Cooperative interaction between ERα and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis

Epithelial to Mesenchymal Transition

EMT is characterized by loss of cellular adhesions and apical polarity associated with epithelial cells. These changes are often due to decreased expression of E-cadherin, an epithelial cell marker, and increased expression of N-cadherin, vimentin, and cellular proteases, which aid in cell motility and trans-endothelial migration. Zeb, Snail, Slug, and Twist are master EMT-inducing transcriptional factors.^1,2

ERα is a marker of breast cancer prognosis

Estrogen receptor alpha (ERα) is a member of a superfamily of transcription factors that bind to small lipid soluble molecules like thyroid and steroid hormones. It can dimerize and bind to estrogen response elements (EREs) in DNA, or ERα can interact with additional proteins to create complexes that recruit transcription machinery to additional response elements.³ Estradiol (E2), which activates and signals through ERα, is required for normal growth and differentiation of breast epithelial cells. ERα is a positive predictor of breast cancer prognosis because it antagonizes EMT signaling pathways and is indicative of response to hormonal therapies.^2,4

While the presence of ERα indicates the likelihood of patients to respond to antiestrogen therapies, not all ERα⁺ breast cancers respond to these therapies and many that initially respond eventually develop resistance. EMT is thought to play a critical role in the development of endocrine resistance. In breast cancer patients high and low ERα-expressing cells coexist. Cells with low ERα expression resemble mesenchymal cells, while cells with high ERα expression maybe epithelial cells or represent a hybrid state. The hybrid state is associated with poor patient prognosis. Even though decreased ERα function correlates with EMT in breast cancers, ERα activation can induce EMT in other types of cancer. In this study, the authors investigated the role of EMT-inducer Zeb1 on ERα activity and ability to modulate EMT in breast cancer to determine whether the two factors can work in concert to promote the progression of more invasive forms of breast cancer.⁵

Short-term ZEB1 expression created a hybrid EMT state

Breast cancer cell lines MCF7, MCF7-V (a variant with enhanced ERα responses), and T-47D, were engineered to express ZEB1 via a doxycycline-inducible lentiviral system. In all three of the cell lines, long-term expression of ZEB1 (8-12 weeks) resulted in decreased ERα expression, as previously reported, and complete EMT. Short-term ZEB1 expression (1-2 weeks), however, maintained ERα expression levels and resulted in partial EMT. ERE luciferase reporter constructs were used to show that these maintained ERα levels correlated with ERα activity. Consistent with the luciferase reporter assays, short-term ZEB1 expression increased mRNA levels of ERα target genes, while long-term expression of ZEB1 resulting in mesenchymal-like phenotype caused reduced ERα target gene mRNA levels.

Using tumor spheroids, researchers found that in the presence of E2, ZEB1 expression increased the size of the spheroid and dissemination into the surrounding matrix. However, FI cocktail (forskolin, to stimulate adenylate cyclase and 3-isobutyl-1-methylxanthine, to block phosphodiesterase) or the antiestrogen ICI, suppressed invasion of the cells into the matrix. 3D invasion assays with T47-D cells showed similar results, indicating that in response to E2 ZEB1 enhances ERα-mediated cell invasion.

The Gene expression-based Outcome for Breast cancer Online (GOBO) tool showed improved overall survival in ERα+ patients with high ZEB1 levels. In ERα- patients, ZEB1 levels did not impact overall survival, but high ZEB1 levels did negatively affect distant metastasis-free survival. Further examination using the GOBO tool suggested that patients with ERα activation due to high levels of ZEB1 were more sensitive to the active tamoxifen metabolite 4-hydroxytamoxifen (4-OHT) during early EMT. This observation was supported by cell cycle assays where 4-OHT treated cells with high levels of ZEB1 were more frequently found arrested in G0/G1 phase than control cells (75% compared to 60%).

ZEB1 directly interacts with ERα

The researchers then wanted to understand how ZEB1 modulates ERα responses. CHIP-seq experiments were performed in MCF7-V-ZEB1 cells after one week of ZEB1 expression. These experiments identified 37,922 ZEB1 binding sites that had significant overlap with ERα binding sites (ERBSs) induced by E2 and the FI cocktail. The Genomic Regions Enrichment of Annotations Tool (GREAT) uncovered functions related to EMT, migration, and WNT signaling for ZEB1-induced ERBSs.

Co-immunoprecipitation experiments confirmed that ZEB1 and ERα are present in the same protein complexes and that binding sites of the AP2 family are highly enriched at the intersections of ZEB1 and ERα binding. Comparing published CHIP-seq data from AP2γ, which regulates transcription at ERBSs, with the ZEB1 and ERα CHIP-seq data identified 6,019 shared sites. To further support AP2γ, ZEB1, and ERα being a part of the same transcription factor complexes, co-IPs found that AP2γ formed a complex with ZEB1 and knockdowns of AP2γ disrupted the ability of ZEB1 and ERα to co-precipitate and reduced ERα chromosomal binding.

ANXA2, KRT8, HSPB1, and TIMP1 were identified as markers for the hybrid EMT state

To further investigate the EMT transitional state, time course experiments of ZEB1 expression were performed in MCF-V cells. Using EpCAM as a marker for EMT, distinctions were made between epithelial and mesenchymal phenotypes across the time course. After ten weeks of ZEB1 expression, a mesenchymal phenotype was marked by EpCAM^low expression and loss of ERα. However, at only five weeks of ZEB1 expression both EpCAM^high and EpCAM^low cell populations were identified, and this population was used to further characterize the EMT hybrid state. Single cell RNA-seq confirmed the presence of additional epithelial cell markers in the EpCAM^high population and genes known to facilitate invasion were present in EpCAM^low cells. ANXA2, KRT8, HSPB1, and TIMP1 were found in both EpCAM^high and EpCAM^low populations and thus might be important in establishing the hybrid EMT state. Depletion of ANXA2, HSPB1, or TIMP1 in the presence of ZEB1 and ERα activation reduced migration in wound-healing assays and cell invasion towards bone, but had no effect on cell invasion toward lung tissue, indicating a role for these genes in tissue tropism of ERα+ breast cancer cells.

CD151 is a novel hybrid EMT marker with therapeutic potential

The single cell RNA-seq data of the EpCAM^high/low cells was unable to detect any previously identified cell surface markers correlating with different EMT stages in primary mammary tumors (CD61^–, CD51^–, and CD106^–). CD151, an integrin involved in metastasis to bone, was significantly enriched in the EpCAM^low/CD61-/CD51-/CD106- cell population and expression correlated with ZEB1 levels. Reduction of CD151 in the presence of ZEB1 and ERα activation decreased cell proliferation, migration, and invasion towards bone. This suggests that CD151 might be a therapeutic target to reduce cell division and migration in ZEB1+/ERα+ breast cancer cells.

While it has been previously shown that EMT results in resistance to antiestrogen therapies, the impact of early or hybrid EMT states on ERα signaling had not been investigated. This study uncovered the ability of ZEB1 to interact with ERα and alter EMT state, cell proliferation, and breast cancer metastasis and provided new insights into possible therapeutic targets to help reduce cancer severity.​⁵​

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References

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5. Mohammadi Ghahhari N, Sznurkowska MK, Hulo N, Bernasconi L, Aceto N, Picard D. Cooperative interaction between ERα and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis. Nat Commun. 2022;13(1):2104. doi:10.1038/s41467-022-29723-5