A Paradoxical Effect of Interleukin-32 Isoforms on Cancer.

The influence of IL-32 in tumor growth through the inactivation of NF-κB and signal transducer and activator of transcription 3 (STAT3) pathways have been mentioned earlier ( 33 ). Moreover, IL-32γ downregulates vital cancer progression proteins including antiapoptotic, cell proliferation, and tumor-promoting genes, while upregulating the apoptotic genes. On the contrary, IL-32γ isoform is shown to diminish the levels of cytokines that promote tumor growth such as TNF-α, IL-1β, and IL-6, whereas the levels of IL-10 cytokine, a tumor growth-inhibiting cytokine, were elevated. The anticancer activity of IL-32γ was found in several cancer cells but not in melanomas like colon, prostate, liver, and lung. It also induces the activation of cytotoxic T cells and NK cells to the tumor site to expand the cancer eradication effect ( 33 , 34 ) as well as recently, it showed better immunotherapy response ( 35 ). Later, IL-32β has been found to play an antitumor activity role as it downregulates vital cancer progression proteins including antiapoptotic proteins, proliferation, and cell growth regulatory proteins through the same pathways, NF-κB and STAT3. In addition, IL-32β was found to induce the expressions of proapoptotic proteins and regulate the release of cytokines in colon and prostate cancer cells ( 15 ). Nevertheless, higher expression of IL-32α has been found to activate NF-κB and STAT3 pathways and induce the production of IL-6, thus supporting the cancer proliferation and progression in MM patients ( 25 ). Therefore, finding the exact function of the IL-32 isoform is still a sensitive consideration and may be influenced not only by its isoform but also with cancer type as well as the whole tumor microenvironment. We have mentioned the anticancer activity of IL-32γ in colon cancers, which is considered through activation of p38 MAPK pathways ( 16 ). Moreover, IL-32α and IL-32θ have been found to suppress the effect on colon cancer, as well ( 17 , 18 , 36 ). In the case of the expression IL-32α, the expression of TNF receptor 1 and the production of reactive oxygen species was increased, thus facilitating apoptosis and prolonged JNK activation. At the same time, several studies have mentioned the contradictory role of IL-32 in colon cancer ( 7 , 37 , 38 ) whereas IL-32 was found to be upregulated and associated with poor survival. In this regard, it is worth mentioning a finding that provides evidence on the contribution of IL-32α in the development of obesity-associated colon cancer by favorably remodeling cytokine for tumor growth ( 39 ). According to the currently available data, we can suppose that in colon cancer, IL-32α has both pro- and antitumor activity depending on other factors such as obesity, gender, and/or age-related factors which have not been studied yet. However, obesity-related IL-32 manipulation indicates that IL-32 could play a role in cancer metabolism as well as inflammation.

