A molecular role for lysyl oxidase in breast cancer invasion

We identified previously an up-regulation in lysyl oxidase (LOX) expression,an extracellular matrix remodeling enzyme, in a highly invasive/metastatic human breast cancer cell line, MDA-MB-231, compared with MCF-7, a poorly invasive/nonmetastatic breast cancer cell line. In this study, we demonstrate that the mRNA expression of LOX and other LOX family members [lysyl oxidase-like (LOXL), LOXL2, LOXL3, and LOXL4] was observed only in breast cancer cells with a highly invasive/metastatic phenotype but not in poorly invasive/nonmetastatic breast cancer cells. LOX and LOXL2 showed the strongest association with invasive potential in both highly invasive/metastatic breast cancer cell lines tested (MDA-MB-231 and Hs578T). To determine whether LOX is directly involved in breast cancer invasion, LOX antisense oligonucleotides were transfected into MDA-MB-231 and Hs578T cells, and found to inhibit invasion through a collagen IV/laminin/gelatin matrix in vitro compared with LOX sense oligonucleotide-treated and untreated controls. In addition, treatment of MDA-MB-231 and Hs578T cells with beta-aminopropionitrile (an irreversible inhibitor of LOX enzymatic activity) decreased invasive activity. Conversely, MCF-7 cells transfected with the murine LOX gene demonstrated a 2-fold increase in invasiveness that was reversible by the addition of beta-aminopropionitrile in a dose-dependent manner. In addition, endogenous LOX mRNA expression was induced when MCF-7 cells were cultured in the presence of fibroblast conditioned medium or conditioned matrix, suggesting a role for stromal fibroblasts in LOX regulation in breast cancer cells. Moreover, the correlation of LOX up-regulation and invasive/metastatic potential was additionally demonstrated in rat prostatic tumor cell lines, and human cutaneous and uveal melanoma cell lines. These results provide substantial new evidence that LOX is involved in cancer cell invasion.     

Genetic manipulation of stromal cell-derived factor-1 attests the pivotal role of the autocrine SDF-1-CXCR4 pathway in the aggressiveness of breast cancer cells

Stromal cell-derived factor-1 (SDF-1), via its receptor CXCR4, has been implicated in metastasis of cancer, including breast cancer. Exogenous SDF-1 is known to regulate locomotion, chemotaxis and adhesion. The knowledge regarding the effect of autocrine SDF-1 on breast cancer cells is not available. The current study evaluated the effects of SDF-1 on the biological behaviour of breast cancer cells by genetically modifying the expression of SDF-1 in breast cancer cells. Two human breast cancer cell lines (MDA-MB-231 and MDA-MB-435s) and a human fetal lung fibroblast cell line (MRC5) were used. The expression of SDF-1 and the SDF-1 receptor, CXCR4 in the cell lines were studied. Expression cassettes of human SDF-1 and hammerhead ribozyme transgenes specifically targeting human SDF-1 were constructed and used to over-express SDF-1 or to knockout the expression of SDF-1 in cancer cells, respectively. Invasiveness, migration and growth of the genetically modified cells were assessed. SDF-1 was expressed in wild-type human breast cancer cell line MDA-MB-435s and fibroblast cell line MRC5, but not in MDA-MB-231 cell line. In contrast, CXCR4 expression was observed in all three cell lines tested. The ability of invasion and migration was significantly reduced in SDF-1 knockout MDA-MB-435s cells, compared with wild-type and vector control cells (p<0.01). On the other hand, SDF-1 transfected MDA-MB-231epsilonSDF1+/+ cells that stably expressed SDF-1 showed a different behaviour from MDA-MB-231SDF1+/- (plasmid control) and wild-type MDA-MB-231 cells, both being SDF-1 negative. MDA-MB-231epsilonSDF1+/+ cells displayed a higher degree of invasiveness and migration, compared with wild-type and MDA-MB-231SDF+/- cells (p<0.01). Furthermore, SDF1-knockout MDA-MB-435s cells showed a slower growth rate over a 7-day period compared with the respective control and wild-type MDA-MB-435s cells. In contrast, the growth of the SDF-1 transfected MDA-MB-231SDF1+/+ cells was markedly enhanced when compared with wild-type and vector control cells. Breast cancer cell lines, when equipped with the autocrine SDF-1-CXCR4 signal pathway, display aggressive behaviour, including an increase in invasiveness, migration together with faster growth. SDF-1, together with its receptor CXCR4 may provide important information for predicting the aggressive nature and constitute important therapeutic targets in human breast cancer.     

