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

Abstract

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.

Identification of Key Regulators in Pancreatic Ductal Adenocarcinoma using Network theoretical Approach

“…Mutations and alterations in the FGFR2 gene have been found to play a significant role in the development and progression of various types of cancer, including endometrial cancer, breast cancer, and gastrointestinal/genitourinary tract cancers. These alterations include somatic hotspot mutations, structural amplifications, and fusions (51).…”

Assessing the validity of driver gene identification tools for targeted genome sequencing data

“…Mutations in many of those genes have previously been linked to the cancer type for which our analyses classified them as drivers. For example, NOTCH1 mutations in bladder cancer ( Maraver et al 2015 ), RAC1 mutations in melanoma ( Lodde et al 2023 ), FGFR2 mutations in endometrial cancer ( Dixit et al 2023 ), and MSH2 and MSH6 mutations in colon cancer ( Haraldsdottir et al 2014 ) were only identified as drivers in the whole-exome analyses. All have published evidence for their role in the corresponding cancer type.…”

Functional Distinctions of Endometrial Cancer-Associated Mutations in the Fibroblast Growth Factor Receptor 2 Gene

“…Ectodomain shedding assays were performed as described earlier [ 25 , 27 , 30 , 31 ]. Briefly, stable clones of COS7 cells expressing WT and mutant FGFR2 growing in a 12-well plate at 65–70% confluency were transfected with Lipofectamine 2000 per the manufacturer’s protocol using AP-HB-EGF plasmids at a concentration of 1 μg per well as described [ 32 ].…”

“…Among these, the pathway leading to activation of mitogen-activated protein kinase (MAPK) signaling has been studied most extensively [ 18 , 19 , 20 ]. Second, FGFR2-mediated phosphorylation of MAPKs is known to stimulate and to regulate the proliferation and migration of epithelial cells in the skin and endometrium [ 21 , 22 , 23 , 24 , 25 ], all of which depends on epidermal growth factor receptor (EGFR)-mediated MAPK signaling and a disintegrin and metalloprotease (ADAM) 17-dependent proteolytic release (shedding) of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) [ 24 , 25 ]. Third, while many of the FGFRs can be activated by several fibroblast growth factors (FGFs), the FGFR2 is specifically activated by FGF7, making it an ideal system to study the crosstalk between FGFR2 and the EGFR signaling.…”

“…Lastly, we have previously shown that the EC-linked S252W and N550K mutations in the FGFR2 gene enhance the sensitivity to FGF7-induced activation of ADAM17 and subsequent transactivation of EGFR. Importantly, we demonstrated that the blockade of ADAM17 activity effectively inhibits FGF7-mediated oncogenic growth induced by FGFR2 in vitro and suppresses the growth of FGFR2-mutant EC in vivo [ 25 ]. These findings highlight the crucial role of ADAM17 in driving the oncogenic effects of FGFR2 mutations in EC.…”