Pakneshan Pouya, Szyf Moshe, Rabbani Shafaat A
Department of Medicine and Department of Pharmacology, McGill University Health Center, Montreal, Canada.
Carcinogenesis. 2005 Mar;26(3):557-64. doi: 10.1093/carcin/bgi009. Epub 2004 Dec 23.
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.
尿激酶型纤溶酶原激活剂（uPA）是一种仅在高侵袭性人类乳腺癌细胞中表达的蛋白酶，其表达受启动子DNA甲基化的抑制。我们验证了以下假说：DNA甲基转移酶1（DNMT1）的上调将导致uPA甲基化和沉默，并抑制转移性乳腺癌细胞的侵袭性。由于之前研究表明RAS可上调DNA甲基化，因此我们检测了组成型活性RAS的异位表达对uPA甲基化和表达的影响。将Ha-RAS转染至MDA-MB-231人乳腺癌细胞中，与对照组相比，细胞倍增时间显著缩短。然而，癌基因RAS显著降低了转移性基因uPA的表达和活性以及细胞的侵袭能力。RAS对uPA的沉默是由uPA启动子的顺式修饰介导的，而非通过对反式作用因子的影响，因为瞬时转染的未甲基化uPA-荧光素酶报告基因在RAS转染细胞和对照细胞中的表达水平相似。然后，我们检测了这些细胞中DNMT1和甲基化DNA结合蛋白2（MBD2）的表达水平，以确定uPA表达的降低是否与DNA甲基化机制的变化有关。我们的结果表明，RAS的异位表达诱导了DNMT1的表达和活性，并抑制了MBD2的表达。与甲基化介导的抑制作用一致，uPA在RAS转染细胞中部分甲基化，用去甲基化剂5'-氮杂胞苷处理RAS转染细胞后可诱导uPA表达。因此，这些结果表明，在许多癌症中促进致癌生长的RAS-DNMT1 DNA甲基化途径，对高侵袭性MDA-MB-231细胞的侵袭能力可产生相反的作用，从而说明了癌症中生长和转移促进途径的差异。这对转移性癌症的新治疗方法具有重要意义。
