Methylation and inhibition of expression of uPA by the RAS oncogene: divergence of growth control and invasion in breast cancer cells

Pouya Pakneshan, Moshe Szyf1 and Shafaat A. Rabbani2

Department of Medicine and 1 Department of Pharmacology, McGill University Health Center, Montreal, Canada

2 To whom correspondence should be addressed at: McGill University Health Centre, 687 Pine Avenue West, Room H4.67, Montreal, Quebec, Canada H3A 1A1. Tel: +1 514 843 1632; Fax: +1 514 843 1712; Email: shafaat.rabbani{at}mcgill.ca


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression of urokinase-type plasminogen activator (uPA), a protease only expressed in highly invasive human breast cancer cells, is inhibited by DNA methylation of its promoter. We tested the hypothesis that up-regulation of DNA methyltransferase 1 (DNMT1) will lead to methylation and silencing of uPA and inhibition of the invasiveness of metastatic breast cancer cells. Since RAS was previously shown to up-regulate DNA methylation, we examined the effects of ectopic expression of constitutively active RAS on methylation and expression of uPA. Transfection of Ha-RAS into MDA-MB-231 human breast cancer cells resulted in a significantly shorter cell doubling time compared with the controls. However, expression and activity of the metastatic gene uPA and invasive capacity of the cells were significantly reduced by the oncogene RAS. Silencing of uPA by RAS is mediated by a cis modification of the uPA promoter and not through an effect on a trans-acting factor, since a transiently transfected unmethylated uPA–luicferase reporter is expressed at a similar level in RAS-transfected and control cells. We then examined the levels of DNMT1 and methylated DNA-binding protein 2 (MBD2) expressions in these cells to determine whether this reduction in uPA expression is associated with changes in the DNA methylation machinery. Our results showed that ectopic expression of RAS induced DNMT1 expression and activity and inhibited MBD2 expression. Consistent with methylation-mediated repression, uPA was partially methylated in RAS-transfected cells and uPA expression was induced upon treatment of RAS transfectants with the demethylating agent 5'-azacytidine. These results therefore imply that the RAS–DNMT1 DNA methylation pathway which promotes oncogenic growth in many cancers can exert an opposite effect on the invasive capacity of the highly invasive MDA-MB-231 cells, thus illustrating the divergence of growth and metastasis promoting pathways in cancer. This has important implications for new therapeutic approaches to metastasizing cancer.

Abbreviations: 5'-azaC, 5'-azacytidine; DNMT1, DNA methyltransferase 1; MBD2, methylated DNA-binding protein 2; MSP, methylation-specific PCR; Neo, neomycin resistance gene; uPA, urokinase-type plasminogen activator


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Urokinase-type plasminogen activator (uPA) is a member of the serine protease family that has been implicated in the process of invasion, metastasis and angiogenesis of several malignancies, including breast cancer, due to its ability to break down different components of the extracellular matrix (1,2). High levels of uPA produced by both tumor cells and the stroma surrounding the tumor are involved in the process of tumor progression in breast cancer (24). Overexpression of uPA and its receptor is directly associated with induction of tumor growth, invasion and metastasis (1). In previous studies we have demonstrated that uPA is expressed only in highly invasive, hormone-insensitive human breast cancer cells, such as MDA-MB-231 (5,6). The lack of uPA expression in normal mammary epithelial cells and in early hormone-responsive stages of cancer is due to transcriptional suppression of uPA gene expression by DNA methylation of the uPA promoter (5). Increased levels of DNA methyltransferase 1 (DNMT1) expression and regional hypermethylation of tumor suppressor genes is generally involved in promotion of oncogenic growth (7,8). However, we have previously found that induction of hypermethylation of uPA in a metastatic breast cancer cell model leads to silencing of uPA gene expression and suppression of metastasis (9). These findings suggest a divergence of oncogenic growth and the metastatic response to increased DNA methylation. There was no evidence, however, that up-regulation of cellular levels of DNMT1 by cellular signaling could lead to methylation of uPA and inhibition of uPA expression and metastasis.