Role for glucose transporter 1 protein in human breast cancer

Glycolysis is increased in cancer cells compared with normal cells. It has been shown that glucose enters cells via a family of five functional glucose transporters (GLUT). However, GLUT expression appears to be altered in human breast cancer, which may serve as a selective advantage and facilitate the metastatic potential of these cells. The relationship of GLUT isoform expression and breast cancer cell invasiveness has not been adequately addressed. Thus, the purpose of this study was to investigate whether an association exists between GLUT expression and human breast cancer cell invasiveness. Invasiveness of the human breast cancer lines MCF-7, MDA-MB-435 and MDA-MB-231 was measured using anin vitro assay and compared with cellular GLUT isoform expression, assessed by Western blot analysis and verified by immunohistochemistry in a poorly differentiated human ductal breast cancer. Cell surface GLUT-1 expression was associated with the invasive ability of MCF-7 (2.0 ± 0.02%), MDA-MB-435 (6.4 ±0.4%), and MDA-MB-231 (19.3 ± 2.0%). However, GLUT-2 and GLUT-5 were inversely associated with invasiveness; GLUT-3 expression was variable; and GLUT-4 was undetected. In a poorly differentiated human ductal breast cancer,in situ GLUT-1 staining was intense. GLUT-1 expression was associated with the in vitro invasive ability of human breast cancer cells which was validatedin situ. If this relationship is found to exist in a larger number of human breast cancer tissues, it may be possible to develop diagnostic and therapeutic strategies based on targeted GLUT isoform expression.     

Tumorigenicity of human breast cancer is associated with loss of the Ca2+-activated chloride channel CLCA2.

The human Ca2+-activated chloride channel-2 (CLCA2) is expressed in normal breast epithelium but not in breast tumors of different stages of progression. Northern analysis of nontransformed and transformed breast epithelial cell lines revealed CLCA2 expression in the nontransformed cell line MCF10A and the nontumorigenic cell line MDA-MB-453, whereas all tumorigenic cell lines were negative (MDA-MB-231, MDA-MB-435, MDA-MB-468, and MCF7). When stably reintroduced into CLCA2-negative MDA-MB-231 and MDA-MB-435 cells, CLCA2 expression reduced Matrigel invasion in vitro and inducibility of s.c. and metastatic tumors of MDA-MB-231 cells in nude mice. Our results suggest that CLCA2 may act as a tumor suppressor in breast cancer.     

Protein kinase Calpha regulates Ets1 transcriptional activity in invasive breast cancer cells

We have previously shown that PKC inhibitors interfere with the Ets1/Smad3-dependent regulation of parathyroid hormone-related protein (PTHrP) P3 promoter activity by TGFbeta in invasive MDA-MB-231 breast cancer cells. By examining PKC expression in a variety of breast cancer cell lines, the protein level of PKCalpha was found to be much higher in Ets1-expressing MDA-MB-231 and MDA-MB-435 breast cancer cells than in Ets1-deficient MCF-7 and SK-BR3 cells. No correlation of Ets1 expression with the expression of other PKC subtypes (PKCbeta1, PKCbeta2, PKCdelta or PKCepsilon) could be observed. In contrast to MDA-MB-231 cells, PKCalpha-deficient MCF-7 cells do not support Ets1-induced activation of the PTHrP P3 promoter suggesting that PKCalpha may be important for Ets1 activity. A constitutively active form of PKCalpha was found to potentiate the P3 promoter activation by Ets1 alone and in synergy with Smad3. PKCalpha, but not PKCepsilon, also induced phosphorylation of the Ets1 protein. Both PKCalpha effects on Ets1 depended on the exon VII domain of Ets1. Using verapamil and ionomycin, we could show that PKCalpha induces Ets1 phosphorylation independent of calcium mobilization. Collectively, our data suggest that PKCalpha may regulate Ets1 activity in invasive breast cancer cells.     

