FGFR1 amplification drives endocrine therapy resistance and is a therapeutic target in breast cancer

Abstract

Amplification of fibroblast growth factor receptor 1 (FGFR1) occurs in approximately 10% of breast cancers and is associated with poor prognosis. However, it is uncertain whether overexpression of FGFR1 is causally linked to the poor prognosis of amplified cancers. Here, we show that FGFR1 overexpression is robustly associated with FGFR1 amplification in two independent series of breast cancers. Breast cancer cell lines with FGFR1 overexpression and amplification show enhanced ligand-dependent signaling, with increased activation of the mitogen-activated protein kinase and phosphoinositide 3-kinase-AKT signaling pathways in response to FGF2, but also show basal ligand-independent signaling, and are dependent on FGFR signaling for anchorage-independent growth. FGFR1-amplified cell lines show resistance to 4-hydroxytamoxifen, which is reversed by small interfering RNA silencing of FGFR1, suggesting that FGFR1 overexpression also promotes endocrine therapy resistance. FGFR1 signaling suppresses progesterone receptor (PR) expression in vitro, and likewise, amplified cancers are frequently PR negative, identifying a potential biomarker for FGFR1 activity. Furthermore, we show that amplified cancers have a high proliferative rate assessed by Ki67 staining and that FGFR1 amplification is found in 16% to 27% of luminal B-type breast cancers. Our data suggest that amplification and overexpression of FGFR1 may be a major contributor to poor prognosis in luminal-type breast cancers, driving anchorage-independent proliferation and endocrine therapy resistance.

Figure 1. FGFR1 amplified breast tumours and cancer cell lines over-express FGFR1

A. 58 ER positive breast cancers distributed in order of FGFR1 mRNA level, expressed relative to the median expression level; Red - tumours with FGFR1 amplification assessed by CISH; Black - tumours without FGFR1 amplification. Right panel example photomicrographs from a tumour without and with FGFR1 amplification. B. FGFR1 amplification status assessed in a second series of 93 invasive breast cancers. FGFR1 gene expression was assessed by quantitative RT-PCR from FGFR1 amplified tumours, and grade and ER matched controls, and expressed relative to the median expression level of controls. FGFR1 amplified tumours had substantially higher median FGFR1 expression than non-amplified controls (13.4 vs 1, p=0.0002 Mann Whitney U test). C. FGFR1 expression assessed by quantitative RT-PCR in a panel of 40 breast cancer cell lines. Six cell lines over-express FGFR1 (indicated by arrows), all off which have high level FGFR1 amplification (Supplementary Figure 3). FGFR1 expression displayed relative to median expression. D. Western blot confirming over-expression of FGFR1 protein in cell lines with FGFR1 amplification compared to non-amplified control cell line MCF7.

Figure 2. Assessment of the FGFR dependence of FGFR1 amplified cell lines

A. Sensitivity of MDA-MB-134 cell lines to FGFR1 siRNA (siFGFR1). Cells were transfected with siFGFR1, or siCON non-targeting control, and survival assessed six days later with Cell Titre-Glo^® cell viability assay (Promega). MDA-MB-134 obtained directly from MD Anderson were sensitive to FGFR1 knockdown (p<0.001, Student's T Test), but not MDA-MB-134 obtained from ATCC. Error bars SEM of three repeat experiments. B. Left: Transfection of FGFR1 amplified cell lines with siCON or siFGFR1, and siPLK1 as a positive toxicity control, with survival assessed at 5-7 days transfection. Right: FGFR1 expression by quantitative RT-PCR in SUM44 cells transfected with siFGFR1, or siCON, 72 hours prior to RNA extraction. C. FGFR1 amplified cell lines were grown for 96 hrs in media supplemented with a range of concentrations of PD173074 pan FGFR tyrosine kinase inhibitor, and survival expressed relative to that of untreated cells. The SUM52PE breast cancer cell line that harbours FGFR2 amplification, and is highly sensitive to FGFR inhibitors, was used a positive control (26). D. CAL120 cells were grown in soft agar with or without continuous exposure to 1μM PD173074. Example micrographs at 4X power from wells with and without PD173074. Bar chart: mean colonies per well from 3 repeats (without 25.3 colonies vs with PD173074 0 colonies, p=0.008, Student's T test).

