NOS2 and COX-2 Co-Expression Promotes Cancer Progression: A Potential Target for Developing Agents to Prevent or Treat Highly Aggressive Breast Cancer

Abstract

Nitric oxide (NO) and reactive nitrogen species (RNS) exert profound biological impacts dictated by their chemistry. Understanding their spatial distribution is essential for deciphering their roles in diverse biological processes. This review establishes a framework for the chemical biology of NO and RNS, exploring their dynamic reactions within the context of cancer. Concentration-dependent signaling reveals distinctive processes in cancer, with three levels of NO influencing oncogenic properties. In this context, NO plays a crucial role in cancer cell proliferation, metastasis, chemotherapy resistance, and immune suppression. Increased NOS2 expression correlates with poor survival across different tumors, including breast cancer. Additionally, NOS2 can crosstalk with the proinflammatory enzyme cyclooxygenase-2 (COX-2) to promote cancer progression. NOS2 and COX-2 co-expression establishes a positive feed-forward loop, driving immunosuppression and metastasis in estrogen receptor-negative (ER^-) breast cancer. Spatial evaluation of NOS2 and COX-2 reveals orthogonal expression, suggesting the unique roles of these niches in the tumor microenvironment (TME). NOS2 and COX2 niche formation requires IFN-γ and cytokine-releasing cells. These niches contribute to poor clinical outcomes, emphasizing their role in cancer progression. Strategies to target these markers include direct inhibition, involving pan-inhibitors and selective inhibitors, as well as indirect approaches targeting their induction or downstream effectors. Compounds from cruciferous vegetables are potential candidates for NOS2 and COX-2 inhibition offering therapeutic applications. Thus, understanding the chemical biology of NO and RNS, their spatial distribution, and their implications in cancer progression provides valuable insights for developing targeted therapies and preventive strategies.

Keywords: biochemistry; breast cancer; nitric oxide; therapy.

Figure 1

The chemical biology of nitric oxide (NO) and reactive nitrogen species (RNS): NO can exert its effects either directly or indirectly. Direct effects are associated with low NO levels (<100 nM). At these concentrations, NO can activate the enzyme cyclic guanylyl cyclase (cGC), initiating the conversion of guanosine triphosphate (GTP) into cGMP. cGMP then serves as a signaling molecule in various physiological processes, such as vasodilation and neurotransmission. Moreover, it contributes to drug detoxification by interacting with proteins from the cytochrome P450 family, which can also eliminate NO itself. In addition, NO can induce the hypoxia-inducible factor (HIF) pathway through the inhibition of prolyl-hydroxylase, while also functioning as a free radical scavenger by inhibiting the Fenton reaction and reacting with reactive oxygen species (ROS). Indirect effects of NO are driven by its reactive nitrogen species (RNSs). These reactive species can undergo various reactions, leading to their recycling back into NO while simultaneously activating diverse oncogenic pathways. Figure Made in BioRender.com (accessed on 21 February 2024).

Figure 2

Nitric oxide (NO) kinetics: (A) NO is enzymatically synthesized through the oxidation of L-arginine by iNOS. Conversely, nitrite (NO₂⁻), the product of NO, acts as an alternative NO reservoir in acidic environments, such as phagosome/lysosome lumens. In these regions, nitrite undergoes a series of reactions, ultimately generating NO and NO₂, which then diffuse from the source to the surroundings in an O₂-dependent manner due to O_2’s impact on NO half-life creating an NO flux gradient with higher levels around the source and lower levels as the distance from the source increases. (B) The extent to which NO can diffuse from its origin and the levels of this free radical in a specific environment depend on the cell density of iNOS-expressing cells. Higher densities of NO-producing cells result in elevated NO levels and an increased diffusion distance. Figure Made in BioRender.com (accessed on 21 February 2024).

Figure 3

Cellular signaling and nitric oxide (NO) levels: NO effects vary based on its concentration. Three distinct nitrosative levels are identified: Level 1 (<100 nM) is linked to cGMP-related cell signaling, contributing to cancer cell proliferation; Level 2 (100–500 nM) involves HIF-1⍺ activation and subsequent pathways promoting cancer progression; Level 3 (>500 nM) is associated with toxic NO effects, primarily triggered by p53 activation, leading to cell cycle arrest and apoptosis. Figure Made in BioRender.com (accessed on 21 February 2024).

Figure 4

NOS2 and PTGS2 (COX2) expression in normal breast and ER- breast cancer. **** p < 0.0001; ** p = 0.0032.

Figure 5

iNOS/COX2 co-expression and its effects on ER^- breast cancer progression: In ER- breast cancer, iNOS and COX2 exhibit orthogonal expression patterns. iNOS niches are located at the stroma-tumor interface, preventing the penetration of CD8⁺ T cells into the tumor, and are associated with a metastatic phenotype. On the other hand, COX2 niches are deeper within the tumor core, near immune-desert regions. There exists a positive feedforward loop between iNOS and COX2, where iNOS-derived nitric oxide (NO) induces COX2 activity, and COX2-derived PGE₂ induces iNOS. The formation of these iNOS and COX2 niches depends on the presence of INF-γ-secreting cells, such as CD8+ T cells, and may be potentiated by other Th1 cytokines like TNF-⍺ and IL-1β. Furthermore, NO and PGE₂ have the potential to induce the production of immunosuppressive cytokines such as IL-10 and TGF-β. Figure Made in BioRender.com (accessed on 24 February 2024).

Figure 6

NOS2 induction in murine and human cells: In murine macrophages, the maximum induction of NOS2 occurs with a combination of INF-y and LPS. However, in both murine and human tumor cells, the highest expression of NOS2 is achieved when INF-y is combined with either TNF-⍺ or IL-1β. The induction of NOS2 in murine macrophages occurs approximately 18 h after treatment, while in murine and human tumor cells, there is a delay, with induction occurring around 24 h to 48 h. Figure Made in BioRender.com (accessed on 24 February 2024).

Figure 7

Direct and indirect approaches for inhibiting iNOS and COX2: Various nonsteroidal anti-inflammatory drugs (NSAIDs), including indomethacin and diclofenac, as well as Celecoxib can directly inhibit COX2. Specific iNOS inhibitors, such as acetamidine compounds like 1400 W, demonstrate highly efficient and targeted effects. In terms of indirect approaches, the focus shifts to factors that induce iNOS and/or COX2 activity. A variety of NSAIDs play a role in inhibiting the production and release of proinflammatory cytokines, such as INF-γ and TNF-α, leading to subsequent inhibition of the NFκB pathway. However, inhibition of these proinflammatory mediators poses a challenge, as they are also crucial to anticancer responses, and their suppression may promote cancer progression. An alternative strategy to disrupt the iNOS/COX2 feedforward loop involves nutraceuticals, such as isothiocyanates and dithiolethione compounds. These compounds exhibit the capability to inhibit carcinogenic phase I enzymes while simultaneously inducing phase II detoxification enzymes, thereby promoting chemoprevention. Phase I enzymes can activate the Aryl hydrocarbon receptor (AhR), leading to COX2 activity induction. These natural compounds also serve as activators of the tumor suppressor protein phosphatase 2A (PP2A). This enzyme acts as an antagonist to various oncogenic pathways activated by nitric oxide. Figure Made in BioRender.com (accessed on 26 February 2024).