Inhibition of FGFR2 in endometrial cancer cells induced apoptosis despite constitutive activation of the PI3K/AKT pathway.

C86

Endometrial carcinoma is the most common gynecological malignancy in the United States. Although most women present with early disease confined to the uterus, the majority of persistent or recurrent tumors are refractory to current chemotherapies. We identified activating mutations in the Fibroblast growth factor receptor 2 (FGFR2) in 10% (19/187) of endometrial cancers and showed these mutations primarily occurred in tumors of the endometrioid histologic subtype (18/115 cases investigated, 16%). Although endometrial cancers presenting with an endometrioid histology are generally considered 'good actors', 25% of all endometrial cancer deaths can be attributed to this histotype. FGFRs have been shown to signal through a variety of downstream signaling pathways including MAPK, PI3K/AKT, Src, PLCg and STATs. In tumors with an endometrioid histology, KRAS activation and PTEN inactivation occur in 15-25% and 55-80% of tumors, respectively. For this reason, we sought to determine if FGFR2 activation occurs simultaneously in tumors with downstream activation of the MAPK pathway via KRAS gain-of-function mutations, or downstream activation of the PI3K/AKT pathway due to loss-of-function PTEN mutations. We therefore sequenced exon one of KRAS and all exons of PTEN in the panel of endometrioid tumors, generating data on 109 samples. We identified KRAS mutations in 12% (13/109) tumors and no tumor was identified with activating mutations in both KRAS and FGFR2. Further analysis revealed PTEN mutations in 69% (75/109) of tumors. Of those tumors with FGFR2 mutations, 12/17 also carried a PTEN mutation, suggesting that FGFR2 drives tumorigenesis primarily through the MAPK rather than the PI3K/AKT pathway. Given the known role of the PI3K/AKT pathway in promoting cell survival it was important to determine if inhibiting FGFR2 in the presence of constitutive downstream AKT signaling could result in cell death. To this end we knocked down FGFR2 via lentiviral transduction with two independent shRNAs and demonstrated a reduction in proliferation and an induction of apoptosis in the AN3CA and MFE296 endometrial cancer cell lines, both of which carry the activating N550K FGFR2 mutation. Consistent with the genetic data, Western blot analysis revealed that this induction of apoptosis following knockdown of FGFR2 correlated with inhibition of phospho-ERK and occurred in the presence of constitutively phosphorylated AKT. To verify that inhibition of FGFR2 with a pan-FGFR inhibitor was a viable therapeutic option in this tumor type, four endometrial cancer cell lines (2 with mutant FGFR2 and 2 with wildtype FGFR2) were treated with the pan-FGFR inhibitor, PD173074 (Calbiochem). The two cell lines with mutant FGFR2 (AN3CA and MFE296) were 10-40x more sensitive to inhibition with PD173074. Notably the most sensitive line (IC50=62nM) has loss-of-function mutations on both PTEN alleles. Western blot data confirmed this cell death occurred in the presence of constitutive AKT signaling in both cell lines. These data suggest that endometrial cancer cells with activated FGFR2 demonstrate oncogene addiction. Moreover, we demonstrate that induction of apoptosis following FGFR2 inhibition is due to inhibition of MAPK, and not the simultaneous inhibition of both MAPK and PI3K/AKT signaling as has previously reported for other receptor tyrosine kinases.