Histone Deacetylase Inhibitors Improve the Replication of Oncolytic Herpes Simplex Virus in Breast Cancer Cells

<div><p>New therapies are needed for metastatic breast cancer patients. Oncolytic herpes simplex virus (oHSV) is an exciting therapy being developed for use against aggressive tumors and established metastases. Although oHSV have been demonstrated safe in clinical trials, a lack of sufficient potency has slowed the clinical application of this approach. We utilized histone deacetylase (HDAC) inhibitors, which have been noted to impair the innate antiviral response and improve gene transcription from viral vectors, to enhance the replication of oHSV in breast cancer cells. A panel of chemically diverse HDAC inhibitors were tested at three different doses (<,  = , and >LD₅₀) for their ability to modulate the replication of oHSV in breast cancer cells. Several of the tested HDAC inhibitors enhanced oHSV replication at low multiplicity of infection (MOI) following pre-treatment of the metastatic breast cancer cell line MDA-MB-231 and the oHSV-resistant cell line 4T1, but not in the normal breast epithelial cell line MCF10A. Inhibitors of class I HDACs, including pan-selective compounds, were more effective for increasing oHSV replication compared to inhibitors that selectively target class II HDACs. These studies demonstrate that select HDAC inhibitors increase oHSV replication in breast cancer cells and provides support for pre-clinical evaluation of this combination strategy.</p></div

HDAC inhibitors enhance oHSV replication in breast cancer cells, but not in normal breast epithelial cells.

<p>MDA-MB-231 human breast cancer, MCF10A normal breast epithelial, and 4T1 murine mammary carcinoma cells were treated with the indicated HDAC inhibitors either 6 hours prior (Pre-) or immediately following (Co-) infection with M002 oHSV at 0.1 PFU/cell. Shown are fold changes in viral titer versus replication in untreated cells, at 48 hours post infection. From left to right, the three sets of bars within each graph indicate inhibitor concentrations below LD₅₀, near LD₅₀ and above LD₅₀.</p

Summary of Increased oHSV Replication in Cancer Cell Lines Pre-treated with HDAC Inhibitors.

<p>No increase (-) or increases in replication of >2 fold (+), >5 fold (++) and >10 fold (+++) are shown.</p>1<p>For breast cancer.</p>2<p>Clinically approved for the treatment of cutaneous T cell lymphoma.</p>3<p>Also inhibits the Class IIa enzyme HDAC 9.</p

Doses of HDAC Inhibitors Used in Viral Replication Experiments.

<p>Doses of HDAC Inhibitors Used in Viral Replication Experiments.</p

Approximate LD₅₀ values determined for a panel of HDAC inhibitors in breast cancer cells.

<p>Proliferating breast cancer (MDA-MB-231), murine mammary carcinoma (4T1) and normal breast epithelial (MCF10A) cells were treated with a panel of histone deacetylase inhibitors at a range of concentrations, and cell viability was assessed after three days. Shown are representative dose-response curves for MDA-MB-231 and MCF10A cells treated with belinostat (upper panels) and a table of approximate LD₅₀ values calculated from dose-response curves for the entire panel of inhibitors.</p

NLS2 is required for metastasis suppression.

<p>Three clones each of NLS1,1, NLS2,2, and NLS2,1 were selected for experimental metastasis assays by injection into the lateral tail vein of athymic mice (10 mice per group). A. Western blot of whole cell lysate showing similar levels of expression for BRMS1 and the BRMS1 mutants (β-actin used as loading control). B. The average number of lung metastasis is shown with SEM. The percentage of metastasis suppression is listed on the right. Mutants lacking NLS2 (NLS1,1 and NLS2,1) did not consistently suppress metastasis. * indicates p≤0.05.</p

NLS1 is required for nuclear localization.

<p>GFP-GST fused constructs listed in Fig. 1 were visualized in COS7 cells. GFP fluorescence from the fused BRMS1 or BRMS1 mutant proteins indicates localization. As predicted, mutants lacking NLS1 were predominantly localized to the cytoplasm. Phalloidin and DAPI were used to visualize the cytoplasm and nucleus respectively.</p

NLS2 is important for cytoplasmic association with SIN3A.

<p>Western blot from co-immunoprecipitated samples of SIN3A in nuclear (top panel) and cytoplasmic (bottom panel) fractions are shown. The antibody used to probe the blot is listed on the right and approximate molecular mass on the left. All BRMS1 mutants were detected in the nuclear fractions but only the wild-type and NLS2,2 mutant were precipitated in the cytoplasm. We note that although BRMS1 is usually undetectable by western blot, endogenous BRMS1 could be detected in the nucleus from the co-immunoprecipitation. L is lysate and IP is immunoprecipitation.</p

A. Domain organization of BRMS1 and the BRMS1 mutants generated for this study.

<p>E-rich is glutamate rich region, CC1 and CC2 are coiled-coil regions, NLS1 and NLS2 are nuclear localization sequences. B. Predicted subcellular localization based on PSORT II and WoLF PSORT (ExPASY proteomics server). The two mutants lacking NLS1 (ΔNLS2 and NLS2,2) have a negative NLS score using PSORT II and high cytoplasmic score using WoLF PSORT. Lamin A/C and tubulin are also listed for proteins representing nuclear and cytoplasmic localization respectively.</p

Endogenous interactions of SIN3A and BRMS1 in the cytoplasm.

<p>Proximity ligation assays were used to clearly show cytoplasmic interactions in the MCF10A normal “immortalized” breast epithelial cells. The interactions of SIN3A with BRMS1 are predominantly nuclear in the metastatic breast cancer cell line MCF10CAa.1. Each red dot indicates an interaction. Representative images are shown. DAPI is shown in blue. Duolink represents images with both primary antibodies and PLA probes, PLA control is without primary antibodies, SIN3A control is without BRMS1 primary antibody and BRMS1 control is without SIN3A primary antibody.</p