Histone acetylation and deacetylation are crucial in the regulation of gene expression. Dynamic changes in gene expression may affect chromatin structure and, consequently, the interaction of chromatin with regulatory factors. In this study, the effects of the antiseizure drug valproic acid (VPA) on the expression of genes that regulate the structure of chromatin and the access of macromolecules to the DNA were investigated. Exposure of breast cancer cells to VPA resulted in rapid dose-dependent hyperacetylation of the histones H3 and H4. VPA further induced a depletion of several members of the structural maintenance of chromatin (SMC) proteins, SMC-associated proteins, DNA methyltransferase, and heterochromatin proteins. Down-regulation of these proteins was associated with chromatin decondensation. The observed alterations of chromatin structure correlated with enhanced sensitivity of DNA to nucleases and increased interaction of DNA with intercalating agents. VPA-induced chromatin decondensation led to a sequence-specific potentiation of DNA-damaging agents in cell culture and xenograft models. Modulation of heterochromatin maintenance proteins was not a direct, but a downstream, effect of histone acetylation. The effects on the chromatin structure were reversible upon drug withdrawal, but obligatory for the potentiation of DNA-damaging agents. In addition to their antitumor activity as single agents, the chromatin decondensation induced by histone deacetylase inhibitors may enhance the efficacy of cytotoxic agents that act by targeting DNA. The proposed mechanism of action suggests an effect of drug sequencing on the antitumor activity of these drugs. (Cancer Res 2005; 65(9): 3815-22)

The maximum-tolerated dose and recommended phase II dose was VPA 140 mg/kg/d for 48 hours followed by epirubicin 100 mg/m2. Sustained plasma concentrations of VPA exceeding those required for in vitro synergy were achieved with acceptable toxicity. Noteworthy antitumor activity was observed in heavily pretreated patients and historically anthracycline-resistant tumors.

Acetylation of histones leads to conformational changes of DNA. We have previously shown that the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), induced cell cycle arrest, differentiation, and apoptosis. In addition to their antitumor effects as single agents, HDAC inhibitors may cause conformational changes in the chromatin, rendering the DNA more vulnerable to DNA damaging agents. We examined the effects of SAHA on cell death induced by topo II inhibitors in breast cancer cell lines. Topo II inhibitors stabilize the topo II-DNA complex, resulting in DNA damage. Treatment of cells with SAHA promoted chromatin decondensation associated with increased nuclear concentration and DNA binding of the topo II inhibitor and subsequent potentiation of DNA damage. While SAHA-induced histone hyperacetylation occurred as early as 4 h, chromatin decondensation was most profound at 48 h. SAHA-induced potentiation of topo II inhibitors was sequence-specific. Pre-exposure of cells to SAHA for 48 h was synergistic, whereas shorter pre-exposure periods abrogated synergy and exposure of cells to SAHA after the topo II inhibitor resulted in antagonistic effects. Synergy was not observed in cells with depleted topo II levels. These effects were not limited to specific types of topo II inhibitors. We propose that SAHA significantly potentiates the DNA damage induced by topo II inhibitors; however, synergy is dependent on the sequence of drug administration and the expression of the target. These findings may impact the clinical development of combining HDAC inhibitors with DNA damaging agents.

Purpose: The aim was to study the biological and molecular effects of the histone deacetylase (HDAC) inhibitor, valproic acid, in patients with solid tumor malignancies. Experimental Design: A phase I dose escalation of valproic acid given on days 1to 3 followed by epirubicin (day 3) was followed by a dose expansion of valproic acid combined with 5-fluorouracil, epirubicin, and cyclophosphamide (FEC100). Pharmacodynamic and pharmacokinetic studies entailed valproic acid and epirubicin plasma levels and their interaction, the effects of valproic acid on histone acetylation in peripheral blood mononuclear cells (PBMC) and tumor cells at baseline and day 3, and baseline expression of HDAC2 and HDAC6 as therapeutic targets. Results: Forty-four patients were enrolled in the phase I part, with a disease-specific cohort expansion of 15 breast cancer patients (median age, 55 years; range, 28-66 years) receiving 120 mg/kg/day valproic acid followed by FEC100. Partial responses were seen in 9 of 41 (22%) patients during the phase I part. Objective responses were seen in 9 of 14 (64%) evaluable patients at the dose expansion with a median number of 6 administered cycles. Predominant toxicities were valproic acid^associated somnolence and epirubicin-induced myelosuppression. Valproic acid plasma levels were associated with short-term, reversible depletion of WBC and neutrophils within 48 hours. Histone acetylation in tumor samples and in PBMCs correlated with valproic acid levels and was further linked to baseline HDAC2 but not to HDAC6 expression. Conclusion: Valproic acid is a clinically relevant HDAC inhibitor, and PBMCs may serve as a surrogate for tumor histone acetylation in solid tumor malignancies. HDAC2 should be further considered as a relevant therapeutic target.