Characterization of variants of a human breast-cancer cell-line isolated from metastases in different organs of nude-mice

The MDA-MB-435 human breast cancer cell line metastasizes from tumors growing in the mammary fatpad of nude mice; metastases are found in the lymph nodes and lungs of 75 to 100% of tumor-bearing mice, and at a lower incidence in other organs such as the heart, skeletal muscle, adrenal gland and brain. Variants of this breast cancer cell line were established from metastases in different organs, including the lungs and brain of nude mice; these lines were designated 435-Lung2 and 435-Br1, respectively. Karyotypic analysis of the new variants suggested a clonal origin for the different organ metastases. The lung-derived cells were as metastatic as the original cell line when re-injected in nude mice, yet the cells from the brain metastasis produced few metastases. Analyses of the invasive potential of the cell lines showed that they did not differ in expression of mRNA for 72-kDa type IV-collagenase, or in gelatinase activity (by zymography), or in ability to invade through a Matrigel-coated filter. The poorly metastatic 435-Br1 cells showed reduced binding to cultured monolayers of mouse lung-endothelial cells, compared with the metastatic 435-Lung2 and MDA-MB-435 cells. When the breast cancer cells were artificially arrested in the lungs of nude mice, by injecting Cytodex beads coated with cells, all three cell lines grew equally well, suggesting that the deficiency found in the metastatic potential of the 435-Br1 cells may be associated with interactions with endothelial cells, rather than growth potential in the lungs. Further comparisons of these two metastasis-derived variants of a heterogeneous cell line will lead to greater understanding of the metastatic phenotype of human breast cancer.     

Semaphorin4C 在乳腺癌子宫内膜癌及前列腺癌中的表达及意义*

目的:研究轴突导向分子Semap horin4C（Sema4C）在乳腺癌、子宫内膜癌及前列腺癌组织和细胞水平的表达情况及其与上述三种恶性肿瘤转移的关系。方法:应用免疫组化技术检测45例乳腺癌、42例子宫内膜癌及49例前列腺癌组织中Sema4C的表达情况,分别采用蛋白免疫印迹法Western Blot及细胞免疫荧光方法检测Sema4C 在三种乳腺癌细胞（MDA-MB-435S、MDA-MB-231、MCF-7）、两种子宫内膜癌细胞株（AN3CA、HEC-1-B）和两种前列腺癌细胞株（PC- 3M-1E8、PC- 3M-2B4）的蛋白表达及其亚细胞定位情况。结果:乳腺癌和子宫内膜癌中,其分别在有淋巴结转移的乳腺癌组和子宫内膜癌组的原发灶中的表达高于无淋巴结转移组,差异有统计学意义（P<0.05）。 前列腺癌中,Sema4C 的阳性表达率随着Gleason评分增加而升高（P<0.05）。 Western blot在七株肿瘤细胞均检测到Sema4C 的表达,且提示Sema4C 的表达与细胞的转移潜能呈正相关。荧光显微镜下观察到Sema4C 主要定位在细胞膜和细胞浆。结论:Sema4C 在乳腺癌、子宫内膜癌和前列腺癌组织和细胞中表达具有普遍性,且与上述三种恶性肿瘤转移关系密切,有望成为上述三种恶性肿瘤治疗的一个新的分子靶点。      

Ectopic expression of RhoBTB2 inhibits migration and invasion of human breast cancer cells

RhoBTB2, or Deleted in Breast Cancer 2 (DBC2), identified as a candidate tumor suppressor gene for breast cancer and other human malignancies, is an atypical member of a novel gene family encoding small GTPases. In this study, we found that ectopic expression of RhoBTB2 inhibits the migration and invasion of the human metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-435 in a dose-dependent manner. Western blotting analysis revealed that ectopic expression of RhoBTB2 induces a significant increase in the breast cancer metastasis suppressor, BRMS1. siRNA suppression of BRMS1 expression markedly reversed the inhibitory effects of RhoBTB2 on the migration and invasion abilities of both cell lines. Ezrin is a member of the ezrin-radixin-moesin cytoskeleton-associated protein family and is a key signaling molecule that regulates cancer migration and invasion. Western blotting analysis demonstrated that ectopic expression of RhoBTB2 results in decreased phosphorylation of ezrin and Akt2 in both MDA-MB-231 and MDA-MB-435 cells. Therefore, we conclude that up-regulation of the breast cancer metastasis suppressor BRMS1 and down-regulation of the phosphorylation of the cancer metastasis-related gene, ezrin, contributed to RhoBTB2-induced inhibition of metastatic breast carcinoma cell migration and invasion. Our findings suggest that understanding RhoBTB2-mediated migration and suppression of invasion is critical to the development of new therapies designed to prevent and treat patients with breast cancer metastasis.     

