TRAIL-induced cytokine production via NFKB2 pathway promotes neutrophil chemotaxis and immune suppression in triple negative breast cancers [neutrophils]

We hypothesized that TRAIL treatment elicits transcriptional changes in triple negative breast cancer cells (TNBC) cells that alter the immune milieu. To test this, we performed a RNAseq analysis of MDA-MB-231 cells treated with TRAIL, followed by validation in various TNBC cell lines. We found that TRAIL significantly induces expression of multiple cytokines, such as CXCLs 1, 2, 3, 8,11 and IL-6, which are known to affect neutrophil function. Gene set enrichment analysis (GSEA) indicated a significant enrichment of NFKB pathway. Concordantly, we confirmed that the non-canonical NFKB2 pathway is primarily responsible for induction of these cytokines. We further found that induction of these cytokines was predominantly mediated by death receptor 5 and caspase-8 protein, but not caspase-8 enzymatic activity. CXCLs and IL-6 produced by the TRAIL-treated TNBC cells enhanced chemotaxis of neutrophils isolated from healthy human donors, demonstrating the functional relevance of these cytokines. In vivo, TNBC xenograft tumors grown in mice treated with TRAIL showed an activation of the NFKB2 pathways, elevated production of CXCLs and IL-6, and an increased neutrophil recruitment into the tumors. Moreover, pre-incubation of neutrophils from human donor with supernatants from TRAIL-treated TNBC cells significantly impaired the cytotoxic effect against TNBC cells and showed a reduced respiratory burst. Transcriptomic analysis of neutrophils incubated with either TRAIL alone or supernatant of TRAIL-treated TNBC revealed significant enrichment of expression of inflammatory cytokines, immune modulatory genes, and immune checkpoint genes along with genes having implication on delayed neutrophil apoptosis. Functional studies with these neutrophils confirmed their suppressive effect on T cell proliferation and an increase in Treg suppressive phenotype. Collectively, our study proposes the novel role of TRAIL-induced NFKB2-dependent cytokine production that promotes neutrophil chemotaxis and immune suppression. Our finding suggests that targeting innate immune system may be a promising strategy to improve the efficacy of TRAIL treatment in TNBC.