Fibroblast growth factor receptor signaling dramatically accelerates tumorigenesis and enhances oncoprotein translation in the mouse mammary tumor virus-Wnt-1 mouse model of breast cancer

Abstract

Fibroblast growth factor (FGF) cooperates with the Wnt/beta-catenin pathway to promote mammary tumorigenesis. To investigate the mechanisms involved in FGF/Wnt cooperation, we genetically engineered a model of inducible FGF receptor (iFGFR) signaling in the context of the well-established mouse mammary tumor virus-Wnt-1 transgenic mouse. In the bigenic mice, iFGFR1 activation dramatically enhanced mammary tumorigenesis. Expression microarray analysis did not show transcriptional enhancement of Wnt/beta-catenin target genes but instead showed a translational gene signature that also correlated with elevated FGFR1 and FGFR2 in human breast cancer data sets. Additionally, iFGFR1 activation enhanced recruitment of RNA to polysomes, resulting in a marked increase in protein expression of several different Wnt/beta-catenin target genes. FGF pathway activation stimulated extracellular signal-regulated kinase and the phosphorylation of key translation regulators both in vivo in the mouse model and in vitro in a human breast cancer cell line. Our results suggest that cooperation of the FGF and Wnt pathways in mammary tumorigenesis is based on the activation of protein translational pathways that result in, but are not limited to, increased expression of Wnt/beta-catenin target genes (at the level of protein translation). Further, they reveal protein translation initiation factors as potential therapeutic targets for human breast cancers with alterations in FGF signaling.

Figure 1

Activation of iFGFR1 signaling synergizes with Wnt-1 signaling, leading to reduced tumor latency with differences in tumor growth rate, multiplicity, and histologic features. A, Kaplan-Meier plot of tumor incidence of Wnt/iR1 (n = 9 of 9) and Wnt-1 (n = 12 of 23) mice. P < 0.0001, log-rank test, Wnt-1 versus Wnt/iR1. B, Wnt/iR1 mice had an average of three tumors per mouse at the time of sacking (n = 9) and Wnt-1 consistently had one tumor per mouse at the time of sacking (n = 12). *, P < 0.0001. Wnt/iR1 (n = 9) and Wnt-1 (n = 12) tumor diameter was measured. *, P < 0.0001. Points, mean; bars, SE. C, H&E of Wnt/iR1 and Wnt-1 tumor sections. Wnt/iR1 tumors are composed of distinct lobules resulting from the expansion of epithelium (arrow). D, immunofluorescence double staining of luminal K18 (green) and myoepithelial K5 markers (red) in the different tumor types. Nuclear staining is shown in blue (DAPI).

Figure 2

Activation of iFGFR1 leads to rapid tumor formation and cell proliferation in the Wnt-1 tumor model. A, hematoxylin-stained whole-mount images of number 4 inguinal mammary glands following dimerizer treatment showing rapid epithelial expansion (red arrow). White arrow, ductal thickening. B, H&E staining of number 4 inguinal mammary gland sections following dimerizer treatment showing a rapid epithelial expansion resulting in zonal organization (asterisk). C, immunofluorescence double staining of mitotic marker phospho-H3 (red) and S-phase marker BrdUrd (green) in number 4 inguinal mammary gland sections following dimerizer treatment. Nuclear staining is shown in blue (DAPI). D, quantification of immunofluorescence double staining of nuclear phospho-H3 and BrdUrd in number 4 inguinal mammary gland sections following dimerizer. Phospho-H3 (n = 3 independent sections): **, P < 0.01; *, P < 0.06, Wnt/iR1 versus Wnt-1. BrdUrd (n = 3 independent sections): **, P < 0.0001; *, P < 0.01, Wnt/iR1 versus Wnt-1. Points, mean; bars, SE.

Figure 3

mRNA expression levels in Wnt/iR1 determined through microarray analysis of tumors identifies changes in genes involved in protein translation. Heat map showing 20 genes differentially expressed in Wnt/iR1 and Wnt-1 tumors. Genes were identified by Ingenuity Pathway Analysis as important in protein translation.

Figure 4

iFGFR1 signaling leads to increased polysomal loading and protein expression of direct Wnt/β-catenin target oncogenes. A, quantitative RT-PCR analysis of cMyc, survivin, and cyclin D1 (*, P < 0.05) normalized to cyclophilin A (n ≥ 4). Columns, mean; bars, SE. B, immunoblot analysis and quantification of three different direct Wnt/β-catenin target oncogenes from three different pools of three Wnt-1 and three Wnt/iR1 tumors. Cyclophilin A was used as a loading control. Densitometry quantification of cMyc (**, P < 0.0001), survivin (*, P < 0.05), and cyclin D1 relative protein levels from multiple tumors (n ≥ 3). Densitometry values from the three genes were normalized to total cyclophilin A protein. Comparisons represent Wnt/iR1 versus Wnt-1 for each gene. Columns, mean; bars, SE. C, schematic illustration of the method used to analyze specific polysome-bound mRNAs in both tumor types. Quantitative RT-PCR results were normalized to 18S rRNA to compensate for global changes in ribosome content per fraction and sample. Figure adapted from del Prete and colleagues (19). Absorbance (260 nm) readings were collected continually across the sucrose gradient to determine the fractions containing the 40S (left arrow), 80S (right arrow), and ribosomal absorbance peaks (asterisks). An equal aliquot of purified RNA from each fraction was run on a 1%agarose gel to verify the sedimentation of rRNAs (18S and 28S) in the deeper sucrose fractions. D, quantitative RT-PCR fold changes of select genes from multiple Wnt/iR1 tumors over multiple Wnt-1 tumors through all nine fractions of the sucrose gradient. Fractions were divided into soluble, nonpolysome bound (fractions 1–4), and polysome bound (fractions 5–9) and compared (Supplementary Fig. S6). cMyc (n = 5): ^‡, P < 0.0005; survivin (n = 3): ^‡, P < 0.02; cyclin D (n = 4): ^‡, P < 0.003. Whole-cell (WC) mRNA was also analyzed. cMyc, survivin, and cyclin D: ^†, P > 0.05, normalized to 18S. Columns, mean; bars, SE.

Figure 5

iFGFR1 dimerization leads to increased phosphorylation of critical translational regulators. A, immunohistochemistry of several critical translational pathway regulators. At least two to three tumors were analyzed per mouse/stain. B, immunoblot analysis of Wnt-1 and Wnt/iR1 mammary gland lysates 0, 3, 6, and 24 h following dimerizer. Protein levels of cMyc, survivin, and cyclin D1 were analyzed as well as phospho-specific phospho-ERK, phospho-MNK, phospho-eIF4E, phospho-AKT, and phospho–S6-RP. Enhanced cMyc and survivin were observed within the 6- to 24-h time period in at least two to three sets of animals. Enhanced phosphorylation of ERK and AKT was observed in at least two to three sets of animals within the 6- to 24-h time period. Phosphorylation of MNK, eIF4E, and S6-RP was observed in one set of four animals per tumor type from 0 to 24 h following injection of dimerizer. Cyclophilin A, total ERK, AKT, MNK, eIF4E, and S6-RP were used as loading controls.

Figure 6

High FGFR expression correlates with the protein translational gene signature in human breast cancer. Heat map comparing FGFR1 and FGFR2 expression levels with protein translational gene signature from Fig. 3 within the Wang data set (37). FGFR1: P = 4E−08, FGFR2: P = 3E−11.