The clinical significance of interleukin 24 and its potential molecular mechanism in laryngeal squamous cell carcinoma

1. Introduction

Laryngeal carcinoma is a common kind of head and neck carcinoma and ranks second in the incidence of head and neck carcinomas [1]. In 100,000 people, an average of nearly 6 have laryngeal carcinoma. The predominant type is laryngeal squamous cell carcinoma (LSCC) [2, 3, 4, 5, 6, 7], seen in 90 of 100 laryngeal carcinoma cases (90%). HPV and smoking are common causes of LSCC [5, 8, 9].

In clinical practice, early LSCC often manifests as chronic laryngitis or leukoplakia. And dysphonia is a major symptom in early LSCC, in particular those glottic LSCCs. With no obvious symptoms, the best time for treatment may be missed and LSCC may already be advanced in many cases. At present, endoscopies and biopsies are performed for diagnosis [10]. Surgery and radiotherapy are used for treatment in clinical work [11, 12, 13]. The potential risks related to LSCC surgery include loss of normal speech and/or swallowing, throat, larynx stenosis, and breathing difficulties. The disadvantages of radiotherapy are that treatment cycles are long and symptoms such as the loss of taste and smell as well as dry mouth may appear [14, 15].

The mechanism of LSCC is not clear. Therefore, screening out relative genes of LSCC and studying their molecular mechanism have great significance for early diagnosis and prognosis of LSCC. Interleukin 24 (IL24) is a newly-identified member of the IL-10 family (IL-10, 19, 20, 22, 24, 26) [16], which is located on human chromosome 1q32-33. Its signaling pathways have many biological functions related to cell differentiation, proliferation, development, apoptosis, and inflammation [17, 18]. Research has shown that IL24 is capable of inhibiting the growth of tumor cells while having no influence on normal cells. Studies have shown that IL24, produced by immune cells, can induce apoptosis of cancer cells in some respects such as inhibiting angiogenesis, inducing specific mitochondrial dysfunction in cancer cells, inducing autophagy of cancer cells, and so on [19, 20, 21, 22]. The authors of some studies have examined IL24 as a potential treatment for cancer such as oral squamous cell carcinoma (OSCC) [23], prostate cancer [24], and hepatocellular carcinoma [25, 26], among others, with consideration for its biological functions [16, 27].

However, few research reports related to the expression of IL24 in LSCC tissues are available. Hence, in this study, we explored the relationship between the occurrence and development of LSCC and IL24 expression from the molecular level and relating to clinical significance, which potentially has diagnostic ability and therapeutic significance in LSCC. The results of our study suggest that IL24 may be used as a prospective diagnostic indicator because its increased expression may indicate the occurrence of cancer and cause immune resistance.

3. Results

In this study, we found for the first time that the mRNA and protein levels of IL24 are both upregulated in laryngeal squamous cell carcinoma tissues as compared to non-cancerous tissues. Multiple detecting methods were used to verify the finding, including immunohistochemistry with clinical samples, gene chips, RNA-sequencing, and an integrated meta-analysis. More importantly, we also observed that the co-expressed genes of IL24 were mostly enriched in the pathways related to the extracellular matrix, which may assist with explaining its clinical significance.

LSCC is a common subgroup of HNSCC. At present, the main well-documented carcinogenic factors of the disease are HPV, smoking, and consuming alcohol [28, 29, 30, 31]. Due to the lack of clinical features of early LSCC, about 60% of patients are diagnosed as having advanced cancer and the five-year survival rate of LSCC is declining [32, 33]. So early diagnosis and timely treatment of LSCC are particularly important.

