Inducible dimerization of FGFR1: development of a mouse model to analyze progressive transformation of the mammary gland - PubMed (original) (raw)
Inducible dimerization of FGFR1: development of a mouse model to analyze progressive transformation of the mammary gland
Bryan E Welm et al. J Cell Biol. 2002.
Abstract
To develop an inducible and progressive model of mammary gland tumorigenesis, transgenic mice were generated with a mouse mammary tumor virus-long terminal repeat-driven, conditional, fibroblast growth factor (FGF)-independent FGF receptor (FGFR)1 (iFGFR1) that can be induced to dimerize with the drug AP20187. Treatment of transgenic mice with AP20187 resulted in iFGFR1 tyrosine phosphorylation, increased proliferation, activation of mitogen-activated protein kinase and Akt, and lateral budding. Lateral buds appeared as early as 3 d after AP20187 treatment and initially consisted of bilayered epithelial cells and displayed apical and basolateral polarity appeared after 13 d of AP20187 treatment. Invasive lesions characterized by multicell-layered lateral buds, decreased myoepithelium, increased vascular branching, and loss of cell polarity were observed after 2-4 wk of treatment. These data indicate that acute iFGFR1 signaling results in increased lateral budding of the mammary ductal epithelium, and that sustained activation induces alveolar hyperplasia and invasive lesions.
Figures
Figure 1.
iFGFR1 dimerization can inhibit apoptosis and induce proliferation. (A) Schematic drawing of FGF-induced dimerization of FGFR and conditional dimerization of the iFGFR1 fusion protein by AP20187. (B) iFGFR1 is phosphorylated in response to AP20187 treatment. Phosphorylated proteins were immunoprecipitated using anti–phospho-tyrosine antibodies and examined by Western analysis with anti-HA epitope antibodies. (C) Western blot of protein extracts isolated from serum-starved NIH3T3 and HC11 cells treated with AP20187 at several time points after treatment (in minutes). Anti–phospho-MAPK and -Akt antibodies show activation of these kinases in response to AP20187 treatment. (D) FRS2/SNT was immunoprecipitated using anti-FRS2 antibodies and tyrosine phosphorylation was analyzed by immunoblotting with anti–phospho-tyrosine antibodies (top). Equal loading of protein on the blot was determined by reprobing the membrane with anti-FRS2 antibodies (bottom). (E) Survival assays were performed using serum-starved NIH3T3 cells. iFGFR1 cells were stably transfected with iFGFR1, whereas pBKneo cells were stably transfected with pBKneo neomycin selection cassette. iFGFR1+ and pBKneo+ cells were treated with 30 pM AP20187, whereas iFGFR1− and pBKneo− cells were treated with ethanol solvent. iFGFR1+ cells remained viable when serum starved. (F) The change in cell number from survival assays was quantitated by MTS bioreduction. The fold-change in cell number was determined by normalizing to the untreated control (−AP20187 or without rFGF8) for each condition. (G) Caspase-3 fluorometric peptide cleavage assay of serum-starved NIH3T3 cells transduced with iFGFR1 or FKBPv alone and treated with increasing concentrations of AP20187. AP20187-treated iFGFR1 cells showed reduced caspase-3 activity when compared with FKBPv control cells. Data points represent quadruplicate wells. (H) Fold difference in proliferation of AP20187-treated serum-starved NIH3T3 and HC11 cells. Spotted bars represent iFGFR1 and solid black bars FKBPv controls. Data were normalized to cells treated with solvent. Proliferating cells were determined by >2 N DNA content as measured by propidium iodide staining and FACS analysis. Only iFGFR1-transduced HC11 cells showed an increase in proliferation in response to AP20187. Data points represent at least three independent analyses. Error bars represent standard error of the mean. Bars, 5 μm.
Figure 1.