Zinc and its transporter ZIP10 are involved in invasive behavior of breast cancer cells

Zinc is an essential element, necessary for sustaining all life. Zinc deficiency causes taste impairments, immune deficiency, skin problems, and growth and mental retardation. Recent reports suggest that zinc is associated with an increased risk of cancer, although it is still unclear whether zinc or its transporters are involved in cancer progression. Here we show that zinc and its transporter ZIP10 are involved in the invasive behavior of breast cancer cells. The screening of clinical samples for ZIP10 mRNA expression suggested that ZIP10 was significantly associated with the metastasis of breast cancer to the lymph node. In addition, the expression of ZIP10 mRNA was higher in the invasive and metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-435S than in less metastatic breast cancer cell lines, such as MCF7, T47D, ZR75-1 and ZR75-30. In in vitro cell migration assays, the depletion of zinc transporter ZIP10 and intracellular zinc inhibited the migratory activity of MDA-MB-231 and MDA-MB-435S cells. These results showed that zinc and ZIP10 play an essential role in the migratory activity of highly metastatic breast cancer cells, and suggest ZIP10 as a possible marker for the metastatic phenotype of breast cancer and a promising target of novel treatment strategies.     

Alterations in the expression of PDCD4 in ductal carcinoma of the breast

Programmed cell death 4 gene (PDCD4), an in vivo repressor of transformation, was originally isolated from a human glioma library by screening it with an antibody against a nuclear antigen in proliferating cells. PDCD4 functions as a transformation repressor by inhibiting the activity of the RNA helicase, eIF4A. We previously showed that retinoids, anti-estrogens and HER2/neu antagonist induce PDCD4 expression in human breast cancer cell lines. Very little is known about the expression of PDCD4 in human breast cancer tissues or the significance of the PDCD4 expression in breast cancer. To gain insight into the pattern of the PDCD4 expression in breast tissues, we performed an immunohistochemical analysis of the PDCD4 expression in 80 archived, normal and ductal breast carcinoma tissues (invasive and carcinoma in situ) (DCIS) and correlated PDCD4 expression with expression of known prognostic markers in breast cancer (ER, PR and HER2/neu). To assess the role of methylation on PDCD4 expression in breast cancer cells, breast cancer cell lines were treated with the demethylating agent 5-deoxy-azacytidine and analyzed for PDCD4 expression. We observed primarily nuclear localization of PDCD4 in ductal carcinoma in situ compared to normal breast tissues where the PDCD4 expression was predominantly cytoplasmic. This was seen more frequently in DCIS cases that were ER positive and HER2/neu negative samples. PDCD4 expression was markedly decreased in the invasive ductal carcinoma. We did not observe any significant relationship between PDCD4 expression and the expression of RAR or PR. In T-47D, MDA-MB-435 and MDA-MB-231 cells, treatment with 5-deoxy-azacytidine did not result in an increased expression of PDCD4. The present study demonstrated altered cellular localization of PDCD4 when comparing normal breast to neoplastic breast tissues. In addition, there was a decreased expression of PDCD4 in breast cancer when compared with normal breast tissue. A loss of the PDCD4 expression in breast cancer cell lines does not appear to result from hypermethylation of the PDCD4 promoter.     

