Interferon-gamma is quintessential for NOS2 and COX2 expression in ER- breast tumors that lead to poor outcome

Abstract

A strong correlation between NOS2 and COX2 tumor expression and poor clinical outcomes in ER breast cancer has been established. However, the mechanisms of tumor induction of these enzymes are unclear. Analysis of The Cancer Genome Atlas (TCGA) revealed correlations between NOS2 and COX2 expression and Th1 cytokines. Herein, single-cell RNAseq analysis of TNBC cells shows potent NOS2 and COX2 induction by IFNγ combined with IL1β or TNFα. Given that IFNγ is secreted by cytolytic lymphocytes, which improve clinical outcomes, this role of IFNγ presents a dichotomy. To explore this conundrum, tumor NOS2, COX2, and CD8+ T cells were spatially analyzed in aggressive ER–, TNBC, and HER2 + breast tumors. High expression and clustering of NOS2-expressing tumor cells occurred at the tumor/stroma interface in the presence of stroma-restricted CD8+ T cells. High expression and clustering of COX2-expressing tumor cells extended into immune desert regions in the tumor core where CD8+ T cell penetration was limited or absent. Moreover, high NOS2-expressing tumor cells were proximal to areas with increased satellitosis, suggestive of cell clusters with a higher metastatic potential. Further in vitro experiments revealed that IFNγ + IL1β/TNFα increased the elongation and migration of treated tumor cells. This spatial analysis of the tumor microenvironment provides important insight into distinct neighborhoods where stroma-restricted CD8+ T cells exist proximal to NOS2-expressing tumor niches that could have increased metastatic potential.

NOS2 and COX2 Provide Key Spatial Targets that Determine Outcome in ER-Breast Cancer

“…Importantly, the tumor core is nearly devoid of NOS2 (Fig. 4E) as well as CD8 + T cells, supporting the idea that NOS2 resides at the tumor/stroma interface as previously described (13). NOS2 was significantly higher in the tumor satellite regions when compared to the tumor core (Fig.…”

“…Fig. 1A-C) (13). When stratifying for survival, the NOS2/COX2 signal intensities in tumors from Deceased vs Alive patients demonstrated a higher fold-change (∼6x) in the strong signal intensities of NOS2 (NOS2s) compared to moderate or weak (∼2-3x) signal intensities (Supplemental Fig.…”

“…Earlier studies identified NOS2/COX2 feedforward signaling that supports many oncogenic pathways in ER-breast cancer (2, 3). Moreover, a strong linear relationship between tumor NOS2/COX2 expression in all patient tumors has been reported (13). Given that the flux of NOS2-derived NO depends on the local density of NO producing cells as well as the concentration, diffusion, and reaction kinetics (16), we used density heat map analyses (Fig.…”

“…Tumor NOS2 density was evaluated at the tumor edge (Fig. 4E) where tumor satellite regions indicative of invasive tumor cells (13) contained significantly higher NOS2 expression while NOS2 was lowest in lymphoid aggregates and the tumor core (Fig. 4E).…”

“…4H). The immune desert is COX2 + but NOS2 - however, NOS2 + and COX2 + tumor satellites form in the inflamed areas near stroma-restricted CD8 + T cells that produce IFNγ (13). Importantly, these NOS2 + tumor satellites exhibit increased elongation and migration consistent with increased tumor metastatic potential (13).…”