Drug-sensitive FGFR2 mutations in endometrial carcinoma

Abstract

Oncogenic activation of tyrosine kinases is a common mechanism of carcinogenesis and, given the druggable nature of these enzymes, an attractive target for anticancer therapy. Here, we show that somatic mutations of the fibroblast growth factor receptor 2 (FGFR2) tyrosine kinase gene, FGFR2, are present in 12% of endometrial carcinomas, with additional instances found in lung squamous cell carcinoma and cervical carcinoma. These FGFR2 mutations, many of which are identical to mutations associated with congenital craniofacial developmental disorders, are constitutively activated and oncogenic when ectopically expressed in NIH 3T3 cells. Inhibition of FGFR2 kinase activity in endometrial carcinoma cell lines bearing such FGFR2 mutations inhibits transformation and survival, implicating FGFR2 as a novel therapeutic target in endometrial carcinoma.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Schematic diagram of novel FGFR2 mutations. Black diamonds indicate each instance of mutations found in endometrial samples; red diamonds indicate squamous lung; blue diamonds indicate cervical carcinoma. Asterisks indicate mutations demonstrated to be oncogenic (data in Table S3). K310R and A389T were not transforming; the remaining mutants without asterisks were not tested. Ig, Ig-like repeat. TM, transmembrane domain.

Fig. 2.

Ectopic expression of FGFR2 S252W in NIH 3T3 cells supports anchorage-independent growth. (A) Pooled NIH 3T3 cells stably expressing the empty vector or the WT, S252W, or kinase-dead S252W/D626A isoform IIIb FGFR2 constructs were assessed for colony formation in soft agar. n = average number of colonies counted in three sets of 10 fields. (B) FGFR2 expression was confirmed by immunoblotting using actin as a loading control. wt, WT FGFR2.

Fig. 3.

Knockdown of mutant FGFR2 expression with shRNA inhibits transformation and survival of endometrial cancer cell lines. Infection with two independent hairpins (shFGFR2#1 and shFGFR2#4) inhibited cell survival as assessed by WST-1 cell survival assay (A and B) and anchorage-independent growth as assessed by colony formation in soft agar (C and D) in the MFE-280 cells harboring an S252W mutation, but not the Hec-1B cells, which express WT FGFR2. shGFP, a hairpin specific for green fluorescent protein, was used as a negative control. All results are normalized to survival or colony formation by cells infected with empty vector.

Fig. 4.

Inhibition of FGFR2 kinase activity inhibits FRS2 phosphorylation and transformation and survival of endometrial cancer cell lines. (A) FGFR2 substrate FRS2 is constitutively phosphorylated in the MFE-280, AN3CA, and MFE-296 endometrial cancer cell lines harboring FGFR2 mutations, as compared with the Hec-1B line, which expresses WT FGFR2. (Upper) Treatment of these cell lines for 40 min with 2 μM FGFR inhibitor PD173074 inhibits both basal and ligand-induced (20-min stimulation with 30 ng/ml FGF7) phosphorylation, as evidenced by immunblotting with anti-phospho-FRS2. (Lower) Similar levels of expression of FRS2 were confirmed by immunoblotting with anti-FRS2. (B) Treatment with the indicated concentrations of PD173074 inhibited soft agar colony formation by the MFE-280, AN3CA, and MFE-296 endometrial cancer cell lines harboring FGFR2 mutations, as compared with the Hec-1B line, which expresses WT FGFR2. Colonies were photographed after 2 weeks. (Magnification: ×2.) (C) Quantification of effects of PD173074 on soft agar colony formation with EC50s indicated. (D) Endometrial cancer cell line WST survival assays performed after 4 days of treatment with PD173074. EC50s are indicated.