To create a comprehensive IL-24 signaling pathway map, a literature survey was carried out on IL-24 signaling. The search terms used were “IL-24 AND Signaling,” and the articles were retrieved from PubMed. The abstract of research articles describing the signaling events induced by IL-24 was manually screened to select the relevant ones. The experimental datasets were limited to the signal transduction events induced by IL-24, excluding the knock-out and co-expression studies. The curated datasets encompass studies pertaining to diseases such as cancer, infectious, and inflammatory conditions.

The data related to signaling events induced by IL-24 were then manually annotated and cataloged according to the PathBuilder criteria ( Figure 1 ) (42). IL-24-induced molecular events such as protein-protein interactions, protein expression, gene regulation events, activation/inhibition events, enzyme catalysis/post-translational modifications (PTM), molecular association, and protein translocation were curated from the research articles (43). The protein and gene expression events were cataloged for overexpressed (fold change >1.3, p-value <0.05) and downregulated (fold change <0.76, p-value <0.05) molecules, as devised in each of the studies. All possible PTM events, including acetylation, ubiquitination, denitrosylation, N-glycosylation, phosphorylation, and dephosphorylation, with their site-specific information, were annotated within the pathway map and are delineated with different colors. Further, the translocation events were categorized based on the transportation of proteins within the cellular organelles comprising the nucleus, plasma membrane, cytoplasm, mitochondria, and Golgi complex. The proteins and genes were mapped to their corresponding Entrez Gene ID to annotate the data in a standardized format. Besides the proteins, the involvement of small molecules or second messengers such as reactive oxygen species (ROS), Ca²⁺, and cAMP was also noted. Individual study-centric signaling events and their related disease conditions and cell type-specific reaction events were documented. The data also included the type of experiments performed to detect and validate the expression of downstream molecules of IL-24 ligand-receptor interaction and the cell line/tissue used. The data on the synergistic activities of IL-24 were curated using the same data annotation strategy and included information on the impact of drugs and other molecules of therapeutic potential.

The annotated molecules, reactions, and disease-specific biological processes were manually drawn based on the topology as a signaling pathway map of IL-24 using the PathVisio (version 3) tool (44). The topology that represents the spatial order of the molecules was devised based on the directionality initiated by the IL-24 and its receptor interaction, as determined by studies using inhibitors or the established cascade of reactions.

The protein-protein interaction network analysis was performed with IL-24, its receptors, and downstream targets. The analysis was carried out using the tool STRING database with a confidence score of >0.7 (45). To enrich the functional clusters, the network was imported into the Cytoscape tool v 3.10.2, and the algorithm Molecular Complex Detection (MCODE) was applied (46, 47). MCODE identifies the clusters based on their local neighborhood density and expands from highly connected hubs. Additionally, publicly available omics datasets were analyzed to provide supporting evidence for the pathway map. The PBMC transcriptomic data were retrieved from the NCBI GEO DataSets and analyzed using GEO2r to assess the expression of IL-24 across various disease conditions. Similarly, differentially expressed proteins were obtained from the publicly available proteomic dataset, and the pathway enrichment analysis was performed using the Reactome database (48).

The distinctive functions of IL-24 in cancer include tumor-specific killing through an integrated effect of apoptosis, leading to a potent “bystander” anti-cancer activity, inhibition of cell proliferation, migration, and angiogenesis (18). This cytotoxic activity could cause either growth arrest or apoptosis through various cellular mechanisms solely in cancer cells at the supraphysiological levels of IL-24. Meanwhile, IL-24, at its natural physiological level, is recognized to function regularly to maintain its cellular processes and immune responses. A potential explanation for this selectively induced apoptosis is the intrinsic biochemical distinctions between normal and cancer cells. This encompasses factors such as high ROS production, altered metabolism, reprogrammed signaling pathways, and oxidative and endoplasmic reticulum (ER) stress (51). The bystander activity of IL-24 induces anti-cancer effects on IL-24 target cells and influences the neighboring untreated cells. This intriguing property makes IL-24 a promising candidate for cancer therapy as it amplifies its therapeutic potential beyond the directly treated cells. The IL-24 expression in tumor cells is primarily derived from tumor-infiltrating immune cells, such as T cells and NK cells, rather than from the tumor cells themselves. Moreover, the cells transduced with IL-24 can secrete other cytokines acting on the adjacent tumor cells, exerting their apoptotic and anti-proliferative effects (11). The tumor suppressive activity of IL-24 has been widely studied in a broad range of cancers, including oral, breast, lung, cervical, and colorectal carcinoma (22, 52–55).

