Estrogen-related receptor alpha (ERRα) promotes gastrointestinal stromal tumor progression and epithelial-mesenchymal transition through Wnt/β-catenin

Technical Summary: Estrogen-related receptor alpha (ERRα) promotes gastrointestinal stromal tumor progression and epithelial-mesenchymal transition through Wnt/β-catenin

Problem Statement
Gastrointestinal stromal tumors (GISTs) are the most common human sarcomas, primarily driven by gain-of-function mutations in the KIT or PDGFRA receptor tyrosine kinases. While tyrosine kinase inhibitors (TKIs) like imatinib have improved outcomes, most patients eventually develop secondary resistance. This resistance often arises from secondary KIT mutations, but non-genetic, reversible adaptive mechanisms occurring early during treatment are also critical. The authors hypothesize that an orphan nuclear receptor, Estrogen-related receptor alpha (ERRα), encoded by ESRRA, may be a previously uncharacterized driver of GIST pathogenesis, promoting tumor growth and the epithelial-mesenchymal transition (EMT), a process linked to metastasis and drug tolerance.

Methodology
The study employed a multi-faceted approach combining in vivo mouse models, in vitro cell line experiments, and human tissue analysis:

• Animal Models: The researchers utilized KitΔV558/+ mice, which spontaneously develop cecal GISTs. They performed ovariectomies and treated mice with tamoxifen or fulvestrant to investigate the role of estrogen signaling. Additionally, human GIST cell lines (T1, HG129, HG130) were xenografted into NSG mice to test the efficacy of the ERRα inverse agonist XCT790.
• Cell Culture and Genetic Manipulation: Human GIST cell lines (including imatinib-sensitive T1/HG129 and resistant HG130) were treated with XCT790 or subjected to siRNA-mediated knockdown of ERRα.
• Omics and Molecular Analysis: Bulk RNA sequencing (RNA-seq) was performed on T1 cells following ERRα knockdown. Differentially expressed genes (DEGs) were analyzed using DESeq2, and pathway enrichment was conducted via Gene Set Enrichment Analysis (GSEA) using Hallmark, KEGG, and Reactome databases. Protein-protein interaction (PPI) networks were constructed using STRING and Cytoscape.
• Molecular Techniques: The study utilized Western blotting, co-immunoprecipitation (IP), quantitative real-time PCR (qPCR), and immunohistochemistry (IHC) on a tissue microarray of 66 human GIST specimens to validate protein expression and physical interactions.
• Functional Assays: Cell viability (CCK-8), colony formation, and scratch assays were used to assess proliferation, clonogenicity, and migration, respectively.

Key Contributions and Results

1. ERRα Expression in Human GIST: Unlike the murine KitΔV558/+ model where ERα drives tumor growth, human GIST cell lines and surgical specimens showed minimal ERα expression but high levels of ERRα. ERRα was confirmed to be robustly expressed across human GIST cell lines and the majority of 66 primary GIST surgical specimens.
2. ERRα Inhibition Suppresses Tumor Growth: Pharmacologic inhibition of ERRα using XCT790 or genetic knockdown via siRNA significantly reduced cell viability, colony formation, and migration in GIST cell lines. In xenograft models, XCT790 treatment markedly suppressed the growth of established T1 and HG130 tumors without significant toxicity.
3. Regulation of EMT: RNA-seq analysis revealed that ERRα knockdown significantly downregulated the "Hallmark Epithelial-Mesenchymal Transition" (EMT) pathway. This was corroborated by a decrease in the EMT signature score and reduced expression of EMT-related genes (e.g., SNAI1, VIM, CDH2). Functional scratch assays confirmed that ERRα inhibition reduced wound closure rates.
4. The ERRα–β-Catenin Axis: Mechanistic investigation identified β-catenin (CTNNB1) as the top hub gene in the PPI network of ERRα-regulated genes. ERRα knockdown reduced levels of active (non-phosphorylated) nuclear β-catenin and its downstream targets, including CCND1 (Cyclin D1), WNT11, and AXIN2. Notably, DKK4, a negative regulator of Wnt signaling, was upregulated upon knockdown.
5. Physical Interaction: Co-immunoprecipitation assays demonstrated a direct physical interaction between ERRα and nuclear β-catenin in both MCF7 (positive control) and HG129 GIST cells.
6. Synergy with TKIs: Combining ERRα inhibition (XCT790 or siRNA) with imatinib resulted in greater suppression of cell viability and phospho-KIT levels than either agent alone. This effect was observed even in the imatinib-resistant HG130 cell line.

Significance and Claims
The paper claims to identify ERRα as a novel oncogenic driver in GIST that operates independently of the canonical KIT/PDGFRA mutations. The authors posit that the ERRα–β-catenin axis represents a therapeutic vulnerability in GIST. Specifically, they suggest that targeting ERRα could:

• Inhibit tumor progression and EMT.
• Overcome or delay adaptive resistance to TKIs by suppressing non-genetic survival mechanisms.
• Provide a complementary strategy to standard TKI therapy, as ERRα inhibition retains efficacy in TKI-resistant settings.

The authors conclude that while ERRα is a druggable target, the current inverse agonist XCT790 has limitations for clinical development, but next-generation agents are under investigation. The study establishes a rationale for combining ERRα inhibitors with standard TKI therapy to suppress EMT activation and improve patient outcomes.