The Interleukin 22 Pathway Interacts with Mutant KRAS to Promote Poor Prognosis in Colon Cancer Sarah Mccuaig1, David Barras2, Elizabeth H
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Published OnlineFirst May 19, 2020; DOI: 10.1158/1078-0432.CCR-19-1086 CLINICAL CANCER RESEARCH | PRECISION MEDICINE AND IMAGING The Interleukin 22 Pathway Interacts with Mutant KRAS to Promote Poor Prognosis in Colon Cancer Sarah McCuaig1, David Barras2, Elizabeth H. Mann1, Matthias Friedrich1, Samuel J. Bullers1, Alina Janney1, Lucy C. Garner1, Enric Domingo3, Viktor Hendrik Koelzer3,4,5, Mauro Delorenzi2,6,7, Sabine Tejpar8, Timothy S. Maughan9, Nathaniel R. West1, and Fiona Powrie1 ABSTRACT ◥ Purpose: The cytokine IL22 promotes tumor progression in Results: Transcriptomic analysis revealed a poor-prognosis murine models of colorectal cancer. However, the clinical subset of tumors characterized by high expression of IL22RA1, significance of IL22 in human colorectal cancer remains the alpha subunit of the heterodimeric IL22 receptor, and KRAS unclear. We sought to determine whether the IL22 pathway mutation [relapse-free survival (RFS): HR ¼ 2.93, P ¼ 0.0006; is associated with prognosis in human colorectal cancer, and to overallsurvival(OS):HR¼ 2.45, P ¼ 0.0023]. KRAS mutations identify mechanisms by which IL22 can influence disease showed a similar interaction with IL10RB and conferred the worst progression. prognosis in tumors with high expression of both IL22RA1 and Experimental Design: Transcriptomic data from stage II/III IL10RB (RFS: HR ¼ 3.81, P ¼ 0.0036; OS: HR ¼ 3.90, P ¼ 0.0050). colon cancers in independent discovery (GSE39582 population- Analysis of human colorectal cancer cell lines and primary tumor based cohort, N ¼ 566) and verification (PETACC3 clinical trial, organoids, including an isogenic cell line pair that differed only in N ¼ 752) datasets were used to investigate the association KRAS mutation status, showed that IL22 and mutant KRAS between IL22 receptor expression (encoded by the genes cooperatively enhance cancer cell proliferation, in part through IL22RA1 and IL10RB), tumor mutation status, and clinical augmentation of the Myc pathway. outcome using Cox proportional hazard models. Functional Conclusions: Interactions between KRAS and IL22 signaling interactions between IL22 and mutant KRAS were elucidated may underlie a previously unrecognized subset of clinically using human colorectal cancer cell lines and primary tumor aggressive colorectal cancer that could benefitfromtherapeutic organoids. modulation of the IL22 pathway. Introduction inflammatory cytokines control several key features of malignancy, including cancer cell proliferation, survival, and dissemination (5). Colorectal cancer is a complex disease driven by the interplay Evidence from preclinical murine models of both sporadic and between tumor mutations, environmental factors, and aberrant immu- colitis-associated colorectal cancer has implicated IL23 and its down- nity (1, 2). Chronic intestinal inflammation (e.g., inflammatory bowel stream effector IL22 in the initiation and progression of colon disease) is a well-known risk factor for colorectal cancer, but colitis- tumorigenesis (6–9). Furthermore, IL22 has been associated with associated cancers account for only a small fraction of total tumor human gastric cancer progression (10) and has been described to incidence (3). However, colorectal cancers that are not associated with þ promote colorectal cancer stemness (11). IL23-responsive CD4 T colitis also elicit inflammatory responses, which are thought to be key cells and innate lymphoid cells secrete IL22 in the tumor microenvi- regulators of tumor progression and therapeutic resistance (4). Indeed, ronment (7, 8), where it signals through a heterodimeric receptor comprised of IL10 receptor beta (IL10RB) and IL22 receptor alpha 1 (IL22RA1; ref. 12). In the intestine, IL22RA1 expression is restricted to 1 the epithelium (13) and activates diverse signal transduction pathways Kennedy Institute of Rheumatology, University of Oxford, Oxford, United in response to IL22, including JAK/STAT3, MAPK, PI3K, and NF-kB, Kingdom. 2SIB Swiss Institute of Bioinformatics, Bioinformatics Core Facility, Lausanne, Switzerland. 3Department of Oncology, University of Oxford, Oxford, which collectively induce expression of genes involved in cell-cycle United Kingdom. 4Nuffield Department of Medicine, University of Oxford, progression and inhibition of apoptosis (14–16). Oxford, United Kingdom. 5Department of Pathology and Molecular Pathology, Oncogenic Ras isoforms modulate inflammatory pathways in University Hospital Zurich, Zurich, Switzerland. 