It is interesting to note, however, that since the exogenous promoter fragment did not contain deacetylation sites, these data suggest that TSA could modulate IRF-8 transcription via mechanisms not necessarily related to HDAC inhibition at the promoter level. We next examined the integrity of events upstream of IRF-8, mainly STAT1 as it is known to be essential for IFN-c-inducible gene regulation, including IRF-8 [19,35]. Phosphorylation of STAT1 plays an important role in regulating IFN-c-mediated gene induction. It has also been reported that HDACi, such as TSA, alters the expression of IFN-c-inducible genes through acetylation of STAT1 in myeloid cells and tumor cells [42?4]. We found that STAT1 silencing in either parental or aggressive CMS4 cells led to a significant reduction in TSA- or IFN-cinduced IRF-8 promoter activity, the latter of which served as a positive control.
These results suggested that TSA-induced IRF-8 promoter activity was STAT1-dependent. However, it is important to emphasize that single agent TSA treatment did not seem to elicit STAT1 phosphorylation, but did promote STAT1 acetylation. Thus, we posit that TSA may impact STAT1 function in an unphosphorylated manner, as previously reported in other systems [36?0]. Overall, our data are consistent with a model that tumor-cell expression of IRF-8 is integral for HDACi-induced antitumor activities (Fig. 6). HDACi exposure may render neoplastic cells more receptive to IRF-8 induction and Fas-mediated death under pro-inflammatory (IFN-c-dependent) conditions. Therefore, IRF8 transcription may be influenced in two ways; one by IFN-c and the other by HDACi (e.g., TSA). In either case, IRF-8 transcription is STAT1-dependent. STAT1 activation, however, may result from both phosphorylation-dependent and �independent (i.e., acetylation) mechanisms, which warrant further study. Moreover, these data do not preclude the possibility that the IRF-8 promoter may be regulated by multiple epigenetic mechanisms, including DNA methylation, and that these mechanisms may impact IRF-8 expression and consequently Fas sensitivity in a direct or indirect manner. Such complex issues, therefore, warrant further study. Nonetheless, the induction of IRF-8, in turn, modulates tumor response to immune attack via Fas-mediated apoptosis. Based on observations in myeloid leukemia, IRF-8 may regulate Fas responsiveness by acting as a transcriptional activator of pro-apoptotic genes, such as caspases, and/or a transcriptional repressor of anti-apoptotic genes, such as PTPN13 (FAP-1) or members of the Bcl-2 family [19,44?6]. Altogether, our results point to IRF-8 expression in tumors as being a potential biomarker for efficacy of response to HDACi and a possible molecular target to improve response to therapy.
Materials and Methods Ethics Statement
All experiments were conducted and approved under our Institutional Animal Care and Use Committee at Roswell Park Cancer Institute under protocol ID number 1117M and in accordance with institutional regulations, NIH and Public Health Service policies.Cell Lines and Reagents
The mouse sarcoma cell line CMS4 was kindly provided by A. DeLeo (University of Pittsburgh, Pittsburgh, PA) and maintained in culture in RPMI-based culture medium [47]. IRF8-deficient (CMS4-shRNA) or control CMS4 cells were previously [16]Figure 4. TSA-mediated IRF-8 transcription is STAT1-dependent. (A) STAT1 mRNA levels in CMS4 or CMS4.met.sel cells after the indicated treatments, as in Fig. 1. (A) Top, real-time PCR. Data presented as fold-change, as in Fig. 1. (A) Bottom, RT-PCR. *P,0.05, based on comparing the single agent treatment to the vehicle-treated control. **P,0.05, based on comparing the combination regimen to the single treatment counterparts. (B) CMS4 or CMS4.met.sel cells were transfected with an IRF-8 promoter reporter construct, followed by treatment with the indicated agents for 6 hr. Results are reported as the mean 6 SEM of the fold-change relative to the vehicle-treated cells from three separate experiments. *P,0.05, based on comparing treatment to matched vehicle control. No activity was observed using the pGL3 vector lacking a promoter. (C) Similar to B, except that CMS4 cells were silenced for STAT1 expression. *P,0.05, based on comparing the indicated treatment group to the matched vehicle-treated control. **P,0.05, based on comparing the STAT1-deficient groups to their matched STAT1-expressing vector controls. (D) Phosphorylated STAT1 (pSTAT1) and total STAT1 protein levels in CMS4.met.sel cells after treatment with the indicated treatments (TSA, 500 nM; IFN-c, 200 U/ml) for 15 min, as measured by Western blot. This experiment is representative of one of three with similar results. (E) Similar to D, except that acetylated STAT1 and total STAT1 levels were measured by IP-Western blot (i.e., IP with anti-STAT1 antibody, followed by Western blot with anti-acetyl-lysine antibody) after treatment with or without TSA (500 nM for 6 hr). Band intensities were quantified, and the data presented as fold-change of TSA-treated vs. untreated samples (mean 6 SEM of triplicate experiments). *P,0.05, based on the TSA-treated group relative to the matched vehicle-treated control. generated by transfection with IRF8-specific or scramble shRNA constructs and maintained in culture containing zeocin (2 mg/ml) (Invitrogen, Carlsbad, CA). Control and CMS4 cells expressing a mutant IRF-8 protein (CMS4-K79E) were previously generated [17] by transfection with an empty vector or a dominant-negative mouse IRF-8 construct that harbors a point mutation in its DNAbinding domain (K to E switch at amino acid site 79) [34], respectively. CMS4-K79E or vector control cells were cultured in media containing G418 (0.75 mg/ml) (Invitrogen). We also used a highly aggressive CMS4 subline, termed CMS4.met.sel, which was selected based on resistance to adoptive immunotherapy with tumor-specific CD8+ CTL [32]. The human colon carcinoma celllines SW480 (CCL-228) and SW620 (CCL-227) were obtained from the American Type Culture Collection (Manassas, VA). SW480 and SW620 are two naturally occurring primary and metastatic colon adenocarcinoma cell lines established from the same patient. The SW620 cell line was derived as a lymph node metastasis identified six months later during disease relapse. [33]. Recombinant mouse IFN-c was obtained from PeproTech (Rocky Hill, NJ). TSA was obtained from Sigma-Aldrich (St. Louis, MO). Depsipeptide (DP) was obtained from the Experimental Therapeutics section of the NCI.carried out as previously described [17]. SYBR green quantification was performed on an ABI7900HT (Applied Biosystems) cycling machine and data analyzed using the DDCT method.
Cell Death Assay
Cell death was measured by propidium iodide (PI) staining as described [16]. Briefly, tumor cells (26105 cells/well) were seeded in 6-well culture plates and incubated for 24 hr with TSA (20 nM or 100 nM where indicated), IFN-c (200 U/ml) or TSA plus IFNc (IFN-c was added 4 hr after TSA treatment). Subsequently, cells were incubated for an additional 24 hr in the absence or presence of recombinant human Fas ligand (FasL; 100 ng/ml; PeproTech). Adherent and suspended cells were collected and treated with PI/ RNase solution (Sigma) for 15 min at room temperature and analyzed immediately by flow cytometry. The percentage of cell death was calculated by the formula: percent cell death = (percent PI+ cells with FasL) ?(percent PI+ cells without FasL). The TSA concentrations (100 nM for CMS4-shRNA cells and 20 nM for K79E cells) chosen for these experiments caused minimal toxicity, as measured by trypan blue exclusion and PI staining.
