In a previous study, we demonstrated decreased survival in patients with high-grade serous carcinomas harboring amplification of 5q31-35.3. Specifically, we observed that overexpression of FGF1, located in this amplicon, was a poor prognostic indicator for these tumors (10
). One of the key receptors for FGF1 is FGFR4, which is located on the same amplicon and preferentially binds to FGF1 (11
). In the present study, we demonstrated that FGFR4 overexpression is associated with a more aggressive high-grade serous carcinoma phenotype in vitro
and in vivo
, suggesting that FGF axis activation through overexpression of both FGFRs and FGF ligands may represent a targetable autocrine signaling loop associated with poor overall survival in ovarian cancer patients.
Birrer and colleagues observed a 25% gain frequency of chromosome segment 5q31-35.3 harboring the FGFR4 gene in ovarian cancer cases (10
). Their data also demonstrated a significant correlation between FGFR4 copy number and overall survival. However, recently reported data from The Cancer Genome Atlas (TCGA) demonstrated a 6% amplification/overexpression rate for FGFR4 with no significant correlation with survival in ovarian cancer cases (23
). The discrepancy between these two sets of data may result from several factors. First, the TCGA tumor samples were bulk tissue samples with stromal cell contamination rates ranging from 5% to 50%. Tissue samples with high levels of stromal contamination will show significantly low levels of FGFR4 gene amplification and/or expression, as the FGFR4 gene and mRNA copies are diluted by the stromal DNA and mRNA. This may impact survival correlation studies. In comparison, in our analyses, we used DNA and RNA extracted from microdissected ovarian tumor cells, which had minimal stromal DNA and RNA contamination. Second, the TCGA samples were collected from multiple institutions, whereas we collected our samples at a single institution, which may have given us a more homogenous patient population. Third, we found a significant correlation between FGFR4 protein expression and survival. Whereas FGFR4 amplification may not correlate with survival as indicated in the TCGA data set, FGFR4 protein expression may correlate with it, as gene amplification and mRNA expression may not correlate with protein expression owing to mechanisms that regulate mRNA and/or protein expression. However, researchers have not immunohistochemically evaluated FGFR4 protein expression in the TCGA data set.
FGFR4 differs from the other three members of the FGFR family in genomic structure, ligand binding, and signal transduction (24
). FGFR activation, either by activating mutations (25
) or overexpression (30
), occurs in multiple solid tumors. FGFR2 and FGFR3 mutations are common in endometrial cancer (36
) and bladder cancer (31
). In comparison, FGFR1 and FGFR4 mutations are not common in carcinoma cells; instead, overexpression of FGFR1 and FGFR4 is more prevalent (37
). To exclude the presence of several rare activating mutations of FGFR4, we sequenced DNA isolated from microdissected high-grade serous carcinoma samples. We did not identify any mutations in either the kinase or intermembrane domain except for the Gly388Arg polymorphism, which has exhibited no effect on cancer prognosis (38
), in 6 of 43 (14%) high-grade serous carcinoma samples. Our data suggest that, similar to breast cancer (39
), FGFR overexpression is the main mechanism implicated in high-grade serous ovarian carcinoma.
Unlike other FGFR family members, FGFR4 preferentially binds to acidic FGF (FGF1) (11
). Our prior work demonstrated that poor survival and related phenotypic changes induced by FGF1 in high-grade serous carcinoma parallel those in FGFR4. Hence, overexpression of both FGF1 and FGFR4 may provide an autocrine loop that drives high-grade serous ovarian carcinoma growth. This may result from activation of the MAPK/ERK signaling pathway by FGF1 as described previously (40
) and confirmed by the pathway reporter and Western blot analysis data in the present study. In addition to activation of the MAPK/ERK pathway, we observed activation of the proliferative NF-κB and WNT signaling pathways in FGF1-treated cells, which our pathway reporter and Western blot analysis confirmed. In addition, our canonical pathway analysis of transcriptome profiling data demonstrated a significant correlation between these pathways and the differentially expressed genes identified by manipulating the level of FGFR4 expression in ovarian cancer cells. These data suggest that multiple pathways can be activated by FGF1, most likely mediated via FGFR4, because the effect of FGF1 on pathways activation can be abrogated by downregulation of FGFR4 expression. Furthermore, our data suggest that the effects of FGFR4 on ovarian cancer cell proliferation and survival result from upregulation of expression of CXCR4, which can regulate cell proliferation and apoptosis upon binding of its ligand CXCL12 in T cells and cancer cells (20
), and from upregulation of expression of BNIP3, which is known to induce apoptosis (22
). We confirmed upregulation of these two proteins in the ovarian tumor samples obtained from mice given the FGF trap protein. However, the molecular mechanisms involved by these two proteins in mediating the effect of the FGFR4 signaling pathway on ovarian cancer cell growth remain to be elucidated.
In the absence of activating mutations of FGFR4, downregulation of FGFR4 expression and prevention of binding of ligands to FGFR4 may be effective in the treatment of serous ovarian cancer. Using two strategies for targeting FGFR4 with an orthotopic mouse model of high-grade serous carcinoma, we demonstrated that decreasing FGFR4 expression leads to a decrease in tumor growth. Although the use of FGFR4 siRNAs in vitro
can completely silence FGFR4 expression, leading to decreased proliferation and survival of ovarian cancer cell lines, ovarian tumors, although small, still developed in mice given FGFR4-specific siRNAs delivered in DOPC nanoliposomes. In addition, the tumors had FGFR4 expression, suggesting that the nanoliposomes may not efficiently deliver the siRNAs to all tumor cells. In addition to the use of siRNAs, our data demonstrated that use of the soluble fusion protein FPT-091 containing the extracellular domain of FGFR4 and an IgG Fc fragment may have significant clinical applications. Replacement of the FGFR4 acid box region with the corresponding FGFR1 region improved the bioavailability and pharmacokinetics of the fusion protein in vivo
. The chimeric FGFR4/FGFR1 extracellular domain in FGFR4mut:Fc is fused to the Fc fragment of IgG1 and can bind to several members of the FGF protein family, including FGF1. The ability to sequester several FGF ligands and inhibit the pathway upstream of the FGF receptor enables the trap protein to inhibit all of the possible effects of overexpression of FGFs and FGFR4. Given the high degree of genetic heterogeneity of high-grade serous carcinomas, activation of multiple pathways and/or targets, such as Notch3 (41
) and NAC1 (42
), may be involved in the pathogenesis of different ovarian cancer patients. A challenge remains in identifying ovarian cancer patients who may benefit from targeting a specific pathway or pathways, possibly via individual tumor expression profiling.
In conclusion, the present study demonstrated that overexpression of FGFR4 is an indicator of poor prognosis for high-grade serous ovarian carcinoma. It also identified mechanisms by which activation of FGFR4 by ligands such as FGF1 may lead to an aggressive cancer phenotype. Successful targeting of FGF axis actication in our orthotopic mouse model suggests that this approach is feasible in clinical trials.