Interleukin‐36α suppresses growth of non‐small cell lung cancer in vitro by reducing angiogenesis

Interleukin (IL)‐36α, a newly recognized IL‐1 family member, has been previously reported to play a pivotal role in autoimmunity diseases and acute inflammatory reactions. Recently, several studies have indicated that IL‐36α has potential anticancer effects against certain types of cancer. However, the expression pattern and functional role of IL‐36α in non‐small cell lung cancer (NSCLC) have not been elucidated. Here, we report that the mRNA and protein levels of IL‐36α are significantly reduced in NSCLC tissues. Low levels of intratumoral IL‐36α are correlated with higher tumor status, advanced TNM stage, increased vascular invasion and shorter overall survival (OS). Intratumoral IL‐36α expression is an independent prognostic factor for OS (hazard ratio = 3.081; P = 0.012) in patients with NSCLC. Overexpression of IL‐36α in lung cancer cells did not disturb cell proliferation, apoptosis or cell‐cycle distribution in vitro, but markedly inhibited tumor growth in vivo. Mechanistically, IL‐36α reduced the expression and secretion of vascular endothelial growth factor A through inhibiting hypoxia‐inducible factor 1α expression. Finally, decreased IL‐36α expression was associated with high microvessel density and vascular endothelial growth factor A in patients with NSCLC. Together, our findings suggest that IL‐36α expression is a valuable marker indicating poor prognosis in patients with NSCLC.

Interleukin (IL)-36a, a newly recognized IL-1 family member, has been previously reported to play a pivotal role in autoimmunity diseases and acute inflammatory reactions. Recently, several studies have indicated that IL-36a has potential anticancer effects against certain types of cancer. However, the expression pattern and functional role of IL-36a in non-small cell lung cancer (NSCLC) have not been elucidated. Here, we report that the mRNA and protein levels of IL-36a are significantly reduced in NSCLC tissues. Low levels of intratumoral IL-36a are correlated with higher tumor status, advanced TNM stage, increased vascular invasion and shorter overall survival (OS). Intratumoral IL-36a expression is an independent prognostic factor for OS (hazard ratio = 3.081; P = 0.012) in patients with NSCLC. Overexpression of IL-36a in lung cancer cells did not disturb cell proliferation, apoptosis or cell-cycle distribution in vitro, but markedly inhibited tumor growth in vivo. Mechanistically, IL-36a reduced the expression and secretion of vascular endothelial growth factor A through inhibiting hypoxia-inducible factor 1a expression. Finally, decreased IL-36a expression was associated with high microvessel density and vascular endothelial growth factor A in patients with NSCLC. Together, our findings suggest that IL-36a expression is a valuable marker indicating poor prognosis in patients with NSCLC.
Lung cancer is the leading cause of cancer-related mortality not only in China but also around the world. Non-small cell lung cancer (NSCLC) covers about 70-80% of all cases and includes specific pathological subtypes, such as squamous and adenocarcinoma cell carcinoma [1]. Despite significant advances in surgical resection, chemotherapy, radiotherapy, targeted therapy and novel immunotherapy, such as nivolumab (Opdivo) and pembrolizumab (Keytruda), the therapeutic effects are limited, and some patients with NSCLC experience cancer recurrence and metastasis [1,2]. Therefore, exploring and developing new agents or therapeutic strategies for patients with NSCLC are critically important and urgent.
In this study, we evaluated the expression pattern and the clinical significance of IL-36a in patients with NSCLC. Moreover, we also investigated the anticancer efficiency of IL-36a and its possible mechanisms in lung cancer cells.

