Clitocine suppresses TNBC progression by boosting CCRL2 to block survival signals and neutrophil-driven inflammation

This study investigates the anti-tumor mechanisms of clitocine, a natural compound, in triple-negative breast cancer (TNBC), with a focus on its role in modulating the immune microenvironment via C-C motif chemokine receptor like 2 (CCRL2). The anti-cancer effects of clitocine were assessed in vitro using TNBC cell lines (MDA-MB-231 and MDA-MB-468) through functional assays, flow cytometry, and immunoblotting. RNA sequencing was performed to identified clitocine-induced transcriptional changes. The subcutaneous 4T1 xenograft models in BALB/c mice were used for in vivo validation. Additionally, mass cytometry (CyTOF) profiled tumor immune infiltration in tumor tissues, and the role of CCRL2 in modulating the tumor immune microenvironment and inflammatory pathways was investigated through database mining (TCGA) and gain/loss-of-function experiments.

Clitocine potently inhibits TNBC cell proliferation, metastasis, and epithelial-mesenchymal transition (EMT), while inducing mitochondrial apoptosis. It alters cancer-related gene expression (upregulating CCRL2), suppresses PI3K-AKT, NFκB, and ERK pathways, and exhibits potent antitumor activity in vivo without systemic toxicity. Mechanistically, clitocine remodels the tumor immune microenvironment by reducing neutrophil infiltration and suppressing neutrophil extracellular traps (NETs) formation, effects mediated through the CCRL2/chemerin axis. Furthermore, CCRL2 is identified as a tumor suppressor, with low expression in TNBC patients correlating with poor survival. Credibly, CCRL2 knockdown in vitro and in vivo significantly abrogates the anti-tumor and immunomodulatory effects of clitocine.

Our findings demonstrate that clitocine exerts potent anti-TNBC effects by upregulating CCRL2 expression, thereby activating the clitocine/CCRL2/chemerin axis. This axis disrupts critical oncogenic signaling pathways and reprograms the immunosuppressive tumor microenvironment.