The tumor microenvironment refers to the surrounding ecosystem that includes extracellular matrix, blood vessels, and an array of cells such as fibroblasts, immune cells, and heterogeneous tumor cells. These components influence one another and thus, contribute to tumor progression and metastasis in either a positive or negative way. Therefore, a better understanding of the tumor microenvironment offers new insights for improving cancer therapies ( 79 , 80 ). Cytokines are one of the key mediators for interactions between immune and nonimmune cells in the tumor microenvironment (TME) ( 81 ). It has been shown to have a different role that is isoform dependent since many cells express IL-32. However, it is not clear yet how IL-32 contributes to the different tumor types including stromal tumor microenvironment. In a study investigating the IL-32 effect in the pathogenesis of endometriosis as an example of stromal cancer, IL-32 showed a correlation in cancer progression. This study revealed that the IL-32 concentration in the peritoneal fluid was drastically greater in patients of advanced-stage endometriosis as compared with the controls. Moreover, they showed that IL-32α and IL-32γ significantly increased cellular viability, proliferating cell nuclear antigen expression, and invasive ability ( 82 ). Several studies showed that the overexpression of IL-32, specifically α, β, and γ were able to reduce tumor growth through inducing apoptosis in tumor cells, which led to CD8 + T-cell responses ( 15 , 17 , 33 ). Nevertheless, other than the antitumor effect, IL-32 demonstrates a monocyte differentiation stimulator as well as cytokine production. Moreover, it has been reported for its ability to activate T cells and therefore stimulate antigen presentation utilizing dendritic cells (DCs). On the contrary, functional studies demonstrated that IL-32γ induced PD-L1 expression on monocytes but not tumor cells, which may contribute to local immunosuppression and therefore are candidates for cotargeting in combination treatment regimens. IL-32γ expression correlates with a treatment-resistant dedifferentiated genetic signature and genes related to T-cell infiltration. This was reported in melanoma cells, suggesting it influences nonmelanoma cells in the tumor microenvironment, such as myeloid cells ( 83 ). More recently, IL-32γ potentiates antitumor immunity in melanoma as the antitumor microenvironment. This result is shown to be enriched in mature DC and M1 macrophages resulting in enhancing the recurrence of activated tumor-specific CD8 + T cells to generate antitumor immunity. Therefore, IL-32 resulted in reducing tumor growth and rendering immune checkpoint blockade resistance ( 35 ). On the other hand, IL-32β stimulates the activation-induced apoptosis of T cells, NK cell cytotoxicity toward tumor cells like IL-32γ in the activation of monocyte differentiation. In addition, IL-32α is shown to be a stimulator of NK cell cytotoxicity, whereas IL-32θ has been shown as an inhibitory effect on monocyte differentiation and cytokine production ( 36 , 84 – 88 ). However, better characterization of the tumor microenvironment is needed to understand how different cell types in the tumor microenvironment are influenced by IL-32. Moreover, how IL-32 isoforms implicated each other is another key factor in overall response to cancer. As we mentioned above, the possibility of IL-32 in exhibiting an isoform switching and self-regulation between IL-32β and IL-32γ was reported ( 41 ). Likewise, isoform δ of IL-32 was found to modulate another isoform, IL-32β, by interacting with it and thus inhibiting its production of IL-10 ( 89 ). Both observations suggest that IL-32 performs its feedback regulation through its isoforms.

The human interleukin-32 (IL-32) is a novel cytokine that exerts both pro and anti-inflammatory roles. IL-32 gene is found in higher primates, and it is located in chromosome 16 at p13.3 encoding for various isoforms. IL-32 plays an essential role in innate and adaptive immune responses, and it induces various cytokines such as tumor necrosis factor (TNF)-α, IL-1β, IL-6, and IL-8 ( 1 ). After its identification, it has been studied in inflammatory disorders including autoimmune diseases and cancers ( 2 , 3 ). In cancer, inflammatory tumor microenvironment such as cytokines, IL-32 plays a crucial role in its progression ( 4 ). Therefore, IL-32 has been studied for its tumor control direction in several cancer types. However, paradoxical effects have been reported regarding IL-32 on cancers, which may be attributed to the dominant isoform, cancer type, and genetic background. On the one hand, IL-32 was reported to augment cancer progression, proliferation, invasion, and metastasis in many tumors including acute myeloid leukemia (AML), hepatocellular carcinoma (HCC), and breast, lung, colon, pancreatic, and gastric cancers ( 5 – 12 ). On the other hand, it was also reported to have anticancer activity in different cancers including acute and chronic myeloid leukemia (AML and CML) and breast, lung, and colon cancers ( 13 – 19 ). IL-32 gene was found to have several isoforms based on different alternative splicing sites. It has eight exons in which the first exon does not translate into amino acids. Mainly, seven isoforms were depicted and were identified separately which are IL-32α, IL-32β, IL-32γ, IL-32δ, IL-32ε, IL-32ζ, and IL-32θ ( 3 ). IL-32α, IL-32β, IL-32γ, and IL-32δ were primarily detected in IL-2-stimulated human NK cells. While IL-32ε and IL-32ζ were observed to be expressed in the activated T cells ( 20 ), and IL-32θ was found within dendritic, Jurkat, human leukemia T cells ( 21 ). Structural characteristics of the seven IL-32 isoforms were reviewed based on the IL-32 eleven protein domains ( 3 ). However, a lot of knowledge is waiting to be revealed regarding IL-32 isoforms, such as their specific receptors. These isoforms displayed distinctive roles and consequences in different conditions although they are deficient in signal peptides. Therefore, a functional comparison between these isoforms as well as specific antibodies to detect IL-32 isoforms is considered necessary. Nevertheless, what has been discovered so far still lacks explicit knowledge about IL-32 function in cancers. It is known that many factors can affect the disease outcome, especially in cancer, yet this much contradiction was not reported to any cytokine other than IL-32. This contradiction is mainly due to not considering IL-32 isoforms in most of the studies. In this review, we aim to analyze previous reports to address the most probable functions of IL-32 on different cancers to provide recommendations for further studies and unravel possible therapeutic options.

