Role of inflammation, innate resistance, and immunity in carcinogenesis

Division Of Basic Sciences - Nci

Investigators

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Abstract

Progress has been made in several of the aims of the project. We studied the role of inflammatory receptors and cytokines in skin and colon chemical carcinogenesis. Signaling through the adaptor protein MyD88 promotes carcinogenesis in several cancer models. In contrast, MyD88 signaling has a protective role in the development of azoxymethane (AOM)/ dextran sodium sulfate (DSS) colitis-associated cancer (CAC). The inability of Myd88-/- mice to heal ulcers generated upon injury creates an altered inflammatory environment that induces early alterations in expression of genes encoding pro-inflammatory and growth factors increasing adenoma formation and progression to infiltrating adenocarcinomas with frequent clonal mutations in the beta-catenin gene. This study revealed a previously unknown level of complexity surrounding MyD88 activities downstream of different receptors that impact tissue homeostasis and carcinogenesis. Constitutively active RAS plays a central role in the development of human cancer and is sufficient to induce tumors in two-stage skin carcinogenesis. RAS-mediated tumor formation is commonly associated with up-regulation of cytokines and chemokines that mediate an inflammatory response considered relevant to oncogenesis. Mice lacking IL-1R or MyD88 are less sensitive to topical skin carcinogenesis than their respective WT controls. MyD88(-/-) or IL-1R(-/-) keratinocytes expressing oncogenic RAS are hyperproliferative and fail to up-regulate proinflammatory genes or down-regulate differentiation markers characteristic of RAS-expressing WT keratinocytes. Using both genetic and pharmacological approaches, we find that the differentiation and proinflammatory effects of oncogenic RAS in keratinocytes require the establishment of an autocrine loop through IL-1alpha, IL-1R, and MyD88 leading to phosphorylation of IkBalpha and NF-kB activation.. Collectively, these results demonstrate that MyD88 exerts a cell-intrinsic function in RAS-mediated transformation of keratinocytes. We recently showed that skin tumor promotion also required intact MyD88/NF-kappaB signaling in keratinocytes and an IL-17+ T cell population. TPA treated MyD88 deficient keratinocytes failed to elaborate cytokines and chemokines required to attract T cells into the skin microenvironment. Conversely, IL-17+ T cells lacking MyD88 failed to infiltrate TPA-treated skin, preventing tumor formation. IL-17 stimulated proliferation and inhibited differentiation of normal keratinocytes and elicited an IKBzeta and protein kinase C dependent expression of molecules essential for tumor promotion. IL-17 elevated IKBzeta and chemokine expression in RAS transformed human and mouse keratinocytes. Deletion of IKBzeta in RAS transformed mouse keratinocytes reduced tumor formation. Expression of transcripts common to RAS transformation were reduced in gene set enrichment analysis of IKBZ deficient RAS transformed keratinocytes. These studies define IKBzeta as an essential mediator of RAS transformation and provide a roadmap through which IL-17 contributes to tumor formation. Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth. Tristetraprolin expression by keratinocytes protects against skin carcinogenesis: Cancer is caused primarily by genomic alterations resulting in deregulation of gene regulatory circuits in key growth, apoptosis, or DNA repair pathways. Multiple genes associated with the initiation and development of tumors are also regulated at the level of mRNA decay, through the recruitment of RNA-binding proteins to AU-rich elements (AREs) located in their 3'-untranslated regions. One of these ARE-binding proteins, tristetraprolin (TTP; encoded by Zfp36), is consistently dysregulated in many human malignancies. Herein, using regulated overexpression or conditional ablation in the context of cutaneous chemical carcinogenesis, we show that TTP represents a critical regulator of skin tumorigenesis. We provide evidence that TTP controlled both tumor-associated inflammation and key oncogenic pathways in neoplastic epidermal cells. We identify Areg as a direct target of TTP in keratinocytes and show that EGFR signaling potentially contributed to exacerbated tumor formation. Finally, single-cell RNA-Seq analysis indicated that ZFP36 was downregulated in human malignant keratinocytes. We conclude that TTP expression by epidermal cells played a major role in the control of skin tumorigenesis. Distinct contributions of cathelin-related antimicrobial peptide (CRAMP) derived from epithelial cells and macrophages to colon mucosal homeostasis: The cathelin-related antimicrobial peptide CRAMP protects the mouse colon from inflammation, inflammation-associated carcinogenesis, and disrupted microbiome balance, as shown in systemic Cnlp(-/-) mice (also known as Camp(-/-) mice). However, the mechanistic basis for the role and the cellular source of CRAMP in colon pathophysiology are ill defined. This study, using either epithelial or myeloid conditional Cnlp(-/-) mice, demonstrated that epithelial cell-derived CRAMP played a major role in supporting normal development of colon crypts, mucus production, and repair of injured mucosa. On the other hand, myeloid cell-derived CRAMP potently supported colon epithelial resistance to bacterial invasion during acute inflammation with exacerbated mucosal damage and higher rate of mouse mortality. Therefore, a well concerted cooperation of epithelial- and myeloid-derived CRAMP is essential for colon mucosal homeostasis. Myd88 in Cd11b+ cells protects from colitis-associated colon cancer through intestinal microbial homeostasis: Myeloid cells arise from the haematopoetic stem cells in the bone marrow and differentiate into natural killer (NK) cells, among others. These cells are an important component of the innate immune system which maintains homeostasis in the gut by maintaining a balance between pro- and anti-inflammatory responses and by promoting the integrity of the barrier function of the epithelium. Armed with an arsenal of pattern-recognition receptors called Toll-like receptors, these cells can respond to the multitudes of microbial antigens on the mucosal surface. Myeloid differentiation factor 88 (Myd88) is an important signaling molecule which contributes to signal transduction triggered by microbial products via recognition mediated by members of the Toll-like receptor and IL-1 receptor families. Using the azoxymethane (AOM)/DSS model of colitis-associated colorectal cancer, we previously showed that Myd88-/- mice are unable to heal colonic ulcers thereby increasing perpetuating the inflammatory microenvironment and fostering the dramatic increase in adenoma and adenocarcinoma formation in the colon. In this study, we decipher the compartment responsible for driving the anti-tumorigenic effects of MyD88. We hypothesized that MyD88 signaling from Cd11b+ myeloid cells mediates anti-tumor responses in the colon by reducing myeloid cell infiltration into the lamina propria and by preventing the expansion of GUS-type beta-glucuronidase- and invasin-containing microbes. Indeed, deletion of Myd88 in Cd11b+ cells (Myd88CD11b-/- mice) increased the number of adenomas formed relative to their corresponding floxed littermate controls. Remarkably, wild type mice co-housed with Myd88CD11b-/- animals or recipients of microbial transfer from these animals developed greater polyps in quantity and size. The contribution of CD11b+ cells to microbial homeostasis includes the negative regulation of E. coli strain O7:H7, which contributes to carcinogenesis through beta-glucuronidase and invasin activity. Taken together, MyD88 signaling in myeloid cells favors the development of a protective anti-tumoral response from the host and the gut microbiota.