Article Figures & Data
Figures
- Figure 1.
The growth of multiple TN cell lines is dependent on FGFR signaling. A, indicated cell lines were grown in 1 μmol/L PD173074 for 72 hours, or vehicle, and growth of PD173074 exposed cells expressed relative to controls. *, P < 0.01 Student's t test. Black bars indicate TN cell lines and gray bars cell lines with expression of estrogen receptor (ER) or HER2 amplification. Below is the cell line subtype as defined by Neve and colleagues (25). TN breast cancer cell lines are more sensitive to PD173074 than other cell lines (P = 0.011 Mann–Whitney U Test). B, relative growth of indicated TN cells lines exposed to 1 μmol/L PD173074 for 72 hours, or vehicle, growing in conventional 2D conditions on cell culture plastic or in anchorage-independent conditions on polyHEMA-coated plates. C, relative growth of indicated cell lines exposed to a range of PD173074 concentrations for 72 hours in anchorage-independent conditions on polyHEMA-coated plates. Black–TN cell lines, Gray–ER and/or HER2 positive. D, growth of CAL51 (TN–sensitive) and T47D (ER positive–insensitive) cells in soft agar exposed to 1 μmol/L PD173074 or vehicle continuously for 2 weeks.
- Figure 2.
FGFR signaling engages down stream signal transduction pathways promoting cell-cycle progression and inhibition of apoptosis. A, indicated cell lines either growing on plastic or on polyHEMA-coated plates were treated for 1 hour prior to lysis with 1 μmol/L PD173074 (+) or vehicle (−). Lysates were subject to SDS-PAGE and Western blotting with antibodies against phosphorylated-RSK-Thr359/Ser363, phosphorylated-AKT-Ser473, phosphorylated-ERK1/2-Thr202/Tyr204, total AKT, total ERK1/2, and β-actin. B, quantification of propidium iodide FACS profiles in BT549, Hs578T, CAL51, and CAL120 cells growing in polyHEMA-coated plates and treated for 48 hours with 1 μmol/L PD173074 (+) or no treatment (−). C, lysates of CAL120, CAL51, and Hs578T cells growing in polyHEMA-coated plates and treated for 48 hours with 1 μmol/L PD173074 or no treatment (−), blotted for PARP1 and β-actin. Arrow indicates cleaved PARP1. D, indicated cell lines growing in polyHEMA-coated plates were treated for 48 hours with a range of PD173074 concentrations or no treatment (−), and apoptosis assessed with an assay of activated caspase3/7. Activated caspase 3/7 was expressed relative to the level in cells with no treatment (−).
- Figure 3.
Basal-like breast cancer cell lines express FGF2. A, FGF2 mRNA expression assessed in 35 breast cancer cell lines by quantitative reverse transcriptase PCR (RT-PCR), with cell lines subtype as defined by Neve and colleagues (25). FGF2 expression is expressed in basal-like breast cancer cell lines (luminal vs. basal median expression 0 vs. 0.37, respectively, P = 0.0001 Mann–Whitney U test). Within basal-like cell lines, FGF2 is expressed at substantially higher levels in cancers of basal B (gray) compared with basal A (black) phenotype (median expression 2.0 vs. 0.10, respectively, P = 0.01 Mann–Whitney U test). FGF2 expression was undetectable in the majority of cell lines, as indicated by lack of a bar. FGF2 expression was expressed relative to the mean expression level. B, FGF2 expression assessed by Western blot on breast cancer cell line lysates along with β-actin. Basal B cell line subtype as indicated by (+) and other subtypes (−). Two nontumorigenic breast epithelial cell lines of basal B phenotype MCF10A and MCF12A are included. C, PD173074 survival fraction (SF) (from Fig. 1A) in cell lines with and without FGF2 protein expression as assessed by Western blot. Cancer cell lines with FGF2 expression have a lower SF (P = 0.006 Mann–Whitney U test). D, FGF2 expression in conditioned media. Indicted cell lines were grown for 72 hours before collection of media. FGF2 expression was assessed by ELISA, and expressed as pg FGF2 protein per mL of media. Basal B cell lines (gray) condition media with FGF2 (median expression basal B 45 pg/mL vs. other subtypes 14 pg/mL, P = 0.001 Mann–Whitney U Test).
- Figure 4.