Patients and specimens
Ninety-one patients diagnosed with NSCLC who underwent surgery between 2014 and 2016 at Department of Thoracic Surgery, West China Hospital of Sichuan University were recruited into this study. None of them received any anticancer therapy prior to surgery. The clinicopathological characteristics, including histological types, differentiation status, smoking status and tumor TNM stages, were recorded and shown in Table 1. Tumor stages were determined by TNM classification according to the 2009 International Union Against Cancer guidelines. The histological diagnosis and grade of differentiation of the tumors were defined by evaluation of the hematoxylin and eosin-stained tissue sections, according to the World Health Organization guidelines of classification (2009). This study was approved by the Ethics Committee of West China Hospital of Sichuan University. Written informed consent was obtained from all patients. The use of human samples complied with the standards stipulated in the Declaration of Helsinki. Tissue specimens were cut into tumor tissues and adjacent normal lung tissues (≥5 cm away from the tumor). Half of them were immediately flash frozen in liquid nitrogen for RNA and protein extraction; the remainder was fixed with formalin for immunohistochemistry (IHC).
The staining patterns of IL-36a and VEGFA were scored based on the intensity and the percentage of positively stained cells. The intensity of the staining was scored as follows: 0 (no staining), 1 (weak staining), 2 (moderate staining) and 3 (strong staining), respectively. The staining extent was scored as follows: 0 (<10%), 1 (10-25%), 2 (25-50%) and 3 (>50%). The final score was calculated using the percentage of positive cells 9 staining intensity, ranging between 0 and 9. Total score ≥4 was defined as high expression, and a score of 0-3 was defined as low expression. Microvessel density (MVD) was recorded by counting CD34-positive endothelial cells [15].

Cell viability, apoptosis and cell-cycle assay
Overexpressing IL-36a-transfected cells, mock-transfected and normal cells were seeded into 96-well plates and cultured for the indicated time. For cell viability assay, 10 lL Cell Counting Kit-8 (CCK-8) solution was added into the culture medium in each well, and absorbance (A) values were read using a microplate reader (Bio-Tek Company, Winooski, VT, USA) after 2-h incubation. For cell apoptosis assay, these cells were collected, washed twice with PBS and stained with Annexin V-FITC (5 lL) and propidium iodide (5 lL) in 500 lL binding buffer for 15 min at room temperature. For cell-cycle assay, these cells were harvested and washed in cold PBS, fixed in 70% cold alcohol for 6 h at 4°C and then stained with propidium iodide solution for 30 min at 4°C. Cells in apoptosis and cell-cycle assays were analyzed immediately by flow cytometry (BD FACSCanto; BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo software (FlowJo, Ashland, OR, USA).

Western blot analysis
Total protein from NSCLC tissues or cell lines was lysed using RIPA buffer with protease inhibitor (Sigma, USA). A total of 20 lg total protein was separated by 10% SDS/ PAGE, transferred onto polyvinylidene fluoride membranes and incubated with rabbit anti-(human IL-36a) IgG

In vivo xenograft model
For evaluation of the tumor growth in vivo, overexpressing IL-36a-transfected H1299 cells (2 9 10 6 ) or A549 cells (3 9 10 6 ) or related mock-transfected cells were injected subcutaneously into the flank region of nude mice (6-8 weeks, female; Charles River Laboratories, Beijing, China). Tumor growth was monitored every week, and tumor volume was measured with digital calipers and was calculated by the following formula: tumor volume = 0.5 9 width 2 9 length. After 4 weeks of tumor inoculation, tumor-bearing mice were sacrificed. The xenografts were extracted, cut into 2-mm 3 cubes, fixed in 10% formalin, and embedded in paraffin. CD34-and VEGFA-positive cells were detected using IHC as described earlier. The animal study was approved by the Research Ethics Committee of West China Hospital of Sichuan University. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health.

Statistical analysis
Statistical analyses were performed using the SPSS-PC package (version 21.0; SPSS, Chicago, IL, USA). The data were expressed as mean AE standard error of the mean. The v 2 test was used to analyze the association between IL-36a expression and clinicopathological variables. Overall survival (OS) was defined as the interval between surgery and death. Kaplan-Meier survival curve was plotted for the analysis of survival rates with log rank test. A multivariate Cox proportional hazards regression model was used to identify independent prognostic factors. A Student's t-test was performed to analyze differences between two groups. One-way ANOVA was performed for three more groups. P < 0.05 was considered statistically significant.