iFGFR1 dimerization can inhibit apoptosis and induce proliferation. (A) Schematic drawing of FGF-induced dimerization of FGFR and conditional dimerization of the iFGFR1 fusion protein by AP20187. (B) iFGFR1 is phosphorylated in response to AP20187 treatment. Phosphorylated proteins were immunoprecipitated using anti–phospho-tyrosine antibodies and examined by Western analysis with anti-HA epitope antibodies. (C) Western blot of protein extracts isolated from serum-starved NIH3T3 and HC11 cells treated with AP20187 at several time points after treatment (in minutes). Anti–phospho-MAPK and -Akt antibodies show activation of these kinases in response to AP20187 treatment. (D) FRS2/SNT was immunoprecipitated using anti-FRS2 antibodies and tyrosine phosphorylation was analyzed by immunoblotting with anti–phospho-tyrosine antibodies (top). Equal loading of protein on the blot was determined by reprobing the membrane with anti-FRS2 antibodies (bottom). (E) Survival assays were performed using serum-starved NIH3T3 cells. iFGFR1 cells were stably transfected with iFGFR1, whereas pBKneo cells were stably transfected with pBKneo neomycin selection cassette. iFGFR1+ and pBKneo+ cells were treated with 30 pM AP20187, whereas iFGFR1− and pBKneo− cells were treated with ethanol solvent. iFGFR1+ cells remained viable when serum starved. (F) The change in cell number from survival assays was quantitated by MTS bioreduction. The fold-change in cell number was determined by normalizing to the untreated control (−AP20187 or without rFGF8) for each condition. (G) Caspase-3 fluorometric peptide cleavage assay of serum-starved NIH3T3 cells transduced with iFGFR1 or FKBPv alone and treated with increasing concentrations of AP20187. AP20187-treated iFGFR1 cells showed reduced caspase-3 activity when compared with FKBPv control cells. Data points represent quadruplicate wells. (H) Fold difference in proliferation of AP20187-treated serum-starved NIH3T3 and HC11 cells. Spotted bars represent iFGFR1 and solid black bars FKBPv controls. Data were normalized to cells treated with solvent. Proliferating cells were determined by >2 N DNA content as measured by propidium iodide staining and FACS analysis. Only iFGFR1-transduced HC11 cells showed an increase in proliferation in response to AP20187. Data points represent at least three independent analyses. Error bars represent standard error of the mean. Bars, 5 μm.
Figure 1.
iFGFR1 dimerization can inhibit apoptosis and induce proliferation. (A) Schematic drawing of FGF-induced dimerization of FGFR and conditional dimerization of the iFGFR1 fusion protein by AP20187. (B) iFGFR1 is phosphorylated in response to AP20187 treatment. Phosphorylated proteins were immunoprecipitated using anti–phospho-tyrosine antibodies and examined by Western analysis with anti-HA epitope antibodies. (C) Western blot of protein extracts isolated from serum-starved NIH3T3 and HC11 cells treated with AP20187 at several time points after treatment (in minutes). Anti–phospho-MAPK and -Akt antibodies show activation of these kinases in response to AP20187 treatment. (D) FRS2/SNT was immunoprecipitated using anti-FRS2 antibodies and tyrosine phosphorylation was analyzed by immunoblotting with anti–phospho-tyrosine antibodies (top). Equal loading of protein on the blot was determined by reprobing the membrane with anti-FRS2 antibodies (bottom). (E) Survival assays were performed using serum-starved NIH3T3 cells. iFGFR1 cells were stably transfected with iFGFR1, whereas pBKneo cells were stably transfected with pBKneo neomycin selection cassette. iFGFR1+ and pBKneo+ cells were treated with 30 pM AP20187, whereas iFGFR1− and pBKneo− cells were treated with ethanol solvent. iFGFR1+ cells remained viable when serum starved. (F) The change in cell number from survival assays was quantitated by MTS bioreduction. The fold-change in cell number was determined by normalizing to the untreated control (−AP20187 or without rFGF8) for each condition. (G) Caspase-3 fluorometric peptide cleavage assay of serum-starved NIH3T3 cells transduced with iFGFR1 or FKBPv alone and treated with increasing concentrations of AP20187. AP20187-treated iFGFR1 cells showed reduced caspase-3 activity when compared with FKBPv control cells. Data points represent quadruplicate wells. (H) Fold difference in proliferation of AP20187-treated serum-starved NIH3T3 and HC11 cells. Spotted bars represent iFGFR1 and solid black bars FKBPv controls. Data were normalized to cells treated with solvent. Proliferating cells were determined by >2 N DNA content as measured by propidium iodide staining and FACS analysis. Only iFGFR1-transduced HC11 cells showed an increase in proliferation in response to AP20187. Data points represent at least three independent analyses. Error bars represent standard error of the mean. Bars, 5 μm.