Aberrant firing of the RAS signaling pathway is a nodal pathway regulating cancerous cell growth and thus has been a target for cancer therapy (10,11). The expression of uPA can be regulated by growth factors that bind to tyrosine kinase receptors and activate the RAS signaling pathway (1,2). It has been reported, on the other hand, that the level of DNA methyltransferase and DNA methylation are controlled by the RAS signaling pathway (1214). Interestingly, it has been known for some time that in human colon adenocarcinoma, where RAS activation is well-established, uPA expression is limited to fibroblast like cells and endothelial cells in the tumor stroma while uPA is not expressed in malignant epithelial cells (15), thus raising the prospect that long-term activation of RAS results in silencing of uPA in malignant cells. We therefore tested the hypothesis that long-term RAS activation leads to inhibition of uPA expression by cis modification of the gene. Ectopic expression of RAS could therefore be used to up-regulate cellular DNMT1 levels and to test the effect of this growth promoting pathway on the invasiveness of breast cancer cells. We introduced ectopic oncogenic RAS into the highly invasive MDA-MB-231 human breast cancer cell line and showed that while RAS promotes DNMT1 expression, DNA methylation and cell growth, it suppresses the expression of uPA and cell invasion, suggesting a divergence of the growth and invasion pathways. While tumor growth is known to be stimulated by methylation of tumor suppressor genes (7,8), metastasis is inhibited by DNA methylation of metastasis promoting genes (5,9,16). The two contradictory effects of activated RAS on cell growth and invasion could therefore be explained by increased DNA methylation of specific genes, suggesting that the RAS–DNMT1 signaling pathway has divergent effects on cell growth and metastasis, which has important therapeutic implications.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
We obtained the MDA-MB-231 human breast cancer cell line from the American Type Culture Collection (Rockville, MD). These cells were maintained in DMEM (Invitrogen) with 10% fetal bovine serum, 2 mM L-glutamine and 100 U/ml penicillin–streptomycin sulfate. MCF-7 and T47D human breast cancer cells were also obtained from the American Type Culture Collection and were maintained according to the provider's recommendations. The cells were transfected with either plasmid pHO6T1 expressing the activated Ha-RAS oncogene or with pHO6 vector alone. Stable transfection was performed using 5 µg/ml DNA and 20 µl/ml LipofectAMINE (Invitrogen) following the manufacturer's protocol. Three independent transfections were performed. The selection medium containing 1 mg/ml Geneticin (G418; Gibco BRL) was added to the cells 72 h after transfection to select for neomycin-resistant transfectants. The resulting polyclonal cell populations were characterized and used for analysis throughout this study. Three independent pools of transfectants were used to rule out idiosyncrasies resulting from exceptional integration sites of the transgenes.

Levels of activated RAS in cell lysates isolated from the control MDA-MB-231 (MDA-231), the vector-transfected MDA-MB-231 (MDA-V) and the RAS-transfected MDA-MB-231 (MDA-RAS) cells were determined using a RAS Activation Assay Kit (Upstate Cell Signaling, Lake Placid, NY; catalog no. 17-218) following the manufacturer's instructions and by using an anti-RAS mouse monoclonal antibody (Upstate Cell Signaling; catalog no. 05-516). For cell growth analysis the cells were cultured at a density of 1.0 x 105 viable cells/plate in triplicate. They were trypsinized and the number of viable cells stained with 0.4% Trypan Blue (Sigma, St Louis, MO) was determined by counting daily for 4 days.

For B-1086 (Eisai Research Institute, Andover, MA), a RAS farnesyltransferase inhibitor, treatment MDA-RAS transfectants were grown to 60% confluence and treated with either vehicle alone or 10 or 50 µg/ml B-1086 for 72 h. MDA-RAS cells were also treated with 25 µM 5'-azacytidine (5'-azaC) (Sigma) for 10 days. Genomic DNA and cellular RNA were extracted from the control and treated cells using DNAzol and TRIzol (Gibco BRL), respectively, following the manufacturer's instructions.

