Abstract P5-10-08: Inflammation drives NOS2-Akt2 signaling in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is aggressive and represents nearly 20% of all breast cancer cases. It is associated with poor prognosis due to its high metastatic capabilities and lack of targeted therapeutic treatments. To improve TNBC treatment, our goal is to better understand the molecular mechanisms that drive TNBC. Recently, inducible nitric oxide synthase (NOS2), a nitric oxide (NO) producing enzyme, has emerged as a key player in TNBC progression. High NOS2 expression in TNBC patient correlates with poor prognosis marked with increased metastasis and disease recurrence. NOS2 catalyzes L-arginine and dioxygen (O2) to produce citrulline and high, sustained levels of NO which has been shown to alter the activity of numerous signaling pathways. Depending on NO concentration and temporal and spatial levels, it has been reported to have both tumor-promoting and tumor-suppressing functions. However, the mechanism by which NOS2 drives TNBC is unclear. Akt is major oncogenic signaling pathway that regulates various aspects of tumor progression, and it has been shown that NO can activate Akt activity. The Akt family has three members, Akt1, Akt2, and Akt3, that seem to have similar and non-redundant functions in cancer. We have discovered for the first time that NOS2 specifically activates Akt2 in TNBC cells. Thus, we hypothesize that Akt2 plays a major role in NOS2 driven TNBC. Inflammatory cytokines (IFN-γ, IL-1β, and TNF-α) highly induce NOS2 in mouse 4T1 TNBC cells. We developed a real-time NO detection assay to measure NO levels in 4T1 cells and confirmed that these cells produce high levels of NO. By pharmacologically and genetically inhibiting NOS2 in the 4T1 cells, we found that NOS2 suppresses Akt2 phosphorylation but not the total levels. Furthermore, NOS2 had no effect on Akt1. The decrease in Akt2 phosphorylation was accompanied by inhibition of its downstream targets, GSK3β and FOXO3a, suggesting that NOS2 is regulating Akt2 activity. Conversely, treatment of 4T1 and human MDA-MD-231 TNBC cells with DETA/NO, a slow releasing NO donor, resulted in increased Akt2 phosphorylation with no effect on Akt1. Akt2 has been shown to promote migration, invasion, and metastasis. Using 4T1 cells, we are presently studying the function of NOS2-Akt2 signaling in TNBC. We have orthotopically injected 4T1 cells into immunocompetent mice and have found that 4T1 tumors express high levels of NOS2 as well as phosphorylated Akt2. We have knocked out NOS2 in the 4T1 cells to examine the effects of NOS2 inhibition in the mouse tumor model. Understanding the link between NOS2 and Akt2 will give further understanding on how NOS2 drives TNBC. Citation Format: Su Chung, William Montfort. Inflammation drives NOS2-Akt2 signaling in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-08.