Background
Colorectal cancer (CRC) is still the third most common cancer, particularly in Western society, despite decades of extensive clinical and basic research [
1]. The incidence in China is also rising, partly due to modernisation and/or industrialisation in food processing, which modifies traditional Chinese food preparations [
2]. The largest clinical challenge is the delay in early detection [
3], compromising the outcomes of CRC patients who consequently may have to be managed with palliative care [
4]. Understanding underlying mechanisms involved in the development of CRC would be beneficial to improve the diagnosis and outcomes of CRC patients.
It is well known that host immunity is critical in the development of cancer(s), for example, the discovery of cancer therapy by inhibition of negative immune regulation [
5].
IL-36α, β and γ (formerly IL-1F6, IL-1F8, and IL-1F9) are IL-1 family members that signal through the IL-1 receptor, i.e. IL-1Rrp2 (IL-1RL2) and IL-1RAcP [
6], via activating the nuclear factor kappa B (NF-κB), Mitogen-activated protein kinase (MAPKs), Jun N-terminal kinases (JNK), and ERK1/2 kinase cascades [
7], which are key signalling pathways for intestinal tumorigenesis [
8] [
9]. The IL-36 isotypes bind to the IL-36 receptor (IL1RL2/IL-1Rrp2/IL-36 receptor dimer) with varying affinities. IL-36 agonists induce various proinflammatory mediators [
6] via activating NF-κB and MAPKs. IL-36 is derived from keratinocytes, human monocytes and myeloid dendritic cells (DCs) [
10]. IL-36 plays an important role in autoimmune diseases, including psoriatic arthritis, systemic lupus erythematosus and Sjogren’s syndrome [
11]. In addition, IL-36 is important in the inflammation of colonic mucosa and promotes inflammation during intestinal diseases, suggesting that IL-36 may be an important therapeutic target for the management of intestinal abnormalities [
11]. Furthermore, IL-36 gene therapy may mediate a therapeutic effect in a fibrosarcoma mouse model [
12].
The relationship between CRC and IL-36α has been reported previously, showing that high colonic production of IL-36α is beneficial for survival of CRC patients [
13]. Interestingly, no non-cancer tissue was included for comparison during the investigation of IL-36α by this research team [
13]. Additionally Weinstein et al investigated the relationship between IL-36γ and tertiary lymphoid structure and inflammatory immunity in CRC [
14], showing that IL-36γ plays a physiological role in the colon, enhancing the development of CRC via inflammation in the tumour microenvironment. However, the relationship between both IL-36β and IL-36γ and clinicopathological factors in CRC has not been examined.
Therefore, it is of great interest to determine whether there is a correlation among these three IL-36 s (IL-36α, IL-36β and IL-36γ) in terms of clinicopathological outcomes in CRC, using univariate and multivariate analysis. In the current study, we substantially increased the number of CRC patients investigated and used objective computerised automated quantification to determine the production of all the colonic IL-36 s. In addition, we have explored the correlation of IL-36α, IL-36β or IL-36γ production in the CRC patients, comparing cancer versus non-cancer tissues, and the relationship between IL-36α, IL-36β and IL-36γ in the CRC patients. Such data may be useful for both basic research as well as for clinical practice.
Discussion
In the current study we demonstrated that colonic IL-36α, IL-36β and IL-36γ were substantially reduced in CRC compared to that of the paired non-CRC tissues. The sensitivity versus specificity of IL-36α, IL-36β and IL-36γ production were determined using ROC curve analysis [
28] and were found to be predictive of the presence of cancer, yielding AUC values of 0.68, 0.73 and 0.65 for IL-36α, IL-36β or IL-36γ, respectively. Although these values were statistically significant, they are not sufficiently high to be used as reliable biomarkers for colorectal cancer.
Survival rate is an objective indicator for evaluating postoperative CRC prognosis [
29]. Based on our current data, IL-36α
high CRC patients have a better survival than IL-36α
low CRC patients. Additionally, IL-36α is an independent factor affecting the survival of CRC patients on multi-variate analysis. This is supported by the finding that IL-36α may exhibit anti-tumour effects in CRC progression [
13], perhaps via activating adaptive T cell immune responses and recruiting CD3
+ and CD8
+ tumour infiltrating lymphocytes (TILs) [
30].
Our survival data from multivariate analysis for IL-36α production is supported by Wang et al. [
13]. However, Wang et al.
, determined colonic IL-36α production only in CRC tissue from CRC patients, without using adjacent non-cancer tissue as a control. In addition, the classification of IL-36α as either high or low was based on a naked eye scoring system. On the other hand, our quantification was performed objectively using computerised software (ImagePro Plus 9.1), which is routinely used in our research group [
21], in addition to the comparison with non-cancer paired colonic tissues. Thus, our rigorous data are probably more convincing and reliable.
Additionally, we observed that CRC patients within the colonic IL-36γ
low production group had a better survival rate than IL-36γ
high. It has been reported that IL-36γ is mainly produced by M1 macrophages in the CRC tumour micro-environment [
14], which may contribute to tumoricidal effects [
31]. Our speculation is that disturbance of differentiation of macrophages may be involve in the development of CRC, which will be clarified in future investigations. We speculate that there are differential regulatory roles of IL-36 during the development of CRC. Our hypothesis is supported by others, showing the distinct expressions of IL-36α, IL-36β, and IL-36γ, and their antagonist (IL-36Ra) in autoimmune disease in human and animal models [
32,
33]. The precise underlying mechanism of these differential roles of IL-36 s remains to be explored in our future experiment.