Northern blot analysis
We fractionated 20 µg of total cellular RNA on a 1.1% agarose–formaldehyde gel in 4-morpholinepropanesulfonic acid buffer and transferred it onto a nylon membrane (Amersham-Pharmacia Biotech, Baie d'Urfé, Canada) using standard protocols. The membranes were then hybridized with either 32P-labeled human uPA, DNMT1, methylated DNA-binding protein 2 (MBD2), neomycin resistance gene (Neo) or 18S rRNA probes for 14 h at 65°C. Autoradiography of the blots was carried out at –80°C using XAR film (Easton Kodak, Rochester, NY). The signals obtained for each of the probes were quantified by densitometric scanning of the autoradiograms (Gel Doc; Bio-Rad, Mississauga, Canada) and were then normalized to the signal obtained for 18S rRNA.

uPA enzyme activity assay
The enzymatic activity of uPA in conditioned medium was examined using Spectrozyme UK (American Diagnostica, Greenwich, CT), a synthetic chromogenic substrate of uPA (16), using high molecular weight recombinant uPA (American Diagnostica) for standardization according to the manufacturer's instructions. The photometric absorbance of the reaction mixtures at 405 nm was measured using a Vmax plate reader (Molecular Devices, Sunnyvale, CA) following 30 min incubation at room temperature.

Boyden chamber Matrigel invasion assay
We examined the invasive capacities of the cells as previously described using two-compartment Boyden chambers (Transwell; Costar, Cambridge, MA) and basement membrane Matrigel (Becton Dickinson, Bedford, MA) (6). We coated the 8 µm pore polycarbonate filters with basement membrane Matrigel (50 µg/filter) and analyzed 5 x 104 cells in each chamber as described (6). The filters were then fixed for 30 min in 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature, washed with phosphate-buffered saline, stained with 1.5% toluidine blue and mounted onto glass slides. The invading cells were then examined and counted in 10 randomly selected fields under a light microscope at x400 magnification. The average number of invading cells was then calculated.

Luciferase reporter assay
The uPA promoter (a gift from Dr Blasi, Milan, Italy) was inserted into a luciferase reporter vector, pGL-3 basic (Promega, Madison, WI), to obtain plasmid uPAP-luc as previously described (6). pGL-3 basic was used as a control. The uPAP-luc and PGL-3 basic plasmids were each co-transfected with PSV-ß-gal (Promega) containing the ß-galactosidase gene to normalize the transfection efficiency into MDA-MB-231 and MDA-RAS cells using 10 µl/ml LipofectAMINE (Invitrogen) according to the manufacturer's instructions. Luciferase reporter activity was determined 72 h after transfection as previously described (6).

DNA methyltransferase activity assay
DNA methyltransferase activity levels were determined as previously described (6) by incubating 5 µg of total nuclear extract isolated from MDA-MB-231, MDA-V and MDA-RAS cells with a hemimethylated oligonucleotide duplex and the labeled methyl donor S-adenosyl-L-methionine-(methyl-3H) (Amersham Biosciences) at 37°C for 3 h. The reaction mixture was precipitated in 10% trichloroacetic acid, passed through GF/C filters (Whatman, Maidstone, UK) and washed with 10% trichloroacetic acid. The filters were then air dried and total radioactivity incorporated into DNA was measured by liquid scintillation counting. Reaction mixtures containing substrate oligonucleotides but no nuclear extract were used as a negative control.

Western blot analysis
To examine changes in MBD2 levels, total nuclear extract was isolated from the untreated and treated MDA-MB-231 cells as previously described (17). Equal amounts of protein were fractionated on SDS–polyacrylamide gels followed by transfer to a nitrocellulose membrane (Amersham-Pharmacia Biotech) using standard protocols. The membranes were blocked in 1% Carnation milk, 2% bovine serum albumin, 150 mM NaCl and 10 mM Tris (pH 7.4). Immunoblotting was performed overnight at 4°C using a 1:200 dilution of the sheep polyclonal antibody MBD2 (Upstate Cell Signaling; catalog no. 07-198) and a 1:2000 dilution of an anti-ß-actin antibody (Sigma). The membranes were washed with Tris-buffered saline and then incubated with a 1:5000 dilution of secondary antibodies purchased from Bio-Rad (Richmond, CA). Proteins were visualized using a chemiluminescence detection kit from Perkin Elmer (catalog no. NEL103001EA).

