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Mechanistic Synergy of HDAC and Topo II Inhibitors
Background: The topo II inhibitors (epirubicin and doxorubicin) are the backbone of breast cancer therapy. While the response rate of tumors to these drugs is high, many tumors are resistant or become resistant. It was reported that the histone deacetylase (HDAC) inhibitor, trichostatin A may enhance the cytotoxic effects of topo II inhibitors in leukemia cells. Inhibition of HDACs leads to histone acetylation and weakening of the DNA-histone interactions, which may increase the access of DNA to topo II inhibitors. We found that pre-exposure of cells to the HDAC inhibitors, valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) increased the binding of DNA by topo II inhibitors and potentiated topo II inhibitor-induced growth arrest, DNA damage, and cell death in breast cancer cells. Synergistic activity between HDAC and topo II inhibitors required the expression of functional topo IIá and/or topo IIâ and was associated with an HDAC inhibitor-induced down-regulation of topo IIá, but not topo IIâ.
Objective/Hypothesis: HDAC inhibitors potentiate topo II inhibitors in a sequence-specific manner. The HDAC inhibitor-induced decondensation of the chromatin increases the access of DNA to topo II inibitors. The expression of topo IIâ and the HDAC inhibitor-induced depletion of topo IIá play an important role in the potentiation of topo II inhibitors by HDAC inhibitors.
Specific Aims: Aim 1: To evaluate HDAC inhibitor-induced sensitization to topo II inhibitors as a function of expression, location, and activity of topo IIá and topo IIâ in cell systems with specific alterations of the topo II isoforms. Aim 2: To define the contributions of the HDAC inhibitor induced modulation of chromatin structure and the depletion of topo IIá in the observed synergy. Aim 3: Evaluate the HDAC inhibitor-induced modulation of topo IIá in tumor samples.
Study Design: We will determine the contributions of topo IIá and topo IIâ in the potentiation of topo II inhibitors by HDAC inhibitors using "single cell systems” where the expression, location, and activity of topo IIá and topo IIâ have been altered. In addition, we will introduce topo IIá, topo IIâ, or both into a topo II depleted cell line and evaluate the transfected cells for increased sensitivity to this drug combination. We will then evaluate the impact of HDAC inhibitor-induced chromatin structural changes and topo IIá depletion on the observed synergy by mimicking the HDAC inhibitor-induced effects by alternative strategies. Lastly, pre- and post- treatment tumor biopsies from a phase I clinical trial involving the HDAC inhibitor, valproic acid, and the topo II inhibitor, epirubicin, will be evaluated for changes in histone acetylation and topo IIá expression to correlate molecular targets with response.
Relevance: Breast cancer is the most common cause of cancer in women and the second most common cause of cancer death. Post-operative therapy has significantly decreased the death rate of breast cancer. The proposed studies will introduce a novel class of drugs, the HDAC inhibitors that may potentiate topo II inhibitors. As the HDAC inhibitor induced sensitization to topo inhibitors occurs predominantly in cells with high expression of topo II, one would expect that the effects be more pronounced in tumor cells, as quiescent cells do not express topo IIá. Therefore, this combination may enhance anti-tumor effects of topo II inhibitors without affecting somatic cells. The development of markers that predict response to topo II inhibitors either alone or in combination with HDAC inhibitors will identify patients most likely to benefit from this therapy. The mechanistic understanding of this synergy could be exploited for the development of novel strategies to potentiate DNA damage and cell death for the treatment of patients with breast cancer.
Type II topoisomerases (topo II alpha and topo II beta) are crucial enzymes required during DNA replication and cell division and therefore are ideal targets for anti-cancer therapy. Topo II alpha is minimally expressed in normal cells. In contrast, overexpression of topo II alpha has been found in breast cancer. Topo II beta is expressed in normal cells, but is expressed at higher levels in cancer cells. The topo II inhibitors, epirubicin and doxorubicin, are among the most active drugs for the treatment of breast cancer. However, many breast cancers are resistant and these drugs have side effects. Modalities that selectively enhance the activity of topo II inhibitors while limiting their side effects would greatly improve therapy.
In this study we propose to evaluate a novel combination of histone deacetylase (HDAC) inhibitors and topo II inhibitors for the treatment of breast cancer. Topo II inhibitors cause cell death by damaging DNA. DNA molecules are normally tightly packed in the nucleus of the cell. HDAC inhibitors are able to unravel the tightly wrapped DNA. We speculated that HDAC inhibitor-induced uncoiling of the DNA renders the DNA a better target for topo II inhibitors.
Our laboratory findings indicate that HDAC and topo II inhibitors worked synergistically to induce DNA damage and cell death in breast cancer cells. However, synergistic activity of these drugs only occurred under very specific conditions. The HDAC inhibitor had to be given at least 48 hours prior to the topo II inhibitor. In fact, if the topo II inhibitor was given first, antagonistic effects were seen. The sensitivity of breast cancer cells to this drug combination seemed to be dependent upon the expression of topo II alpha and topo II beta. Furthermore, after treatment with the HDAC inhibitor, the topo II alpha levels in these cancer cells were markedly decreased. The significance of these findings is unknown, but will be investigated in this proposal.
We will determine the importance of topo II alpha and II beta expression in the sensitivity of cancer cells to HDAC inhibitor-topo II inhibitor drug combinations using cell lines that have been altered in the expression or function of topo II alpha or topo II beta. We will study how the unraveling of DNA by the HDAC inhibitor may increase the activity of the topo II inhibitor. We will further determine if the HDAC inhibitor-induced loss of topo II alpha in cancer cells is required for topo II inhibitor-induced cell death.
The mode of action of HDAC inhibitors provides a strong rationale to combine these drugs with topo II inhibitors. This study will provide an in-depth understanding of the mechanism of this synergy which may help to establish a new paradigm for sequence-specific combinations of HDAC inhibitors and topo II inhibitors and may define predictive markers of response.