Methylation and inhibition of expression of uPA by the RAS oncogene: divergence of growth control and invasion in breast cancer cells
Authors: Pouya Pakneshan
Moshe Szyf
Shafaat A Rabbani
Journal: Carcinogenesis (2004)
Discipline: CLINICAL MEDICINE
Keywords: uPA; DNMT1; RAS; DNA methylation; breast cancer
Affiliations: Department of Medicine and Department of Pharmacology, McGill University Health Center, Montreal, Canada.

Abstract:  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. 

Retracted: No

PubPeer comments:
 Figure 5A. Much more similar than expected.  , 
 I agree with comment #1, and I have an additional concern about Figure 5A, which I have marked with red boxes.  , 
 Figure 3. * Green and orange boxes: Some lanes look more similar than expected.  , 
 Figure 6A. * Pink boxes: Some lanes look more similar than expected.  , 
 Figure 7A. * Brown, cyan, and yellow boxes: Some lanes look more similar than expected.  , 
 Figure 7B. * Purple boxes: Some lanes look more similar than expected.  , 
 Figure 6A. Much more similar than expected.  , 
 Figure 7. Much more similar than expected.  , 
 Figure 5A. Much more similar than expected.  , 
 Figure 2A. Much more similar than expected.  , 
 Figure 3. Much more similar than expected.  , 
 Figure 6B. Much more similar than expected. Note that the backgrounds are very similar in lanes 1 and 2 of the MBD2 panel, yet the left ends of the bands are slightly different. All 3 lanes in the beta-actin panel are very similar, one light spot on the upper surface of the band in lane 3 does not make that lane different.  ,