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

Abstract

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2009.Contributed by V. Craig Jordan, September 14, 2011 (sent for review June 21, 2011) 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.aromatase inhibitor | antihormonal resistance | estrogen receptor | gene expression microarrays | selective estrogen receptor modulator E lucidation of the basic structure function relationships of synthetic estrogens based on either stilbene (1) or triphenylethylene (2) was a landmark achievement that continues to have major therapeutic implications to this day. The first successful chemical therapy for the treatment of any cancer was the use of high-dose synthetic estrogen for the treatment of metastatic breast cancer (3). Response rates for patients who were more than a decade beyond menopause were about 30%. Importantly, treatment near menopause was ineffective, and therefore, tumor responsiveness was related to the duration of estrogen deprivation. In 1970, Alexander Haddow commented that "the extraordinary extent of tumor regression observed in...

Breast Cancer Cell Apoptosis with Phytoestrogens Is Dependent on an Estrogen-Deprived State

“…The expected growth/apoptotic responses to E2, biomarker statuses of ERa, progesterone receptor (PgR), and HER2, and ER-regulated transcriptional activity were confirmed in both cell lines. Protein levels of ERa, PgR, and HER2 were characterized by semiquantitative immunoblot analysis and ER transcriptional activity was evaluated using an estrogen-responsive elementregulated dual luciferase reporter gene system (36). The last characterization was reported in (37) and the DNA fingerprinting patterns of the cell lines were consistent with the report by the American Type Culture Collection.…”

“…MCF7:WS8 cells were derived from MCF7 cells as previously described (35) and maintained in RPMI media supplemented with 10% FCS, 6 ng/mL bovine insulin and penicillin and streptomycin. The MCF7:WS8 and the MCF7:5C cells have been fully characterized and experiments were done as previously reported (36). The expected growth/apoptotic responses to E2, biomarker statuses of ERa, progesterone receptor (PgR), and HER2, and ER-regulated transcriptional activity were confirmed in both cell lines.…”

“…Microarray analysis indicates that endoplasmic reticulum stress (ERS) and inflammatory response genes are top scoring pathways associated with E 2 -induced apoptosis (36). To investigate whether phytoestrogens induce ERS genes, we used RT-PCR to quantitate mRNA levels.…”

Proven value of translational research with appropriate animal models to advance breast cancer treatment and save lives: the tamoxifen tale

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The function of membrane-associated molecules in acquired resistance to antiestrogens in breast cancer

“…Long-term therapy generates selection pressure for cell populations that evolve from acquired SERM resistance, ubiquitously observed in metastatic breast cancer, to eventually expose a vulnerability that is expressed as E2-induced apoptosis [3][4][5] . Laboratory observations further show that three phases of acquired SERM-resistance exist (Figure 1), which depend on the length of SERMs exposure [4,6,7] .…”