Methylation-specific PCR (MSP)
Genomic DNA was treated with sodium bisulfite as previously described (16,18). Two sets of MSP primers were used to amplify the region from –123 to –28 of either the methylated (5'-AGC GTT GCG GAA GTA CGC GG-3' and 5'-AAA CCC GCC CCG ACG CCG CC-3') or unmethylated (5'-AGT GTT GTG GAA GTA TGT GG-3' and 5'-AAA CCC ACC CCA ACA CCA CC-3') uPA promoter using Platinum Taq DNA polymerase (Invitrogen) according to the manufacturer's recommendations under the following cycle conditions: 95°C for 3 min; 10 cycles of 95°C for 30 s, 52°C for 30 s and 72°C for 45 s; 20 cycles of 95°C for 30 s, 50°C for 30 s and 72°C for 45 s; a final extension of 72°C for 5 min. The amplified products were fractionated on 2% agarose gels.

Statistical analysis
All experiments were performed with three independent polyclonal populations of MDA-RAS transfectants and control cells. Results are presented as means ± SE. Statistical comparisons were performed using Student's t-test analysis of variance and ANOVA. A probability value of P < 0.05 was considered to be significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Stable transfection of MDA-MB-231 cells with the RAS oncogene
MDA-MB-231 cells were transfected with either plasmid pHO6T1 expressing the activated Ha-RAS oncogene (MDA-RAS) or with plasmid pHO6 alone (MDA-V) (19,20). Three neomycin-resistant polyclonal cell populations were obtained from three independent transfections and were characterized for levels of activated RAS in their cell lysates using a RAS Activation Assay Kit. The assay confirmed a significant induction of RAS activity in the MDA-RAS cells as compared with the control MDA-MB-231 and MDA-V cells (Figure 1A). To determine whether ectopic expression of activated RAS affected cell growth parameters, the cell doubling time of the MDA-RAS transfectants was examined. As shown in Figure 1B, the MDA-RAS transfectants exhibited a significantly shorter cell doubling time as compared with the MDA-MB-231 and MDA-V control cells. This effect is consistent with the known growth promoting effects of the RAS oncogene.



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Fig. 1. Stable transfection of MDA-MB-231 cells with the activated RAS oncogene. (A) MDA-MB-231 cells were transfected with a vector expressing constitutively active RAS. The MDA-MB-231 RAS transfectants (MDA-RAS) as well as MDA-MB-231 cells transfected with vector alone (MDA-V) and untransfected control (MDA-231) cells were characterized for levels of activated RAS in their cell lysates using a RAS Activation Assay Kit. The blot presented is representative of three independent polyclonal cell populations. (B) Cell doubling time of the MDA-RAS transfectants and controls was examined by cell growth curve analysis. The cells were plated at a similar density in triplicate, trypsinized and counted daily for 4 days. Results represent the means ± SE of three independent polyclonal cell populations. Significant difference from controls was determined by Student's t-test and is indicated by an asterisk (P < 0.05).

 
Effect of RAS overexpression on uPA expression
To examine the effect of RAS on uPA expression, total cellular RNA was isolated from MDA-MB-231, MDA-V and MDA-RAS cells and levels of uPA mRNA expression were examined by northern blot analysis using uPA and 18S rRNA probes. Hybridization to the Neo probe was used to verify the presence of the vector in both vector alone and RAS transfectants. The results of the northern blot analyses show a significant decrease in uPA mRNA levels in the MDA-RAS transfectants as compared with the control MDA-MB-231 cells and MDA-V transfectants (Figure 2A). The enzymatic activity of uPA was determined by Spectrozyme UK, which is a direct chromogenic uPA activity assay. The assay detected a significant decrease in uPA enzymatic activity in the MDA-RAS transfectants as compared with the control cells (Figure 2B). Overexpression of RAS therefore significantly reduced uPA mRNA expression and activity in the MDA-RAS cells. In order to examine whether this reduction in uPA expression had any effect on the invasive capacity of MDA-RAS cells, we used the Boyden Chamber Matrigel invasion assay. The results of this assay, presented in Figure 2C, show that a reduction in uPA expression in the MDA-RAS transfectants was accompanied by inhibition of their invasive capacity.



