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.

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NOS2 and COX2 expression is significantly correlated with poor prognosis in ER- breast cancer. Multiple pro-oncogenic pathways involving metastasis, proliferation, angiogenesis, immunosuppression, and chemoresistance are stimulated in a synergistic manner by NOS2 and COX2, resulting in much more aggressive cancers. In this study, our single-cell RNAseq data demonstrated that certain combinations of cytokines, IFNg with TNFA or IL1B, drove NOS2 and COX2 expressions in vitro in the MDA-MB-231 cell line after 48 hours. Analysis of t-SNE plots indicates that the clustering patterns of NOS2 and COX2 are also related to cytokine expression. Moreover, according to our spatial proteomic analysis, CD8 that are associated with IFNg are more likely to have a positive response, indicating a dichotomy in the requirement for IFNg. To better understand this mystery, the spatial distribution of NOS2 and COX2 with CD8 cells in 21 ER- breast tumors was evaluated. In the presence of tumor-excluded CD8, high expression and clustering of NOS2-positive cells were seen near the tumor-stromal interface, showing that NOS2-positive cells were excluded from the tumor. Within the tumor nest, strong expression and population clustering of COX2 reached into the immune desert. In contrast, tumor-penetrant CD8 cells were few and poorly expressed. Metastasis and chemoresistance were among the leading causes of death in this cohort, and it was shown in vitro that IFNg/IL1B or TNFA increased tumor cell migration, suggesting that the interface of tumor with stromal restricted lymphoid aggregates provides the unique spatial environment to increase NOS2 expression and increase metastatic potential. The spatial study of the TME (tumor microenvironment) provides vital insight into how NOS2 and restricted CD8 lead to a higher probability of poor survival. Citation Format: Yuk Sing Robert Cheng, Lisa Ridnour, Abigail Walke, Noemi Kedei, Adelaide Wink, Elijah Edmonston, Donna Butcher, Tiffany Dorsey, William Heinz, Richard Bryant, Robert Kinders, Stanley Lipkowitz, Stephen Wong, Milind Pore, Stephen Hewitt, Daniel McVicar, Stephen Anderson, Jenny Chang, Stefan Ambs, Stephen Lockett, David Wink. Interferon-gamma induces NOS2 and COX2 in ER- breast cancer that drives increase metastatic potential [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 2863.

Triple negative breast cancer (TNBC) is notoriously known for poor prognosis due to a lack of actionable molecular target(s). Remarkably, the majority of TNBC patients suffer the cancer relapse even they receive treatment given for early stage breast cancers. Up-to-date, lots of therapeutic agents have been developed and actionable molecular targets have been proposed. Nevertheless, the survival rate for metastatic invasive breast cancer is low because of poor prognosis. In this study, we determined whether different classes of chemo-agents (or compounds) interact with different cellular and molecular pathways they might share some common pathway(s) if their therapeutic outcome is similar. We first tested the effect of aminoguanidine (AG), APOE mimetics (COG), and nitroxyl (HNO) compounds on carcinogenesis and metastasis using an orthotopic breast cancer model. In vivo imaging analysis indicated that these compounds significantly reduced the tumor loading in the xenografts (MDA-MB-231-GFP). Moreover, compounds AG and COG are able to reduce brain metastasis significantly. RNA-Seq analysis identified only one gene which is commonly altered in all treatment groups. This gene encodes marginal zone B and B1 cell specific protein (MZB1). MZB1 was downregulated after treatment and its expression was inversely correlated with the efficacy of brain metastasis. In addition, pathway comparison analysis of RNA-Seq data revealed there are three top canonical pathways including (1) Role of NFAT in Regulation of the Immune Response, (2) PKC theta Signaling in T Lymphocytes, and (3) iCOS-iCOSL Signaling in T Helper Cells, downregulated in all treatment groups. In conclusion, our findings demonstrated three different classes of compounds can reduce not only tumor loading but metastasis. MZB1, identified from our transcriptome analysis, may serve as an actionable molecular target for TNBC. All-in-all, it provides new insights into the development of new therapeutic targets and biomarkers for TNBC. Citation Format: Yuk Sing Robert Cheng, Debashree Basudhar, Rdinour Lisa, Veena Somasundaram, David Wink. Downregulation of MZB1 by aminoguanidine, APOE mimetic and nitroxyl compounds associated with reduction in invasive breast tumor volume and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2015.