Triple-negative breast cancer (TNBC) represents a more aggressive and difficult subtype of breast cancer where responses to chemotherapy occur, but toxicity is significant and resistance often follows. Immunotherapy has shown promising results in various types of cancer, including breast cancer. Here, we investigated a new combination strategy where histone deacetylase inhibitors (HDACi) are applied with immune checkpoint inhibitors to improve immunotherapy responses in TNBC.Testing different epigenetic modifiers, we focused on the mechanisms underlying HDACi as priming modulators of immunotherapy. Tumor cells were co-cultured with human peripheral blood mononuclear cells (PBMCs) and flow cytometric immunophenotyping was performed to define the role of epigenetic priming in promoting tumor antigen presentation and immune cell activation. We found that HDACi up-regulate PD-L1 mRNA and protein expression in a time-dependent manner in TNBC cells, but not in hormone responsive cells. Focusing on TNBC, HDACi up-regulated PD-L1 and HLA-DR on tumor cells when co-cultured with PBMCs and down-regulated CD4+ Foxp3+ Treg in vitro. HDACi significantly enhanced the in vivo response to PD-1/CTLA-4 blockade in the triple-negative 4T1 breast cancer mouse model, the only currently available experimental system with functional resemblance to human TNBC. This resulted in a significant decrease in tumor growth and increased survival, associated with increased T cell tumor infiltration and a reduction in CD4+ Foxp3+ T cells in the tumor microenvironment. Overall, our results suggest a novel role for HDAC inhibition in combination with immune checkpoint inhibitors and identify a promising therapeutic strategy, supporting its further clinical evaluation for TNBC treatment.

IntroductionThe emergence of hormone therapy resistance, despite continued expression of the estrogen receptor (ER), is a major challenge to curing breast cancer. Recent clinical studies suggest that epigenetic modulation by histone deacetylase (HDAC) inhibitors reverses hormone therapy resistance. However, little is known about epigenetic modulation of the ER during acquired hormone resistance. Our recent phase II study demonstrated that HDAC inhibitors re-sensitize hormone therapy-resistant tumors to the anti-estrogen tamoxifen. In this study, we sought to understand the mechanism behind the efficacy of this combination.MethodsWe generated cell lines resistant to tamoxifen, named TAMRM and TAMRT, by continuous exposure of ER-positive MCF7 and T47D cells, respectively to 4-hydroxy tamoxifen for over 12 months. HDAC inhibition, along with pharmacological and genetic manipulation of key survival pathways, including ER and Bcl-2, were used to characterize these resistant models.ResultsThe TAMRM cells displayed decreased sensitivity to tamoxifen, fulvestrant and estrogen deprivation. Consistent with previous models, ER expression was retained and the gene harbored no mutations. Compared to parental MCF7 cells, ER expression in TAMRM was elevated, while progesterone receptor (PGR) was lost. Sensitivity of ER to ligands was greatly reduced and classic ER response genes were suppressed. This model conveyed tamoxifen resistance through transcriptional upregulation of Bcl-2 and c-Myc, and downregulation of the cell cycle checkpoint protein p21, manifesting in accelerated growth and reduced cell death. Similar to TAMRM cells, the TAMRT cell line exhibited substantially decreased tamoxifen sensitivity, increased ER and Bcl-2 expression and significantly reduced PGR expression. Treatment with HDAC inhibitors reversed the altered transcriptional events and reestablished the sensitivity of the ER to tamoxifen resulting in substantial Bcl-2 downregulation, growth arrest and apoptosis. Selective inhibition of Bcl-2 mirrored these effects in presence of an HDAC inhibitor.ConclusionsOur model implicates elevated ER and Bcl-2 as key drivers of anti-estrogen resistance, which can be reversed by epigenetic modulation through HDAC inhibition.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0533-z) contains supplementary material, which is available to authorized users.

Understanding the contribution of dysregulated gene silencing to epigenomic alterations in cancer development provides the rationale for the use of epigenetic modulators, such as histone deacetylase (HDAC) inhibitors, in cancer therapy. HDAC inhibitors have been approved as single agents for cutaneous and peripheral T-cell lymphoma and have shown promising activity in reversing therapy resistance in other tumor types. The effects of HDAC inhibitors on immune modulation have created a recent interest in their potential role in immunotherapy. This review describes the current understanding on integrating HDAC inhibitors into various immunotherapeutic approaches, such as cancer vaccines, adoptive T-cell transfer and immune checkpoint inhibitors. Furthermore, it summarizes promising treatment strategies in epigenetic immune priming from clinical trials that are currently underway.

Broad use of germline testing has identified an increasing number of women at risk for breast cancer with a need for effective chemoprevention. We report a novel method to selectively deliver various anti-estrogens at high drug levels to the breast tissue by implanting a device comprised of silastic tubing. Optimized tubing properties allow elution of otherwise poorly bioavailable anti-estrogens, such as fulvestrant, into mammary tissue in vitro and in vivo with levels sufficient to inhibit estrogen receptor activation and tumor cell proliferation. Implantable silastic tubing delivers fulvestrant selectively to mouse mammary fat tissue for one year with anti-tumor effects similar to those achieved with systemic fulvestrant exposure. Furthermore, local delivery of fulvestrant significantly decreases cell proliferation, as assessed by Ki67 expression, most effectively in tumor sections adjacent to tubing. This approach may thereby introduce a potential paradigm shift and offer a promising alternative to systemic therapy for prevention and early interception of breast cancer.

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