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Fig. 2. Effect of activated RAS expression on uPA levels and invasive capacity of MDA-MB-231 cells. (A) The level of uPA mRNA expression was examined by northern blot analysis of total cellular RNA isolated from MDA-MB-231, MDA-V and MDA-RAS cells. All blots were probed with random primed 32P-labeled uPA, 18S rRNA or Neo probes and mRNA expression was quantified by densitometric scanning (upper panel). The signals were normalized to the intensity of signal obtained with the 18S rRNA probe and are plotted as the ratio of uPA mRNA expression to 18S rRNA expression (lower panel). The Neo probe was used to verify the presence of the transfected plasmid in the transfectants. (B) The enzymatic activity of uPA in conditioned medium of these cells was determined by Spectrozyme UK, which is a direct chromogenic uPA activity assay. (C) The invasive capacity of the cells was examined by Boyden Chamber Matrigel invasion assay. All results are representative of the means ± SE of triplicate analyses of three independent polyclonal cell populations. Significant difference of the results obtained for the transfectants from MDA-MB-231 control cells was determined by Student's t-test and is indicated by an asterisk (P < 0.05).

 
Stable transfection of Ha-RAS into low invasiveness MCF-7 and T47D human breast cancer cells, which do not express uPA due to silencing of its promoter by DNA methylation (5), did not result in induction of uPA gene expression (Figure 3). This suggests that Ha-RAS expression might affect expression of the unmethylated uPA gene in metastatic MDA-MB-231 breast cancer cells but not expression of the methylated uPA gene in these non-metastatic cancer cell lines.



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Fig. 3. Effect of activated RAS expression on uPA levels in MCF-7 and T47D cells. Both MCF-7 and T47D human breast cancer cells, which do not normally express uPA, were transfected with the vector expressing constitutively active RAS (RAS) or empty vector (V). The level of uPA mRNA expression was examined by northern blot analysis of total cellular RNA isolated from these cells. All blots were probed with either 32P-labeled uPA, 18S rRNA or Neo probes. Results are representative of triplicate analyses of three independent polyclonal cell populations.

 
To determine whether RAS activity was responsible for suppression of uPA in MDA-MB-231 cells, we treated the MDA-RAS transfectants with different concentrations of B-1086, which is an inhibitor of the enzyme farnesyltransferase. As shown in Figure 4A, B-1086 treatment significantly induced uPA mRNA expression in MDA-RAS transfectants in a dose-dependent manner, almost completely reversing the effects of RAS overexpression in these cells at the highest concentration. This suggests that inhibition of uPA expression and activity in MDA-RAS is caused by RAS activity.



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Fig. 4. Effects of RAS on uPA expression and uPA promoter activity. (A) The MDA-RAS cells were treated with different concentrations of B-1086, a RAS farnesyltransferase inhibitor, for 72 h. Total cellular RNA was isolated at the end of the treatment and the level of uPA mRNA expression was examined by northern blot analysis. Blots were probed with labeled uPA and 18S probes (upper panel). The mRNA expression levels were quantified by densitometric scanning and normalized to 18S rRNA (lower panel). (B) A uPA promoter–luciferase construct was transfected into MDA-MB-231 and MDA-RAS cells to examine the effects of RAS overexpression on uPA promoter activity. Bars 1, 2 and 3 correspond to three independent transfections. Results are representative of the means ± SE of triplicate analyses. Significant difference from the control MDA-MB-231 cells was determined by Student's t-test and is indicated by an asterisk (P < 0.05).

 
RAS expression does not affect transactivation of the uPA promoter
To determine whether RAS activity affected the trans-acting factors required for uPA promoter activity we determined whether RAS expression affects the activity of an unmethylated uPA promoter–luciferase construct transiently transfected into the cells. The results of this experiment show that there was no significant difference in uPA promoter activity in MDA-MB-231 and MDA-RAS cells, suggesting that inhibition of uPA expression in MDA-RAS transfectants is not due to indirect effects of RAS on the trans-acting machinery acting on the uPA promoter (Figure 4B). The lack of a trans effect of RAS on uPA supports the hypothesis that Ha-RAS expression results in a cis modification of the uPA gene resulting in its silencing. The fact that exogenous unmethylated promoter is not affected by RAS overexpression is consistent with previous observations which demonstrated that ectopic DNA transiently transfected into either human (21,22) or murine cells (23) is not subjected to de novo methylation. DNMT1 is a resident of the replication fork (24) and therefore does not target ectopic plasmid DNA, which does not bear an origin of replication. Methylation of such DNA would be possible only after integration into the genome. Even after integration, de novo methylation of exogenously transfected DNA is a protracted process and occurs after multiple passages in culture (23,25).

