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Antiestrogen Resistant Cell Lines Expressing Estrogen Receptor ? Mutations Upregulate the Unfolded Protein Response and are Killed by BHPI.

ABSTRACT: Outgrowth of metastases expressing ER? mutations Y537S and D538G is common after endocrine therapy for estrogen receptor ? (ER?) positive breast cancer. The effect of replacing wild type ER? in breast cancer cells with these mutations was unclear. We used the CRISPR-Cas9 genome editing system and homology directed repair to isolate and characterize 14 T47D cell lines in which ER?Y537S or ER?D538G replace one or both wild-type ER? genes. In 2-dimensional, and in quantitative anchorage-independent 3-dimensional cell culture, ER?Y537S and ER?D538G cells exhibited estrogen-independent growth. A progestin further increased their already substantial proliferation in micromolar 4-hydroxytamoxifen and fulvestrant/ICI 182,780 (ICI). Our recently described ER? biomodulator, BHPI, which hyperactivates the unfolded protein response (UPR), completely blocked proliferation. In ER?Y537S and ER?D538G cells, estrogen-ER? target genes were constitutively active and partially antiestrogen resistant. The UPR marker sp-XBP1 was constitutively activated in ER?Y537S cells and further induced by progesterone in both cell lines. UPR-regulated genes associated with tamoxifen resistance, including the oncogenic chaperone BiP/GRP78, were upregulated. ICI displayed a greater than 2 fold reduction in its ability to induce ER?Y537S and ER?D538G degradation. Progestins, UPR activation and perhaps reduced ICI-stimulated ER? degradation likely contribute to antiestrogen resistance seen in ER?Y537S and ER?D538G cells.

SUBMITTER: Mao C

PROVIDER: S-EPMC5054422 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Antiestrogen Resistant Cell Lines Expressing Estrogen Receptor α Mutations Upregulate the Unfolded Protein Response and are Killed by BHPI.

Mao Chengjian C Livezey Mara M Kim Ji Eun JE Shapiro David J DJ

Scientific reports 20161007

Outgrowth of metastases expressing ERα mutations Y537S and D538G is common after endocrine therapy for estrogen receptor α (ERα) positive breast cancer. The effect of replacing wild type ERα in breast cancer cells with these mutations was unclear. We used the CRISPR-Cas9 genome editing system and homology directed repair to isolate and characterize 14 T47D cell lines in which ERαY537S or ERαD538G replace one or both wild-type ERα genes. In 2-dimensional, and in quantitative anchorage-independent ...[more]

PMID: 27713477

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Project description:Breast cancer resistance to the antiestrogens tamoxifen (OHT) and fulvestrant is accompanied by alterations in both estrogen-dependent and estrogen-independent signaling pathways. Consequently, effective inhibition of both pathways may be necessary to block proliferation of antiestrogen-resistant breast cancer cells. In this study, we examined the effects of apigenin, a dietary plant flavonoid with potential anticancer properties, on estrogen-responsive, antiestrogen-sensitive MCF7 breast cancer cells and two MCF7 sublines with acquired resistance to either OHT or fulvestrant. We found that apigenin can function as both an estrogen and an antiestrogen in a dose-dependent manner. At low concentrations (1 mumol/L), apigenin stimulated MCF7 cell growth but had no effect on the antiestrogen-resistant MCF7 sublines. In contrast, at high concentrations (>10 mumol/L), the drug inhibited growth of MCF7 cells and the antiestrogen-resistant sublines, and the combination of apigenin with either OHT or fulvestrant showed synergistic, growth-inhibitory effects on both antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. To further elucidate the molecular mechanism of apigenin as either an estrogen or an antiestrogen, effects of the drug on estrogen receptor-alpha (ERalpha); transactivation activity, mobility, stability, and ERalpha-coactivator interactions were investigated. Low-dose apigenin enhanced receptor transcriptional activity by promoting interaction between ERalpha and its coactivator amplified in breast cancer-1. However, higher doses (>10 mumol/L) of apigenin inhibited ERalpha mobility (as determined by fluorescence recovery after photobleaching assays), down-regulated ERalpha and amplified in breast cancer-1 expression levels, and inhibited multiple protein kinases, including p38, protein kinase A, mitogen-activated protein kinase, and AKT. Collectively, these results show that apigenin can function as both an antiestrogen and a protein kinase inhibitor with activity against breast cancer cells with acquired resistance to OHT or fulvestrant. We conclude that apigenin, through its ability to target both ERalpha-dependent and ERalpha-independent pathways, holds promise as a new therapeutic agent against antiestrogen-resistant breast cancer.

Project description:The most frequently diagnosed cancers in women are the estrogen receptor (ER)-positive breast cancer subtypes, which are characterized by estrogen dependency for their growth. The mainstay of clinical treatment for this tumor relies on the modulation of ERα action or on the suppression of estrogen biosynthesis via the administration of Selective ERα Modulators/Down-regulators (SERMs/SERDs) or aromatase inhibitors, respectively. Nevertheless, de novo and acquired resistance to these therapies frequently occurs and represents a major clinical concern for patient survival. Recently, somatic mutations affecting the hormone-binding domain of ERα (i.e., Y537S, Y537N, D538G) have been associated with endocrine resistance, disease relapse and increased mortality rates. Hence, devising novel therapies against these ERα isoforms represents a daunting challenge. Here, we identified five molecules active on recurrent Y537S ERα polymorphism by employing in silico virtual screening on commercial databases of molecules, complemented by ER-transactivation and MTT assays in MCF7 and MDA-MB-231 breast cancer cells expressing wild type or mutated ERα. Among them, one molecule selectively targets Y537S ERα without inducing any cytotoxicity in breast cell lines. Multi-microseconds (4.5 μs) of biased and unbiased molecular dynamics provided an atomic-level picture of the structural, thermodynamics (i.e., binding free energies) and the kinetic (i.e., dissociation free energy barriers) of these active ligands as compared to clinically used SERM/SERDs upon binding to wild type and distinct ERα variants (Y537S, Y537N, D538G). This study contributes to a dissection of the key molecular traits needed by drug-candidates to hamper the agonist (active)-like conformation of ERα, normally selected by those polymorphic variants. This information can be useful to discover mutant specific drug-candidates, enabling to move a step forward toward tailored approaches for breast cancer treatment.

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Antiestrogen Resistant Cell Lines Expressing Estrogen Receptor ? Mutations Upregulate the Unfolded Protein Response and are Killed by BHPI.

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Antiestrogen Resistant Cell Lines Expressing Estrogen Receptor α Mutations Upregulate the Unfolded Protein Response and are Killed by BHPI.

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