Immunity Article Interleukin-17 Receptor A Signaling in Transformed Enterocytes Promotes Early Colorectal Tumorigenesis Kepeng Wang,1 Min Kyoung Kim,2 Giuseppe Di Caro,1 Jerry Wong,1 Shabnam Shalapour,1 Jun Wan,3,4 Wei Zhang,5 Zhenyu Zhong,1 Elsa Sanchez-Lopez,1 Li-Wha Wu,6 Koji Taniguchi,1,7 Ying Feng,8 Eric Fearon,8 Sergei I. Grivennikov,9 and Michael Karin1,* 1Laboratory of Gene Regulation and Signal Transduction, Departments of Pharmacology and Pathology, School of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0723, USA 2Division of Hematology-Oncology, Department of Medicine, Yeungnam University College of Medicine, 317-1, Daemyung-5 dong, Namgu, Daegu 705-717, South Korea 3Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-Hong Kong University of Science and Technology Medical Center, Shenzhen 518036, Guangdong Province, China 4Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, 100044 China 5Shenzhen Key Laboratory for Translational Medicine of Dermatology, Biomedical Research Institute, Shenzhen Peking University-Hong Kong University of Science and Technology Medical Center, Shenzhen 518036, Guangdong Province, China 6Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, 1 University Rd, Tainan 70101, Taiwan, ROC 7Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan 8Departments of Internal Medicine, Human Genetics and Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA 9Cancer Prevention and Control Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, USA *Correspondence: karinoffi[email protected] http://dx.doi.org/10.1016/j.immuni.2014.11.009 SUMMARY Chronic inflammation associated with infection and autoimmune disease increases cancer risk and accelerates progression of Interleukin-17A (IL-17A) is a pro-inflammatory cyto- many malignancies, including stomach, liver, and colon cancers kine linked to rapid malignant progression of colo- (Balkwill and Mantovani, 2001; Grivennikov et al., 2010). Pro-in- rectal cancer (CRC) and therapy resistance. IL-17A flammatory cytokines and tumor-infiltrating myeloid and immune exerts its pro-tumorigenic activity through its type cells play critical roles in almost every stage of tumorigenesis, A receptor (IL-17RA). However, IL-17RA is expressed from initiation and tumor promotion to malignant progression in many cell types, including hematopoietic, fibro- and metastatic spread. Even in cancers that do not arise in the context of underlying inflammation, a tumor-evoked inflamma- blastoid, and epithelial cells, in the tumor microenvi- tory response plays an important promoting role in malignant ronment, and how IL-17RA engagement promotes progression (Grivennikov et al., 2012). colonic tumorigenesis is unknown. Here we show Among inflammatory cytokines that promote tumor develop- that IL-17RA signals directly within transformed ment, the interleukin-17 (IL-17) family, which includes IL-17A, colonic epithelial cells (enterocytes) to promote early B, C, D, E, and F (Dungan and Mills, 2011), occupies an important tumor development. IL-17RA engagement activates position in both mouse models and human cancer. IL-17A and F ERK, p38 MAPK, and NF-kB signaling and promotes are the closest members of this family, and both bind to IL-17 the proliferation of tumorigenic enterocytes that just receptors A (IL-17RA) and C (IL-17RC), whose engagement lost expression of the APC tumor suppressor. activates mitogen-activated protein kinases (MAPKs), nuclear Although IL-17RA signaling also controls the pro- factor-kappa B (NF-kB), and CCAAT-enhancer-binding protein duction of IL-6, this mechanism makes only a par- (C/EBP) signaling pathways through the adaptor proteins Act1 and TRAF6 (Iwakura et al., 2011; Reynolds et al., 2010). IL-17A tial contribution to colonic tumorigenesis. Combined and F are produced by Th17 cells, gdT cells, natural killer T treatment with chemotherapy, which induces IL-17A (NKT) cells, and subsets of innate lymphoid cells (ILCs) (Rey- expression, and an IL-17A neutralizing antibody nolds et al., 2010; Sutton et al., 2012; Zou and Restifo, 2010). enhanced the therapeutic responsiveness of estab- Initial evidence for the involvement of IL-17 cytokines in cancer lished colon tumors. These findings establish IL- development came from studies of mouse colonic tumorigen- 17A and IL-17RA as therapeutic targets in colorectal esis. With the ApcMin model, it was shown that infection of cancer. mice with the human enterotoxigenic Bacteroides fragilis (ETBF) bacteria triggers colitis and accelerates IL-17A-depen- dent tumor development (Wu et al., 2009). Neutralization of INTRODUCTION IL-17A with a specific antibody prevented ETBF-induced accel- eration of colonic tumorigenesis (Wu et al., 2009). Retrospective A link between inflammation and cancer has long been sus- clinical studies revealed that high IL-17A expression in stage I or pected, but direct experimental evidence linking the two patho- II