Although increasing evidence implicates FGFR in the etiology of human breast cancer, the mechanisms underlying its involvement have yet to be elucidated. Mouse models, like the ones used in these studies, provide valuable tools to decipher the function of conserved pathways such as FGF and Wnt in human cancer. The fact that random insertion of the MMTV provirus in the mouse genome consistently generates tumors with simultaneous activation of both Wnt and FGF ligands suggests that these two pathways are highly collaborative in the initiation of mammary tumorigenesis (10
). Although previous studies provide evidence for the existence of FGF and Wnt pathway cooperation, they fail to elucidate potential mechanisms responsible for this cooperativity. Previous studies in nulliparous animals showed that chronic iFGFR1 activation resulted in hyperplasia but was not sufficient to cause palpable tumor formation (16
). Thus, the rapidity at which palpable tumors developed in the Wnt/iR1 bigenic mice on dimerizer treatment was quite unexpected. Based on these results, we decided to examine the changes, which occurred immediately following dimerizer treatment, and uncovered an unprecedented expansion of the mammary epithelium resulting from activation of these two pathways. This, therefore, provided an excellent model in which to investigate the potential mechanisms involved.
Several studies investigating tumor initiation in the MMTV–Wnt-1 mouse model have implicated secondary activating mutations in Hras1
in ~50% of MMTV–Wnt-1–induced tumors (43
). These mutations were shown to result in greater sustained activation of phospho-ERK and phospho–phosphatidylinositol 3-kinase (PI3K)/AKT pathways (44
). In the current studies, the tumors did not originate from a clonal cell population. The rapid and widespread activation of cell proliferation observed throughout the gland suggests that secondary mutations are not required in this case for tumorigenesis, highlighting the importance of cooperativity between these two pathways. Importantly, the FGF signaling pathway, known to activate both ERK and PI3K/AKT, seems to have obviated the requirement for secondary mutations needed to activate these pathways. In fact, a very strong correlation was observed between ERK pathway activation and cell proliferation in the Wnt/iR1 tumors (data not shown).
We initially hypothesized that increased β-catenin stabilization and resultant canonical Wnt target gene transcription might be the primary mechanism contributing to the FGF/Wnt cooperativity. Previous studies have shown that the inhibition of glycogen synthase kinase 3 as a consequence of insulin-like growth factor-I/protein kinase B/AKT or ERK-mediated phosphorylation of Ser21/9
residues resulted in enhanced β-catenin stabilization (45
). However, microarray analysis did not reveal any significant change in Wnt/β-catenin target gene expression as a result of iFGFR signaling in bigenic mice, nor did we observe any differences in β-catenin stabilization. Interestingly, this suggested that much of the cooperativity between these two pathways may have been through posttranscriptional mechanisms. The highly significant protein translational gene signature identified through pathway analysis of the microarray results also suggested that iFGFR1 activation of protein synthesis might enhance mammary tumorigenesis in the bigenic mice. However, it is not clear if this gene signature is the result of direct FGFR signaling inducing gene expression or an indirect effect as a result of a positive feedback mechanism. In support of the first hypothesis, a 2-hour treatment with dimerizer in iFGFR1-expressing HC11 cells resulted in the transcriptional activation of several translational regulators identified in our signature, such as EIF1α, eIF4E-BP, eIF4E, and EIF5b (data not shown). Alternatively, increased levels of cMyc have been reported to exert a general effect on translational efficiency (46
). Thus, it is possible that this translational signature reflects both direct and indirect effects of FGFR1 signaling.