Effect of RAS overexpression on methylation of the uPA promoter in MDA-MB-231 cells
We have previously shown that the state of uPA promoter methylation and uPA gene expression correlate with increased levels of DNMT1 expression and reduced levels of MBD2 in breast cancer cells (6). Since it has been previously shown that RAS activation increases DNMT1 mRNA levels and activity as well as DNA methylation (1214), we examined the levels of DNMT1 expression and activity in MDA-RAS transfectants. Both DNMT1 mRNA levels (Figure 5A) and DNA methyltransferase activity (Figure 5B) are significantly elevated in MDA-RAS transfectants as compared with control cells. Since the expression of MBD2 was also previously shown to correlate with hypomethylation and expression of uPA and since inhibition of MBD2 led to hypermethylation and inhibition of uPA expression (6,9), we also examined the levels of MBD2 expression in the MDA-RAS transfectants by both northern blot (Figure 6A) and western blot analysis (Figure 6B). These results indicate that overexpression of RAS in MDA-MB-231 cells results in a significant decrease in MBD2 mRNA and protein levels as compared with the control cells.



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Fig. 5. Effect of RAS overexpression on DNMT1 expression and activity. (A) DNMT1 mRNA expression was examined by northern blot analysis of total cellular RNA isolated from either control or RAS-transfected MDA-MB-231 cells. Blots were probed with labeled DNMT1, 18S rRNA or Neo probes (upper panel). The mRNA expression levels were quantified by densitometric scanning and normalized to 18S rRNA (lower panel). The Neo probe was used to verify the presence of the plasmid in the transfectants. (B) The DNA methyltransferase activity in these cells was determined by incubating the total nuclear extract isolated from the cells with a hemimethylated oligonucleotide duplex and the labeled methyl donor S-adenosyl-L-methionine-(methyl-3H). Reaction mixtures containing substrate oligonucleotides but no nuclear extract were used as a negative control (CTL). Results are representative of the means ± SE of triplicate analyses. Significant difference from the MDA-MB-231 control was determined by Student's t-test and is indicated by an asterisk (P < 0.05).

 


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Fig. 6. Effect of RAS overexpression on MBD2 expression. (A) Total cellular mRNA extracted from MDA-MB-231, MDA-V and MDA-RAS cells was examined for level of MBD2 expression by northern blot analysis. All blots were probed with labeled MBD2 and 18S rRNA probes (upper panel). The mRNA expression levels were quantified by densitometric scanning and the results normalized to18S rRNA expression (lower panel). (B) Levels of MBD2 protein in MDA-MB-231, MDA-V and MDA-RAS cells were examined by western blot analysis of the total nuclear extracts isolated from these cells. ß-Actin expression was determined as a control to assure equal protein loading (upper panel). The intensity of the bands was quantified and plotted as the ratio of MBD2 expression to ß-actin expression (lower panel). Results are representative of the means ± SE of triplicate determinations of three independent polyclonal MDA-RAS cell populations. Significant difference from the MDA-MB-231 control was determined by Student's t-test and is indicated by an asterisk (P < 0.05).

 
Our findings described above suggest that RAS overexpression brings about a change in expression and activity of components of the DNA methylation machinery. To determine whether these changes in the DNA methylation machinery trigger methylation of the uPA promoter, we performed a MSP analysis to determine the state of methylation of the uPA promoter in RAS and control transfectants using primers specific for either methylated or unmethylated CpGs within the uPA promoter, as previously described (9). The MSP results presented in Figure 7A confirm that the uPA promoter was partially methylated in MDA-RAS cells as compared with control MDA-MB-231 cells, in which the uPA promoter was completely unmethylated, as previously reported (5). To determine whether DNA methylation played a causal role in the silencing of uPA caused by activation of RAS, we treated MDA-RAS transfectants with the demethylating agent 5'-azaC. This treatment reversed the hypermethylation (Figure 7A) and inhibitory effects (Figure 7B) of RAS overexpression on uPA expression, resulting in restoration of uPA mRNA levels to those observed in control transfectants (Figure 7B). These data are consistent with the hypothesis that suppression of uPA by RAS is mediated by increased DNA methylation. Thus RAS expression leads to increased DNMT1 expression and DNMT activity, which in turn results in cis modification of the uPA gene by DNA methylation.



