ERα is an established therapeutic target for breast cancer treatment, but the development of subtype selective estrogenic ligands has gained interest with the identification of ERβ [1
]. ERβ opposes the actions of ERα suggesting that it may be a potential therapeutic target. Exogenous ERβ expression in ERα positive breast cancer cells impaired E2 stimulated proliferation [24
] and tumor growth in xenografts [25
]. In support of the anti-proliferative role of ERβ, MCF7 cells were more proliferative when ERβ was knocked down [6
]. Activation of ERβ by subtype selective ligands may enhance ERβ growth repression without stimulating proliferation through ERα; indeed ERβ selective ligands inhibited growth of HC11 mouse mammary cells [5
]. Here, we have also shown that ERβ ligands can inhibit the growth of breast cancer cells when ERβ is expressed. In breast cancer, however, ERβ expression is thought to decline during progression [26
] so ligands aimed at targeting ERβ must be highly selective and used only in patients that lack ERα or those with low ERα:ERβ ratios of expression. The rate of ERβ positivity in breast cancer has been reported to range from 13% to 83% [29
]. In order to effectively target ERβ for cancer treatment, there is an imminent need to: a) identify ERβ selective ligands with minimal side effects and better in vivo
efficacy and selectivity, and b) design clinical trials to recruit patients with low ERα:ERβ ratios in earlier stages of disease progression.
Although ERβ selective ligands have not yet been used for cancer treatment, the therapeutic value of ERβ has been assessed in other diseases. Two of the most promising ERβ selective therapies are the ERβ selective ligand ERB-041 and the herbal extract MF-101 [33
]. Clinical trials have been completed to determine the efficacy of ERB-041 for treatment of Crohn’s disease, endometriosis, interstitial cystitis, and rheumatoid arthritis. Although results have not been published for most of the clinical trials, results of the rheumatoid arthritis trial showed ERB-041 was well tolerated but did not improve arthritis symptoms [34
]. MF-101 also showed a relatively safe profile and reduced the frequency of hot flashes in a phase II clinical trial for treatment of post-menopausal symptoms [35
]. Liquiritigenin is the most active estrogenic component of MF-101[11
], suggesting ERβ selective ligands may prove useful for treating post-menopausal symptoms.
Strategies to identify ER subtype selective ligands include competitive ligand binding, dimerization, transcriptional reporter, and proliferation assays [21
]. Competitive ligand binding assays provide insight into binding affinities and are useful for high throughput small molecule screening [37
], but they are limited because ligands can act as agonists or antagonists and binding affinity does not often reflect transcriptional potency. BRET assays to measure receptor dimerization have been used to identify subtype selective ligands [38
], but also cannot differentiate between agonists or antagonists [39
]. Agonists can be characterized using proliferation assays in MCF7 cells, which are highly sensitive and provide a biologically relevant endpoint in the context of estrogen-sensitive cells [40
]. However, this assay is limited by a lack of specificity, as non-estrogenic mitogens can stimulate proliferation, and cannot be used to detect subtype selective agonists.
Transcriptional assays can differentiate between agonists and antagonists, overcoming limitations of binding and dimerization assays. Mammalian reporter cell lines useful for identifying subtype selective ligands have been created from HeLa cervical carcinoma cells [12
] and HEK293 kidney cells [13
]. HELN-ERα and HELN-ERβ were generated from HeLa cells in two steps: 1) stable integration of ERE-luciferase to generate HELN cells, 2) stable expression of ERα or ERβ to generate HELN-ERα and HELN-ERβ [12
]. 293/hERα and 293/hERβ cells were generated by a similar strategy. Only one breast cancer reporter cell line, T47D-KBLuc, is available to characterize agonists in the context of breast cancer cells [14
], but both ERα and ERβ are expressed, preventing identification of subtype selective ligands.