IL24 is a newly-identified member of the IL-10 family [16]. Research has shown that IL24 produced by immune cells is capable of inhibiting the growth of tumor cells by different ways, such as inhibiting angiogenesis, inducing specific mitochondrial dysfunction in cancer cells, inducing autophagy of cancer cells, and so on [19, 20, 21]. For example, recombinant IL24 could synergize with cisplatin to suppress the angiogenesis by down-regulating the expressions of VEGF (vascular endothelial growth factor), VEGF-C and PDGF-B (Platelet-derived growth factor) [34]. Recombinant sIL24 (smallest isoform of IL24) peptide could mediate the apoptosis of cancer cells by regulating Bax-2 and Bcl-2 [22]. A hybrid gene delivery vector called AdLTR2EF1 α -based vector was used to transfer IL-24 (AdLTR2EF1 α -IL-24) to treat a human oral squamous cell carcinoma cell line KB and immortal human keratinocyte cell line HaCaT [23]. This AdLTR2EF1 α -IL-24 treatment could lead to autophagy, which was interrupted by autophagy inhibitor 3-MA [23]. Similar effect of IL24 was reported [35], that a viral vector expressing mda-7/IL-24 (Ad. IL-24) infection could reduce miR-221, which subsequently increased toxic autophagy in MDA-MB-231 breast cancer cells [35]. Besides the direct effect on the cellular function of tumor, IL24 has also been reported to own therapeutically potential to treat various tumor cells [20, 36], including melanoma, carcinomas of breast, liver, prostate, ovarian, and nasopharyngeal [37]. For instance, the combination of oncolytic adenovirus armed with IL24 (ZD55-IL24) and docetaxel (DTX) showed synergistic inhibitory effects on prostate cancer cell viability and invasion [38]. Lentivirus-mediated IL24 expression was found to inhibit hepatocellular carcinoma (HCC) cells proliferation and colony formation [39]. Adenovirus-mediated human interleukin 24 (Ad-hIL24) transfection reduced cisplatin resistance in lung carcinoma cell line A549/DDP [40]. Thus, IL24 itself, or combined with other therapeutic methods, could become a target for the treatment of malignant tumors. There were also some similar studies that explored the treatment of human laryngeal cancer with adenovirus-mediated IL24 (Ad-IL24) in vivo and in vitro. IL24 (Ad-IL24) could reduce the expression of a proangiogenic factor: vascular endothelial growth factor which was involved in the inhibition of tumor angiogenesis and thus led to apoptosis of laryngeal cancer cells via intrinsic apoptotic pathways. Ad-IL24 had a strong selective killing activity in laryngeal cancer cells and was a potential candidate for laryngeal cancer gene therapy [41, 42]. But there have been no studies of the relationships between IL24 expression and LSCC in the protein or mRNA expression level so far, the purpose of this study was to explore the clinical significance and molecular mechanism of IL24 expression in LSCC, also to provide potential indicator for the prediction of prognosis and treatment effect of LSCC.

First, we mainly evaluated the clinical significance of IL24 in LSCC from the protein expression level and the mRNA expression level, respectively. In the protein expression level, a total of 49 LSCC samples and 26 normal samples were collected for immunohistochemical staining. The results showed that the IL24 protein expression was significantly higher in LSCC tissues than that in the controls ( 0.0001). In the mRNA expression level, by mining the data from TCGA, GEO, SRA, ArrayExpress, and Oncomine databases and retrieving a large number of references, we obtained four microarrays and an RNA-Seq dataset that met the inclusion criteria of this study. A total of 172 LSCC and 81 non-LSCC cases were achieved to compare the IL24 mRNA expression. IL24 was consistently highly expressed ( 0.0001) in LSCC. Furthermore, up-regulation of IL24 level had good diagnostic significance for distinguishing cancer from non-cancer patients by use of the sROC curve [AUC = 0.89 (95% CI = 0.86–0.92)]. IL24 is produced by a variety of immune cells, such as myeloid cells, lymphocytes, and monocytes, in response to treatment with lipopolysaccha-rides inhibitors or specific cytokines. In Th2 lymphocytes and T cells, in particular CD4 + naive cells and memory cells activated by anti-CD3 monoclonal antibody, IL24 is caused by stimulation by phorbol myristate acetate and ionomycin. In monocytes, IL24 is induced by antigenic stimulation of lipopolysaccharide, concanavalin A and cytokines. In B-lymphocytes, IL24 expression can also be triggered by stimulating B-cell receptor signaling. In addition, nonlymphoid cells can also express IL24 by responding to cytokines secreted by immune cells [16, 21]. When cancer occurs, the body may protect itself by expressing IL24 and producing related cytokines. Therefore, IL24 was determined to be highly expressed at both the mRNA and protein levels in some LSCC patients; this could provide a potential reference for the screening of LSCC. We also analyzed the clinical role of IL24 expression in LSCC by using independent samples t-tests for clinicopathological factors such as age, sex, grades, stages, etc. With statistically significant difference, we found in these clinical parameters, the IL24 protein expression tended to be higher in LSCC patients who were in the stage III–IV ( P = 0.007) or pathologic stage N1 ( P = 0.044). It is interesting to observe that IL-24 expression in LSCC tissue was enhanced in advanced stages, which contracted its inhibiting cancer capacity in other reports. Consistent reports from literature could partially explain the enhancing effect of IL-24 in LSCC development. Treatment of human endothelial HECV cell with recombinant human interleukin IL-24 (rhIL-24) could boost cell migration [43]. It is possible that IL-24 can also promote the LSCC cell migration and cause tumor progression into more advanced clinical stage. Another example was in cutaneous squamous cell carcinoma (CSCC). Remarkably increased expression level of IL-24 was reported in invasive CSCC as compared to non-invasive ones, and the authors proposed a probable involvement of IL-24 to CSCC invasion via promoting focal expression of MMP7 [44]. Our current data also provide evidence that IL-24 may play a pivotal role in the metastasis of LSCC cells to lymph node, so as to accelerate LSCC to advanced stage. Survival analysis showed that LSCC patients with low IL24 expression might have a poor prognosis according to Kaplan-Meier estimator, but these were not statistically significant [P (log-rank) = 0.5743]. The prognostic value of IL24 in LSCC remains to be first investigated with larger sample size. To summarize the above findings, IL24 may show higher level in the LSCC tissues, and it increases when the tumor is progressing.