HOXD3在乳腺癌细胞干性和化疗耐药性中的作用及机制研究

  目的  探讨HOXD3表达对乳腺癌细胞干性的影响,并研究HOXD3表达与乳腺癌细胞化疗耐药的关系。  方法  收集2006年1月至2008年12月哈尔滨医科大学附属肿瘤医院87例乳腺癌患者组织标本。采用免疫组织化学染色法检测乳腺癌细胞和组织中HOXD3表达;采用RT-PCR、Western blot和免疫荧光染色法检测HOXD3在顺铂或阿霉素耐药细胞系MDA-MB-231和MDAMB-435中的表达水平,分析HOXD3过表达对乳腺癌细胞系MDA-MB-231和MDA-MB-435的干细胞生物标志物表达水平的影响;采用MTT法和集落形成实验分析HOXD3在乳腺癌细胞化疗耐药中的作用。  结果  乳腺癌组织中HOXD3 mRNA相对表达量显著高于癌旁正常组织,乳腺癌细胞系MDA-MB-231、MDA-MB-435和MCF-7的HOXD3 mRNA相对表达量均高于正常乳腺上皮细胞系MCF-10A（均P < 0.05）。顺铂或阿霉素耐药的细胞系MDA-MB-231和MDA-MB-435的半抑制浓度（half maximal inhibitory concentration,IC₅₀）分别为（20.82±0.05）μmol/L和（19.69±0.47）μmol/L,或（32.26±0.23）mmol/L和（26.08±0.55）mmol/L,均高于对应原始细胞系（均P < 0.05）;耐药倍数分别为2.47和3.10倍,或1.86和2.08倍。HOXD3过表达MDA-MB-231、MDA-MB-435的肿瘤球体数目、干细胞生物标志物的表达水平均明显增加（均P < 0.05）。  结论  HOXD3过表达对乳腺癌细胞干性的维持及化疗耐药性的发生发挥重要的作用,为制定针对肿瘤干细胞的分子靶向治疗提供理论参考。      

Regulation of breast cancer cell motility by insulin receptor substrate-2 (IRS-2) in metastatic variants of human breast cancer cell lines.

Insulin-like growth factors (IGFs) regulate breast cancer cell proliferation, protect cells from apoptosis, and enhance metastasis. In this study, we examined the IGF signaling pathway in two breast cancer cell lines selected for metastatic behavior. LCC6 was selected for growth as an ascites tumor in athymic mice from parental MDA-MB-435 cells (435P). The MDA-231BO cell line was derived from osseous metastases that formed after intracardiac injection of the MDA-MB-231 cell line in athymic mice. Compared to the parental cell lines, IGF-I treatment enhanced IRS-2 phosphorylation over IRS-1 in the metastatic variants. IGF-I stimulated cell migration in the variant cells, but not in the parental cells. To determine the role for IRS-2 in IGF-mediated motility, we transfected MDA-231BO cells with an anti-sense IRS-2 construct. Transfected cells had decreased levels of IRS-2 with diminished IGF-mediated motility and anchorage independent growth when compared to control cells. However, adherence to fibronectin was enhanced in the transfected cells compared to MDA-231BO cells. Our data show that breast cancer cells selected for metastatic behavior in vivo have increased IRS-2 activation and signaling. In these cells, IGF-I enhances cell adhesion and motility suggesting that IRS-2 may mediate these aspects of the malignant phenotype.     

上皮性肿瘤细胞中Snail与E-cadherin的表达及其与体外侵袭性的关系

目的 研究上皮性肿瘤细胞Snail与E-cadherin的表达以及与瘤细胞表型、转移潜能的关系。方法 采用Northern blot、共聚焦激光显微镜研究6株不同组织来源、不同分化程度及转移潜能的上皮性肿瘤细胞、1株正常上皮细胞、1株成纤维细胞中Snail与E-cadherin mRNA和蛋白表达与定位;Boyden小室体外侵袭实验反映细胞的转移潜能。结果 在分化程度较高的癌细胞和对照正常上皮细胞中,E-cadherin mRNA与蛋白表达较强,而Snail表达缺如;在分化程度低、转移潜能高的癌细胞和对照成纤维细胞中,E-cadherin、Snail mRNA及蛋白表达与上述情况相反。E-cadherin多定位于细胞胞浆和胞膜,Snail主要定位于胞核和胞浆。结论 在上皮性肿瘤细胞中E-cadherin和Snail在mRNA与蛋白表达水平上存在逆反关系,且与细胞分化、转移潜能相关。     

Breast cancer cell invasiveness: Correlation with protein kinase C activity and differential regulation by phorbol ester in estrogen receptor-positive and -negative cells