On the other hand, surprisingly, there was no statistically significant difference in survival rate between IL-36β
high and IL-36β
low production groups in CRC patients, suggesting that IL-36β may not be a good marker for predicting prognosis of CRC. However, our observation that colonic mucosal IL-36β was ~ 80% reduced in CRC tissue suggests that IL-36β may participate in inhibiting the development of CRC. Interestingly, IL-36α and IL-36γ, but not IL-36β, are upregulated at the molecular and cellular levels in inflammatory bowel disease (IBD) [
34], although IL-36α, IL-36β and IL-36γ are all pro-inflammatory cytokines [
11]. This observation from Nishida et al. suggests that only IL-36α and IL-36γ, but not IL-36β, contribute to the pathogenesis of intestinal inflammation. The discrepancy in IL-36β production between Nishida, in intestinal inflammation where little change was observed, and our current findings in CRC where a substantial reduction in IL-36β was observed, may be due to the different severity of the diseases and/or different pathogenesis. Nevertheless, based on the observation from Nishida et al. and ourselves, we speculate that, to contribute to pathogenesis, the production of IL-36β may require substantial alteration, i.e. a moderate to minor change in IL-36β in IBD, but a substantial change in CRC. The precise mechanism of IL-36β in oncogenesis, particularly in the development of CRC, will be further investigated, particularly its signalling pathways.
Interestingly, a stratification into sub-groups using a combination of IL-36α plus IL-36γ production provided a better prognostic outcome than when IL-36α or IL-36γ production only was evaluated in CRC patients. Specifically, a larger and more distinct difference was observed between the sub-group containing IL-36αhigh plus IL-36γlow patients compared to the sub-group containing IL-36αlow plus IL-36γhigh patients, when evaluated using survival curves. Such an observation might be useful in clinical decision making in the management of CRC patients.
It has been reported that 4.5 cm is the optimal cut-off value for the whole colon, receiver-operator characteristic (ROC) analysis has been applied to different parts of the large bowel, and has determined the following cut-off values of 5 cm, 5.3 cm, 3.9 cm, and 3.4 cm have the strongest discriminatory capacity for the whole colon, right-sided colon, left-sided colon, and rectal cancers, respectively [
35]. Thus, a 5 cm cut-off is a good size to utilise. We will, of course, use such defined location sizing for our future studies.
We are wary that the different cut offs that have been chosen may influence the statistical significance of the results. Thus, the interpretation of our current study should be cautious. More highly powered studies should serve to clarify this potential issue.
A recent paper on TNM staging in CRC questioned the validity of full TNM staging as a prognostic indicator, rather suggesting that simple evaluation of mesenteric node spread was more highly predictive. Thus, the TNM system is quite controversial [
36]. Thus, we have elected to use both simple nodal involvement and full TNM to circumvent this controversy and also to improve our statistical correlation. Notably, both nodal involvement and TNM score were strongly statistically correlated with IL36α expression by univariate analysis, but not by multivariate analysis. While the referee is correct in stating that T contributes to TNM, TNM contains considerable additional contributions to its score, that potentially could mask a correlation with IL-36 s expression. Indeed, we observed that only T remained significant in multivariate analysis.
The interval between malignant transformation at the cellular level and tissue diagnosis is difficult to determine and may vary depending on the pathogenic pathway underpinning the CRC (microsatellite instability/mismatch repair gene mutations versus Wnt/APC pathway) [
15,
16]. The extent to which pathway pathogenesis influences the development of variation in IL-36 s expression is therefore difficult to formulate. We elected to exclude mucinous adenocarcinoma CRC samples typically associated with mismatch repair gene mutations to minimise this potential factor.
Furthermore, we observed that IL-36α and IL-36γ were good indicators for prognosis for sub-groups of CRC patients when stratified by clinicopathological characteristics, particularly when comparing the more advanced stages of CRC to earlier stages. The TNM staging system incorporates the depth of invasion and lymph node metastasis of tumours, which are known to be two of the influential factors determining prognosis of CRC patients [
37] [
36]. Although there was no significant difference between IL-36γ
high and IL-36γ
low in patients that were T1–3, there was a trend, which may be due to the relatively small number of followed-up patients, especially the T1–3 patients, who generally had survived well, with very few deaths. We also acknowledge that the speculative role of IL-36α, β or γ in predicting survival based on different T status, TNM staging, LN metastasis and right vs left side cancer in the current study. However as stated above, the precise differential regulatory role of IL-36 s in the carcinogenesis is being investigated in genetically manipulated animals.
Finally, there is often both a difference in pathogenesis and stage at diagnosis between left-sided and right-sided CRC, in part due to different clinical presentation and treatment [
38]. This is consistent with our finding that the CRC patients with right-sided colon CRC, versus left-sided colon CRC, mostly presented at a late stage by TNM [
39].
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.