Oncogenic ras, AKT, and ERK signaling have all been shown to contribute to the selective translation of oncogenic mRNAs such as cMyc and cyclin D1 in human glioblastoma tissue and cell lines (32
). Downstream phosphorylation of critical translational regulatory components such as mammalian target of rapamycin (mTOR), eIFs, ribosomal proteins such as S6-RP, and their inhibitors are key regulators involved in the specific recruitment of progrowth mRNA species to polysomes. In fact, many of these factors and their phosphorylated forms are upregulated in various human cancers, including breast cancer (33
). Phosphorylation of these components through AKT-mTOR or ERK-MNK signaling has been shown to enhance the specific translation of oncogenic “inefficiently translated” mRNAs with G/C-rich 5′ untranslated regions and reduces the translation of more “efficiently translated” mRNA species not involved in cell cycle progression or survival (33
). Interestingly, in recent studies using iFGFR1-transformed MCF10A cells and a cell line derived from a human lobular carcinoma, known to have a copy number amplification of FGFR1, a dependence on ribosomal S6 kinase (RSK) activity for FGFR1-incurred growth and survival was observed (49
). In agreement with these results, we now show that iFGFR1 enhances phosphorylation of S6-RP, a direct RSK phospho-target. Whether the RSK dependence observed in breast cancers with activated FGFR1 requires the selective translational recruitment of progrowth and prosurvival genes remains to be established.
In summary, the current studies show that FGFR signaling leads to increased phosphorylation and transcription of key translational regulators and recruitment of several Wnt/β-catenin target mRNAs to polysomes on sustained signaling, along with the rapid appearance of palpable tumors. The expression of oncogenes, such as cMyc, survivin, and cyclin D1, is regulated by multiple mechanisms and signaling pathways in addition to the translational mechanisms described in this study. Furthermore, FGF signaling is known to regulate many factors important for mammary tumorigenesis, including macrophage recruitment and angiogenesis (17
). However, because of the rapid effects observed on tumorigenesis in these bigenic mice, FGFR-induced protein synthesis, including translational upregulation of Wnt/β-catenin target oncogenes, already induced at the transcriptional level may play a critical role in driving and sustaining the rapid tumor growth observed. These translational effects are most likely not limited to Wnt/β-catenin target genes alone. Future bioinformatic studies of large-scale changes to the proteome and next-generation sequencing of polysomal mRNAs performed at different times following FGFR activation using the bigenic mouse model will be required to provide a more complete picture of translationally regulated mRNAs.
It is not surprising that rapid and sustained induction of cell proliferation may be dependent on new protein synthesis. In fact, the effects of AKT signaling on polysomal recruitment of specific mRNAs have been shown previously to occur as rapidly as 2 hours following activation before major changes in gene transcription (34
). In addition, cells proliferating at a very high rate are dependent on the continued production of new proteins necessary for both basic cellular functions as well as cell cycle regulators and oncogenes, which would otherwise be lost after successive cell divisions. It is likely that the general inhibition of protein translation will attenuate tumor growth in our mouse model as observed for other studies of tumor growth both in vitro
and in vivo
. Interestingly, early clinical trials with the first generation of protein synthesis inhibitors targeting eIF4E in several cancers, including breast cancer, are currently being initiated (50
Traditionally, most studies investigating changes in gene regulation involved in tumorigenesis have focused almost exclusively on gene expression profiling, with much less attention on changes in protein synthesis and activity. Interestingly, novel high-throughput studies using nextgeneration sequencing and ribosomal recruitment assays recently have shown that it is possible to interrogate global changes in the yeast proteome on starvation conditions (36
). In the future, these types of studies should provide new insights into the role translational regulation plays in the etiology of breast cancer, potentially providing additional targets for adjuvant therapy. The current studies further extend our understanding of FGFR signaling in the etiology of breast cancer and provide an excellent model to undertake future studies of global changes in the breast cancer proteome as a function of oncogenic FGF signaling. Fortunately, FGFRs, like other receptor tyrosine kinases, notably ErbB2, may provide druggable targets for adjuvant therapy, and there are several known FGFR inhibitors currently in clinical trials. Additionally, small-molecule inhibitors regulating the key pathways involved in translation initiation also may provide new therapeutic targets for human breast cancers with alterations in FGFR signaling.