Beyond the intrinsic property of IL-24 as a cytotoxic and tumor-suppressant agent, numerous studies have investigated the utility of IL-24 with other drugs as a combinatorial drug administration approach in various diseases ( Figure 3 ). Research findings have demonstrated that combining multiple chemotherapeutic agents and radiation with IL-24 in cancer treatment has amplified the effectiveness of chemotherapy and radiotherapy in numerous cancers (79–81). Also, studies have shown that IL-24 promotes apoptosis in various cancer types, boosting its anticancer activity when used in conjunction with some anti-inflammatory drugs, pleiotropic cytokines, including IL-20, IFNG, IL-6, vitamin E, gamma-secretase, and other small-molecule inhibitors (82–84).

IL-24 can be a potent therapeutic agent against viral and bacterial infections like influenza, Human immunodeficiency virus (HIV), Mycobacterium, and Salmonella (85). As well, in inflammatory conditions such as endometriosis, inflammatory bowel diseases (IBD), asthma, and psoriasis, IL-24 has been observed to modulate inflammation-associated events (86–90). Higher expression levels of IL-24 have always been associated with chronic inflammation and autoimmune disorders such as psoriasis, arthritis, vascular permeability, and IBD (91). In chronic inflammatory conditions such as asthma, IL-24 contributes to inflammation by promoting EMT of alveolar cells and neutrophils (89, 90). While in pathogenic infections, IL-24 induces an immune response through the secretion of cytokines such as IL-12, IL-23, IL-27, and IFNG (20, 92). IL-24 induces cytokine secretion by the activation of SOCS proteins through the JAK-STAT pathway. The SOCS proteins are known to play an important role in IL-24 signaling cascades for various inflammatory and infectious conditions (14).

The role of IL-24 in inflammatory diseases is context-specific, where it has been implicated in promoting inflammation in certain pathophysiological conditions. Similar to the case of cancer, IL-24 functions to reduce the proliferation and invasiveness of endometrial squamous cells (ESCs) in endometriosis, marked by the downregulation of MKI67 and PCNA. In addition, IL-24 also suppresses the macrophage-induced high viability and invasiveness of ESCs (88). Whereas, in IBD, recent studies have found that peripheral blood mononuclear cells (PBMCs) and epithelial cells secrete higher amounts of IL-24, resulting in the activation of extracellular matrix (ECM) and mucin proteins (collagens, fibronectins) contributing to the remodeling of the mucous layer in IBD (93, 94). In other inflammatory conditions, such as psoriasis, IL-24 is highly stimulated and induces the expression of the inflammatory mediators, including Psoriasin, chemokine ligands (87).