6Ludwig Center for Cancer cancer by directly influencing inflammatory cytokine expression and 7 Research, University of Lausanne, Lausanne, Switzerland. Department of signal transduction (5). IL23, IL17, and IL22 are elevated in human Oncology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, 8 colorectal cancer, and their expression is modulated by the presence Switzerland. Molecular Digestive Oncology, KU Leuven, Leuven, Belgium. KRAS 9CRUK/MRC Oxford Institute for Radiation Oncology, University of Oxford, of mutant Ras (17). Activating mutations in , a major Ras Oxford, United Kingdom. isoform, occur in 40% to 45% of colorectal cancers and drive constitutive activation of the Ras-Raf-MEK-ERK pathway (18). Note: Supplementary data for this article are available at Clinical Cancer KRAS Research Online (http://clincancerres.aacrjournals.org/). Although mutations are strongly associated with resistance to EGFR-targeted therapy, they do not correlate with resistance to Corresponding Author: Fiona Powrie, University of Oxford, Roosevelt Drive, chemotherapy (19–21). However, given the cytokine-modulating Headington, Oxford OX3 7YF, United Kingdom. Phone: 01865-612-600; E-mail: KRAS fi[email protected] capacity of the Ras family, mutations may be clinically signif- icant in tumors with active cytokine signaling. Oncogenic KRAS Clin Cancer Res 2020;XX:XX–XX could influence virtually all signal transduction pathways downstream doi: 10.1158/1078-0432.CCR-19-1086 of IL22, but a specific interaction between IL22 signaling and KRAS Ó2020 American Association for Cancer Research. mutation has not been described. AACRJournals.org | OF1 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst May 19, 2020; DOI: 10.1158/1078-0432.CCR-19-1086 McCuaig et al. arrayexpress) on the A-AFFY-101 platform (customized Affymetrix Translational Relevance chip) and is a merge of E-MTAB-863 and E-MTAB-864 (26). Clinical IL22 promotes tumor progression in preclinical models of information on overall survival (OS) was available for 1,734 patients and colorectal cancer, but its importance in human colorectal cancer on relapse-free survival (RFS) for 1,499 patients. Gene expression remains unclear. Using a rigorous discovery/verification analysis of profiles of the patients in the combined dataset were merged at the tumor gene expression from over 1,000 patients, we have discov- gene level and then normalized and corrected for batch effects using the ered that among patients with colorectal cancer with high expres- Combat R package. For the individual analyses of the GSE39582 and sion of either or both subunits of the heterodimeric IL22 receptor, PETACC3 datasets, gene expression profiles from each of the datasets KRAS mutation confers poor prognosis. Functional studies were used independently (not normalized to the others). Tumors revealed that this association is due to an interaction between IL22 proximal to the splenic flexure were defined as proximal, and tumors signaling and oncogenic KRAS that enhances tumor cell prolifer- distal to the splenic flexure were classified as distal. ation through induction of the Myc pathway. These findings All stage II/III patients determined to have a KRAS-mutant tumor demonstrate that cell-intrinsic drivers of colorectal cancer can were included in the analysis. The KRAS mutation sites by codon interact with cell-extrinsic factors from the inflammatory micro- (where available) are detailed for each of the datasets in Supplementary environment to influence disease progression. Our data further Fig. S1B. justify the assessment of KRAS mutations in patients with colo- The interaction between mutant KRAS and all available cytokine/ rectal cancer, which has until now been clinically beneficial only for cytokine receptor genes was assessed by Cox proportional hazards tests prediction of cetuximab responsiveness, and suggest that for for interaction using the combined dataset. The top tertile was KRAS-mutant colorectal cancers with high IL22 receptor expres- considered high expression, and genes which yielded an uncorrected sion, closer monitoring and more aggressive or alternative ther- P value of less than 0.05 were considered for further analysis. apeutic strategies (e.g., antibody-based blockade of IL22) could be beneficial. Histotype analysis Tissue composition of the TCGA and an independent cohort, Stratification in COloRecTal cancer (S:CORT), was assessed by visual pathology review. The S:CORT cohort is composed of 385 In light of the strong evidence pointing toward a protumorigenic formalin-fixed, paraffin-embedded (FFPE) tumors from the FOCUS- role for IL22 in murine models of colorectal cancer, we sought to randomized clinical trial which assessed response to different chemo- determine the clinical importance of this cytokine in human disease therapy regimens in patients with advanced colorectal cancer (27). and to identify subtypes of colorectal cancer in which IL22 signaling Hematoxylin and eosin slides