Article Figures & Data
Figures
- Figure 1.
FGFR1-amplified breast tumors and cancer cell lines overexpress FGFR1. A, 58 ER-positive breast cancers distributed in order of FGFR1 mRNA level, expressed relative to the median expression level. Red, tumors with FGFR1 amplification assessed by CISH; black, tumors without FGFR1 amplification. Right, example photomicrographs from a tumor 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 tumors, and grade- and ER-matched controls, and expressed relative to the median expression level of controls. FGFR1-amplified tumors had substantially higher median FGFR1 expression than nonamplified controls (13.4 versus 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 overexpress FGFR1 (indicated by arrows), all off which have high-level FGFR1 amplification (Supplementary Fig. S2). FGFR1 expression displayed relative to median expression. D, Western blot confirming overexpression of FGFR1 protein in cell lines with FGFR1 amplification compared with nonamplified 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 nontargeting control, and survival was assessed 6 d later with CellTiter-Glo cell viability assay. MDA-MB-134 cells obtained directly from M.D. Anderson were sensitive to FGFR1 knockdown (P < 0.001, Student's t test), but not MDA-MB-134 obtained from ATCC. Columns, mean of three repeat experiments; bars, SE. B, left, transfection of FGFR1-amplified cell lines with siCON or siFGFR1, and siPLK1 as a positive toxicity control, with survival assessed at 5 to 7 d after transfection; right, FGFR1 expression by quantitative RT-PCR in SUM44 cells transfected with siFGFR1, or siCON, 72 h before RNA extraction. C, FGFR1-amplified cell lines were grown for 96 h in medium supplemented with a range of concentrations of PD173074 pan-FGFR tyrosine kinase inhibitor, and survival was expressed relative to that of untreated cells. The SUM52PE breast cancer cell line that harbors FGFR2 amplification, and is highly sensitive to FGFR inhibitors, was used as a positive control (27). D, CAL120 cells were grown in soft agar with or without continuous exposure to 1 μmol/L PD173074. Example micrographs at 4× power from wells with and without PD173074. Bar chart, mean colonies per well from three repeats [without PD173074 (25.3 colonies) versus with PD173074 (0 colonies); P = 0.008, Student's t test].
- Figure 3.
FGFR1 amplification drives both ligand-dependent and ligand-independent signaling. A, indicated cell lines growing in 10% serum were treated for 15 min before lysis with 1 ng/mL FGF2 (+) or not (−). Lysates were subjected 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 was established with empty vector (T47D-EV) or FGFR1 expression vector (T47D-FGFR1). Western blots of T47D-EV or T47D-FGFR1 cells treated for 15 min before lysis with 1 ng/mL FGF2, or no treatment (−), and blotted with indicated antibodies. C, indicated cell lines were serum starved for 24 h, and lysates were made after 1-h exposure to 1 μmol/L 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 M.D. Anderson, were transfected with siCON, siFGFR1, or two individual siRNA targeting FGFR1 (siFGFR1-A and siFGFR1-B). Starting at 48 h after transfection, cells were treated with range of concentrations of 4-OHT, and survival was assessed after 6 d exposure. Points, mean of three repeat experiments; bars, SE. B, SUM44 cells were transfected with siCON or siFGFR1 and, 48 h after transfection, treated with range of concentrations of 4-OHT in the presence of 10 ng/mL FGF2 or with no FGF2. Survival was assessed after 6-d exposure. C, propidium iodide FACS profiles in SUM44 cells transfected 6 d earlier with siCON, or siFGFR1, and treated for 72 h with 10 ng/mL FGF2, 10 nmol/L 4-OHT, 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%) versus FGF2/4-OHT treated (13.4%; P = 0.3, Student's t test); siFGFR1 transfected, 7.2% versus 4.5% (P = 0.02).
- Figure 5.
Signaling in SUM44 cells in 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 h before lysis with 100 nmol/L 4-OHT, 100 nmol/L ICI-182780, or no treatment (−), with or without 10 ng/mL FGF2. Phosphorylated AKT1 was not detected. B, Western blots of phosphorylated PLCγ1 (Tyr783), phosphorylated AKT, phosphorylated p90-RSK (Thr359/Ser363), phosphorylated ERK1/2, and β-actin on SUM44 cell lysates treated for either 10 min or 24 h with 10 ng/mL FGF2 before lysis. C, quantitative RT-PCR analysis of CCND1 (top) and PR (bottom) expression in SUM44 cells treated with or without 10 ng/mL FGF2 for 24 h before RNA isolation, without (black columns) or in the presence of 100 nmol/L ICI-182780 (gray columns). D, SUM44 cells were cotransfected with EREIItkLuc (ERE-luciferase reporter construct) and pCH110 (β-galactosidase reporter construct) and treated for 48 h with 10 ng/mL FGF2, or no treatment, with 100 nmol/L ICI-182780 as positive control. Luciferase activity was expressed relative to β-galactosidase activity. Columns, mean of three repeats; bars, SE. P values, Student's t test.
- Figure 6.
FGFR1 overexpression is common in high-risk ER-positive breast cancer. A, Kaplan-Meier curves of distant metastasis-free survival from Guy's series of ER-positive tumors with FGFR1 overexpression (n = 10) versus normal FGFR1 expression (76). FGFR1-overexpressing tumors have substantially worse survival (hazard ratio, 7.4; 95% CI, 1.8–30.5; P = 0.0053, log-rank test). B, proportion of tumors with PR expression in the same cohort (all tumors were ER positive; P = 0.03, Fisher's exact test). C, Ki67 was assessed by immunohistochemistry in the same cohort. FGFR1-overexpressing tumors have higher Ki67 (P = 0.021, Mann-Whitney U test). Of tumors with high proliferation [≥14% Ki67 as a surrogate for luminal B subtype (2)], 16% have FGFR1 overexpression compared with 3.5% of low-proliferating cancers. D, left, incidence of FGFR1 overexpression in breast cancers according to intrinsic subtype (23% luminal B overexpress FGFR1); right, incidence of 8p11-12 amplification, defined by co-overexpression of neighboring genes (Supplementary Materials and Methods), according to intrinsic subtype (27% luminal B have 8p11-12 amplification). Analysis of data on 295 invasive breast cancers from van de Vijver and colleagues (32). Statistical comparison across groups was with the χ2 test.
Additional Files
Supplementary Data, Pearson et al.
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Volume 70, Issue 5