Furthermore, studies have proven that IL-24 can act as a protective agent and reverse the molecular events caused by the injuries induced by specific agents, such as H₂O₂ and β-glycerophosphate (95, 96). Similarly, in liver fibrosis, IL-24 functions to protect the liver from inflammation and injury. IL-24 protects the liver cells through the activation of anti-oxidant proteins and the reduction of ER stress-related DNA-damaging proteins, pro-apoptotic and pro-inflammatory proteins (97, 98). Also, its potential as a therapeutic agent in central nervous system autoimmunity has been recently investigated, revealing that IL-24 suppresses ocular inflammation by markedly inhibiting the Th1 and Th17 cells differentiation and cytokine secretion from the ocular infiltrating pathogenic Th1 and Th17 cells (99). In inflammatory conditions, IL-24 plays a dual role in Th17 cells by promoting mitochondrial recruitment of STAT3, enhancing oxidative phosphorylation, and dampening the nuclear transcriptional activity of STAT3, thereby regulating the IL-10 expression and restraining the Th17-mediated immunopathology (100). Additionally, IL-24 also acts as a downstream regulator in the IL-17A-mediated signaling loop within Th17 cells, controlling the expression of other Th17-associated cytokines such as IL-17F and GM-CSF (101). Thereby, IL-24 effectively modulates the balance between inflammation and regulation in autoimmune diseases by orchestrating Th17 cell function and cytokine expression. Therefore, the intricate involvement of IL-24 in modulating immune responses and inflammatory processes unravels its potential as a therapeutic target in a range of inflammatory conditions.

IL-24 is widely recognized as an immunomodulatory cytokine with the property to enhance the tumor-specific immune response and disrupt the tolerance toward tumor antigens (102). Recent reports indicate that IL-24 can augment CD4+ and CD8+ T cell populations and reduce T regulatory cells within tumor-infiltrating lymphocytes (TILs), suggesting IL-24-mediated anti-tumor immune responses in colorectal adenocarcinoma (22). Also, Ma et al. (2016) have reported IL-24 mediates tumor suppression through increased percentages of CD45+CD4+ and CD45+CD8+ cells and decreased percentage of CD45+CD4+Foxp3+ cells in IL-24-treated colon cancer (102). The enhanced cytolytic activity of CD8+ T cells was marked by the IFNG, granzymes, and perforins secretion stimulated by IL-24 (22). Moreover, IL-24 markedly enhanced the mRNA expression of the transcription factor T-bet (Th1 transcriptional factor) to activate CD4+ T cells. It also inhibits the expression of a T regulatory cell activation marker, transcriptional factor Forkhead Box P3 (FOXP3), ultimately reducing the T regulatory cells in colorectal carcinoma (22, 102). Alongside, it also activates and enhances the functions of the cluster of differentiation 80 and 86 (CD80, CD86) proteins that are known for the transduction of secondary signals for T-cell activation. Furthermore, it also stimulates innate and adaptive immune responses in myelogenous leukemia cells through the production of ligands (MICA and MICB) for Killer Cell Lectin Like Receptor K1 (KLRK1) on NK and T cells (103).

Besides its intrinsic immunoregulatory functions, IL-24 can induce the secretion of other cytokines and modulate its activity in diverse pathological conditions (104, 105). IL-24 stimulates the secretion of IL-6, IL-12, IL-27, and IFNG in cancer and infectious disease conditions. Thereby, IL-24 plays a significant role in regulating the immune response against tumorigenesis by reversing the immunosuppressive tumor microenvironment and promoting anti-tumor immunity (103). Additionally, in infectious disease conditions, it stimulates the immune response through the recruitment of CD4+, CD8+ T cells, and neutrophils by the activation of T-Cell-Specific T-Box Transcription Factor (TBX21), leading to the secretion of IFNG and several other interleukins (106). This orchestrated immune activation by IL-24 plays a crucial role in combating pathogens, external antigens, and tumor cells, thereby promoting the host-defense mechanisms.