Figure 3. FGFR1 amplification drives both ligand dependent and independent signalling

A. Indicated cell lines growing in 10% serum were treated for 15 minutes prior to lysis with 1ng/ml FGF2 (+), or not (−). Lysates were subject to SDS-PAGE and western blotting with antibodies against phosphorylated-FRS2-Tyr196, phosphorylated-AKT1-Ser473, phosphorylated-ERK1/2-Thr202/Tyr204, and β-ACTIN. Two different exposures of FRS2-Tyr196 are shown. B. Stable polyclonal pool of T47D cells were established with empty vector (T47D-EV) or FGFR1 expression vector (T47D-FGFR1). Western blots of T47D-EV or T47D-FGFR1 cells treated for 15 minutes prior to lysis with 1ng/ml FGF2, or no treatment (−), and blotted with indicated antibodies. C. Indicated cell lines were serum starved for 24 hours, and lysates were made after 1hr exposure to 1μM PD173074 (+), or no exposure (−), as indicated. Lysates were subjected to western blotting and blotted with indicated antibodies.

Figure 4. FGFR1 drives endocrine therapy resistance in amplified lines

A. FGFR sensitive MDA-MB-134 cells, obtained from MD Anderson, were transfected with siCON, siFGFR1, or two individual siRNA targeting FGFR1 (siFGFR1-A and siFGFR1-B). Starting at 48hours post transfection, cells were treated with range of concentrations of 4-hyrdoxytamoxifen (4-OHT), and survival assessed at 6 days exposure. Error bars represent SEM of 3 repeat experiments. B. SUM44 cells were transfected with siCON, or siFGFR1, and 48 hours after transfection treated with range of concentrations of 4-OHT in the presence of 10ng/ml FGF2, or with no FGF2. Survival was assessed after 6 days exposure. C. Propidium Iodide FACS profiles in SUM44 cells transfected six days earlier with siCON, or siFGFR1, and treated for 72 hours with 10ng/ml FGF2, 10nM 4OH-tamoxifen, the combination, or no treatment (−). D. Quantification of S phase fraction from three independent experiments. Fraction in S phase; siCON transfected no treatment 14.4% vs FGF2/4OH-T treated 13.4% (p=0.3, Student's T test); siFGFR1 transfected 7.2% vs 4.5% respectively (p=0.02).

Figure 5. Signalling in SUM44 cells is response to endocrine therapies

A. Western blots of PR, ER, phosphorylated-ERK1/2, ERK1/2, CCND1, β-ACTIN, and FGFR1. SUM44 cell lysates treated for 24 hours prior to lysis with 100nM 4-OHT, 100nM ICI-182780, or no treatment (−), with or without 10ng/ml FGF2. Phosphorylated-AKT1 was not detected. B. Western blots of phosphorylated-PLCγ1 (Tyr783), phosphorylated-AKT, phosphorylated-p90RSK (Thr359/Ser363), phosphorylated-ERK1/2, and β-ACTIN on SUM44 cell lysates treated for either 10 minutes or 24 hours with 10ng/ml FGF2 prior to lysis. C. Quantitative RT-PCR analysis of cyclin D1 (CCND1 - Top) and (PR - Bottom) expression in SUM44 cells treated with or without 10ng/ml FGF2 for 24 hours prior to RNA isolation, without (Black bars) or in the presence of 100nM ICI-182780 (Grey bars). D. SUM44 cells were co-transfected with EREIItkLuc (ERE-luciferase reporter construct) and pCH110 (β-galactosidase reporter construct) and treated for 48 hours with 10ng/ml FGF2, or no treatment, with 100nM ICI-182780 as positive control. Luciferase activity was expressed relative to β-galactosidase activity. Error bars SEM of 3 repeats, p values Student's T-test.

Figure 6. FGFR1 over-expression is common in high risk ER positive breast cancer

A. Kaplan-Meier curves of distant metastasis free survival from Guys series of ER positive tumours with FGFR1 over-expression (n=10) versus normal FGFR1 expression (76). FGFR1 over-expressing tumours have substantially worse survival (HR 7.4, 95% CI 1.8 to 30.5, p=0.0053, log rank test). B. Proportion of tumours with progesterone receptor expression in the same cohort (all tumours were ER positive, p=0.03 Fisher's exact test). C. Ki67 was assessed by IHC in the same cohort. FGFR1 over-expressing tumours have higher Ki67 (P=0.021, Mann Whitney U Test). Of tumours with high proliferation (≥14% Ki67 as a surrogate for luminal B subtype(2)) 16% have FGFR1 over-expression, compared to 3.5% of low proliferation cancers. D. Left: Incidence of FGFR1 over-expression in breast cancers according to intrinsic subtype (23% Luminal B over-express FGFR1). Right: Incidence of 8p11-12 amplification, defined by co-overexpression of neighbouring genes (Supplementary methods), according to intrinsic subtype (27% Luminal B have 8p11-12 amplification). Analysis of data on 295 invasive breast cancers from van de Vijver et al 2002(31). Statistical comparison across groups was with the Chi squared test.