The TEB is located exclusively in the developing mammary glandand is the key driving force for mammary gland development

Similar results were observed in parental CMS4 cells in response to the different treatments, indicating that the lack of TSAinduced STAT1 phosphorylation did not reflect subline-specific differences. These results indicate that the ability of TSA to enhance IRF-8 promoter activity is STAT1-dependent ; albeit, it does not coincide with STAT1 phosphorylation status. These data are consistent with the ability of TSA to affect STAT1 activity via unphosphorylated-based mechanisms, such as acetylation. To explore that possibility, the experiment was repeated and the lysates examined for STAT1 acetylation via IP for total STAT1 protein, followed by Western blot for acetylated lysine residues on STAT1. Importantly, we Talsupram hydrochloride showed that TSA treatment led to a significant increase in acetylated STAT1 levels compared to the vehicle-treated control preparation. Furthermore, TSA treatment led to a significant increase in total STAT1 protein compared to the vehicle-treated control, which is consistent with the effect of TSA on STAT1 mRNA levels. Our data indicate TSA treatment in vitro can facilitate Fasmediated killing via an IRF8-dependent mechanism. Moreover, it has been shown that TSA, depending upon drug dose, can mediate antitumor activity in vivo. Thus, we hypothesized that tumor-cell expression of IRF-8 is also important for response to TSA-mediated antitumor activity in vivo. To test this hypothesis, we investigated the effects of TSA treatment in mice bearing either IRF8-competent or IRF8-deficient tumor cells. The schema involved several daily peritumoral injections of TSA to mice once tumors became palpable. We showed that TSA treatment of mice bearing IRF8- competent tumor cells led to dramatic tumor growth inhibition, suggesting that this TSA-based schema can indeed facilitate antitumor activity in vivo. In contrast, we showed that TSA treatment of mice bearing the IRF8-deficient tumor cells failed to promote significant antitumor effects, suggesting that ‘tumor response to therapy’ in vivo was IRF8-dependent. Epigenetic modifiers, such as HDACi, have achieved encouraging results in both hematologic and non-hematologic cancer clinical trials. Understanding key molecular features for response to such systemic therapies is critical to improving the way disease status is monitored and potentially how patients are selected for treatment. Since HDACi generally impact the expression of numerous genes, it becomes difficult to determine which ones are relevant for ‘response to therapy’. Here, we took a more focused approach to elucidate molecular determinants required for HDACi-mediated antitumor effects. Our model focused on Fas-induced death in response to HDACi treatment. Based on previous work that established that HDACi can enhance Fas sensitivity and that IRF-8 expression was required for response to Fas killing, we tested the hypothesis that tumor-cell expression of IRF-8 was required for Fas-induced death following HDACi treatment. We demonstrated that loss of IRF-8 expression led to a concomitant loss of Fas sensitivity to TSA-treated tumor cells in vitro. Moreover, we showed that TSA-mediated suppression of tumor growth in vivo was dependent on tumor expression of IRF-8. These new findings extend our previous work showing that tumor cell susceptibility to Fas-based effector mechanisms was IRF-8- dependent in vivo. Indeed, in mice lacking functional FasL expression, both control and IRF-8-deficient tumors grew at similar rates, whereas in wild-type mice, IRF-8-deficient tumors grew at a significantly higher rate than control tumors. Together, these results indicate that HDACi promote Fasmediated tumor cell death, in part, through IRF-8-dependent pathways. It is likely that response to TSA in vivo involves a complex set of host-dependent and tumor-dependent interactions that require further elucidation. Nonetheless, it is important to emphasize that tumor-cell expression of IRF-8 was crucial for therapeutic response to HDACi. We also showed that TSA in combination with IFN-c boosted IRF-8 expression. Similar results were observed with DP, suggesting that modulation of IRF-8 expression was not limited to TSA. Moreover, similar results with TSA were observed in a second tumor cell model, suggesting that the effects of HDACi on IRF-8 expression were not tumor model-specific. These results support the notion that HDACi, potentially in concert with certain innate or adaptive inflammatory signals, can enhance sensitivity to apoptosis in otherwise refractory or resistant tumor subpopulations.

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