A Novel Strategy to Co-target Estrogen Receptor and Nuclear Factor κB Pathways with Hybrid Drugs for Breast Cancer Therapy

Abstract

Nearly 75% of breast tumors express estrogen receptor (ER), and will be treated with endocrine therapy, such as selective estrogen receptor modulator (SERM), tamoxifen, or aromatase inhibitors. Despite their proven success, as many as 40-50% of ER+ tumors fail to respond to endocrine therapy and eventually recur as aggressive, metastatic cancers. Therefore, preventing and/or overcoming endocrine resistance in ER+ tumors remains a major clinical challenge. Deregulation or activation of the nuclear factor κB (NFκB) pathway has been implicated in endocrine resistance and poor patient outcome in ER+ tumors. As a consequence, one option to improve on existing anti-cancer treatment regimens may be to introduce additional anti-NFκB activity to endocrine therapy drugs. Our approach was to design and test SERM-fumarate co-targeting hybrid drugs capable of simultaneously inhibiting both ER, via the SERM, raloxifene, and the NFκB pathway, via fumarate, in breast cancer cells. We find that the hybrid drugs display improved anti-NFκB pathway inhibition compared to either raloxifene or fumarate. Despite some loss in potency against the ER pathway, these hybrid drugs maintain anti-proliferative activity in ER+ breast cancer cells. Furthermore, these drugs prevent clonogenic growth and mammosphere formation of ER+ breast cancer cells. As a proof-of-principle, the simultaneous inhibition of ER and NFκB via a single bifunctional hybrid drug may represent a viable approach to improve the anti-inflammatory activity and prevent therapy resistance of ER-targeted anti-cancer drugs.

Keywords: Breast cancer; Co-targeting drugs; Estrogen receptor; NFκB pathway.

Fig. 1

The effect of ER antagonists, SERMs (a) and SERDs (b), on NFκB activity in ER+ breast cancer cells. MCF-7 cells were transfected with NFκB-RE and renilla reporter plasmids. Cells were then pretreated with various concentrations of drugs for 1 h, followed by TNFα (10 ng/mL) for 4 h to activate the NFκB pathway. Each drug’s inhibitory activity was calculated as percentage of TNFα alone, which is set to 100%. Data are presented as mean ± SEM

Fig. 2

Hybrid drug co-targeting strategy and chemical structures are indicated. a Schematic representation of a hybrid drug capable of targeting simultaneously both ER and NFκB and its potential benefit in breast cancer. b Chemical structures of the parent drugs, raloxifene and dimethyl fumarate (DMF), along with the co-targeting drugs are shown. The fumarate moiety of Ral-Fum hybrids is highlighted

Fig. 3

Co-targeting Ral-Fum hybrids have anti-NFκB activity in breast cancer cells. a MCF-7 cells were transfected with NFκB-RE and renilla reporter plasmids. Cells were then pretreated with various concentrations of drugs for 1 h, followed by TNFα (10 ng/mL) for 4 h to activate the NFκB pathway. Each drug’s inhibitory activity was calculated as percentage of TNFα alone, which is set to 100%. b MCF-7 cells were pretreated with various concentrations of drugs for 1 h, followed by TNFα (10 ng/mL) for 2 h to activate the NFκB pathway. mRNA expression of ICAM1 was measured by RT-QPCR. Each drug’s inhibitory activity was calculated as percentage of TNFα alone, which is set to 100%. IC₅₀s are calculated with GraphPad Prism software

Fig. 4

Co-targeting Ral-Fum hybrids have anti-ER activity in breast cancer cells. a MCF-7 cells were transfected with ERE and renilla reporter plasmids. Cells were then treated with various concentrations of drugs for 1 h, followed by E2 (10 nM) for 4 h to activate ER. Each drug’s inhibitory activity was calculated as percentage of E2 alone, which is set to 100%. b MCF-7 cells were treated with various concentrations of drugs for 1 h, followed by E2 (10 nM) for 2 h. mRNA expression of PR was measured by RT-QPCR. Each drug’s inhibitory activity was calculated as percentage of E2 alone, which is set to 100%

Fig. 5

Ral-Fum 2 inhibits ER and NFκB crosstalk genes. MCF-7 cells were treated with 10 μM Ral-Fum 2 or 10 μM DMF for 1 h, followed by E2+TNFα for 2 h. mRNA expression of crosstalk genes, BIRC3 and PTGES, was measured by RT-QPCR

Fig. 6

Co-targeting Ral-Fum hybrids block proliferation, clonogenic growth, and mammosphere formation of breast cancer cells. a MCF-7 cells were treated with various drug concentrations in the presence of E2 (10 nM) for 72 h. Each drug’s inhibitory activity was calculated as percentage of E2 alone, which is set to 100%. b MCF-7 cells are seeded as single cells in estrogenized growth media (GM), and then treated with the indicated concentration every 3–4 days for 2 weeks. Colonies were stained and quantified with ImageJ. Each drug’s inhibitory activity was calculated as percentage of GM alone, which is set to 100%. c MCF-7 cells were seeded as single cells on low attachment plates in MS media. DMSO (Veh) or drugs were added next day, and 7 days later the number of MS (MS >75 μm in diameter) was quantified. Double asterisks indicate (**) P < 0.01