Results
Decreased IL-36a expression is associated with poor prognosis in patients with NSCLC Decreased IL-36a expression has been previously observed in HCC [12], colorectal cancer [13] and EOC [14]. However, its expression in lung cancer remains undecided. Here, we found that the mRNA and protein levels of IL-36a in NSCLC tissues were significantly decreased compared with corresponding normal tissues (Fig. 1A,B). IHC staining further confirmed that IL-36a was mainly located in normal lung tissues and was restricted to the cytoplasm (Fig. 1C). In addition, through IHC score, we found that IL-36a expression in the cytoplasm was significantly lower in NSCLC tissues. As shown in Fig. 1D, the low expression of IL-36a was covered 39 of 91 NSCLC tissues (42.8%), while high expression of IL-36a was covered in 69 of 91 normal tissues (75.8%) (P < 0.01).
To investigate the prognostic value of IL-36a in patients with NSCLC, we assessed OS for patients with high or low IL-36a expression by Kaplan-Meier survival analysis. The results indicated that patients with NSCLC with lower cytoplasmic IL-36a expression had significantly worse OS than those with high IL-36a expression (P = 0.016, log rank test; Fig. 1E). Low IL-36a expression was found to be significantly correlated with higher tumor status (P = 0.012), advanced TNM stage (P = 0.006) and vascular invasion (P = 0.019) ( Table 1). Moreover, univariate and multivariate Cox regression analysis revealed that cytoplasmic IL-36a expression was an independent prognostic factor for OS (hazard ratio = 3.081; 95% confidence interval, 1.231-3.992; P = 0.012; Table 2).

IL-36a suppresses NSCLC growth in vivo
Based on the relationship between IL-36a and NSCLC mentioned earlier, we next explored the functional role of IL-36a in lung cancer. First, we demonstrated that the expression levels of IL-36a in NSCLS cell lines (H1299, A549, H1975) were decreased compared with normal lung bronchial epithelial cells ( Fig. 2A). We then chose the H1299 cell line and transfected overexpressing IL-36a in H1299 cells. The overexpression of IL-36a was validated by qPCR, immunoblotting and ELISA (Fig. 2B,C). Through a series of analyses including CCK-8 assay, apoptosis and cell-cycle detection, we found that there were no significant differences in the cell proliferation, apoptosis induction and cell-cycle arrest between overexpressing IL-36a-transfected H1299 cells and mock-transfected H1299 cells (Fig. 2D-F). Notably, we also observed no effect on cell proliferation by rhIL-36a treatment in vitro (Fig. 2G), suggesting that IL-36a might not directly affect tumor growth in vitro. To further confirm our findings, we used another cell line, A549, to repeat the in vitro experiments. The data showed that overexpression of IL-36a in A549 cells also had no effect on cell proliferation and apoptosis in vitro (Fig. 2H,I and Fig. S1A,B).
Our findings demonstrate that overexpression of IL-36a did not affect cell growth of NSCLC in vitro. The To explore the role of endogenous IL-36a in the growth of NSCLC cells, we knocked down the endogenous IL-36a in H1299 cells by siRNA (Fig. S1C,D). Functional assay showed that knockdown of IL-36a also had no obvious effect on tumor proliferation and apoptosis in vitro (Fig. 2J,K). Further, we elucidated the effect of IL-36a on NSCLC-bearing mice in vivo. As shown in Fig. 2L,M, the xenografts in overexpressing IL-36a-transfected H1299 cell-bearing mice grew more slowly than the mock-transfected and normal control groups. Moreover, the tumor weights of the overexpressing IL-36a-transfected xenografts were markedly smaller than the mocktransfected and normal control groups (Fig. 2N). Similar results were observed using A549 cells with overexpressed IL-36a in vivo (Fig. 2O,P). Collectively, these results indicate that the anticancer effects of IL-36a might be closely involved with the tumor growth in vivo.

IL-36a suppressed tumor angiogenesis through inhibiting HIF-1a-VEGFA signaling
Angiogenesis contributes to tumor growth, and VEGFA is a potent inducer of angiogenesis in vivo  [16]. We speculate that IL-36a-suppressed tumor growth of NSCLC in vivo might be associated with reducing angiogenesis. As expected, we found that overexpressing IL-36a-transfected xenografts had lower CD34 expression, which indicates the MVD and VEGFA expression compared with mock-transfected and normal control groups (Fig. 3A,B). Moreover, we found that VEGFA mRNA and protein levels were also significantly reduced in overexpressing IL-36atransfected H1299 xenografts and A549 xenografts,  (Fig. 3C-F). Furthermore, we found that rhIL-36a treatment could significantly inhibit HUVEC growth in a time-and dose-dependent manner (Fig. 3G,H) and repress the formation of vessel-like tubes in a dose-dependent manner (Fig. 3I). Importantly, these effects could be abolished by adding VEGFA protein (Fig. 3J,K), suggesting that IL-36a suppresses tumor angiogenesis by regulating VEGFA expression. IL-36a binds to the IL-36 receptor and initiates signal transduction and activation through NF-jB and MAPK pathways [17]. However, we found that the expressions of phosphorylated (p)-p65 and p-p38 MAPK were not changed after IL-36a treatment in HUVECs (Fig. 3L,M), suggesting that IL-36a inhibits angiogenesis in a NF-jB and/or MAPK signaling-independent manner. HIF-1a is a critical transcription factor that regulates VEGFA expression [18]. We found that the mRNA and protein levels of HIF-1a were significantly reduced after IL-36a treatment in HUVECs (Fig. 3N,O), indicating that IL-36a suppresses VEGFA production by down-regulation of HIF-1a expression.

