# Deciphering ERR family genes as prognostic and immunological biomarkers through pan-cancer analysis with validation in gallbladder cancer

## TL;DR

This study explores the role of ERR family genes in various cancers, showing their impact on tumor progression and immunity, with ESRRG identified as a key driver in gallbladder cancer.

## Contribution

The study provides a pan-cancer analysis of ERR genes and validates ESRRG's role in gallbladder cancer through experimental and immunological investigations.

## Key findings

- ERR family genes show distinct expression patterns and prognostic value across multiple cancers.
- ESRRG is linked to poor prognosis, immune evasion, and cancer progression in gallbladder cancer.
- ESRRG knockdown reduces cancer cell proliferation and PD-L1 expression in gallbladder cancer.

## Abstract

The estrogen-related receptor family genes (ERRs), including ESRRA, ESRRB, and ESRRG, have been implicated in a few tumors, exhibiting distinct roles through diverse mechanisms. The purpose of our research is to explore the commonalities and underlying mechanism of ERRs in malignancies from a pan-cancer perspective and to validate the role and mechanisms of ESRRG in gallbladder cancer (GBC).

We leveraged public databases such as TCGA and GTEx to systematically investigate the potential functions of ERRs in malignancies. ESRRG expression was analyzed through immunohistochemical staining in gallbladder cancer and cholecystitis tissues. For functional validation, ESRRG was knocked down in GBC cell lines, followed by CCK-8, colony formation, scratch wound healing, Transwell migration, and invasion assays. Western blot, qPCR, and immunofluorescence were performed to evaluate the relationship between ESRRG, PD-L1, and CD8+ T cells.

Compared to adjacent normal tissues, ESRRA is overexpressed in most tumors, ESRRB is generally underexpressed, and ESRRG exhibits significant expression alterations across various tumors. All three ERRs demonstrate significant prognostic value across different cancers. Notably, the strong associations of ERRs with key immunological features—stromal scores, immune cell infiltration, microsatellite instability (MSI), and tumor mutational burden (TMB)—suggest their involvement in immune evasion and their potential utility in guiding immunotherapy strategies. All three ERRs display a positive correlation with advanced tumor stages in cholangiocarcinoma (CHOL). Specifically, in CHOL, ESRRG expression is closely associated with lymphatic metastasis, poorer overall survival, reduced immune infiltration, elevated PD-L1 expression, epithelial-mesenchymal transition (EMT), and DNA damage response. In GBC tissues, we subsequently confirmed that ESRRG expression positively correlates with pathological staging and PD-L1 expression, while negatively correlating with prognosis and CD8+ T cell infiltration. Knockdown of ESRRG in gallbladder cancer cells results in decreased proliferation, migration, and invasion. Moreover, the expression of PD-L1, MSH2, BRCA1, MMP2, and VIMENTIN decreased with ESRRG knockdown.

Our pan-cancer analysis reveals ERRs as critical regulators of tumor immunity and progression, with ESRRG emerging as a key oncogenic driver in GBC. The mechanistic link between ESRRG and PD-L1/EMT suggests its potential as a therapeutic target to enhance immunotherapy efficacy. These findings underscore the need for tissue-specific targeting strategies for ERR family members in precision oncology.