For many years, the search for new cancer therapy targets focused on genetic changes associated with the transformation of normal cells into malignant cells, and with resistance to therapy. However, it is now clear that epigenetic mechanisms are also essential targets in cancer therapy. Epigenetic changes are frequent events, and unlike genetic mutations, epigenetic modifications are reversible events; thus, the inhibition of these mechanisms could be a potential therapeutic strategy for treatment of breast cancer, either through direct effects on epigenetic changes, or by modulating known targets of other therapies (e.g. ER).
Several inhibitors of enzymes controlling epigenetic modifications, specifically DNMT and HDACs, have been developed, and show promising anti-tumorigenic effects. The DNMT inhibitors include Vidaza (AZA), Decitabine (DAC), and zebularine. A number of HDAC inhibitors including hydroxamates (e.g. TSA, SAHA, LBH-589), cyclic peptides (e.g. Depsipeptides), aliphatic acids (e.g. phenylbutyrate, valproic acid), and benzamides (e.g. SNDX-275, p-N-acetyl dinalin, MGC0103) have been clinically investigated [87
]. The DNMT inhibitors Vidaza (AZA) and Decitabine (DAC), and the HDAC inhibitor Vorinostat (SAHA) have been FDA-approved in hematological malignancies, where the in general the greatest success has been achieved. For example, treatment of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients with Vidaza and Decitabine resulted in very encouraging response rates and improved survival outcomes compared with cytotoxic chemotherapy [88
]. Whether these epigenetic drugs could be used in other forms of cancer, and in particular in solid malignancies, is being evaluated. In breast cancer, there are only a few studies which have investigated the use of demethylating or HDAC inhibitors alone, or in combination with other agents such as endocrine therapies, cytotoxic agents, or novel targeted therapies. Early reports suggest that there may be stabilization of disease or some response; however, thus far these agents have shown a less impressive clinical track record in solid tumors (including breast cancer) compared to hematological malignancies. One problem is decreased drug efficacy in solid tumors, due to a pool of low replicating cells [88
] limiting the use of current demethylating agents in non-hematological malignancies.
Several phase I and II clinical trials are ongoing using DNA methylation and/or HDAC inhibitors in breast cancer patients (summarized in ). Decitabine and Vidaza are currently in clinical trials in breast cancer patients with advanced and metastatic disease. The HDAC inhibitors Vorinostat, Panobinostat, and Entinostat are being tested in trials with patients with ductal carcinoma in situ, as well as and advanced and metastatic tumors. In a phase II study, 14 patients received oral vorinostat, 200 mg twice daily for 14 out of 21 day cycles. Although stable disease was observed in 4 patients (time to progression of 4, 8, 9, and 14 months), the study was terminated due to lack of complete or partial responses in the first stage [90
]. In a phase I study of Decitabine and Vorinostat in patients with advanced solid tumors or relapsed/refractory non-Hodgkin's lymphoma, Canadian investigators studied two different schedules and reported results from a sequential schedule. The investigators reported disease stabilization for 4 or more cycles in 7 of 22 (31.8%) evaluable patients, which included two patients with breast cancer [91
]. Dose-limiting toxicities included mainly myelosuppression, constitutional and gastrointestinal symptoms in 7 of 27 (26%) participants. In the HDAC inhibitor trial, the most common adverse events included fatigue, nausea, diarrhea, and lymphopenia [90
]. These side effects were expected, since they have been previously reported to be associated with targeting HDACs. In general, various HDAC inhibitors differ in their toxicity profile when comparing the side effects described in the available clinical studies of HDAC inhibition in the treatment of cancer. There are potentially other, more serious side effects including cardiotoxicity, and effects on chromosomal stability. Finally, these drugs may also affect normal hematopoiesis; hematologic toxicity is common to many drugs but stimulation of hematopoiesis seems to occur for others. The observed side effects might result from effects on epigenetic regulation of various target genes, but more likely they are caused by inhibition of other targets of acetylases—acetylation is an important posttranslational modulation of several proteins (in addition to histone) involved in the regulation of cell proliferation, differentiation and apoptosis in normal as well as cancer cells.
Epigenetics clinical trials in breast cancer.
Few studies incorporated HDAC inhibitors with cytotoxic agents. In a proof-of-principle study, the demethylating agent hydralazine and the HDAC inhibitor magnesium valproate were given in combination with neoadjuvant doxorubicin and cyclophosphamide to locally advanced breast cancer patients to assess their safety and biological efficacy [92
]. It was found that hydralazine and valproate exerted their proposed molecular effects by significantly decreasing global 5mC content and HDAC activity. There was an up-regulation of 1,091 and down-regulation of 89 genes in primary tumors. Importantly, this treatment was safe and well-tolerated, and appeared to increase the efficacy of doxorubicin and cyclophosphamide. Also, a recent phase II single-arm study of hydralazine and magnesium valproate added to the same schedule of chemotherapy on which patients were progressing, suggesting that hydralazine and valproate overcome epigenetic changes, mediating chemotherapy resistance regardless of chemotherapy drug and tumor type [93
]. In another phase I/II trial, women with metastatic breast cancer received first line chemotherapy with paclitaxel 90 mg/m2
on days 1, 8, 15, bevacizumab 10 mg/kg on days 1, 15 every 28 days, and oral vorinostat 200 mg (N
=3) or 300 mg (N
=41) twice daily for 3 days given the day before, day of, and day after each paclitaxel dose. The dose level recommended phase II dose of vorinostat was 300 mg twice a day, and a sufficient number of responses occurred in the first stage of the phase II trial (15/28) to continue accrual to the second stage. Toxicity was not different than expected for paclitaxel and bevacizumab therapy. Preliminarily, at least 25 of 43 evaluable patients at both dose levels have had either a confirmed (N
=22) or unconfirmed (N
=3) objective response (ORR 58%, 95% C.I. 42%,73%). Two patients had tumor biopsies and peripheral blood mononuclear cells collected before and 4 h after the third dose of Vorinostat, which revealed increased acetylation of the lysine residue K69 of the chaperone protein Hsp 90, as well as up-regulation of Hsp70, and down-regulation of AKT [94
Another promising approach is the combination of epigenetic and endocrine therapy. In a recent phase II trial, the HDAC inhibitor Vorinostat and tamoxifen were given in combination to patients with ER+ metastatic breast cancer that had progressed on prior hormonal and chemotherapy regimens [95
]. Of 29 participants, 6 (21%) patients had an objective response, and 3 (10%) had disease stabilization for 6 or more months. Since most of the patients had received prior adjuvant tamoxifen or up to two aromatase inhibitors, these results support the hypothesis that adding an HDAC inhibitor to tamoxifen may restore hormone sensitivity. This might be a result of re-expression of silenced ER, a main rationale for combining epigenetic and endocrine treatment. It is however very likely that HDAC inhibitors will not only alter the “histone code” (i.e. affecting both histone acetylation and methylation) at the ESR1
(and other) genes, but also impact a host of transcription factors, and thus might act via other pathways. Measurement of target gene expression, and identification and measurement of other potential biomarkers is a critical task for ongoing and future trials with epigenetic drugs.
In summary, early reports of combining HDAC inhibitors and other standard agents suggest that there is a role for further investigation in breast cancer and in other solid malignancies. Challenges include agent selection as well as dose, schedule, and identification of biomarkers.