Figure 1.
iFGFR1 dimerization can inhibit apoptosis and induce proliferation. (A) Schematic drawing of FGF-induced dimerization of FGFR and conditional dimerization of the iFGFR1 fusion protein by AP20187. (B) iFGFR1 is phosphorylated in response to AP20187 treatment. Phosphorylated proteins were immunoprecipitated using anti–phospho-tyrosine antibodies and examined by Western analysis with anti-HA epitope antibodies. (C) Western blot of protein extracts isolated from serum-starved NIH3T3 and HC11 cells treated with AP20187 at several time points after treatment (in minutes). Anti–phospho-MAPK and -Akt antibodies show activation of these kinases in response to AP20187 treatment. (D) FRS2/SNT was immunoprecipitated using anti-FRS2 antibodies and tyrosine phosphorylation was analyzed by immunoblotting with anti–phospho-tyrosine antibodies (top). Equal loading of protein on the blot was determined by reprobing the membrane with anti-FRS2 antibodies (bottom). (E) Survival assays were performed using serum-starved NIH3T3 cells. iFGFR1 cells were stably transfected with iFGFR1, whereas pBKneo cells were stably transfected with pBKneo neomycin selection cassette. iFGFR1+ and pBKneo+ cells were treated with 30 pM AP20187, whereas iFGFR1− and pBKneo− cells were treated with ethanol solvent. iFGFR1+ cells remained viable when serum starved. (F) The change in cell number from survival assays was quantitated by MTS bioreduction. The fold-change in cell number was determined by normalizing to the untreated control (−AP20187 or without rFGF8) for each condition. (G) Caspase-3 fluorometric peptide cleavage assay of serum-starved NIH3T3 cells transduced with iFGFR1 or FKBPv alone and treated with increasing concentrations of AP20187. AP20187-treated iFGFR1 cells showed reduced caspase-3 activity when compared with FKBPv control cells. Data points represent quadruplicate wells. (H) Fold difference in proliferation of AP20187-treated serum-starved NIH3T3 and HC11 cells. Spotted bars represent iFGFR1 and solid black bars FKBPv controls. Data were normalized to cells treated with solvent. Proliferating cells were determined by >2 N DNA content as measured by propidium iodide staining and FACS analysis. Only iFGFR1-transduced HC11 cells showed an increase in proliferation in response to AP20187. Data points represent at least three independent analyses. Error bars represent standard error of the mean. Bars, 5 μm.
Figure 2.
iFGFR1 signaling can induce lateral buds in mammary glands of transgenic mice. Whole-mount and histological analyses of MMTV-iFGFR1 transgenic and wild-type littermates injected i.p. with AP20187 or diluent. Whole mounts of mammary glands at 4× magnification (A, C, E, G, and I) and 10× magnification (B, D, F, H, and J). (A and B) Wild-type mouse treated with AP20187. (C and D) Transgenic mouse treated with diluent. (E and F) Transgenic mouse treated with AP20187. (G and H) Ovex wild-type mouse treated with AP20187. (I and J) Ovex transgenic mouse treated with AP20187. Arrows indicate regions of increased branching and arrowheads show lateral budding at distal regions of ducts. (K) H&E stain of transgenic mouse treated with diluent. (L) Transgenic mouse treated with AP20187. (M) Wild-type mouse stained with anti-HA antibody and Texas red secondary and DAPI-stained nuclei. (N) Transgenic mouse stained with anti-HA antibody and DAPI showing transgene localization in the lateral buds. Bars, 5 μm.
Figure 2.
iFGFR1 signaling can induce lateral buds in mammary glands of transgenic mice. Whole-mount and histological analyses of MMTV-iFGFR1 transgenic and wild-type littermates injected i.p. with AP20187 or diluent. Whole mounts of mammary glands at 4× magnification (A, C, E, G, and I) and 10× magnification (B, D, F, H, and J). (A and B) Wild-type mouse treated with AP20187. (C and D) Transgenic mouse treated with diluent. (E and F) Transgenic mouse treated with AP20187. (G and H) Ovex wild-type mouse treated with AP20187. (I and J) Ovex transgenic mouse treated with AP20187. Arrows indicate regions of increased branching and arrowheads show lateral budding at distal regions of ducts. (K) H&E stain of transgenic mouse treated with diluent. (L) Transgenic mouse treated with AP20187. (M) Wild-type mouse stained with anti-HA antibody and Texas red secondary and DAPI-stained nuclei. (N) Transgenic mouse stained with anti-HA antibody and DAPI showing transgene localization in the lateral buds. Bars, 5 μm.
Figure 3.
