Abstract 2539: Tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces cytokine release via the alternative NFKB2 pathway in triple negative breast cancer cells (TNBC) and modulates neutrophil chemotaxis
Authors: Kundu Manjari , Greer Yoshimi Endo , Ridnour Lisa A. , Wink David A. , Lipkowitz Stan 
Published: April 2023

Abstract
TRAIL induces apoptosis in many preclinical cancer models including breast cancers and has been extensively studied as a potential cancer therapeutic. However, its efficacy in clinical trials is limited, suggesting that there are unknown modulatory mechanisms responsible for its lack of TRAIL activity in vivo. We hypothesized that TRAIL treatment elicits transcriptional changes in TNBC cells that alter the immune milieu, modulating the therapeutic efficacy. To investigate the hypothesis, we performed RNAseq analysis of MDA-MB231 cells treated with TRAIL for different time points, followed by validation with RT-PCR in various TNBC cells. TRAIL treatment of the TNBC significantly induced expression of a number of cytokines, such as CXCL1, CXCL2, CXCL3, CXCL8, CXCL11, IL6, which are known to affect neutrophil function. Mechanistically, induction of these cytokines was predominantly mediated by Death Receptor 5 and Caspase-8 protein, but not Caspase-8 enzymatic activity. Gene Set Enrichment Analysis of the RNAseq indicated that NFKB pathway-mediated TNF-alpha signaling was significantly enriched. Concordantly, we confirmed that both canonical NFKB1 and non-canonical NFKB2 pathways were activated by TRAIL. However, siRNA knockdown experiments indicated that the induction of the cytokine mRNAs was primarily dependent on the NFKB2 pathway. Neutrophils isolated from healthy human donors incubated with supernatants from TNBC cells in vitro indicated that TRAIL-induced CXCLs’ (1, 2, 3, 8) and IL6 significantly increased neutrophil chemotaxis in a NFKB2-dependent manner. Moreover, neutrophils pre-incubated with supernatants from TRAIL-treated TNBC significantly inhibited its cytotoxic effect in TNBCs in a NFKB2-dependent pathway. Further RNAseq and RT-PCR of neutrophils incubated with by either TRAIL or supernatant of MDA-MB231 cells treated with TRAIL revealed significant enrichment of inflammatory pathway-related genes as well as increased expression of immune modulating cytokines. These results suggested that TRAIL exerts a pro-inflammatory role towards immune cells in tumor microenvironment. CODEX (CO-Detection by indEXing) analysis of in vivo TNBC xenografts from mice treated with the TRAIL agonist MEDI3039 confirmed that TRAIL treatment increases the number of neutrophils in the tumor. Using TNBC organoids and humanized mice models, the changes in tumor immune environment caused by TRAIL are currently under investigation. Collectively, our study suggests the novel role of TRAIL-induced NFKB2-dependent cytokine production that affects neutrophil functions, leading to modulation of the immune response in TNBC.
Citation Format: Manjari Kundu, Yoshimi Endo Greer, Lisa A. Ridnour, David A. Wink, Stan Lipkowitz. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces cytokine release via the alternative NFKB2 pathway in triple negative breast cancer cells (TNBC) and modulates neutrophil chemotaxis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2539.