Interleukin-32 (IL-32) was originally identified in natural killer (NK) cells activated by IL-2 in 1992. Thus, it was named NK cell transcript 4 (NK4) because of its unknown function at that time. The function of IL-32 has been elucidated over the last decade. IL-32 is primarily considered to be a booster of inflammatory reactions because it is induced by pro-inflammatory cytokines and stimulates the production of those cytokines and
Interleukin 32 (IL-32) was initially identified in activated natural killer (NK) and T cells, and its expression is strongly augmented by microbes, mitogens, and other pro-inflammatory cytokines. Accumulating evidences has shown that IL-32 is an amplifier of inflammation through its stimulatory effects on pro-inflammatory cytokines, including IL-1β, IL-6, and tumor necrosis factor α (TNF-α). Thus, it is likely that prolonged or over-activation of IL-32 would play a role in some chronic inflammation-related diseases, including rheumatoid arthritis (RA), Crohn’s disease, and cancer. Indeed, since many studies have uncovered the molecular mechanisms by which IL-32 exerts anti-tumor or pro-tumor effects, it is appropriate to summarize the detailed functions of IL-32 in various cancers to understand a precise role of IL-32, and to present the direction for future research. Thus, we will review the effects of IL-32 on various cancers in three aspects: first, a role of IL-32 in tumors which are highly related to inflammation; second, a role of IL-32 in tumors which are not related to inflammation; and lastly, effects and functions of IL-32 in pro- or anti-tumor immune cells.
Development of malignant tumors follows a dynamic sequential progression that includes initiation, malignant conversion, and metastasis. As tumor mass increases progressively, tumor cells need to communicate with components of the tumor microenvironment for maintenance of tumor mass, consisting of infiltrating immune cells and stromal cells. Interactions between tumor and immune cells in tumor development are also crucial factors of the tumor microenvironment (1). Therefore, tumor infiltrating immune cells are considered accurate independent prognostic factors, superior to tumor node metastases (TNM) tumor stage (2). The most common subsets of tumor infiltrating lymphocytes (TILs) are clusters of CD3+, CD4+, CD8+, and Forkhead box P3+ (Foxp3+) T lymphocytes. CD4+ T lymphocytes were classified into Th1 and Th2 cells in the early 1980s, based on different T cell functions (3). Th1 cells promote the toxic effects of cytotoxic T lymphocytes (CTLs) and Th2 cells enhance the antibody-mediated humoral immune response. Recently, CD4+Foxp3+-expressing T lymphocytes were identified as regulatory T lymphocytes (Treg). Tregs play a role in suppressing the immune system and promoting cancer progression (4). Therefore, it is conceivable that several cancers are caused by chronic infection and inflammation, after acquiring various harmful genetic alterations. Chronic inflammation of the esophagus can increase incidence of esophageal carcinoma (5). Cirrhosis and inflammation of the liver can develop into hepatocellular carcinoma (6). Chronic pancreatitis increases the incidence rate of pancreatic cancer (7). Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC) have been linked to the development of colon adenocarcinoma (8). Since IL-32 expression is increased in inflammatory diseases, such as IBD and RA, and amplifies inflammatory cytokines, it is conceivable that IL-32 is strongly associated with various cancers related to inflammation.
Even though most infected individuals remain asymptomatic throughout their life (9), infection with
Several isoforms of IL-32 can be generated through alternative splicing (13, 14). The complete transcript that contains all exons is IL-32γ. IL-32γ mRNA can be spliced to produce shorter isoforms, including IL-32α, IL-32β, and IL-32δ (15–17). Other isoforms of IL-32 have not been well-studied yet. These isoforms show context-dependent function. For example, IL-32γ and IL-32β have been shown to induce caspase-8-dependent apoptosis in thyroid cancers (16), but not in breast cancers (18). Human gastritis tissue, as well as a gastric cancer cell line AGS cells expressed mostly IL-32β and marginally IL-32ɛ, without detection of other isoforms (11).
