Synergistic anti-breast cancer effect of a combined treatment with the methyl donor S-adenosyl methionine and the DNA methylation inhibitor 5-aza-2'-deoxycytidine.

DNA-demethylating agents activate tumor suppressor genes that are silenced by DNA methylation in cancer and are therefore emerging as a novel approach to cancer therapy. 5-azacytidine (VIDAZA), the first representative of this class of drugs was approved for treatment of myelodysplastic syndromes and is currently being tested on other cancers including solid tumors. However, 5-azacytidine or its deoxy-analog, 5-aza-2'-deoxycytidine (5-azaCdR) could also induce methylated prometastatic genes by DNA demethylation and induce cancer cell invasiveness. Since 5-azacytidine is a potent cancer growth inhibitor, we tested whether combining it with a DNA-methylating agent, the methyl donor S-adenosyl methionine (SAM), would block the adverse demethylating activity of 5-azaCdR while maintaining its growth suppression effects. We show here using several invasive and non-invasive breast cancer cell lines that SAM inhibits global- and gene-specific demethylation induced by 5-azaCdR, prevents 5-azaCdR activation of prometastatic genes uPA and MMP2, resulting in inhibition of cell invasiveness while augmenting the growth inhibitory effects of 5-azaCdR and its effects on tumor suppressor genes. Combination of drugs acting on the DNA methylation machinery at different levels is proposed as a new strategy for epigenetic therapy of cancer.

Synergistic effects of combined DNA methyltransferase inhibition and MBD2 depletion on breast cancer cells; MBD2 depletion blocks 5-aza-2'-deoxycytidine-triggered invasiveness.

5-Aza-2'-deoxycytidine (5-azaCdR) not only inhibits growth of non-invasive breast cancer cells but also increases their invasiveness through induction of pro-metastatic genes. Methylated DNA binding protein 2 (MBD2) is involved in silencing methylated tumor suppressor genes as well as activation of pro-metastatic genes. In this study, we show that a combination of MBD2 depletion and DNA methyltransferases (DNMT) inhibition in breast cancer cells results in a combined effect in vitro and in vivo, enhancing tumor growth arrest on one hand, while inhibiting invasiveness triggered by 5-azaCdR on the other hand. The combined treatment of MBD2 depletion and 5-azaCdR suppresses and augments distinct gene networks that are induced by DNMT inhibition alone. These data point to a potential new approach in targeting the DNA methylation machinery by combination of MBD2 and DNMT inhibitors.

Effects of specific DNMT gene depletion on cancer cell transformation and breast cancer cell invasion; toward selective DNMT inhibitors.

A hallmark of cancer is aberrant DNA methylation, consisting of global hypomethylation and regional hypermethylation of tumor suppressor genes. DNA methyltransferase inhibitors have been recognized as promising candidate anticancer drugs. Drug development has focused on DNA methylation inhibitors with the goal of activating tumor suppressor genes silenced by DNA methylation. 5-azacytidine (5-AC; Vidaza), a global DNA methyltransferase inhibitor, was Food and Drug Administration approved to treat myelodysplastic syndromes and is clinically tested for solid tumors. In this paper, it was demonstrated that 5'-aza-2'-deoxycytidine (5-azaCdR) activated both silenced tumor suppressor genes and pro-metastatic genes by demethylation, raising the concern that it would promote metastasis. 5-AzaCdR treatment increased the invasiveness of non-invasive breast cancer cell lines MCF-7 cells and ZR-75-1 and dramatically induced pro-metastatic genes; Urokinase plasminogen activator (uPA), matrix metalloproteinase 2 (MMP2), metastasis-associated gene (H-MTS1; S100A4) and C-X-C chemokine receptor 4 (CXCR4). The hypothesis that the blocking of cellular transformation activity of DNA methyltransferase inhibitor could be separated from the pro-metastatic activity was tested using short interfering RNA (siRNA) targeted to the different DNA methyltransferase (DNMT) genes. Although depletion of DNMT1 had the strongest effect on colony growth suppression in cellular transformation assays, it did not result in demethylation and activation of uPA, S100A4, MMP2 and CXCR4 in MCF-7 cells. Depletion of DNMT1 did not induce cellular invasion in MCF-7 and ZR-75-1 non-invasive breast cancer cell lines. These data have implications on the design of new DNA methyltransferase inhibitor and on the proper utilization of current inhibitors.

Role of epigenetics in cancer initiation and progression.

The epigenome which comprises DNA methylation, histone modifications, chromatin structures and non-coding RNAs controls gene expression patterns. In cancer cells, there are aberrant changes in the epigenome. The question in cancer epigenetics is that whether these changes are the cause of cell transformation, or rather the consequence of it. We will discuss the epigenetic phenomenon in cancer, as well as the recent interests in the epigenetic reprogramming events, and their implications in the cancer stem cell theory. We will also look at the progression of cancers as they become more aggressive, with focus on the role of epigenetics in tumor metastases exemplified with the urokinase plasminogen activator (uPA) system. Last but not least, with therapeutics intervention in mind, we will highlight the importance of balance in the design of epigenetic based anti-cancer therapeutic strategies.

Pharmacological methyl group donors block skeletal metastasis in vitro and in vivo.

BACKGROUND AND PURPOSE: DNA hypomethylation was previously implicated in metastasis. In the present study, we examined whether methyl supplementation with the universal methyl donor S-adenosylmethionine (SAM) inhibits prostate cancer associated skeletal metastasis. EXPERIMENTAL APPROACH: Highly invasive human prostate cancer cells PC-3 and DU-145 were treated with vehicle alone, S-adenosylhomocysteine (SAH) or SAM and their effects on tumour cell proliferation, invasion, migration and colony formation were monitored. For in vivo studies, control (SAH) and SAM-treated PC-3 cells were injected into the tibia of Fox chase SCID mice and skeletal lesions were determined by X-ray and µCT. To understand possible mechanisms involved, we delineated the effect of SAM on the genome-wide methylation profile of PC-3 cells. KEY RESULTS: Treatment with SAM resulted in a dose-dependent inhibition of tumour cell proliferation, invasion, cell migration, colony formation and cell cycle characteristics. Animals injected with 250 µM SAM-treated cells developed significantly smaller skeletal lesions, which were associated with increases in bone volume to tumour volume ratio and connectivity density as well as decreased trabecular spacing. Genome-wide methylation analysis showed differential methylation in several key signalling pathways implicated in prostate cancer including the signal transducer and activator of transcription 3 (STAT3) pathway. A selective STAT3 inhibitor decreased tumour cell invasion, effects which were less pronounced as compared with SAM. CONCLUSIONS AND IMPLICATIONS: These studies provide a possible mechanism for the role of DNA demethylation in the development of skeletal metastasis and a rationale for the use of hypermethylation pharmacological agents to impede the development and progression of skeletal metastasis.