Identification of a distinct AREG+ cell population that promotes the progression, recurrence, and metastasis of endometrioid endometrial cancer at single-cell resolution

Abstract Endometrial cancer (EC) is the second most common malignant tumor of the female reproductive system worldwide. Endometrioid endometrial cancer (EEC) constitutes the most prevalent subtype, representing approximately 75% of all EC cases. Most patients diagnosed with EEC present at an early stage and generally exhibit a favorable prognosis. However, a small subset of low-grade, early-stage, well-differentiated EECs may exhibit metastatic behavior and recurrence. Early alterations in the microenvironment of the peritumoral region, particularly concerning the proliferation, migration, and invasion of tumor cells within this area, are critical factors influencing tumor progression, metastasis, and recurrence. In this study, single-cell RNA sequencing (scRNA-seq) technology was used to sequence five EEC tumors and four peritumoral tissues from patients diagnosed with early-stage EEC. For the first time, a population of cells exhibiting high expression of AREG was identified in the peritumoral region of EECs. Bioinformatics analyses revealed that this population of cells demonstrated significant capabilities for proliferation, migration, and invasion. The immunohistochemistry (IHC) analysis of the tissue microarray (TMA) revealed elevated AREG expression in EECs, which correlated with various clinical parameters, including FIGO stage, degree of differentiation, pelvic lymph node metastasis, muscular layer infiltration, Ki67 expression, and PAX-2 expression. Both in vitro and in vivo experiments confirmed that AREG promotes malignant biological behaviors in EECs, including proliferation, migration, invasion, and regulation of apoptosis and the cell cycle. Additionally, AREG was found to activate the EGFR-Smad2/3 signaling pathway through phosphorylation, inducing epithelial-mesenchymal transition (EMT) and thus promoting the migration and invasion abilities of EECs. This study identified a high AREG-expression cell population in EECs, providing valuable insights into the pathological mechanisms of EEC progression, recurrence, and metastasis, and proposing AREG as a potential biomarker for the prognostic prediction and targeted therapeutic strategies in EECs.