Characterisation of the role of the IL-36 family of cytokines in the pathogenesis of colon cancer

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Baker, Kevin J.
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University College Cork
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The IL-36 cytokines are a recently described subset of the IL-1 family of cytokines. These cytokines have now been identified to play a role in the pathogenesis of many inflammatory diseases and are increasingly being implicated in tumourigenesis. Given the pluripotent nature of other IL-1 family members and the relationship between inflammation and tumorigenesis, here we have investigated the effects of IL-36 signalling in colorectal cancer. In this study we demonstrate that expression of IL-36 family member mRNA and protein is significantly increased in colorectal cancer tissue compared to adjacent colonic non-tumour tissue. Colon cancer cell lines express IL-36 family genes differentially, and these genes are inducible with Toll-like Receptor ligands and pro-inflammatory cytokines. Following stimulation with IL-36 agonists, colon cancer cell lines increase expression of pro-inflammatory genes, especially genes involved in myeloid cell chemotaxis. Colon cancer cells lines are more responsive to IL-36β and IL-36γ in comparison to IL-36α. In vitro assays showed stimulation of colon cancer cell lines with IL-36 agonists augmented several pro-tumorigenic phenotypes such as cellular migration, invasion and proliferation in both 2D and 3D models. In pre-clinical models of colon cancer, intraperitoneal injection of the IL-36 Receptor antagonist (IL-36Ra) significantly reduced tumour burden using the subcutaneous CT26 tumour model in syngeneic Balb/mice. This was associated with a decrease in Ki-67 expression by tumour cells in the IL-36Ra-treated group relative to untreated control tumours, suggesting the inhibition of the pro-proliferative signalling of IL-36 agonists resulted in the decreased tumour size. Moreover, colon cancer cells lacking the IL-36R also showed reduced tumour growth and reduced Ki-67 expression in vivo. IL-36 agonist administration also resulted in a tumour reduction in mice, although this was not as effective as IL-36Ra administration and did not alter Ki67 expression levels in tumour tissue but rather acted through immune infiltration of tumours. Taken together, this data suggests that targeting IL-36R signalling may be a useful targeted therapy for colorectal cancer patients with IL-36R+ cancer cells. In order to further understand the effects of IL-36 cytokine signalling in the context of immune cells, co-cultures of macrophages and colon cancer cells were completed in vitro. The THP-1 model of macrophages showed minimal changes in response to IL-36 agonist stimulation. M1 macrophage cells significantly reduced spheroid formation of HT29 cells, with addition of IL-36 agonists facilitating recovery of spheroid size back to untreated size, indicating colon cancer cells are more responsive to IL-36 stimulation than macrophages when in co-culture in this model. Preliminary work using HL-60 cells as models of neutrophils showed IL-36 can augment cancer-cell induction of neutrophil NETosis, potentially contributing to immune evasion and metastasis. Transcriptomic analysis of publicly available patient cohorts revealed increased expression of IL-36 family members in malignant intestinal tissue in comparison to paired healthy tissue. Moreover, IL-36R expression is associated with poorer patient survival rates in colon cancer. Our DEG analysis of tumours expressing high levels of IL-36R mRNA revealed a possible role for the IL-17/IL-22/IL-23 signalling axis in colon cancer involving IL-36 signalling. Together, this study demonstrates that IL-36 signalling in colon cancer may contribute to disease progression and that inhibition of this signalling, in subgroups of patients stratified according to cancer cell expression of the IL-36R, may benefit survival rates, as shown in our in vivo pre-clinical models of colon cancer.
Immunology , Cancer , Inflammation , Immunotherapy
Baker, K. J. 2022. Characterisation of the role of the IL-36 family of cytokines in the pathogenesis of colon cancer. PhD Thesis, University College Cork.