In addition to the possibility that IL-32β is associated with GC carcinogenesis, the role of IL-32 in GC progression and metastasis was also revealed by analysis of 120 patients diagnosed with GC (21). Cytoplasmic IL-32 expression was stronger in malignant stages of GC, and IL-32β was the major isoform found, with minor expressions of IL-32α and IL-32γ. Moreover, both IL-32 isoforms were highly expressed in patients with invaded serosal surface of the gastric walls and lymph node metastasis. This study supports the possibility that IL-32 could be a prognostic marker for GC. The underlying molecular mechanism of IL-32 on GC invasion has also been elucidated. Human GC TSGH9201 cells overexpressing IL-32γ showed increases in both intra- and extracellular levels of IL-32β, and augmented migration and invasion capacities. Furthermore, increased activation of AKT-β-catenin signaling pathway by IL-32β was accompanied by increased production of IL-8, vascular endothelial growth factor (VEGF), and active matrix metalloproteinase 2 and 9 (MMP2 and 9) (15). Clarification is needed regarding whether this IL-32β-mediated GC invasion is mediated by intra- or extracellular IL-32 or both. Although several studies showed the positive relationship between expression levels of IL-32 and metastasis and invasion of GC in patients (21, 22), one study showed that IL-32 had a non-significant relationship with the GC malignancy marker TNF-α in patients with GC (23). This discrepancy suggests that experiments with a larger GC patient population are needed in future studies.
In general, tumor infiltrating immunosuppressive cells, including tumor associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and Tregs are correlated with poor GC prognosis, whereas immune activator and effector cells, including CTL, dendritic cells (DCs), and CD45RO memory T cells are associated with an improved GC prognosis. Those immune cells affect the progression and malignancy of GC as they produce and secrete various immunoregulatory soluble factors into the tumor microenvironment. Chang
IL-32 is more highly expressed in inflamed lesions of chronic pancreas, compared to those in normal pancreatic duct cells. In addition, a strong expression of IL-32 has been observed in pancreatic cancer tissues. Pro-inflammatory cytokines IL-1, interferon γ (IFN-γ), and TNF-α stimulated IL-32 production in pancreatic cancer cell lines, including PANC-1, MIA PaCa-2, and BxPC-3 cells, which weakly express IL-32 without inflammatory stimulation. Activation of the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway was required to induce IL-32 expression by those cytokines. Inhibition of NF-κB and activated protein-1 (AP-1) signaling pathways markedly suppressed the IL-1, IFN-γ, and/or TNF-α-induced IL-32 mRNA expression (25). Collectively, IL-32 expression was induced by PI3K-Akt signaling pathway-dependent NF-κB-AP-1 signaling activation. Since small interfering IL-32 RNA (siIL-32) reduced pro-survival proteins (B cell lymphoma 2, Bcl-2; B cell lymphoma extra large, Bcl-xL; and myeloid cell leukemia sequence 1 protein, Mcl-1) without changing pro-apoptotic proteins (Bcl-2 associated X protein, Bax; Bcl-2 antagonist killer 1, Bak; BH3 interacting domain death agonist, Bid; Bcl-2 associated death promoter, Bad), IL-32 is likely to promote growth and survival of pancreatic cancer cells. On the other hand, a functional isoform of IL-32 in pancreatic cancer cell has not been reported. Therefore, further studies are needed to determine the isoform of IL-32 responsible for pro-survival effects in pancreatic cancer cells.
The risk of colorectal cancer (CRC) is positively proportional to the extent and duration of IBD, such as UC and CD (26). Since IL-32 is a pro-inflammatory cytokine and CRC is one of well-known inflammation-induced cancers, the effect of IL-32 on CRC was determined in an azoxymethane (AOM)-induced CRC model using human IL-32α transgenic (TG) mice. The expression of IL-32α showed protective effects on AOM-induced CRC incidence (27). The underlying mechanism was reported that the induction of TNF receptor 1 (TNFR1) expression by IL-32α in tumors, which causes apoptosis of tumor cells. Even though the IL-32 gene is not found in rodents, human IL-32α seems to be able to induce the apoptotic death of mouse tumor cells. This result suggests a role for human IL-32 in the mouse cells. The study also confirmed that TNF-α induced cell death in IL-32α-expressing human SW620 colon cancer cells. IL-32α induced sustained c-Jun N-terminal kinases (JNK) activation via reactive oxygen species (ROS) production, resulting in the apoptotic death of SW620 cells. A relationship between IL-32α and TNFR1 has also been suggested from studies of tumor tissue from CRC patients, where IL-32α and TNFR1 were co-expressed. Expression of IL-32α and TNFR1 increased until CRC reached stage II, and decreased in stage III and IV. This suggests that IL-32α exerts suppression of CRC growth at the initial stage of tumor progression. On the other hand, why and how IL-32α expression decreases as CRC stage progresses remains to be investigated. In a study by Yun
Hepatic fibrosis is initiated upon hepatocyte damage, followed by the recruitment of inflammatory cells and can further progress to chronic liver diseases and cirrhosis, and even to hepatocellular carcinoma (HCC) (33). Hepatic fibrosis is characterized by the progressive accumulation of extracellular matrix (ECM) secreted by hepatic stellate cells (HSCs), and the balance between ECM production and degradation, mediated by matrix metalloproteinases (MMPs), and endogenous inhibitors of metalloproteinases (TIMPs). IL-32 induces cytokines, including IL-1β, IL-6, IL-8, and TNF-α. (34, 35), which are highly increased in the liver with severe fibrosis (34, 36, 37). Moreover, recombinant IL-32γ treatment of LX-2 cells, an ECM-producing human hepatic stellate cell line, was enough to induce TIMP-1 expression by activation of the AP-1 signaling pathway (34). Moreover, the increase of TIMP-1 expression promoted the migration of LX-2 cells. In that study, the authors suggested that IL-32 might be a new therapeutic target for hepatic fibrosis as well as hepatitis, because hepatitis B virus (HBV) and hepatitis C virus (HCV) infections could induce IL-32 expression, as well as liver fibrosis in hepatocytes (38, 39).