In this report, we described the development of a new set of breast cancer reporter cell lines to characterize subtype selective estrogenic ligands. Hs578T-ERαLuc and Hs578T-ERβLuc cells were highly sensitive to E2 with EC50
values of 1 pM and 6.5 pM, respectively (). Similar E2 sensitivity was observed in T47D-KBLuc cells, which showed an approximate EC50
of 3 pM [14
]. Hs578T-ERαLuc and Hs578T-ERβLuc cells were more sensitive to E2 than HELN-ER and 293/ER reporter cells, but all reporter cell lines showed greater E2 sensitivity in ERα expressing cells. HELN-ERα cells were approximately 3 times more sensitive to E2 than HELN-ERβ cells (EC50
of 0.017 nM and 0.068 nM, respectively) [12
] and 293/hERα cells were approximately 4 times more sensitive to E2 than cells expressing ERβ (EC50
of 50 pM and 200 pM, respectively) [13
]. Although Hs578T-ERαLuc and Hs578T-ERβLuc cells were not created using the same strategy as HELN-ER or 293/hER reporter cells and likely have unique genomic integration of the reporter, similar sensitivities observed in all reporter cell lines suggest that this does not inhibit comparison of subtype selectivity.
Reporter assays with two ER subtype selective ligands confirmed that Hs578T-ERαLuc and Hs578T-ERβLuc cells could be used to differentiate between ERα and ERβ selective ligands. The ERβ selective agonist DPN maintained 33-fold selectivity in Hs578T-ERLuc cells (EC50
of 0.26 nM for ERβ and 8.5 nM for ERα, ). Dose response assays with the ERα selective agonist PPT revealed the sensitivity of Hs578T-ERβLuc cells (). Although PPT was unable to activate reporter expression in HEC-1 cells transfected with ERβ [12
], PPT did activate reporter expression in Hs578T-ERβLuc cells at high concentrations, although not to the full extent induced by E2. PPT reporter activation was blocked by ICI 182,780 co-treatment () and did not occur in the absence of Dox treatment (data not shown), verifying reporter activation was mediated by ERβ. Despite activation of ERβ at high concentrations, PPT could not fully activate reporter expression in Hs578T-ERβLuc cells and maintained 1000-fold selectivity for ERα.
Subtype selectivity of two natural phytoestrogens, cosmosiin and liquiritigenin, was also assessed in Hs578T-ERαLuc and Hs578T-ERβLuc cells. Liquiritigenin maintained selectivity for ERβ but to a lesser extent than expected, as it has been shown to minimally activate ERα in other cell lines [11
]. The discrepancy in the selectivity of liquiritigenin may be due to the enhanced sensitivity of Hs578T-ERαLuc cells, differences in cofactor expression in Hs578T cells, or purity of the compound (our studies utilized commercially available liquiritigenin and Mersereau and coworkers [11
] used extract from G. uralensis
). The selectivity of cosmosiin could not be assessed using luciferase assays due to supramaximal induction (). Supramaximal activation of estrogen responsive reporters have been described in many systems [21
]. Here, we showed that supramaximal induction by cosmosiin was not due to enhanced transcriptional activation of the reporter (). Despite limitations of the reporter system, the subtype selectivity of cosmosiin could be characterized by assessing target gene regulation in Hs578T-ERα and Hs578T-ERβ cells. While DPN and liquiritigenin maintained similar extents of selectivity as measured by reporter assays, cosmosiin activated both ERα and ERβ as measured by endogenous gene regulation (). Cosmosiin and liquiritigenin induced similar growth inhibitory effects as E2 in Hs578T-ERβ cells, indicating the phytoestrogens could elicit ERβ activation to a similar extent as E2 ().
Hs578T-ERαLuc and Hs578T-ERβLuc cells have several advantages for identifying ERβ selective agonists in comparison to available mammalian reporter cell lines. First, the Hs578T reporter cell lines have inducible expression of ERα and ERβ, allowing determination of off-target reporter activation by assessing reporter expression in the absence of Dox. Second, Hs578T-ERαLuc and Hs578T-ERβLuc cells are highly sensitive to estrogenic ligands. Third, endogenous gene regulation can be used to validate subtype selectivity. Finally, growth inhibition assays using Hs578T-ERβ cells in the presence and absence of Dox can be used to determine the biological endpoint of ERβ activation and validate specificity of ligands to ensure they do not have off-target cytotoxic effects. High throughput screening may be possible using Hs578T-ERαLuc and Hs578T-ERβLuc cells, and luciferase assay optimization using Hs578T-ERβLuc cells has shown a Z factor of 0.5 (data not shown), an acceptable range for high throughput screening [41
]. Therefore, Hs578T-ERαLuc and Hs578T-ERβLuc cells are useful for the identification and characterization of ER subtype selective ligands that may hold therapeutic promise.