The demonstration and visualization of the mechanisms of IL-24-mediated immunoregulation facilitate and aid in developing and utilizing IL-24 therapeutic approaches. Recent reports have proven the efficacy of IL-24 in enhancing CAR-T cell therapy against cancer stem cells (CSCs) that exhibit therapeutic resistance (25). The study by Hu et al. (2021) has reported that IL-24 potentially enhances the vitality of T cells, which is a common cell resource for CAR-T products, and significantly inhibits the viability and proliferation of cancer cells in leukemia and lymphoma (26). In addition, it was found that IL24-potentiated T-cell expansion and increased tumor infiltration significantly inhibited the cancer progression. Therefore, arming T cells with IL-24 shows the capabilities of tumor eradication and T-cell expansion within the tumor microenvironment. This highlights a promising approach of cellular immunotherapy that inhibits tumor escape (27). Moreover, through the annotation of IL-24 signaling mechanisms, we identified that IL-24 has the potency to significantly suppress the expression and activity of the T regulatory cell transcription factor, FOXP3, in cancer cells and infectious disease conditions. This transcription factor is well known for activating T regulatory cells that impair the treatment response in cancer immunotherapy (107). Extensive research activities are carried out to suppress this transcription factor as a cancer therapeutic approach (108). However, the ability of IL-24 to specifically target and suppress the function of FOXP3 remains inadequately investigated. Therefore, with this signaling pathway map, we were able to showcase all possible signaling mechanisms, primarily in cancer as well as in other inflammatory and infectious diseases. IL-24 stands out as a potent therapeutic agent, with supporting evidence highlighting its potential in developing advanced and personalized cancer therapeutics.

The current pathway model serves as a tool that integrates molecular-level information derived from individual studies and does not represent a temporally orchestrated network within a single cell; rather, it represents standalone findings from each study. This applies to all pathway models available to date and is further augmented by the limitations in their effective visualization (37, 43, 109). In a way, most maps are static, considering the signaling network highly spatiotemporal and dynamic. However, the pathway model represents the collective information on the molecules and their reactions that are identified to be perturbed upon IL-24 stimulation into a computationally encoded form for incorporation into enrichment tools. These details, which are otherwise scattered across the literature, would be time-consuming for individual researchers to compile into pathway models. Additionally, the present study does not provide new in vivo or clinical validation. However, all the signaling events incorporated into the pathway were curated from experimentally validated studies, reflecting the available literature up to the time of publication. Even though challenged by the above limitations, the current model serves as a reference platform and database to update molecular data as more studies on IL-24 become available. We also aim to refer to notable studies in the map to ensure transparency and to support future updates as more data becomes available.

Furthermore, to demonstrate the utility of the annotated signaling pathway map serving as a framework for applying gene set enrichment analysis, we mapped it into the protein-protein interaction network comprising IL-24, its receptors, and downstream targets. The analysis highlighted the various hub genes, including key signaling modules such as JAK-STAT, SOCS, PI3K, PTK2, and MAPK, together with apoptosis and cell cycle regulator proteins ( Figure 4 ). This emphasizes the role of the curated signaling map in the detection of enriched signaling modules and the selection of candidate molecules for further analysis. Additionally, we looked into the publicly available transcriptomic profiles of peripheral blood mononuclear cells in cancer and infectious disease conditions. We identified three transcriptomic datasets where IL-24 expression was detected (110–112). Notably, IL-24 was consistently downregulated in cancer and inflammatory conditions, together with the suppression of anti-tumor cytokine genes within the immunosuppressive microenvironment. Whereas, in the infectious disease condition of tuberculosis, IL-24 was found to be overexpressed, indicating its anti-infectious property. This reinforces the immunomodulatory role of IL-24 and represents the utility of this curated pathway map to elucidate disease-gene association and its expression changes for further functional analysis.

Further, to complement the signaling pathway, we performed a pathway enrichment analysis with the differentially expressed proteins identified from the publicly available proteomic dataset of IL-24-treated cervical cancer cells (113). The analysis revealed significantly enriched pathways, including apoptosis, immunomodulatory pathways, and cell cycle regulations ( Supplementary Figure S1 ). These enriched pathways align with the key signaling modules annotated in the pathway map of IL-24, further supporting the role of IL-24 in tumor suppression and immunoregulation. This integration demonstrates the utility of the application of this signaling pathway map in gene set enrichment analysis pipelines, offering a valuable resource for future studies in the analysis of high-throughput data.