Targeting CLIC6 with theaflavin enhances radiotherapy sensitivity in ER+/HER2- breast cancer.

BACKGROUNDRadiotherapy is a cornerstone of breast cancer treatment; however, estrogen receptor-positive (ER+)/human epidermal growth factor receptor 2-negative (HER2-) tumors frequently exhibit intrinsic radioresistance. Understanding the molecular mechanisms underlying this resistance is crucial for improving therapeutic outcomes. This study aims to identify key molecular determinants of intrinsic radioresistance in ER+/HER2- breast cancer and to explore potential radiosensitization strategies targeting these determinants.

METHODSWe integrated single-cell RNA sequencing (scRNA-seq) data from ER+/HER2- breast tumors before and after radiotherapy with transcriptomic profiles from The Cancer Genome Atlas (TCGA). A high-dimensional weighted gene co-expression network analysis (hdWGCNA) was employed to identify gene modules associated with clinical traits. Functional assays were conducted in ER+ breast cancer cells with chloride intracellular channel 6 (CLIC6) overexpression or knockdown. A molecular docking analysis revealed that theaflavin specifically interacts with CLIC6. Further, clonogenic assays and Cell Counting Kit-8 (CCK-8) proliferation assays were performed to validate the biological effects of theaflavin and CLIC6 following radiotherapy.

RESULTSThe hdWGCNA identified a luminal A-like gene module correlated with progesterone receptor (PR) positivity and low Ki67 expression, within which CLIC6 emerged as a key hub gene. Elevated CLIC6 expression enhanced radioresistance, whereas CLIC6 knockdown increased radiosensitivity. Molecular docking confirmed that theaflavin binds to CLIC6 with high affinity (binding energy -8.8 kcal/mol). Theaflavin treatment significantly enhanced radiosensitivity in wild-type and CLIC6-overexpressing cells but had no effect in CLIC6-knockdown or ER-negative (ER-) cells.

CONCLUSIONSThis study identified CLIC6 as a novel gene associated with radioresistance in ER+/HER2- breast cancer and showed that theaflavin functions as a natural radiosensitizer in a CLIC6-dependent manner, consistent with its high-affinity interaction with CLIC6. Targeting CLIC6 may provide a promising strategy to enhance radiotherapy efficacy in this common breast cancer subtype.