NOD1 and NOD2: intracellular pattern recognition receptors implicated in gynaecological malignancies and their therapeutic potential

Introduction

The innate immune system, present in all multicellular organisms, is the primary defence barrier against the invasion of pathogenic microorganisms. It functions by recognising conserved structures of pathogens through specific pattern recognition receptors (PRRs), such as pathogen-associated molecular patterns. These receptors can activate downstream intracellular signalling pathways, triggering the organism’s immune response.1 PRRs, located either on the cell membrane, like Toll-like receptors (TLRs) or within the cytoplasm, such as nucleotide-binding oligomerisation domains (NODs), bind pathogen-associated fragments to initiate the intrinsic immune response.2 In addition to activating host defence mechanisms, PRRs have been identified to be significant for the development and progression of multiple malignancies, especially in cancers associated with chronic inflammation.3 4

NODs are cytoplasmic PRRs that have been identified in recent years. They are genetically highly conserved, lack signal peptides and transmembrane domains and are key members of the NOD-like receptor (NLR) family. Also known as the CATERPILLER family (comprising caspase activation and recruitment domain (CARD), transcription enhancer, R(purine)-binding, pyrin and leucine repeats), this group includes 22 members associated with various human diseases, such as apoptotic protease activating factor 1 (Apaf-1), NOD1 (nucleotide-binding oligomerisation domain containing 1 (NLRC1), CARD domain containing 4 (CARD4)), NOD2 (NLRC2, CARD15), ice-associated protease-activating factor (Ipaf), class II transactivator. (CIITA), NACHT, LRR and PYD domains containing proteins (NALPs), neuronal apoptosis inhibitory protein (Naip), cold-induced autoinflammatory syndrome (CIAS1) and Pyrin.5 Among these, NOD1, NOD2 and NALPs have been the focus of extensive research. This review systematically examines the pathophysiological roles of NOD1 and NOD2 in gynaecologic malignancies. A deeper understanding of these proteins’ function may open new avenues for the development of novel and effective anticancer therapeutics.6

Structure of NOD1/NOD2

The genes encoding NOD1 (NLRC1, CARD4) and NOD2 (NLRC2, CARD15) are located on human chromosomes 7p14 and 16q12, respectively. Each receptor is composed of three functional domains. The N-terminal effector-binding domain contains a CARD, which is a signalling hub to activate downstream pathways, and its integrity is essential for the activation of nuclear factor kappa B (NF-κB) mediated by NOD2. The central nucleotide-binding site includes Walker A and B motifs, which facilitate self-oligomerisation necessary for receptor activation. The C-terminal domain, comprised 10 leucine-rich repeats, is crucial for ligand sensing; its absence renders NOD2 unresponsive to its ligand. A key structural difference between NOD1 and NOD2 lies in their CARD domains: NOD1 has a single CARD, whereas NOD2 possesses two tandemly arranged CARDs7 (figure 1).

General functions of NOD1/NOD2

NOD1 and NOD2, as PRRs within the NLR family, can detect conserved bacterial motifs and activate intracellular signalling pathways, thereby triggering proinflammatory and antimicrobial responses. 8 The bacterial motifs recognised by NOD1 and NOD2 differ significantly.9–11 NOD1 identifies peptidoglycan fragments containing the γ-D-Glu-meso-DAP motif, which is predominantly found in Gram-negative bacteria and some Gram-positive bacteria, establishing NOD1’s specificity for these organisms.12 In contrast, NOD2 recognises muramyldipeptide (MDP) in almost all bacterial peptidoglycans,9 13 including MDP with L-alanylmuramic acid and structurally similar molecules like MurNAc-L-Ala-D-Glu (M-Di), M-TriLys and M-TriOm.14 However, NOD2 cannot detect N-acetylglucosamine-tri-diaminopimelic acid (GM-triDAP), which is specifically recognised by NOD1.

