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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.

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.

Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including “sawtooth” patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes. Electronic supplementary material The online version of this article (doi:10.1007/s10549-015-3468-2) contains supplementary material, which is available to authorized users.

Background Adipose fat transfer is increasingly used for contour corrections of the tumor bed after lumpectomy and breast reconstructions after mastectomy. The lipophilic nature of the fat tissue may render adipocytes an ideal vehicle with which to deliver a high boost of an antiestrogen to the tumor bed to serve as an adjunct systemic hormonal therapy. The authors therefore tested whether adipocytes could safely be loaded with an antiestrogen and allow for release at therapeutic concentrations to treat breast cancer. Methods Adipose tissue was collected from patients undergoing autologous fat grafting. The influence of adipose tissue on tumorigenesis was determined both in vitro and in vivo using breast cancer cell lines. Ex vivo, adipose tissue was assessed for its ability to depot fulvestrant and inhibit the growth of breast cancer cell lines. Results Adipose tissue harvested from patients did not promote breast cancer cell growth in vitro or in an in vivo mouse model. Adipose tissue was successfully loaded with fulvestrant and released at levels sufficient to inhibit estrogen receptor signaling and growth of breast cancer cells. Conclusions This work supports the hypothesis that adipose tissue used for autologous fat grafting can serve as a novel method for local drug delivery. As this technique is used to reconstruct a variety of postsurgical defects following cancer resection, this approach for local drug delivery may be an effective alternative in therapeutic settings beyond breast cancer.

Aberrations in histone acetyltransferases and histone deacetylases (HDAC) have been associated with tumor development and tumor progression. Pharmacological interference with histone acetylation and methylation has shown to be promising for the treatment and prevention of cancer. Several structurally and functionally different HDAC inhibitors have been developed as a novel modality to reverse aberrant epigenetic changes in cancer cells. With a growing body of literature, there is ample preclinical rationale to test these drugs in various stages and settings of cancer, either as single agents or in rationally designed combinations as a therapeutic strategy in hematological as well as solid tumor malignancies. The first HDAC inhibitor vorinostat (Zolinza) was approved for the treatment of patients with cutaneous T-cell lymphoma. HDAC inhibitors have also demonstrated great promise in treating a variety of noncancer diseases, including epilepsy, cystic fibrosis, spinal muscular atrophy, and human immunodeficiency virus infection.

Background Treatment options for sarcoma are limited. Histone deacetylase inhibitors increase the efficacy of topoisomerase II inhibitors by promoting access to chromatin and by down-regulating DNA repair. Thus, combined panobinostat and epirubicin therapy was evaluated to treat refractory sarcoma. Patients and methods Patients with advanced solid tumors were enrolled in a 3 + 3 dose-escalation phase I trial of panobinostat given on days 1, 3, and 5 followed by 75 mg/m2 of epirubicin on day 5 in 21-day cycles, with a dose expansion at maximum tolerated dose (MTD) in 20 sarcoma patients. Peripheral blood mononucleocyte histone acetylation was also evaluated. Results Forty patients received 20–60 mg panobinostat. Dose-limiting toxicities included thrombocytopenia, febrile neutropenia, and fatigue at 60 mg, defining a panobinostat MTD at 50 mg. Four responses were seen in 37 assessable patients, all after progression on prior topoisomerase II inhibitors. For those with sarcoma, 12 of 20 derived clinical benefit (1 partial response and 11 stable disease, median overall survival 8.3 months), including 8 of 14 previously progressed on topoisomerase II therapy. Treatment benefits correlated with increased histone acetylation and decreased neutrophil count on day 5. Conclusions Panobinostat and epirubicin treatment is well tolerated and may reverse anthracycline resistance. Changes in histone acetylation and associated decrease in neutrophil count correlated with clinical benefit and warrant investigation as predictive biomarkers. Clinical trial This trial is registered at www.Clinicaltrials.gov , Identifier: NCT00878904.

Introduction The 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. Methods We 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. Results The 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. Conclusions Our 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 material The online version of this article (doi:10.1186/s13058-015-0533-z) contains supplementary material, which is available to authorized users.