FGF2 expression is associated with the core basal-like phenotype in breast cancers. A, assessment of FGF2 expression by IHC, with a cancer negative for FGF2 expression (left), with FGF2 cytoplasmic expression (middle), and FGF2 nuclear expression (right). B, validation of FGF2 IHC staining, with assessment of FGF2 expression by quantitative RT-PCR on mRNA extracted from a random selection of tumors positive and negative for FGF2 expression (n = 28). FGF2 mRNA was expressed at higher levels in FGF2 IHC–positive cancers (P < 0.0001 Kruskal–Wallis one way ANOVA), and at higher levels in cancers with FGF2 cytoplasmic expression (n = 6, P = 0.018) and FGF2 nuclear expression (n = 3, P = 0.013 Mann–Whitney U test) compared with FGF2 negative cancers (n = 15). C, Cytoplasmic FGF2 expression according to tumor subtype, defined using the IHC criteria of Nielsen and colleagues (28), with the percentage of tumors in each subtype positive for cytoplasmic FGF2 expression (P < 0.0001 comparison of all subtypes with χ2 test and P = 0.0015 comparison of core basal-like and nonbasal TN cancers with Fisher's exact test). Number in each group luminal n = 138, HER2 n = 26, core basal-like n = 26, TN nonbasal n = 9. D, Kaplan–Meier curves of overall survival for breast cancers with cytoplasmic FGF2 expression (n = 24) compared with cancers without cytoplasmic FGF2 expression (n = 175; P = 0.035 log-rank test, P = 0.007 Gehan–Breslow–Wilcoxon test).
- Figure 5.
FGF2 autocrine signaling promotes growth and downstream signaling. A, CAL120, CAL51, and Hs578T cell lines were transfected with siCON nontargeting control, 2 individual siRNA targeting FGF2 (siFGF2-A and siFGF2-B) and SMARTpool targeting FGF2 (siFGF2), with survival assessed after 5 days. Comparison between siCON and FGF2 siRNA with Student's t test *, P < 0.01, +, P < 0.05. B, CAL120 cells were transfected with siCON, 3 individual siRNA targeting FGFR1 (siFGFR1 A–C), and FGFR1 SMARTpool (siFGFR1) and survival was assessed 5 days post-transfection. Comparison between siCON and FGFR1 siRNA with Student's t test *, P < 0.01. C, Hs578T cells were transfected with siCON and siFGF2, lysates were made 72 hours post-transfection and subject to Western blotting with indicated antibodies. D, Hs578T cells in polyHEMA-coated plates were exposed to various concentrations of control IgG antibody or FGF2-neutralizing antibody, with survival assessed after 72 hours exposure. E, FGFR signaling presents a potential therapeutic target. CAL51 cells were injected into the flanks of 20 nude mice (3 × 106 cells per flank), and xenografts established over 21 days. Half mice were treated with 25 mg/kg PD173074 and half vehicle, for 5 days of 7, and tumor growth assessed over 2 weeks. PD173074 substantially reduced the growth of CAL51 xenografts (P < 0.0001 ANOVA). Arrows indicate the treatment days with PD173074 or vehicle.
Tables
- Table 1.
Tumor features associated with cytoplasmic FGF2 expression assessed by IHC in an invasive breast cancer tissue microarray
FGF2 positive FGF2 negative P n 24 175 Pathology IDC 24 125 0.013 ILC 0 44 Other 0 6 Median tumor size (mm) 20.5 20 1a Histologic grade 1 0 23 0.0005b 2 1 56 3 23 91 Vascular invasion 75% (18/24) 69% (120/175) 0.64 Axillary node positive 63% (16/24) 67% (117/175) 1 ER positive 21% (5/24) 86% (151/175) <0.0001 PR positive 25% (6/24) 77% (135/175) <0.0001 HER2 positive 13% (3/24) 13% (23/175) 1 EGFR 42% (10/24) 5% (8/175) <0.0001 CK5/6 58% (14/24) 5% (8/175) <0.0001 CK14 46% (11/24) 4% (7/175) <0.0001 Any basal marker 79% (19/24) 9% (15/175) <0.0001 Tumor subtype Luminal 5 133 <0.0001b HER2 3 23 Core basal-like 16 10 TN nonbasal 0 9 Abbbreviations: IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; CK5/6, cytokeratin 5/6; CK14, cytokeratin 14, any basal-marker (positive for any of EGFR, CK5/6 or CK14).
NOTE: Statistical analysis was with Fisher's exact test, unless indicated. Tumor subtype as defined by Nielsen and colleagues (ref. 28; HER2–HER2 amplified, Luminal–HER2 negative/ER positive, core basal-like–TN expressing CK5/6 or EGFR, TN nonbasal–TN with no expression of CK5/6 and EGFR).
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aMann–Whitney U Test or
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bχ2 test.
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Supplementary Data
Files in this Data Supplement:
- Supplementary Figures 1-7, Methods - PDF file - 1.5MB
Volume 17, Issue 16