It conflicts in targeting therapy for IL-32 in cancer because IL-32 roles remain unclear, thus there is no specific direction for IL-32 in cancer therapy. However, some isoforms showed an inhibitory effect that can be administered exogenously to stop or reverse cancer progression such as IL-32θ for cytokine-based immunotherapy. Moreover, it was found that patients with higher expression of IL-32 demonstrated more aggressive cancers. In these cases, IL-32 can be targeted precisely to stop its progression role. There is a great gap in this matter even after selecting the IL-32 isoform for cancer therapy. A lot more studies are needed before this knowledge can be used clinically. This difficulty regarding IL-32 was addressed in a recent review considering interleukins in improving cancer therapies ( 4 ). Again, this is due to IL-32 showing no clear effect on cancer which differs based on IL-32 isoforms, cancer type, and genetic background.

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Changes in the genetic material provide different effects within individuals and populations. Recently, several studies have demonstrated the impact of IL-32 polymorphisms on cancer progression. Moreover, IL-32 SNPs were studied and reviewed with their association to disease outcome ( 90 – 95 ), and by 2021, one review performed a meta-analysis to evaluate the SNPs in malignancy ( 96 ). Up to now, three polymorphisms of IL-32 were found to be associated with the progression of several cancers that are rs28372698, rs12934561, and rs2015620 ( Table 1 ). IL-32 polymorphisms and their associated cancers. Based on human genome build 38: GRCh38. SNP rs28372698 was found in many cancers including thyroid carcinoma and lung, endometrial, ovarian, gastric, bladder, and colorectal cancers that are related to the higher expression of IL-32 resulting in cancer progression ( 96 – 104 ). In thyroid carcinoma, this polymorphism revealed higher expression of isoform IL−32γ that increased the risk of tumor development ( 104 ). In a study to evaluate cytokine polymorphisms and their association with gastric cancer, this SNP (rs28372698) of IL-32 has shown no association. However, when the patient has another SNP, IL-8 rs4073, there was an interaction between both SNPs and thus suggested increased gastric cancer risk ( 103 ). Interestingly, another study on the Chinese population revealed that IL-32 SNP rs2015620 is highly associated with the risk of gastric cancer by interacting with two more SNPs, IL-18RAP rs917997 and IL-22 rs1179251 ( 101 ). However, these studies were subjected to two different populations, Chinese and Chilean; the reason why IL-32 SNP has a different effect. Although studies on IL-32 SNPs are not dispersed in the world, yet according to the published data, SNP rs28372698 showed high cancer influence on the Chinese population. Moreover, this SNP was linked to colorectal cancer in the Swedish cohort but not reported in the Chinese colorectal cancer patients ( 99 ). Both IL-32 SNPs of rs28372698 and rs12934561 have been correlated with bladder cancer processes ( 102 ). However, only rs12934561 was related to poor survival status in squamous carcinoma ( 98 ). Overall, these association studies were subjected to some limitations due to the limited population and selected population. A large-scale study must include more than one kind of population and ethnicity to discover the role of IL-32 SNPs in cancers.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This paper was written as part of Konkuk University’s research support program for its faculty on sabbatical leave in 2022. This work was supported by the National Research Foundation of Korea (NRF-2021R1F1A1057397). This research was supported by the Main Research Program (E0210602-02) of the Korea Food Research Institute (KFRI) funded by the Ministry of Science and ICT. S-YK and Y-GK were supported by NRF-2021M3A9G1026605. SL was supported by NRF-2019R1I1A1A01057699.

Conceptualization: SS, SL, YH, SK, TN, AT, JH, HJ, YL, SY, Y-GK, and SHK. Funding acquisition: HJ, SHK. Supervision: SHK. Writing—original draft: SS, SL, YH, and SHK. Writing—review and editing: AT and SHK. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.