Beyond mediating innate immune responses, NOD1 and NOD2 also play a significant role in regulating adaptive immunity, particularly through the production of interleukin-1β, which is vital for initiating and amplifying immune responses against pathogens.15 Activation of these receptors has been shown to promote antigen-specific immunity, primarily resulting in a T-helper 2 (Th2) polarisation profile. However, in combination with TLR stimulation, NOD1 is necessary for inducing Th1, Th2 and Th17 immune pathways.16 17 Furthermore, research by Kasimsetty et al demonstrated that the absence of both NOD1 and NOD2 in mice accelerated T-cell death on encountering alloantigens, highlighting these receptors as potential therapeutic targets for ameliorating T-cell responses in various inflammatory conditions.18

NOD1/NOD2 in multiple inflammation-related malignancies

Recent evidence has strongly linked inflammation to tumour development.19 As early as 1863, Rudolf Virchow observed significant inflammatory cell infiltration in tumour tissues and proposed that tumours might originate from inflammatory processes. Current research shows that up to 20% of malignant tumours are associated with chronic inflammation.20 This persistent inflammatory microenvironment contributes to cancer development by generating reactive oxygen species and reactive nitrogen species, which may cause genetic instability and epigenetic alterations during ongoing tissue damage and repair.21 Additionally, inflammatory mediators like tumour necrosis factor-alpha (TNF-α) and macrophage migration inhibitory factor might exacerbate DNA damage, further implicating inflammation in oncogenesis.22

The innate immune system, as the first line of defence against infection, non-specifically recognises pathogenic substances through PRRs like TLRs and intracytoplasmic NOD receptors. Studies have shown that the NOD family, including NOD1 and NOD2, plays a crucial role in regulating immune function. The typical example is Helicobacter pylori infection, which is a key risk factor for stomach malignancy. Peptidoglycans from this bacterium are recognised by NOD1 and NOD2, leading to the activation of receptor-interacting protein kinase 2 (RIPK2), which subsequently facilitates the phosphorylation of extracellular signal-regulated kinase (ERK) and p38. This process involves the mitogen-activated protein kinase (MAPK) and NF-κB signalling pathways.23 Furthermore, human papillomavirus (HPV), the main cause of head and neck cancers, is known to induce chronic infections that contribute to lesion development.24 Literature reports indicated that NOD1 could be a prognostic marker for identifying head and neck squamous cell carcinoma (HNSCC) patients with poor survival trends. Additionally, evidence has shown that C-X-C motif chemokine ligand 1 (CXCL1) plays an important role in HNSCC progression via the NOD1-MAPK pathway.25 In summary, NOD1 and NOD2 are involved in multiple inflammation-related malignancies. Understanding their underlying mechanisms could provide novel insights into preventing pathogenesis (figure 2).

NOD1/NOD2 in gynaecological malignancies

In gynaecological malignancies, cervical cancer is a prominent example of HPV-related cancer,26 which can activate immune defence responses. Our research group27 has found that the NOD1/2-NF-κB/ERK signalling axis significantly influences the progression of squamous cervical carcinoma. Upregulation of NOD1 and NOD2 has been associated with increased tumour cell proliferation, invasion and migration. Specifically, high expression levels of NOD1 were correlated with poor prognosis due to lymph-vascular space invasion and lymph node metastasis.

These results were consistent with the conclusions of Bruno et al, who reported that a higher systemic immune-inflammation index and response index were associated with a significant risk of early-stage cervical cancer recurrence.28 Similarly, the neutrophil-to-lymphocyte ratio, an inflammatory marker, has been correlated with cervical cancer metastasis.29 Another study identified HCLS1-associated protein X-1 (HAX-1) as a novel interaction partner of NOD1 in the migration of cervical carcinoma cell line HeLa.30

NOD1 has been implicated in TNF-α-induced apoptosis and shown to inhibit the growth of oestrogen-sensitive tumours by reducing oestrogen receptor expression in experimental models.31 The anatomical connection between the endometrium and the external environment suggests a potential association with infections. Consequently, studies on PRRs in endometrial carcinoma have been relatively abundant. Modugno et al32 found that risk factors associated with the development of endometrial cancer, such as early menarche, late menopause and prolonged exposure to an unopposed oestrogenic environment, might increase inflammation. Thus, these risk factors played an important role in the pathogenesis of endometrial cancer. King et al33 found that NOD1 and NOD2 were expressed in the endometrial mesenchymal cells and glandular epithelium, with NOD1 expression remaining stable and persistent, whereas NOD2 expression was significantly increased during the late secretory phase of the menstrual cycle. Multiple studies34–36 focus on molecular classification involving the genetic variants in endometrial cancer, which currently guides clinical decision-making. Till now, there were no significant associations observed for the specific polymorphisms in NOD1 and NOD2.34 Nevertheless, additional discoveries have been identified: the expression of NOD2 (p=0.013) has been proved to be associated with overall survival, which was identified as one of the prognostic differentially expressed pyroptosis-related genes and also verified as protective genes (HR=0.583, <1).37 This result has been further validated. Lower expression levels of NOD2 were associated with lower progression-free survival (p=0.021) and advanced tumour stage (p=0.0024); and NOD2 presented a positive correlation with the tumour-infiltrating lymphocytes (TILs) and the immune checkpoints (p<0.001). NOD2 mechanistically promoted the macrophages transformation from the M2 phenotype to the M1 phenotype38 39 and influenced endometrial cancer prognosis.40

However, this association was not statistically significant after adjusting for other risk factors, such as body mass index, diabetes mellitus and hypertension. Further research is needed to explore this potential link in greater depth.