Purpose Despite initial sensitivity to chemotherapy, ovarian cancers (OVCA) often develop drug-resistance, which limits patient survival. Using specimens and/or genomic data from 289 patients and a panel of cancer cell lines, we explored genome-wide expression changes that underlie the evolution of OVCA chemo-resistance and characterized the BCL2 antagonist of cell death (BAD) apoptosis pathway as a determinant of chemo-sensitivity and patient survival. Experimental Design Serial OVCA cell cisplatin treatments were performed in parallel with measurements of genome-wide expression changes. Pathway analysis was performed on genes associated with increasing cisplatin-resistance (EC50). BAD-pathway expression and BAD-protein phosphorylation were evaluated in patient samples and cell lines as determinants of chemo-sensitivity and/or clinical outcome and as therapeutic targets. Results Induced in vitro OVCA cisplatin-resistance was associated with BAD-pathway expression (P < 0.001). In OVCA cell lines and primary specimens, BAD-protein phosphorylation was associated with platinum-resistance (n = 147, P < 0.0001) and also with overall patient survival (n = 134, P = 0.0007). Targeted modulation of BAD-phosphorylation levels influenced cisplatin sensitivity. A 47-gene BAD-pathway score was associated with in vitro phosphorylated-BAD levels and with survival in 142 patients with advanced-stage (III/IV) serous OVCA. Integration of BAD-phosphorylation or BAD-pathway score with OVCA surgical cytoreductive status was significantly associated with overall survival by log-rank test (P = 0.004 and <0.0001, respectively). Conclusion The BAD apoptosis pathway influences OVCA chemo-sensitivity and overall survival, likely via modulation of BAD-phosphorylation. The pathway has clinical relevance as a biomarker of therapeutic response, patient survival, and as a promising therapeutic target.

Modulation of estrogen signaling is one of the most successful modalities for the treatment of estrogen receptor (ER)-positive breast cancer, yet de novo and acquired resistance are frequent. Recent data suggests that the induction of autophagy may play a considerable role in promoting tumor cell survival and resistance to anti-estrogen therapy. Hence, bypassing autophagy may offer a novel strategy to enhance the anti-tumor efficacy of anti-estrogens. Histone deacetylases (HDAC) are involved in the regulation of steroid hormone receptor mediated cell signaling and their inhibition potentiates the anti-tumor effects of anti-estrogens. However, the mechanism underlying this anti-tumor activity is poorly understood. In this report, we show that the addition of an HDAC inhibitor redirects the response of ER-positive breast cancer cells when treated with tamoxifen from growth arrest to apoptotic cell death. This redirection requires functional ER signaling and is mediated by a depletion of Bcl-2 and an induction of Bax and Bak, manifesting in cytochrome C release and PARP cleavage. With combined treatment, a subpopulation of cells are refractory to apoptosis and exhibit a strong induction of autophagy. Inhibition of autophagy in these cells, using siRNA directed against Beclin-1 or treatment with chloroquine, further promotes the induction of apoptosis. Thus, supporting prior reports that autophagy acts as a survival mechanism, our findings demonstrate that HDAC and autophagy inhibition directs autophagy-protected cells into apoptotic cell death, which may impair development of tamoxifen resistance.

Several histone deacetylases (HDAC) are involved in estrogen receptor (ER)-mediated gene transactivation, and HDAC inhibitors have been reported to restore sensitivity to antihormonal therapy. The modulation of ER is the most promising approach to ER-expressing breast cancers. Recent studies further suggest a critical role of the progesterone receptor (PR) on ER signaling. Although HDAC inhibitors modulate ER, little is known about their effects on PR. We evaluated the roles of specific HDAC isoenzymes and their inhibition on both ER and PR signaling and their importance in response to endocrine therapy. The roles of individual HDAC isoenzymes on ER and PR expression and their functions were evaluated by depletion of select HDAC enzymes using siRNA or pharmacologic inhibition. Cotreatment of breast cancer cell lines with HDAC inhibitors and the antiestrogen, tamoxifen, resulted in synergistic antitumor activity with simultaneous depletion of both ER and PR. Selective inhibition of HDAC2, but not HDAC1 or HDAC6, was sufficient to potentiate tamoxifen-induced apoptosis in ER/PR-positive cells. Depletion of HDAC1 and HDAC6 was associated with down-regulation of ER but not PR. Only the selective depletion of HDAC2 siRNA down-regulated both ER and PR expression, and was sufficient to potentiate tamoxifen. Selective depletion of HDAC2 resulted in simultaneous depletion of ER and PR, and potentiated the effects of antihormonal therapy in ER-positive cells. A more effective pharmacologic inhibition of HDAC2 and evaluation of HDAC2 and PR as therapeutic targets or as predictive markers in hormonal therapy may be considered when combining HDAC inhibitors and hormonal therapy. [Cancer Res 2008;68(5):1513-9]

Purpose: The novel topoisomerase I inhibitor karenitecin (KTN) shows activity against melanoma.We examined whether histone deacetylase inhibition could potentiate the DNA strand cleavage, cytotoxicity as well as the clinical toxicity, and efficacy of KTN in melanoma. Experimental Design: Apoptosis, COMET, and xenograft experiments were carried out as described previously. A phase I/II trial of valproic acid (VPA) and KTN was conducted in patients with stage IV melanoma, with any number of prior therapies, Eastern Cooperative Oncology Group performance status 0-2, and adequate organ function. Results: VPA pretreatment potentiated KTN-induced apoptosis in multiple melanoma cell lines and in mouse A375 xenografts. VPA increased KTN-induced DNA strand breaks. In the phase I/II trial, 39 patients were entered, with 37 evaluable for toxicity and 33 evaluable for response. Somnolence was the dose-limiting toxicity.The maximum tolerated dose forVPA was 75 mg/kg/d; at maximum tolerated dose, serumVPA was f200 Ag/mL (1.28 mmol/L). At the dose expansion cohort, 47% (7 of 15) of patients had stable disease; median overall survival and time to progression were 32.8 and10.2 weeks, respectively. Histone hyperacetylation was observed in peripheral blood mononuclear cells at maximum tolerated dose. Conclusion: VPA potentiates KTN-induced DNA strand breaks and cytotoxicity. VPA can be combined at 75 mg/kg/d for 5 days with full-dose KTN without overlapping toxicities. In metastatic poor prognosis melanoma, this combination is associated with disease stabilization in 47% of patients. Further testing of this combination appears warranted.