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Fig. 7. Effect of RAS overexpression on methylation of the uPA promoter. (A) Methylation status of the uPA promoter was examined by MSP analysis. Genomic DNA isolated from these cells was treated with sodium bisulfite and then amplified using primers specific for methylated or unmethylated uPA promoter sequence, indicated as m and u, respectively. Results are representative of triplicate analyses. (B) MDA-RAS cells were treated with the demethylating agent 5'-azaC for 10 days. Total cellular RNA was extracted from these cells at the end of the treatment as well as from untreated MDA-RAS and MDA-MB-231 cells to examine the levels of uPA mRNA expression by northern blot analysis. Blots were probed with labeled uPA and 18S rRNA probes (upper panel). The level of mRNA expression was quantified by densitometric scanning and results were plotted as the ratio of uPA mRNA expression to 18S rRNA expression (lower panel). Results are representative of triplicate analyses and significant difference from the MDA-MB-231 control cells was determined by Student's t-test and is indicated by an asterisk (P < 0.05).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One of the key mediators in the process of tumor cell invasion and metastasis in several malignancies, including breast cancer, is uPA (1). It has been shown in many studies over the years that induction of uPA expression is directly associated with highly invasive characteristics of cancer cells (1,2). We have previously shown that uPA expression is regulated by DNA methylation (5,6,16). The uPA promoter is silenced by DNA methylation in normal human mammary epithelial cells and in non-invasive human breast cancer cell lines, while it is active and unmethylated in highly invasive human breast cancer cell lines (5). This gene is therefore regulated in an opposite manner to tumor suppressor genes, which are repressed by DNA methylation in cancer cells but are unmethylated in normal cells (7,26,27). This suggests that the two main processes involved in tumorigenesis, uncontrolled growth and metastasis, are inversely regulated by DNA methylation, representing two different stages in tumor development. However, it was not clear whether the divergent methylation patterns of tumor suppressor genes and prometastatic genes result from independent and stochastic gene-specific events or whether the state of methylation of uPA could be modulated by central cellular signaling pathways which also affect cellular growth. We have previously shown that DNMT1 is a downstream effector of the RAS signaling pathway (12,13), which also controls cellular growth, thus raising the possibility that activation of the same pathway could lead to the two opposite results of increased growth and decreased invasiveness.

In this paper we show that ectopic expression of constitutively active RAS in the highly metastatic breast cancer cell line MDA-MB-231 results in inhibition of uPA gene expression which is commensurate with the increased cell growth parameters. Thus, RAS can concurrently act to increase cell growth and reduce invasive capacity by inhibiting uPA expression and activity, suggesting that cell invasion and cell growth could be inversely controlled by activated RAS and, perhaps, by additional signaling pathways in the advanced stages of cancer. This effect of RAS is mediated by the RAS–DNMT1 DNA methylation pathway and requires a cis modification of the uPA gene. This conclusion is supported by the following results presented here. First, the effect of RAS is abrogated by 5'-azaC, suggesting that DNA methylation must be involved. Second, RAS does not affect the trans-acting machinery needed to activate an unmethylated uPA promoter. The unmethylated uPA promoter fires at similar rates in both RAS transfectants as well as control cells, suggesting that a cis modification is responsible for silencing of the endogenous gene. Third, the uPA promoter is methylated in RAS transfectants relative to controls. Fourth, RAS has no effect on uPA expression in non-metastatic cells in which the promoter of the uPA gene is methylated. This suggests that RAS effects are mediated by increased methylation. RAS would not affect uPA in normal breast cancer cells since its promoter is methylated in these cells, thus implying that the main model in which excess RAS activity affects uPA gene expression is highly metastatic cancer cells, which is the example analyzed in this paper.

