ING1 and 5-Azacytidine Act Synergistically to Block Breast Cancer Cell Growth

Abstract

BackgroundInhibitor of Growth (ING) proteins are epigenetic “readers” that recognize trimethylated lysine 4 of histone H3 (H3K4Me3) and target histone acetyl transferase (HAT) and histone deacetylase (HDAC) complexes to chromatin.Methods and Principal FindingsHere we asked whether dysregulating two epigenetic pathways with chemical inhibitors showed synergistic effects on breast cancer cell line killing. We also tested whether ING1 could synergize better with chemotherapeutics that target the same epigenetic mechanism such as the HDAC inhibitor LBH589 (Panobinostat) or a different epigenetic mechanism such as 5-azacytidine (5azaC), which inhibits DNA methyl transferases. Simultaneous treatment of breast cancer cell lines with LBH589 and 5azaC did not show significant synergy in killing cells. However, combination treatment of ING1 with either LBH589 or 5azaC did show synergy. The combination of ING1b with 5azaC, which targets two distinct epigenetic mechanisms, was more effective at lower doses and enhanced apoptosis as determined by Annexin V staining and cleavage of caspase 3 and poly-ADP-ribose polymerase (PARP). ING1b plus 5azaC also acted synergistically to increase γH2AX staining indicating significant levels of DNA damage were induced. Adenoviral delivery of ING1b with 5azaC also inhibited cancer cell growth in a murine xenograft model and led to tumor regression when viral concentration was optimized in vivo.ConclusionsThese data show that targeting distinct epigenetic pathways can be more effective in blocking cancer cell line growth than targeting the same pathway with multiple agents, and that using viral delivery of epigenetic regulators can be more effective in synergizing with a chemical agent than using two chemotherapeutic agents. This study also indicates that the ING1 epigenetic regulator may have additional activities in the cell when expressed at high levels.

Reduced ING1 levels in breast cancer promotes metastasis

“…Since ING1 and ING2 are very closely related evolutionarily [43] and functionally [4], it is likely that both target the Sin3A HDAC complex to chromatin locales containing relatively higher density of the H3K4Me3 chromatin mark. This mechanism is consistent with recent observations that the epigenetic targeted drugs 5-azacytidine and the LBH589 HDAC inhibitor can act additively, or in some cases synergistically with ING1 in killing cells in breast cancer cell and animal models [20]. Data generated in this study provide mechanistic insight into why breast cancer cells may be selectively sensitive to HDAC inhibitors compared to normal breast epithelium; down-regulation of ING1 would already reduce the ability of cancer cells to accurately target the Sin3A complex and so treatment with HDAC inhibitors such as SAHA that selectively target ING2 and/or ING1 would be expected to have greater effects upon the epigenomes of cancerous versus normal epithelial cells.…”

Src Regulates the Activity of the ING1 Tumor Suppressor

“…MDA-MB-468 breast cancer cell line that we have shown is sensitive to ING1-induced apoptosis [40]. As shown in Figure 7, transfection with a GFP expression construct resulted in 20% of transfected cells undergoing apoptosis while GFP plus ING1 expression induced apoptosis in 80% of the cell population.…”

5-Azacytidine inhibits human rhabdomyosarcoma cell growth by downregulating insulin-like growth factor 2 expression and reactivating the H19 gene product miR-675, which negatively affects insulin-like growth factors and insulin signaling

“…the methylation of genomic Dna in cells is catalyzed by Dna methyltransferases (DnMts). recent studies have shown that application of methyltransferase inhibitors, such as 5-azacitidine (azac), may become a clinically relevant strategy for tumor growth inhibition (29,30). DnMt inhibitors, which are usually analogues of the cytidine nucleoside, bind to Dna methyltransferase 1, which results in Dna hypomethylation.…”