IL-32α levels in the tumor region from patients with HCC were markedly higher than in the non-tumor region, and HCC patients also showed high levels of circulating IL-32α levels (40, 41). In tumor tissue, IL-32α localized in the cytoplasm and IL-32-expressing tumor cells were located at the invasion site of blood vessels. IL-32α stimulated cell survival and growth through NF-κB activation and maintenance of the Bcl-2 anti-apoptotic protein in the SK-Hep1, HCC cell line. Additionally, tumor promoting activity of IL-32α was confirmed using an HCC xenograft mouse model with various cell lines. One study investigated whether IL-32 would be an accurate marker for early diagnosis of HCC using 50 HCC patients and 15 healthy volunteers (42). The mean serum level of IL-32 was higher in patients with HCC than in the control subjects (P < 0.001), but did not correlate with survival rate. Those experimental data support IL-32 as a prospective diagnostic marker for HCC along with alpha-fetoprotein (AFT) and IL-18, but not as a prognostic marker. Further studies are needed to determine the levels of IL-32 in the sera of patients with HCC, cirrhosis, and stage-dependent HCC patients to validate the possible application of IL-32 as an early diagnosis marker for HCC. Recently, Kim
Initiation and progression in lung cancer, as in other cancers, are primarily driven by genetic alterations (44) and are strongly linked to inflammation. IL-32 expression is highly up-regulated in most adenocarcinomas, but not in squamous-cell carcinomas (SCC). IL-32–expressing TILs, mainly composed of CD68+ macrophages, CD4+ T lymphocytes, and dendritic cell (DC)-specific intracellular adhesion molecule-3-grabbing non-integrin DC (DC-SIGN+), and IL-32-expressing tumor cells are frequently observed in lymph node metastases, along with IL-6, IL-8, and VEGF (45, 46). Thus, it is likely that IL-32 significantly correlates with lung tumor progression stages, lymph node and distant metastases with poor prognosis (45). IL-32 expression is associated with acquisition of an invasive and metastatic phenotype of lung cancers. As an underlying mechanism, amplification of the loop of IL-32 and NF-κB is related with enhanced migration via up-regulation of MMP2/9. On the other hand, IL-32 polymorphisms (rs12934561 and rs28372698) are closely associated with poor survival in SCC, and with poor prognosis in moderate and well-differentiated lung cancer patients (47). On the other hand, Yun
We previously reported that IL-32β and IL-32γ mRNAs were highly increased in MDA-MB-231 malignant breast cancer cells, but only that IL-32β protein was detected by Western blotting (49), consistent with a previous report showing that IL-32γ mRNA splices into IL-32β, followed by mainly expression of IL-32β protein (15). The expression level of IL-32β was positively correlated with tumor volume, metastasis to lymph nodes, and tumor stage. MDA-MB-231 cells expressing IL-32β exhibited increased migration and invasion capacities, with increases in epithelial mesenchymal transition (EMT) markers, including vimentin and slug. As an underlying signal transduction mechanism, we showed that IL-32β enhanced migration and invasion through increase of soluble factor VEGF production by signal transducer and activator of transcription 3 (STAT3) activation. Later, Wang
The pro-inflammatory cytokines IL-1β and IL-18 are important amplifiers of the inflammatory process with IL-32. These cytokines are produced as pro-IL-1β and pro-IL-18 and inflammasomes facilitate maturation of pro-IL-1β and pro-IL-18 under infection and tissue damage conditions. Without inflammasome signals, pro-IL-1β is degraded through ubiquitination. Upon generation of an activation signal, it is de-ubiquitinated and processed into its mature form (51). IL-32β is another interleukin which is regulated by ubiquitination.