RAS has previously been proposed to elevate DNMT1 expression through the action of FOS and JUN on regulatory AP-1 sequences in both the murine and human DNMT1 genes (12). Activated oncogenic RAS was also shown to be associated with increased methylation in tissue culture (13). Associated with this induction of DNMT1 expression and activity, RAS activation might have also led to methylation of certain tumor suppressor genes, which is a common mechanism of their silencing and the promotion of cell growth, as previously reported (7). On the other hand, we and others have shown that prometastatic genes silenced by methylation are activated by demethylation in metastatic cancer cells (16,28). Thus, activated RAS might have these opposite effects on tumor cell growth and invasion by stimulating DNA methylation of both tumor suppressor and prometastatic genes. The data presented here are consistent with this hypothesis. Interestingly, another member of the DNA methylation machinery, MBD2, which was previously suggested to be required for demethylation of uPA, is here shown to be down-regulated by RAS (9). Down-regulation of MBD2 in addition to DNMT1 up-regulation by RAS might cooperate in altering the methylation profile of uPA and perhaps other critical genes in the cell. Our data are consistent with two different methylation programs in breast cancer cells which might be required for different stages of tumorigenesis, uncontrolled growth versus metastasis. These programs might be either controlled or disrupted by nodal oncogenic signals such as activated RAS. This long-term effect of RAS activation on silencing of uPA through cis modification of the gene might explain a long-standing observation that in many tumors, especially colon adenocarcinoma, uPA is expressed by fibroblasts in the stroma and not by the malignant cells (15). The mechanism described in this study might provide an explanation. We hypothesize that RAS activation, which is well established in colon adenocarcinoma, results in an increase in DNMT1, increased methylation of the uPA gene promoter and silencing of expression through DNA methylation. Although future experiments are required to prove this point, the data presented here provide a testable and universal mechanism for uPA silencing in many tumors.

These data illustrate that by manipulating central signaling pathways we can cause hypermethylation of genes that are critical for metastasis. Our results also suggest that metastasis and increased cell growth might be inversely correlated under certain conditions. This might unfold into a therapeutic strategy which might be used at stages of advanced metastasis to silence prometastatic genes, which might seem a paradoxical concept since activation of signaling pathways is also growth promoting. Nevertheless, it is possible that in certain stages of the disease, inhibition of metastasis would be paramount. The RAS–DNMT1 pathway is a prime target for anticancer therapy. Inhibitors of DNMT1 were previously shown to inhibit cell growth. Therapeutic strategies that are directed at reducing cell growth, such as inhibition of RAS signaling or DNMT1, might increase the invasiveness of metastatic cells under some conditions, as indicated by the increased uPA expression in MDA-MB-231 Ha-RAS transfectants treated with the RAS inhibitor B-1086 (Figure 4A). In accordance with this hypothesis, we have previously shown that 5'-azaC, an inhibitor of DNMT1, causes demethylation and induction of uPA expression in MCF-7 cells (16,28).

Another interesting issue that needs to be discussed is previous data showing increased expression of uPA upon binding of growth factors to their tyrosine kinase receptors and activation of the RAS signaling pathway (1,29,30). This seems to contradict the main argument of this paper that RAS inhibits metastasis. However, our data do not necessarily contradict these previous observations. To resolve this contradiction we propose that whereas the short-term direct effects of RAS activation on an unmethylated uPA promoter might well be activation of expression, the long-term consequences of RAS activation would result from its indirect effects brought about by increasing DNA methylation, as illustrated in this paper. DNA methylation caused indirectly by RAS activation would lead to stable and long-term inhibition of uPA and perhaps to its unresponsiveness to firing of the RAS signaling pathway. Thus, activation of growth factor-responsive pathways might have opposite short-term and long-term effects on expression of critical metastatic genes such as uPA. Our data presented here showing that activation of signaling pathways might lead to changes in DNA methylation suggest that the long-term effects of drugs which modulate signaling must be taken into account when considering their clinical utility.


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 Abstract
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 Discussion
 References
 

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Received October 19, 2004; revised December 7, 2004; accepted December 15, 2004.