required for detection of protein by Western blot analysis. A potential role for PDEF as a tumor suppressor has been shown by studies showing reduced PDEF protein expression and/or loss by immunohistochemistry in invasive cancer (3), as well as the repression of survivin (33), uPA (3), and slug.4 Furthermore, recent studies showed PDEF protein expression inhibited xenograft tumor formation in vivo (33). Identifying the mechanisms of PDEF protein loss provides novel insight into the transcriptional networks that are active during metastatic progression. It is generally accepted that multiple mechanisms of regulation of any protein are active in normal cells, and each of these represent pathways that can be altered during pathogenesis. Indeed, we show that regulatory elements are also present within the 5' UTR of PDEF, supporting a previous model that the 5' UTR of PDEF is involved in its posttranscriptional regulation in prostate cancer cells through inhibition of translation initiation (5). Interestingly, a recent study found that mRNA are repressed by engineering miRNA-binding sites in the 5' UTR as efficiently as in the 3'UTR, although no endogenous targets regulated in this manner have been found (34). In addition, both PDEF mRNA (4) and protein expression5 are lost upon stimulation of cells with transforming growth factor-β (TGF-β), a soluble growth factor able to induce EMT (35). The mechanism of TGF-β–dependent loss of PDEF mRNA and protein expression may include activation of inhibitory miRNAs, as a recent study illustrated a TGF-β–induced EMT miRNA signature in human keratinocytes (36). Indeed, the EMT-like phenotype observed in MCF7 cells stably transfected with miR-204 or after transient expression of miR-510 supports the notion that growth factors, like TGF-β, that induce EMT may play a role in the regulation of this and other miRNAs during breast cancer progression.

This and other studies have shown the importance of PDEF regulation during breast cancer progression. We also show that miR-204 and miR-510 levels are elevated in breast cancer compared with nontumor tissue. Until now, the expression levels of miR-204 and miR-510 have not been specifically examined in breast cancer specimens, although a high throughput microarray study has been performed for miRNA profiling on breast cancer biopsies (37). Whereas miR-204 levels were not specifically highlighted in this study, the datasets show a significant elevation of miR-204 in those breast cancer biopsy samples with a tumor cell percentage of 70% or more when compared with the normal controls. miR-510 levels were not evaluated in this study. Of interest, the genomic locations of these two miRNAs have been associated with cancer. Amplification of a noncoding region mapped to chromosome 9q21 (miR-204) has been associated with prostate cancer (38). Furthermore, amplification of Xq27 (miR-510) occurs during the process of cell transformation and tumorigenesis using breast cancer as a model system and in sporadic breast cancer (39, 40). Future studies directed toward elucidating the mechanism of activation of these miRNAs will provide valuable insight into the complex pathways involved in metastatic breast cancer progression. This is exemplified by the recent study that identified the metastatic specific induction of miRNA-10b through the action of the transcription factor Twist (41), another known regulator of EMT, emphasizing the fundamental role for miRNAs in cancer progression and their representation as a new class of tumor suppressors and oncogenes.

The function of the majority of miRNAs is still currently unknown, although a plethora of predicted targets exist. We show here for the first time a validated target, PDEF, for two miRNAs, miR-204 and miR-510, as well as functional consequences of their interaction. These studies support the model that miR-204 and miR-510 are potential oncogenes and that their elevated expression contributes to the loss of PDEF protein expression during breast cancer progression. It is likely that additional miRNAs are involved in the posttranscriptional regulation of PDEF, and their identification will increase our understanding of the mechanisms that exist to regulate PDEF during breast cancer progression. Furthermore, future studies involved in the identification of additional targets for these miRNAs will provide greater insight into the complex biological pathways involved in metastatic progression.


   Disclosure of Potential Conflicts of Interest
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: NIH grant P01CA78582.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Richard Neve for the kind gift of cell lines.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

1 Unpublished data. Back

2 Unpublished data. Back

3 D.P. Turner et al., manuscript in preparation. Back

4 D.P. Turner et al., manuscript in preparation. Back

5 V.J. Findlay et al., unpublished observation. Back

Received 3/13/08. Revised 7/10/08. Accepted 7/31/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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