human colorectal tumors is associated with rapid progression logical processes has only become available in recent decades. to lethal metastatic disease and thus serves as a strong indicator 1052 Immunity 41, 1052–1063, December 18, 2014 ª2014 Elsevier Inc. Immunity IL-17RA Promotes Early Colorectal Tumorigenesis of poor clinical outcome (Tosolini et al., 2011). Subsequent IL-17RA (Chae and Bothwell, 2011; Chae et al., 2010; Grivenni- studies demonstrated that IL-17A also enhances development kov et al., 2012; Hyun et al., 2012; Song et al., 2014; Tanaka of colitis-associated cancer (CAC) induced by the pro-carcin- et al., 2003; Tong et al., 2012; Wu et al., 2009). However, it ogen azoxymethane (AOM) and the irritant dextran sulfate so- is not known whether IL-17RA engagement exerts its pro- dium (DSS) (Hyun et al., 2012; Tanaka et al., 2003; Tong et al., tumorigenic effect directly within epithelial cells or whether, 2012). Although IL-17A and IL-17F are related and signal through akin to IL-23R (Grivennikov et al., 2012), it acts by controlling the same receptors and effector mechanisms, IL-17F was re- production of other pro-tumorigenic cytokines by myeloid cells ported to attenuate CAC development (Tong et al., 2012). The or cancer-associated fibroblasts (CAFs), which also express IL- divergent roles of IL-17A and IL-17F in CAC can be explained 17RA (Iwakura et al., 2011; Korn et al., 2009). To understand by their distinct functions in autoimmune- and chemically- how IL-17RA promotes colorectal tumorigenesis, we employed induced inflammation, which is a critical step in CAC induction the CPC-APC model (Hinoi et al., 2007) as previously described (Yang et al., 2008). Other studies, however, have shown that (Grivennikov et al., 2012). An absence of IL-17RA in all cells of genetic ablation of either IL-17A or IL-17F attenuates tumor CPC-APC mice resulted in a marked reduction in tumor cell development in ApcMin mice, although the effect of IL-17A is proliferation and increased apoptosis (Figures 1A and 1B). much more pronounced (Chae and Bothwell, 2011; Chae et al., Loss of IL-17RA also resulted in reduced IL-6 expression and 2010). consequently decreased signal transducer and activator of A useful mouse model of colorectal tumorigenesis is provided transcription (STAT3) activation in adenoma epithelial cells (Fig- by the so-called CPC-APC mouse, in which one allele of the Apc ures 1A–1C and Figure S1 in the Supplemental Information tumor suppressor gene is deleted in the colon and in which available online), whereas stimulation of CAFs isolated from subsequent Apc loss-of-heterozygocity (LOH) results in devel- CPC-APC tumors with recombinant IL-17A resulted in IL-6 opment of large colonic adenomas that progress to invasive mRNA induction (Figure 1D). Activation of Akt and, to a lesser carcinomas (Hinoi et al., 2007). Using this model, we found extent, ERK signaling in tumors was also reduced after that early colonic adenomas exhibit substantial upregulation of IL-17RA ablation (Figure 1E). Expression of other inflammatory IL-23 expression by tumor-associated myeloid cells (TAMs) as cytokines, including IL-21 and TNF, in tumors was elevated a result of the loss of both protective mucin expression and tight upon loss of IL-17RA, although expression of the anti-inflam- junctions between intestinal epithelial cells (IECs), which result in matory and anti-tumorigenic cytokines IL-10 and transforming invasion of the barrierless adenomas by components of the mi- growth factor-b1 (TGFb1) (Becker et al., 2004; Mumm et al., crobiome (Grivennikov et al., 2012). A similar process can occur 2011) was also elevated (Figure S1). The expression of in human colonic adenomas, which also exhibit loss of mucins IL-17C, and the IL-17-related chemokines CXCL1 and and junctional adhesion molecules. IL-23 induces tumoral CXCL2, was significantly elevated in tumors compared to expression of IL-17A, and ablation of IL-17RA inhibited colon normal colon tissue and was reduced in Il17ra-null mice (Fig- tumor development and progression in CPC-APC mice. These ure S1). IL-17A, IL-17F, and IL-11 were barely affected by results established the pro-tumorigenic function of a cytokine Il17ra ablation (Figure S1). The amounts of IL-17RC and cascade in which IL-23 produced by TAM controls IL-17A pro- IL-17RE mRNAs, which interact with IL-17RA to mediate IL- duction by Th17 and other lymphoid cells within the tumor micro- 17A, C, and F signaling, were upregulated in Il17ra-ablated environment and in which IL-17A stimulates tumor development tumors, possibly as a result of compensation (Figure S1). through IL-17RA (Grivennikov et al., 2012). More recently, it was The expression of several cytotoxic-response markers was shown that IL-17C also stimulates tumor development in ApcMin elevated in the absence of IL-17RA (Figure S2A). Loss of mice (Song et al., 2014), presumably through IL-17RA as well.