In order to comprehensively and systematically explore the molecular mechanism of IL24, we not only calculated the IL24-related genes but also screened the differentially-expressed genes in LSCC by the microarrays and RNA-Seq datasets mentioned above. Then we took the intersection of the two because these intersection genes could more accurately illustrate the molecular mechanism of the link between IL24 expression and LSCC. The intersection genes were then used for KEGG pathway enrichment analysis and GO annotation. In the GO annotation, the top terms of BP, CC, and MP were extracellular matrix (ECM) organization (GO: 0030198), extracellular matrix (GO: 0031012), and cytokine activity (GO: 0005125), respectively. The top three items of KEGG pathway enrichment were ECM-receptor interaction (hsa04512), rheumatoid arthritis (hsa05323), and hematopoietic cell lineage (hsa04640). The GO and KEGG analyses indicated that the pathways related to extracellular matrix was the most powerful one for the biological function of IL24. As a crucial component of the cancer cell niche, the ECM provides the mechanical support for the tissue and mediates the cell-microenvironment interactions. Significantly, collagens which have been implicated in many aspects of neoplastic transformation, are one of the major proteins found within the ECM [45, 46], so ECM pathways play a key role in tumor shedding, adhesion, degradation, metastasis and proliferation [47]. Thus, the up-regulation of IL24 could play its accelerating role in the development of LSCC related to ECM.

Some well-known oncogenic or tumor suppressive genes may work together with IL24 by their interactions. The PPI network indicated that the hub genes of the co-expressed genes were SERPINE1 (serpin family E member 1), TGFB1 (transforming growth factor beta 1), MMP1 (matrix metallopeptidase 1), MMP3 (matrix metallopeptidase 3), CSF2 (colony stimulating factor 2), and ITGA5 (integrin subunit alpha 5). Among these hub genes, SERPINA1 has been reported with high abundance in saliva of patients with oral squamous cell carcinoma (OSCC), suggesting that SERPINA1 may be related to the occurrence of OSCC [48]. As OSCC and LSCC both originate from squamous cells, SERPINA1 may play consistent function in LSCC. The expression of TGFB1 was related to the pathological grade and clinical stage of LSCC [49]. Furthermore, inhibition of MMP1 could reduce the invasion ability of HNSCC cells [50]. Hence, the interactions between IL24 and SERPINA1, TGFB1 and MMP1 may exist in the progress of LSCC. However, due to the lack of direct evidence of the functions of the other three hub genes MMP3, CSF2, and ITGA5, their relationships with IL24 in LSCC remains to be explored.

It can be argued that IL24 is an immunomodulatory cytokine and an anti-tumor molecule with a wide range of activities including tumor-specific induction of apoptosis and inhibition of tumor angiogenesis. IL24 can be used as a tumor treatment drug to induce apoptosis, enhance the activation and function of T cells, and play a certain adjuvant role in the chemotherapy of many tumors. This also provides a new research idea for future clinical trials of IL24 as an adjuvant therapy for LSCC. In conclusion, our study displays that high expression IL24 has a certain relationship with the occurrence and progression of LSCC, which has potential diagnostic ability and therapeutic significance in LSCC.