Increased protein kinase C (PKC) activity in malignant breast tissue and in most aggressive breast cancer cell lines has suggested a possible role of PKC in breast carcinogenesis and tumor progression. We have investigated here the involvement of PKC in the in vitro invasiveness and motility of several breast cancer cell lines. Modulation of PKC activity by treatment with a phorbol ester (TPA), drastically increased the invasiveness of 2 estrogen receptor-positive (ER⁺) lines (MCF7 and ZR 75.1), whereas it markedly decreased the invasiveness of 2 ER⁻ cell lines (MDA-MB-231 and MDA-MB-435). A PKC inhibitor (H7) reversed the TPA effects in MCF7 cells, whereas it mimicked TPA action in MDA-MB-231 cells. All of these effects of TPA also were observed to a similar extent for cell chemotaxis, and they were not dependent on protein neo-synthesis. In parallel, short TPA treatment induced cell spreading and microtubule organization in MCF7 cells and inverse morphological changes in MDA-MB-231 cells. In ER⁺ cells, constitutive PKC activity and PKCα expression were very low as compared to ER⁻ cells, and this correlated with the invasive potential of the cells. The opposed effects of TPA in ER⁺ and ER⁻ cells could be due to the abnormal TPA regulation of PKCα observed in ER⁻ cells. Int. J. Cancer 75:750–756, 1998.© 1998 Wiley-Liss, Inc.     

Autocrine human growth hormone reduces mammary and endometrial carcinoma cell sensitivity to mitomycin C

Drug resistance is a major cause of chemotherapy failure in breast cancer patients with metastatic disease. We previously demonstrated that autocrine human growth hormone (hGH) plays a key role in oncogenic transformation and progression of mammary carcinoma. The present study investigated the role of autocrine hGH in the development of resistance to mitomycin C (MMC), an alkylating agent utilised in the treatment of advanced metastatic breast cancer. Stable forced expression of the hGH gene was established in the mammary carcinoma cell lines MDA-MB-231, MCF-7 and T47D. Autocrine hGH reduced the sensitivity of mammary carcinoma cells to MMC in cell viability assays and reduced MMC-induced apoptotic cell death when compared to a control cell line. In addition, autocrine hGH enhanced MDA-MB-231 clonogenic survival, anchorage independent cell growth, growth in 3D Matrigel and protected MDA-MB-231 cells from induction of DNA double-strand breaks following MMC treatment. Functional antagonism of hGH in the endometrial carcinoma cell line RL95-2, which endogenously expresses hGH, significantly increased the sensitivity of these cells to MMC-induced DNA damage and cell death. Thus, autocrine hGH promotes mammary and endometrial carcinoma cell resistance to MMC. These studies indicate a potential role for antagonism of autocrine hGH in chemoresistant breast cancer.     

High Expression of the RECK Gene in Breast Cancer Cells is Related to Low Invasive Capacity

OBJECTIVE To investigate the expression of the RECK gene in human breast (cancer) cell lines, and to determine the relationship between RECK gene expression and the invasive capacity of the breast cancer cell lines. METHODS The invasive capacity of breast (cancer) cell lines including HBL-100, MCF-7 and MDA-MB-435S were determined by the Tran-swell method. The protein expression levels of RECK, MMP-2 and MMP-9 genes in these three cell lines were measured by immunocytochemical methods. The expressions of the RECK gene and protein level were measured by RT-PCR and Western blots in the cell lines respectively. RESULTS The order of the invasive capacity of the breast (cancer) cell lines was MDA-MB-435S, being the highest, and HBL-100, being the lowest. The invasive capacity difference between any two groups among the three groups was significant (P<0.01). The protein expression level of the RECK gene in the HBL-100 cell line was highest, and no expression was detected in MDA-MB-435S cells. Moreover, the expression of the RECK gene was negatively correlated with the expression of the MMP-2 and MMP-9 genes. The mRNA level of the RECK gene in HBL-100 cells was the highest, but no expression was found in the MDA-MB-435S cells (P<0.001). CONCLUSION There was a significant negative correlation between the expression level of the RECK gene and invasive capacity in vitro, and the RECK gene expression showed an inverse proportion to that of the MMP-2, MMP-9 genes.     

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

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.