Indeed, entinostat upregulates the expression of three and transgenic model, only the coding sequence of the transgene rat was inserted downstream of MMTV promoter.48 Thus, the exogenously expressed erbB2 (or rat erbB2/neu) and erbB3 may not be regulated by miRNAs. often interacts with other RTKs, such as erbB3, to activate cell signaling. SCH28080 Numerous studies have established the critical role of erbB3 as a co-receptor of erbB2, and the expression of erbB3 is a rate-limiting factor for erbB2-induced breast cancer cell survival and proliferation.8, 9 Thus, novel strategies/agents targeting both erbB2 and erbB3 receptors should be more effective to treat the breast cancer patients whose tumors overexpress erbB2. Numerous studies indicate that deregulation of histone acetylation and deacetylation has an important role in aberrant gene expression in human cancers.10, 11 Histone deacetylases (HDACs) are relatively easier tractable enzymes, and have recently become attractive therapeutic targets. Inhibitors of HDACs exhibit anticancer SCH28080 activity in a variety of tumor cell models via influencing cell cycle progression, apoptosis, differentiation, and tumor angiogenesis.12, 13 Many HDAC inhibitors (HDACi) are currently under clinical investigations as potential anticancer agents.14, 15 Entinostat (also known as MS-275, SNDX-275, Syndax Pharmaceuticals, Inc., Waltham, MA, USA) is a synthetic benzamide derivative class I HDACi. It inhibits cancer cell growth with an IC50 in the submicromolar range, and exhibits both and activities against various cancer types, including solid tumors and hematologic malignancies.16 In breast cancers, entinostat has been shown to inhibit cell proliferation and/or promote apoptosis.17, SCH28080 18, 19, 20, 21 Recent studies suggest that entinostat exerts different effects towards distinct subtypes of human breast cancers. Entinostat increases expression of estrogen receptor (ERand/or in erbB2-overexpressing breast cancer cells. Results Entinostat does not affect the mRNA levels of and in breast cancer cells To explore the molecular mechanism by which entinostat downregulates erbB2 and erbB3 in breast cancer cells, we first studied whether entinostat might modulate and mRNA levels. While treatment with 1?and in MDA-MB-453 and BT474 breast cancer cells (Figure 1). To confirm the results, we designed additional primers amplifying distinct cDNA fragments of human and mRNA expression upon entinostat treatment in both SKBR3 and BT474 cells (Supplementary Figure S1). Thus, our findings suggested that Angpt2 entinostat downregulated erbB2/erbB3 receptors through a transcription-independent mechanism. Open in a separate window Figure 1 Treatment with entinostat does not affect mRNA levels of both and in breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells untreated or treated with entinostat (ent) at indicated concentrations for 24?h were subjected to total RNA extraction. (a) First-strand cDNA was synthesized using a reverse transcription kit from Applied Biosystems. The partial coding sequence of was amplified with specific primers. The PCR products were separated on a 2% agarose gel containing ethidium bromide and visualized under a UV light. (b) The mRNA levels of and were measured by qRT-PCR. Bars, S.D. The data are representative of three independent experiments Entinostat reduces the protein levels of endogenous, but not exogenous, erbB2 and erbB3 We next investigated whether entinostat might alter erbB2/erbB3 protein stability. In our previous report, we observed an interesting phenomenon that entinostat specifically reduced the levels of endogenous, but not exogenous, erbB3 in breast cancer cells.24 Additional studies confirmed that entinostat did not lower the expression of exogenous erbB3 via transient transfection, although the levels of endogenous erbB2 and erbB3 were clearly reduced by entinostat in both MDA-MB-453 and BT474 cells (Figure 2a). Similar results were also observed in SKBR3 cells (Supplementary Figure S2). We then reasoned if entinostat might possess the similar discrimination effects on endogenous and exogenous erbB2. MDA-MB-435 is a human cancer cell line with erbB2 low SCH28080 expression. We generated its erbB2-high expressing clone (435.eB1) in our previous studies.36 Entinostat reduced the levels of endogenous erbB3 in both lines; however, it did not reduce exogenous erbB2 in 435.eB1 cells (Figure 2b). In fact, the expression levels of exogenous erbB3 and erbB2 were clearly increased upon treatment with entinostat (Figures 2a and b). This is possibly because both and cDNAs are driven by the CMV promoter in the expression vectors,24, 36 as recent studies show that HDAC inhibitors are capable of enhancing CMV SCH28080 promoter activity.37, 38 Furthermore,.