Abstract 4266: Investigating the functional effects of oncogenic FGFR2 mutations in endometrial cancer.
Authors: Fearon Abbie E. , Cutillas Pedro R. , Grose Richard P. 
Published: August 2014

Background:

Fibroblast growth factors (FGFs) signal by binding to FGF receptors (FGFRs), eliciting a diverse range of cellular responses. FGFR mutations are frequently found in endometrial cancer and may be a driving force in tumorigenesis. We are characterizing endometrial cancer cell lines containing common FGFR2 mutations and assessing their effects on cellular transformation.

Methods:

Three endometrial cancer cell lines, containing at least one common FGFR2 mutation, were selected. FGF and FGFR isoform expression and downstream signalling was established using 2D culture and further assessed in a 3D organotypic model.

Immortalized endometrial cells have been generated using a BMI-1 lentivirus. Protein expression and cell behaviour have been characterised and compared to endometrial cancer cells.

FGFR inhibitor treatment and siRNA-mediated FGFR2 knock down in endometrial cancer cells has also been investigated. The effects of these treatments on cell signalling have been assessed using a mass spectrometry (MS) approach, looking at the global downstream phosphoproteome.

The role of FGFR2 mutations in endometrial cancer is being assessed further using a zinc finger nuclease approach. Here, we investigate mutant FGFR2 constructs in immortalized endometrial cells under the control of the endogenous FGFR2 promoter. FGFR2 mutations have also been reversed in endometrial cancer cells and signalling impact assessed using MS.

Results:

FGFR2c and its ligand, FGF2, were expressed strongly in all cell lines. ERK phosphorylation increased upon stimulation with FGF2, indicative of MAP Kinase pathway activation. This was abolished by an FGFR inhibitor. Treatment with FGF7, specific to FGFR2b, had no effect on ERK phosphorylation. AKT phosphorylation remained at basal levels throughout stimulation assays. FGFR inhibitor treatment did not decrease levels of p-AKT.

3D culture data indicate endometrial cancer cells do not invade into the stroma. FGFR inhibitor treatment has cell line specific effects; MFE-296 cells acquire resistance to FGFR inhibition over time, while treatment of AN3CA cells leads to cell death.

Conclusions:

High levels of FGFR2c in these epithelial cell lines indicates receptor class switch from FGFR2b, resulting in increased proliferation and survival, perhaps via autocrine stimulation. Endometrial cancer cells signal via both MAPK and AKT pathways with MAPK predominating when the FGFR pathway is stimulated.

Results of 2D proliferation assays, taken with results of the 3D culture model, indicate: i. MFE-296 cells are sensitive to FGFR inhibition initially but acquire resistance to FGFR inhibitor treatment over prolonged exposure, ii. AN3CA cells are oncogene addicted; inhibition of FGFR2 leads to cell death.

Acknowledgments:

We thank Cancer Research UK for funding.

Citation Format: Abbie E. Fearon, Pedro R. Cutillas, Richard P. Grose. Investigating the functional effects of oncogenic FGFR2 mutations in endometrial cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4266. doi:10.1158/1538-7445.AM2013-4266