On the other hand, hypoxia-induced IL-32β interacts with protein kinase C δ (PKCδ) and this interaction results in the suppression of PKCδ-induced apoptosis (52). We also showed that the accumulated IL-32β translocates to the mitochondria under hypoxic conditions. To investigate the role of mitochondrial IL-32β, metabolic alteration was examined. The hypoxia-induced IL-32β enhanced glycolysis through activation of lactate dehydrogenase through the sustained activation of proto-oncogene tyrosine-protein kinase Src (50). This was the first report showing that IL-32β plays a role in the regulation of breast cancer cell metabolism. A recent report suggested that IL-32 is a prospective therapeutic target for triple negative breast cancer, since IL-32 was more highly expressed in cancer tissue from patients with triple negative breast cancer (53), implying a critical role of IL-32 in the progression of the breast cancer.
IL-32 was highly expressed in tissues showing classical morphological features of human papillomavirus (HPV) infection, including koilocytosis, acanthosis, and papillomatosis from cervical cancer patients, but not in tissues lacking HPV-associated nuclear atypia. However, IL-32 expression did not correlate with survival rates of cervical cancer patients. When HPV E7 oncoprotein was induced in cervical cancer cells, such as SiHa and C33A cells, IL-32 expression was induced by cyclooxygenase 2 (COX2), which is an enzyme producing inflammatory mediators, and suppression of COX2 failed to induce IL-32 by HPV E7 oncoprotein (54). Interestingly, IL-32γ overexpression inhibited E7 oncoprotein and COX2 expressions in SiHa and CaSki cells. These results imply that E7-induced IL-32γ suppresses E7 and COX2 expression by a negative feedback loop. In addition, Lee
IL-32 and COX2 expressions were immunohistochemically detected in 31 primary gastric B-cell lymphoma patients and 19 chronic gastritis patients, and were significantly higher in
Cutaneous T-cell lymphoma (CTCL) is characterized by clonal expansion of malignant T-cells, and the most common type of CTCL is mycosis fungoides (MF). MF progresses from flat erythematous patches to the tumor stage by expansion of malignant T-cells. An
IL-32 was originally identified as a cytokine produced and secreted from NK cells and NK cells are well known innate immune cells which display tumor-killing capacity. Thus, it is conceivable that IL-32 affects tumor growth by modulating NK cell activity. Indeed, Cheon
To determine a systemic role of IL-32, Yun
One study tested the possibility of whether IL-32 could be developed as an anti-tumor drug. To evaluate the anti-tumor effect of IL-32 gene therapy, CMS4 sarcoma-bearing Balb/c mice were established and they were received intratumoral injections of syngeneic DCs engineered to overexpress human IL-32β. IL-32β-overexpressing DCs more potently activated the type 1 T cell responses
It is well known that octamer-binding transcription factor 4 (OCT4) expression, one of stemness factors, is increased in cancer stem cell (CSC)-like cancer cells and enhances CSC-like characteristics, including sphere formation, cell colony formation, cell migration, invasiveness, and drug resistance. Patient-derived CRCs enforced to express OCT4 resulted in high expression of IL-8 and IL-32. Neutralization of either IL-8 or IL-32 in those CRCs partially inhibited the tumorigenic effects exerted by OCT4 overexpression. Altogether, these data indicate that IL-32 affects the regulation of CSC-like properties with IL-18, through both autocrine and paracrine manners (74). To furtherly expand on this concept, more patient-derived CRCs should be established because the study developed CRCs from only two patients. Future studies should be undertaken to determine whether OCT4 is a direct transcription factor inducing IL-32, and which IL-32 isoform is associated with induction by OCT4.
In an effort to identify a new growth factor which stimulates proliferation of hematopoietic progenitor cells (HPCs), Moldenhauer
In various tumor cells, IL-32 is highly increased and mainly located in the cytoplasm. Presently, IL-32β is considered the primary isoform in cancers. By thorough review of published studies on the biological roles of IL-32 in various cancers, we found several points that should be considered for further studies. First, whether the functional roles of IL-32 in cancer are dependent on cytosolic or extracellular IL-32 should be investigated. Second, it is important to elucidate the critical signal that allows IL-32 to act as a pro-tumor or anti-tumor cytokine. Third, it is important to determine whether IL-32 originates from stromal or tumor or immune cells to comprehensively understand the role of IL-32 in the formation of tumor microenvironments.
We especially thank Professor Soo Hyun Kim (Konkuk University, Seoul, Korea) for comments that greatly improved the manuscript. This work was supported by the National Research Foundation of Korea grant funded by the Korean government, MSIT (Ministry of Science and ICT: 2016R1A2B2011683 and 2016R1A6A3A11931083), and SRC (Science Research Center) program (Cellular Heterogeneity Research Center: 2016R1A5A1011974). We apologize for missing precious works, even though the authors tried to mention all published works.