Estrogen induces apoptosis in estrogen deprivation-resistant breast cancer through stress responses as identified by global gene expression across time

Abstract

In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17β-estradiol (E(2)) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E(2) paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E(2)-induced apoptosis by analysis of gene expression across time (2-96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E(2)-induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E(2) in 5C cells compared with both WS8 and 2A cells and hence, were associated with E(2)-induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E(2) inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E(2)-induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E(2)-inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E(2) interacted to superadditively induce apoptosis. Therefore, these data indicate that E(2) induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.

Fig. 1.

Examples of estrogen signaling genes. Full annotation, dAUC values, and P values of all estrogen signaling genes are given in Dataset S2.

Fig. 2.

Examples of apoptosis genes. Full annotation, dAUC values, and P values of all apoptosis genes are given in Dataset S3.

Fig. 3.

Examples of inflammatory response genes. Full annotation, dAUC values, and P values of all inflammatory response genes are given in Dataset S4.

Fig. 4.

Functional interrogation of E₂-induced apoptosis. (A) AA and E₂ interact to superadditively induce apoptosis. 5C cells were treated with combinations of AA and E₂ as indicated for 72 h. (B) Screening of selective CASP inhibitors. The selectivity of the inhibitors for individual caspases is indicated according to the manufacturer. 5C cells were treated with 10⁻⁹ M E₂ and 10 μM of each CASP inhibitor as indicated for 96 h. (A and B) Apoptosis according to altered plasma membrane permeability was determined by flow cytometric analysis of cells stained with the DNA-specific binding dyes YO-PRO-1 and 7-aminoactinomycin D. Double-negative staining cells were defined as viable, double-positive staining cells were defined as dead, and intermediately staining cells were defined as apoptotic. Data shown in B represent triplicates and associated SDs.

Fig. 5.

Functional involvement of CASP4 in E₂-induced apoptosis. E₂-induced CASP4 at the (A) mRNA and (B) protein levels in 5C cells but not in WS8 or 2A cells. CASP4 mRNA and protein levels were measured by qPCR and immunoblotting, respectively. (B) In 5C cells, E₂ led to cleavage of the apoptotic marker PARP, which was blocked by the CASP4 inhibitor z-LEVD-fmk. C, control; E, E₂; I, inhibitor (z-LEVD-fmk); c-CASP4, cleaved CASP4; NS, nonspecific band. (C) E₂-inhibited growth of 5C cells was completely reversed by z-LEVD-fmk. Proliferation was determined after 6 d of 10⁻⁹ M E₂ exposure and measured by DNA mass per well. CASP4 Inh, CASP4 inhibitor z-LEVD-fmk. (D) Morphologic alterations after 96 h of 10⁻⁹ M E₂ in 5C cells were completely reversed by z-LEVD-fmk (LEVD). (Scale bar: 100 microns.) (A–D) E₂ was used at 10⁻⁹ M and z-LEVD-fmk at 2 × 10⁻⁵ M. Data in A and C represent the average and SDs of four and eight replicates, respectively.