FGFR2 mutations promote endometrial cancer progression through dual engagement of EGFR and Notch signalling pathways.

BACKGROUND: Mutations in the receptor tyrosine kinase gene fibroblast growth factor receptor 2 (FGFR2) occur at a high frequency in endometrial cancer (EC) and have been linked to advanced and recurrent disease. However, little is known about how these mutations drive carcinogenesis. METHODS: Differential transcriptomic analysis and two-step quantitative real-time PCR (qRT-PCR) assays were applied to identify genes differentially expressed in two cohorts of EC patients carrying mutations in the FGFR2 gene as well as in EC cells harbouring mutations in the FGFR2. Candidate genes and target signalling pathways were investigated by qRT-PCR assays, immunohistochemistry and bioinformatics analysis. The functional roles of differently regulated genes were analysed using in vitro and in vivo experiments, including 3D-orthotypic co-culture systems, cell proliferation and migration protocols, as well as colony and focus formation assays together with murine xenograft tumour models. The molecular mechanisms were examined using CRISPR/Cas9-based loss-of-function and pharmacological approaches as well as luciferase reporter techniques, cell-based ectodomain shedding assays and bioinformatics analysis. RESULTS: We show that common FGFR2 mutations significantly enhance the sensitivity to FGF7-mediated activation of a disintegrin and metalloprotease (ADAM)17 and subsequent transactivation of the epidermal growth factor receptor (EGFR). We further show that FGFR2 mutants trigger the activation of ADAM10-mediated Notch signalling in an ADAM17-dependent manner, highlighting for the first time an intimate cooperation between EGFR and Notch pathways in EC. Differential transcriptomic analysis in EC cells in a cohort of patients carrying mutations in the FGFR2 gene identified a strong association between FGFR2 mutations and increased expression of members of the Notch pathway and ErbB receptor family. Notably, FGFR2 mutants are not constitutively active but require FGF7 stimulation to reprogram Notch and EGFR pathway components, resulting in ADAM17-dependent oncogenic growth. CONCLUSIONS: These findings highlight a pivotal role of ADAM17 in the pathogenesis of EC and provide a compelling rationale for targeting ADAM17 protease activity in FGFR2-driven cancers.

Loss of iRhom2 accelerates fat gain and insulin resistance in diet-induced obesity despite reduced adipose tissue inflammation.

BACKGROUND: Low-grade inflammation and metabolic dysregulation are common comorbidities of obesity, both of which are associated with alterations in iRhom2-regulated pro-inflammatory cytokine and epidermal growth factor receptor (EGFR) ligand signaling. OBJECTIVE: Our objective was to determine the role of iRhom2 in the regulation of low-grade inflammation and metabolic dysregulation in a murine model of diet-induced obesity. METHODS: Wild type (WT) and iRhom2-deficient mice were fed normal chow (NC) or a high-fat diet (HFD) starting at 5 weeks of age for up to 33 weeks. Body composition, glucose and insulin tolerance, feeding behavior, and indirect calorimetry were measured at defined time points. Adipose tissue cytokine expression and inflammatory lesions known as crown-like structures (CLS) were analyzed at the end-point of the study. RESULTS: iRhom2-deficient mice show accelerated fat gain on a HFD, accompanied by insulin resistance. Indirect calorimetry did not demonstrate changes in energy expenditure or food intake, but locomotor activity was significantly reduced in HFD iRhom2-deficient mice. Interestingly, CLS, macrophage infiltration, and tumor necrosis factor (TNF) production were decreased in adipose tissue from HFD iRhom2-deficient mice, but circulating cytokines were unchanged. In inguinal and perigonadal fat, the EGFR ligand amphiregulin was markedly induced in HFD controls but completely prevented in iRhom2-deficient mice, suggesting a potentially dominant role of EGFR-dependent mechanisms over TNF in the modulation of insulin sensitivity. CONCLUSIONS: This study elucidates a novel role for iRhom2 as an immuno-metabolic regulator that affects adipose tissue inflammation independent of insulin resistance.

The Threshold Effect: Lipopolysaccharide-Induced Inflammatory Responses in Primary Macrophages Are Differentially Regulated in an iRhom2-Dependent Manner.

A well-controlled innate immune response is characterized by a rapid yet self-limiting inflammatory response. Although much is known about the range of inflammatory stimuli capable of triggering an innate immune response, the mechanisms which govern the degree of inflammation induced by inflammatory insults and the mechanisms in place to reset or maintain homeostasis are poorly understood. Tumor necrosis factor (TNF) is a potent early response pro-inflammatory cytokine produced by immune cells following a broad range of insults spanning autoimmunity and metabolic diseases to pathogenic infections. Previous studies have shown that a disintegrin and metalloproteinase (ADAM) 17 controls the release of soluble TNF and epidermal growth factor receptor signaling. Utilizing a genetic model of ADAM17 deficiency through the deletion of its regulator, the inactive rhomboid 2 (iRhom2), we show that loss of ADAM17 activity in innate immune cells leads to decreased expression of various cytokines in response to low levels of pathogen-associated molecular pattern (PAMP) stimulation but not at high-dose stimulation. In addition, TNF receptor (TNFR) 1/2-deficient bone marrow-derived macrophages yielded significantly reduced TNF expression following low levels of PAMP stimulation, suggesting that signaling through the TNFRs in immune cells drives a feed-forward regulatory mechanism wherein low levels of TNF allow sustained enhancement of TNF expression in an iRhom2/ADAM17-dependent manner. Thus, we demonstrate that inflammatory expression of TNF and IL1ß is differentially regulated following high or low doses of PAMP stimulation, invoking the activation of a previously unknown regulatory mechanism of inflammation.

Loss of RHBDF2 results in an early-onset spontaneous murine colitis.

Inflammatory bowel disease (IBD) is a heterogeneous group of inflammation-mediated pathologies that include Crohn's disease and ulcerative colitis and primarily affects the colon and small intestine. Previous studies have shown that a disintegrin and metalloprotease (ADAM) 17, a membrane-bound sheddase, capable of cleaving the proinflammatory cytokine TNF and epidermal growth factor receptor ligands, plays a critical role in maintaining gut homeostasis and modulating intestinal inflammation during IBD. Rhomboid 5 homolog 2 (RHBDF2), a catalytically inactive member of the rhomboid family of intramembrane serine proteases, was recently identified as a crucial regulator of ADAM17. Here, we assessed the role of RHBDF2 in the development of colitis in the context of IL10 deficiency. Il10-/- /Rhbdf2-/- mice developed spontaneous colitis and experienced severe weight loss starting at 8 wk of age, without the need for exogenous triggers. Severity of disease pathology in Il10-/- /Rhbdf2-/- mice correlated with a dysbiotic gut microbiota and elevated Th1-associated immune responses with increased interferon gamma and IL2 production. In addition, Il10-/- /Rhbdf2-/- mice failed to maintain their epithelial cell homeostasis, although the intestinal epithelial barrier of Rhbdf2-/- mice is intact and loss of Rhbdf2 did not significantly exacerbate sensitivity to dextran sulfate sodium-induced colitis, suggesting differences in the underlying disease pathway of intestinal inflammation in this model. Taken together, our results demonstrate a critical regulatory role for RHBDF2 in the maintenance of the unique homeostasis between intestinal microbiota and host immune responses in the gut that is dysregulated during the pathogenesis of IBD.