Abstract 3804: Interferon gamma induces NOS2 and COX2 expression in estrogen receptor negative breast cancer leading to immunosuppressive tumor microenvironment

Abstract

Breast cancer is the second leading cause of cancer death among U.S. women although the mortality is on a steadily decreasing trend since early 90s. We previously demonstrated a strong correlation between elevated NOS2 and COX2 expressions suggesting these genes can serve as markers of poor prognosis in estrogen receptor negative (ER-) breast cancer. In other words, NOS2 and COX2 is generally associated with the immune response. Induction of NOS2 and COX2 has also been shown to orchestrate multiple pro-oncogenic pathways that may contribute to the immunosuppressive nature of cancer cells. To better understand the interactions between cytokines and breast cancer cells and develop effective therapeutics for breast cancer, we examined the effects of selected cytokines on MDA-MB-231, a human (ER-) breast adenocarcinoma cell line. We found that in combination with cytokines, such as IFNG plus TNF, IL1B, or IL17, significantly induced NOS2 and COX2. IFNG plus TNF or IL1B most strongly induced NOS2 and COX2 where IFNG or IL17 to a lesser extent. It indicates that expressions of NOS2 and COX2 required IFNG but at a low dose in presence of other cytokines. Single-cell RNAseq analysis shows that cells treated with IFNG plus TNF or IL1B clustered with those expressed NOS2 and COX2 as revealed in tSNE plots. Expression of other factors such as IL8 and IL6 (suggested markers for prognosis of breast cancer) or PDL1 and IDO1 (suggested cancer therapeutic targets) also co-clustered with NOS2 and COX2 expressions. In addition, we found increased IL1A and IL1B expressions provide fortification to NOS2 and COX2 expressions. Taken together, our current findings indicate the interactions between breast cancer cells and cytokines. We believe further spatial transcriptomic and proteomic studies may be beneficial for clarifying the complex networks of the immunosuppressive tumor microenvironment. Citation Format: Yuk Sing Robert Cheng, Lisa Ridnour, Veena Somasundaram, Dana Bhattacharyya, Christopher McGinity, Daniel McVicar, Stephen Anderson, Stephen Lockett, David Wink. Interferon gamma induces NOS2 and COX2 expression in estrogen receptor negative breast cancer leading to immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3804.

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

“…These results suggest that NOS2 fluorescence signal intensity has higher predictive value than COX2, which is supported by NOS2 and COX2 HR of 6 and 2.5, respectively as reported by Glynn et al (4,14) 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.…”

“…Our earlier findings demonstrated correlations between tumor NOS2/COX2 and the spatial orientation of CD8 + T cell in breast tumors, as well as tumor budding or satellitosis, which is an index of tumor invasiveness. We extended these earlier findings by examining the spatial localization of CD8 + T cells in Deceased vs Alive patient tumors (10,13). Because NOS2/COX2 products NO/PGE2 have antagonistic effects on T effector cell function, NOS2/CD8 and COX2/CD8 ratios were quantified and found to be significantly elevated in tumors from Deceased patients, for both total and cytolytic (CD8 + PD1 -) CD8 + T cell populations (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.…”

“…In contrast, tumors with stroma-, margin-restricted, or abated CD8 + T cells (immune cold or immune desert, respectively) are immunosuppressed and predictive of poor clinical outcomes (12). As previously shown by us, elevated tumor COX2 expression may correlate with abated CD8 + T cell infiltration into breast tumor, whereas stroma restricted CD8 + T cells may correlate with both increased tumor NOS2 expression and tumor budding or satellitosis (13). Thus, we hypothesized that spatial relationships exist between tumor NOS2/COX2 expression and infiltrating CD8 + T cells that could influence clinical outcomes by promoting immunosuppression and increased disease aggressiveness.…”

“…Previous studies have demonstrated the requirement of IFN and IL1/TNF cytokines for tumor NOS2/COX2 expression where correlations between tumor NOS2 and CD8 expressions were observed at the tumor stroma interface (3,13,22). To further explore this relationship relative to survival, spatial distribution and density heat maps were compared between Deceased vs Alive patient tumors, which demonstrated that tumor NOS2/COX2 and CD8 + T cell phenotypes occupy distinct regions in Deceased patient tumors (Fig.…”