Relatively few studies have investigated the roles of NOD1 and NOD2 in ovarian cancer. One study reported the increased expression levels of NOD1 and RIPK2 in ovarian cancer, particularly in metastatic disease; NOD1 facilitated the expression level of proliferation-related proteins such as PCNA and Ki67, also increased NF-κB protein and phosphorylation. These phenomena illustrated that NOD1 might improve the capacity of proliferation and invasion in ovarian cancer cells, suggesting a potential role for NOD1 in promoting tumour proliferation and invasion.41 Recently, a study demonstrated that TRPM2, as a poor prognostic predictor in ovarian carcinoma, was involved in pyroptosis and showed a strong positive correlation with pyroptosis-related genes (including NLRP3, NLRC4, NOD2 and NOD1).42

The potential therapeutic targeted values of NOD1 and NOD2

20 years ago, studies demonstrated that SB203580, a p38 MAPK inhibitor, could suppress dextran sulfate sodium-induced experimental colitis in a mouse model by inhibiting RIPK2 (NOD2 effector) in the NOD2/RIPK2/NF-κ B signalling pathway. This model reflected human chronic inflammatory bowel diseases.43 Based on these findings, a highly selective 4-aminoquinoline-based RIP2 inhibitor (GSK583) was identified as effective in the intestinal mucosa of inflammatory bowel diseases by blocking NOD2 signalling.44

In serious ovarian cancer, the high expression of RIPK2 has been linked to Taxol resistance through the activation of the NOD1/RIPK2/NF-κB pathway and tumour microenvironment changes.45 As a result, a series of dual NOD1/NOD2 antagonists have been developed: (1) A 1,4-benzodiazepine-2,5-dione derivative (26bh) can inhibit both the NF-κB and MAPK inflammatory signalling pathways and can sensitise paclitaxel to suppress Lewis lung carcinoma proliferation46; (2) A quinazolinone derivative (36b), a novel NOD1/NOD2 dual antagonist, significantly sensitises B16 tumour-bearing mice to paclitaxel treatment by inhibiting both NF-κB and MAPK signalling47; (3) Compound 14k, a new derivative of dual NOD1/NOD2 antagonists with novel benzofused five-membered sultams, has been identified as the most potent molecule in inhibiting NF-κB and MAPK signalling stimulated by NOD1 and NOD2.48 These findings demonstrated the potential of targeting both NOD1 and NOD2 signalling as a valuable adjunctive treatment.

Discussion

In summary, NOD1 and NOD2, as cytoplasmic PRRs within the NLR family, play multifaceted roles in innate immunity and inflammation-associated oncogenesis. Structurally, their distinct CARD domains and ligand-specific recognition mechanisms enable differential activation of downstream signalling pathways, generally including NF-κB and MAPK, which are pivotal in both immune regulation and carcinoma progression. In gynaecological malignancies, these receptors exhibit context-dependent roles: NOD1 and NOD2 upregulation in cervical cancers correlates with tumour proliferation, invasion and poor prognosis, whereas their involvement in ovarian cancer highlights potential contributions to metastasis and chemoresistance. Notably, although direct associations between NOD1/NOD2 polymorphisms and endometrial carcinoma outcomes remain inconclusive, emerging evidence delineates the expression of NOD2 associated with overall survival as protective biomarkers and presented a positive correlation with the TILs and the immune checkpoints.

Emerging evidence underscores the therapeutic potential of targeting NOD1/NOD2 signalling. Dual antagonists of NOD1/NOD2 inhibitors have demonstrated efficacy in preclinical models by suppressing inflammatory pathways and sensitising tumours to chemotherapy. However, inconsistencies in statistical significance after adjusting for confounders, such as metabolic comorbidities, emphasise the need for further mechanistic exploration and validation in diverse cohorts. Future research should prioritise elucidating the interplay between NOD1/NOD2-mediated immune modulation, tumour microenvironment dynamics and therapeutic resistance, ultimately paving the way for novel adjuvant strategies in gynaecological oncology.