ERα-negative breast cancers remain difficult to manage and treatment options are limited to chemotherapy since the tumors are more aggressive and resistant to endocrine therapy. Genetic alternations such as mutations, loss of heterozygosity or homozygous deletions are rare (6
). Studies performed by Davidson and colleagues suggested that the loss of ERα protein expression is the result of the hypermethylation of the CpG islands within the ERα promoter (5
). Deacetylated histones were associated with the inactive ERα promoter in MDA-MB-231 cells, whereas acetylated histones were associated with the active ERα promoter in MCF-7 cells (25
). Treatment with HDAC inhibitors reversed repression of ERα in MDA-MB-231 cells (27
). In this paper, we show that the HDACi, ENT was effective in causing expression of ERα and aromatase, the enzyme that is key in the production of estrogen. Targeting the upregulated aromatase using letrozole caused reduced cell viability in vitro and tumor regression in vivo. Our studies investigated both biochemical and biological consequences of these treatments and provide a strong rationale for use of AIs in combination with HDAC inhibitors for the treatment of ER-negative breast cancers.
Several studies have now confirmed that gene silencing by methylation involves generation of inactive chromatin structure, characterized by deacetylated histones (4
). The HDACs deacetylate lysine groups of histones H3 and H4, allowing ionic interactions between positively charged lysine residues on histones and negatively charged DNA. This results in compaction of the nucleosomes, which prevents transcription (3
). An endogenous interaction exists between HDAC 1 and ERα in the absence of estrogen in breast cancer cells (28
). HDAC 2 and 3 have been indicated to associate with ERα-regulated genes such as c-Myc and Cathepsin D (27
). ENT was used in this study due to its specificity for class I HDACs such as HDAC 1, 2 and 3. ENT also exhibited a long half-life (~100 hrs) and favorable pharmacodynamic effects in humans (29
Our novel findings show that HDACi ENT can lead to an increase in functional tumoral aromatase activity and an increase in ERα mediated transcription (pS2 upregulation), both in vitro and in vivo. Our previous study with letrozole resistant LTLT-Ca cells showed that upregulation of ERα led to activation of aromatase gene transcription in a ligand dependent manner (24
). However, in MDA-MB-231 cells treated with ENT, aromatase was upregulated in ERα independent manner.
Among the three HDAC inhibitors tested, ENT, SAHA and butyric acid, ENT was the most potent. These effects were not specific to just one cell line, MDA-MB-231. In vitro studies with two-other ERα-negative cell lines (SKBr-3 and Hs578T) confirmed the results obtained with MDA-MB-231. Based on these findings, we hypothesized that treatment with an HDAC inhibitor would convert ER-negative tumors to ER-positive tumors thereby rendering them sensitive to estrogens, and consequently, the inhibitory effects of AIs. Treatment of xenografts with MDA-MB-231 () and HS578T tumors () confirmed this hypothesis. In initial dose finding studies, reduction in tumor growth was observed in ovariectomized mice treated with ENT alone (). However, when the mice were supplemented with Δ4A to produce estrogen, tumor growth was slightly stimulated thus negating the inhibitory effect of ENT.
The induction of aromatase in the tumor leads to the production of estrogen via aromatization of Δ4A. This results in the activation of ERα and transcription of ERα regulated genes, leading to tumor growth, thereby counteracting the tumor inhibitory effect of ENT (Figure- and ). In this setting, combining the AI, letrozole with ENT inhibited production of estrogen. This resulted in inhibition of tumor growth and lung metastases.
ERα -positive cancer cell lines respond to HDACi differently. In these cells, in contrast to our findings in ERα-negative cell lines, Chen et al
have shown that the HDACi, LBH589, specifically inhibits aromatase activity and downregulates gene and protein expression through suppression of promoters I.3/PII (31
). SAHA acts in a similar manner in MCF-7 and BT-474 cells (32
). SAHA down-regulates ERα through hyperacetylation of HSP-90, a chaperone protein that maintains the stability of ERα (32
). HDAC6 is HSP90 deacetylase and inhibition of HDAC6 is responsible for HDACi-mediated HSP90 inhibition (33
). This complex regulation of ERα by HDACi in ER-positive versus ER-negative breast cancer warrants further investigation. There is also some evidence of partial restoration of functional ERα in cells that have lost ERα and PgR expression as result of acquired resistance to endocrine therapy (35
). In this model system, expression of PgR could not be restored with re-expression of ERα.
The detailed molecular mechanism of the conversion of MDA-MB-231 cells from hormone-independent to hormone-dependent cells expressing ER and aromatase is unknown at this time. However, it suggests phenotypic plasticity of the cells (37
) that enables them to adapt (11
) to changes in their microenvironment. Further studies are needed to elucidate the precise mechanisms underlying this phenomenon.
In conclusion, our study using both biological and biochemical assays demonstrates that HDAC inhibitor ENT increases both ERα and aromatase expression and activity, thereby converting ER-negative tumors to ERα-positive tumors. The breast cells are now sensitive to the growth stimulatory effects of estrogens synthesized locally by the aromatization of Δ4A in the tumor. Thus, when AI, letrozole, was combined with the HDAC inhibitor, the ERα expressing tumors were deprived of estrogen. This resulted in suppression of tumor growth. Furthermore, the combination treatment was also effective in inhibiting tumor cell colonization in the lungs. This novel approach could potentially provide a new treatment strategy for the management of ERα-negative breast cancer.