Decreased IL-36a expression is associated with high MVD and VEGFA in patients with NSCLC
To further confirm the functional role between IL-36a and angiogenesis in patients with NSCLC, we analyzed 91 NSCLC tissues through IHC staining with CD34 and VEGFA. The results demonstrated that those patients with low IL-36a expression exhibited higher MVD levels (Fig. 4A,B), as well as higher VEGFA expression (Fig. 4A,C), compared with patients with high IL-36a expression, suggesting that IL-36a expression might be negatively associated with MVD and VEGFA levels in patients with NSCLS.

Discussion
Recent reports demonstrate that IL-36a exerts potential anticancer function in certain types of cancer, including HCC, colorectal cancer and EOC. However, there is little knowledge about the relationship between IL-36a and NSCLC. Here, we provided the evidence that IL-36a might be a poor prognosis marker for patients with NSCLC and play potent anticancer efficiency through suppressing tumor angiogenesis.
In this study, we first investigated the expression pattern of IL-36a in NSCLC tissues of patients and analyzed its clinical significance based on the supported data, including histological types, tumor status and TNM stage. Our results demonstrated that IL-36a, located in cytoplasm, was mainly expressed in nontumor tissues and was decreased in NSCLC tissues. In addition, reduced IL-36a expression was observed to be remarkably negatively associated with higher tumor status, advanced TNM stage and vascular invasion. Patients with low cytoplasmic IL-36a expression were associated with poor overall prognosis. Our findings are consistent with prior studies, where IL-36a expression was decreased and closely associated with tumor progression in HCC [12], colorectal cancer [13] and EOC [14].
Next, the functional role of IL-36a on lung cancer cells was explored. As expected, the expression levels of IL-36a were decreased in NSCLC cells compared with the normal lung bronchial epithelial cells, which was similar with our previous results verified in NSCLC tissues. Through transfecting overexpressing IL-36a in lung cancer cells, we did not observe significant effects of IL-36a on the cell proliferation, apoptosis induction and cell-cycle arrest. Indeed, in HCC, Pan et al. [12] also clarified that there was no significant difference of cell proliferation in vitro between overexpressing IL-36a-transfected HepG2 cells and the control vector. On the contrary, Chang et al. [14] reported that overexpressing IL-36a suppressed proliferation of EOC cells in vitro, which indicated that the functional role of IL-36a might be of difference in diverse types of tumors. Interestingly, in lung cancerbearing mice, the growth of overexpressing IL-36atransfected xenografts was suppressed obviously compared with the mock-transfected and control groups. Based on our previous results mentioned earlier, we speculate that IL-36a-inhibited tumor growth is possibly associated with the regulation of the tumor microenvironment.
VEGFA is one of the most potent proangiogenic factors, which is closely involved with angiogenesis in the tumor microenvironment and significantly promotes tumor growth [19]. In our next study, through IHC staining, we found that IL-36a was negatively correlated with MVD and VEGFA expression in tumor tissues of lung cancer-bearing mice and NSCLC tissues. In addition, in overexpressing IL-36a-transfected H1299 cells, the expression and secretion of VEGFA had decreased. This effect was further verified by the time-or dose-dependent proliferation and formation of vessel-like tubes of HUVECs in vitro treated with rhIL-36a, indicating that IL-36a regulated tumor angiogenesis by targeting VEGFA.  The protein levels of HIF-1a in HUVECs treated with rhIL-36a. Data shown are mean AE SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with PBS; # P < 0.05, ## P < 0.01, compared with rhIL-36a + VEGFA.

Conclusion
In summary, our findings demonstrate that IL-36a expression is associated with poor prognosis in patients with NSCLC, and IL-36a exerts potent anticancer efficiency in NSCLC involved with, or partly, reducing tumor angiogenesis via inhibiting VEGFA expression. Therefore, IL-36a might be a valuable prognostic marker and therapeutic target for patients with NSCLC.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article.