Histology of iFGFR1-induced lesions in the mammary gland. Three histologically distinct lesions are observed in AP20187-treated transgenic mouse mammary glands. (A) Panel shows gross mammary gland morphology by whole mounts (10× magnification), cellular detail by H&E stain (20 and 40×), and cellular polarity by immunofluorescence analysis (100×) with anti-ZO-1 (Texas red) and anti-laminin (FITC). Type I lesions initially appeared by day 3 of treatment, and are characterized by the punctate appearance of lateral buds lining the ductal epithelium (Type I, whole mount). Type I lateral buds contain a single layer of polarized mammary epithelial cells with large distinct lumens (Type I, H&E and ZO-1/Laminin). Type II lesions appear starting at week 2 of AP20187 treatment, and are distinguished by uniform multicellular epithelium with small collapsed lumens (Type II). Type III lesions are multicellular, invasive, well vascularized, and have lost ZO-1 and laminin expression (Type III). Bars, 5 μm.
Figure 4.
AP20187 treatment of transgenic mice induces proliferation and altered cell polarity in the mammary gland. (A) Immunoprecipitation with anti-HA epitope antibodies and Western analysis using anti–phospho-tyrosine and HA-epitope antibodies of extracts from transgenic mice treated with AP20187 (+) or diluent (−) for 2 wk. iFGFR1 shows increased phosphorylation levels in AP20187-treated transgenic mice. (B) Western blot analysis showing increased phosphorylation levels of MAPK and Akt in AP20187-treated transgenic mice (MMTV-iFGFR1) over wild-type mice treated with diluent (wt). The positive control was iFGFR1-transduced NIH3T3 cells (NIH3T3-iFGFR1) treated with AP20187. Immunofluorescence analysis with anti–phospho-MAPK antibody (Texas red) and DAPI in wild-type (C) and transgenic (D) mice treated with AP20187 for 2 wk. Anti-BrdU immunofluorescence from wild-type (E) and transgenic (F) mice treated with AP2087 for 4 wk and pulsed with BrdU for 2 h. To determine reversibility, iFGFR mice were treated for 3 d with AP20187, and treatment was stopped 120 h before tissue biopsy. Phosphorylated MAPK (Texas red) and proliferation (anti–BrdU-FITC) were detected in the AP20187-treated (G) and AP20187-withdrawal biopsy (H). Confocal microscopic and immunofluorescence analysis of midpregnant wild-type (I) and 2-wk AP20187-treated transgenic (J) mammary glands using anti–E-cadherin (Texas red, arrows) and anti-laminin (FITC, arrowhead) antibodies. AP20187-treated transgenic mice show multi-cell layering (distance between arrowhead and arrow), collapsed lumens (asterisks), and peripheral localization of E-cadherin. Bars, 5 μm.
Figure 4.
AP20187 treatment of transgenic mice induces proliferation and altered cell polarity in the mammary gland. (A) Immunoprecipitation with anti-HA epitope antibodies and Western analysis using anti–phospho-tyrosine and HA-epitope antibodies of extracts from transgenic mice treated with AP20187 (+) or diluent (−) for 2 wk. iFGFR1 shows increased phosphorylation levels in AP20187-treated transgenic mice. (B) Western blot analysis showing increased phosphorylation levels of MAPK and Akt in AP20187-treated transgenic mice (MMTV-iFGFR1) over wild-type mice treated with diluent (wt). The positive control was iFGFR1-transduced NIH3T3 cells (NIH3T3-iFGFR1) treated with AP20187. Immunofluorescence analysis with anti–phospho-MAPK antibody (Texas red) and DAPI in wild-type (C) and transgenic (D) mice treated with AP20187 for 2 wk. Anti-BrdU immunofluorescence from wild-type (E) and transgenic (F) mice treated with AP2087 for 4 wk and pulsed with BrdU for 2 h. To determine reversibility, iFGFR mice were treated for 3 d with AP20187, and treatment was stopped 120 h before tissue biopsy. Phosphorylated MAPK (Texas red) and proliferation (anti–BrdU-FITC) were detected in the AP20187-treated (G) and AP20187-withdrawal biopsy (H). Confocal microscopic and immunofluorescence analysis of midpregnant wild-type (I) and 2-wk AP20187-treated transgenic (J) mammary glands using anti–E-cadherin (Texas red, arrows) and anti-laminin (FITC, arrowhead) antibodies. AP20187-treated transgenic mice show multi-cell layering (distance between arrowhead and arrow), collapsed lumens (asterisks), and peripheral localization of E-cadherin. Bars, 5 μm.
