DNA methylation inhibitor induces invasiveness and metastasis of breast cancer cells

in vitro and in vivo

Bushra Ateeq1,2, Gaoping Chen1,2, Alexander Unterberger1,3, Moshe Szyf1,3 and Shafaat A. Rabbani1,2

1Department of Medicine, 2Oncology and 3Pharmacology, McGill University, Montreal, Quebec, Canada

The DNA methylation inhibitor 5-azadeoxycytidine (5-aza-CdR) is currently being tested as candidate anti-cancer agent. The mechanism of action is believed to be activation by demethylation of tumor suppressor genes. We tested here the hypothesis that demethylating agents also demethylate and activate genes involved in tumor invasion and metastasis resulting in adverse effect of conversion of non metastatic breast cancer to metastatic breast cancer. Treatment of the non-invasive human breast cancer cells MCF-7 cells with 5-aza-CdR shows as predicted a dose dependent inhibition in cell proliferation, induction of the tumor suppressor gene RASSF1Aand G2/M phase arrest of the cell cycle which slows the growth of MCF-7 tumors when inoculated in nude mice. However, in contrast to these growth inhibitory effects of DNA demethylation, 5-aza-CdR induced a battery of pro-metastatic genes such as uPA, CXCR4, HEPARANASE, SYNUCLEIN γ and TGF-βby a mechanism, which involves demethylation of their promoters as determined by semi-quantitative RT-PCR and further confirmed by bisulfite genomic sequence analyses. Induction of these pro-metastatic genes resulted in increased tumor cell migration and invasion as determined by Matrigel Boyden chamber invasion assay. For in vivo studies inoculation of control and experimental MCF-7 cells into the mammary fat pad of female BALB/c nu/nu mice implanted with slow release estrogen pallet resulted in the development of tumors of significantly smaller volume in animals receiving cells which were treated with 5-aza-CdR for 8 days. Lungs from control animals showed no evidence of tumor metastasis. In contrast lungs from 25% of experimental animals showed the presence of micro metastasis. Immunohistochemical analyses on the 5-aza-CdR treated and control tumor tissues further confirmed the up-regulation in the levels of expression of pro-metastatic (uPA, CXCR4 and heparanase), angiogenesis (CD31) and proliferation (Ki67) markers. Collectively our findings reveal that although demethylating agents activate as expected the expression of tumor suppressor genes and slow tumor growth they also induce the expression of metastasis promoting genes leading to metastasis in vivo. These results draw attention to the critical role of demethylation as a mechanism promoting metastasis and to the risks associated with DNA demethylation therapy in cancer.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA