RELB Overexpression Induced by DNA Hypomethylation Contributes to Perioperative Immunotherapy Resistance in Resectable Esophageal Squamous Cell Carcinoma by Modulating the mregDC-Treg Axis.

BACKGROUND

Perioperative immunotherapy has significantly improved outcomes for patients with resectable esophageal squamous cell carcinoma (ESCC). Nevertheless, a substantial proportion of patients develop resistance to immunotherapy. This study aimed to identify biomarkers predictive of perioperative immunotherapy efficacy and elucidate the mechanisms underlying treatment resistance in non-responders with resectable ESCC.

METHODS

RELB expression in ESCC clinical samples and prognosis was first assessed using tissue microarrays. Bisulfite sequencing and methylation-specific PCR were employed to explore the mechanisms underlying RELB overexpression in ESCC. Subsequently, functional assays and western blot analysis were performed to evaluate the biological role of RELB in ESCC cells. Fifty-nine tissue samples were collected from patients who received perioperative immunotherapy for ESCC at our center and categorized into progressive disease (PD) and stable disease (SD) groups. Triple-label immunofluorescence staining, single-cell RNA sequencing, and bulk-RNA sequencing analyses were conducted to investigate the role of RELB in perioperative immunotherapy resistance.

RESULTS

A correlation was revealed between perioperative immunotherapy resistance in ESCC and RELB expression. RELB overexpression in ESCC tissues, which is regulated by DNA hypomethylation, exhibited a significant positive correlation with poor prognosis and advanced tumor stage. RELB knockdown inhibited the migration and proliferation of Eca109 and KYSE450 cells while promoting their apoptosis. The elevated RELB expression in patients with ESCC increased perioperative immunotherapy resistance by driving the mature dendritic cells enriched in immunoregulatory molecules (mregDCs)-regulatory T cells (Tregs) axis for Treg recruitment and activation in the tumor microenvironment. Importantly, RELB was mainly enriched in and strongly correlated with the infiltration of mregDCs and Tregs. RELB+ mregDCs exhibited stronger interaction with Tregs and upregulation of IDO1 as well as activation of tryptophan metabolism.

CONCLUSIONS

DNA hypomethylation-induced RELB overexpression promoted perioperative immunotherapy resistance in ESCC by driving the mregDC-Treg axis and is a potential predictive biomarker for perioperative immunotherapy response.