Figure 5.
iFGFR1 activation can regulate MMPs and induce invasive lesions. (A) iFGFR-transduced HC11 cells were serum starved for 16 h and treated with AP20187 or diluent in serum-free media for 24 h. Media was collected, concentrated, and loaded in equal volumes on a gelatin zymography gel. Media isolated from AP20187-treated cells showed increased levels of MMP-9 and MMP-2 activity. APMA treatment of media for 1 h before gel loading increased the mobility of MMP-9, demonstrating conversion of proMMP-9 to active MMP-9. No APMA induced mobility shift was observed with MMP-2, suggesting only active MMP-2 was present. EDTA (5 mM) treatment in the incubation buffer inhibited MMP activity. (B and C) Indirect immunofluorescence analysis using anti-keratin-14 antibodies (Texas red) demonstrates that AP20187-treated iFGFR1 mice have reduced myoepithelium surrounding lateral buds. (D) Masson's trichrome stain of untreated transgenic mouse mammary gland epithelium showing blue stained collagen (arrow) surrounding the duct. (E) AP20187-treated mouse mammary gland with reduced collagen matrix surrounding the duct and lateral buds (arrows). (F and G) Confocal microscopy of the vascular network (FITC-lectin) surrounding the mammary epithelium (Texas red phalloidin) from AP20187-treated mice, showing increased vessel branching (arrows) associated with lateral buds. Bars, 5 μm.
Figure 5.
iFGFR1 activation can regulate MMPs and induce invasive lesions. (A) iFGFR-transduced HC11 cells were serum starved for 16 h and treated with AP20187 or diluent in serum-free media for 24 h. Media was collected, concentrated, and loaded in equal volumes on a gelatin zymography gel. Media isolated from AP20187-treated cells showed increased levels of MMP-9 and MMP-2 activity. APMA treatment of media for 1 h before gel loading increased the mobility of MMP-9, demonstrating conversion of proMMP-9 to active MMP-9. No APMA induced mobility shift was observed with MMP-2, suggesting only active MMP-2 was present. EDTA (5 mM) treatment in the incubation buffer inhibited MMP activity. (B and C) Indirect immunofluorescence analysis using anti-keratin-14 antibodies (Texas red) demonstrates that AP20187-treated iFGFR1 mice have reduced myoepithelium surrounding lateral buds. (D) Masson's trichrome stain of untreated transgenic mouse mammary gland epithelium showing blue stained collagen (arrow) surrounding the duct. (E) AP20187-treated mouse mammary gland with reduced collagen matrix surrounding the duct and lateral buds (arrows). (F and G) Confocal microscopy of the vascular network (FITC-lectin) surrounding the mammary epithelium (Texas red phalloidin) from AP20187-treated mice, showing increased vessel branching (arrows) associated with lateral buds. Bars, 5 μm.
Figure 6.
Model for FGFR-induced lateral buds and hyperplasia in the mammary gland. (A) Acute iFGFR signaling in the mammary epithelium induces lateral buds (type I) within 72 h of treatment with AP20187. Continuous treatment for 2 wk results in multicellular epithelium (type II) that can progress into invasive lesions (type III) after ∼4 wk of treatment. (B) Normal mammary epithelial cells are polarized (box) with apical localization of ZO-1 at tight junctions, and lateral localization of E-cadherin. The ECM and myoepithelium at the basement membrane can regulate the bioavailability of growth factors and inhibit angiogenesis. Acute iFGFR activity in the mammary epithelium induces proliferation and upregulation of ECM proteases resulting in epithelial invasion into the stroma and the formation of lateral buds. Disruption of the ECM, through MMP upregulation (•), may induce vascular branching by increasing the bioavailability of endothelial growth factors (▴), thus supporting epithelial growth. However, chronic iFGFR activation results in disorganized cell polarity including peripheral localization of E-cadherin and loss of ZO–1 localization at tight junctions. Additionally, the loss of anti-angiogenic factors associated with the myoepithelium may contribute to the invasive characteristics of the iFGFR- induced lesions. Through these mechanisms, FGFR signaling during ductal morphogenesis may function in the proliferation and invasion of mammary epithelium to establish a ductal network, whereas its aberrant regulation may play a role in breast cancer.
References
- Batsakis, J.G., and A.K. el-Naggar. 1999. Myoepithelium in salivary and mammary neoplasms is host-friendly. Adv. Anat. Pathol. 6:218–226. - PubMed
- Benaud, C., R.B. Dickson, and E.W. Thompson. 1998. Roles of the matrix metalloproteinases in mammary gland development and cancer. Breast Cancer Res. Treat. 50:97–116. - PubMed
- Burke, D., D. Wilkes, T.L. Blundell, and S. Malcolm. 1998. Fibroblast growth factor receptors: lessons from the genes. Trends Biochem. Sci. 23:59–62. - PubMed
- Chodosh, L.A., H.P. Gardner, J.V. Rajan, D.B. Stairs, S.T. Marquis, and P.A. Leder. 2000. Protein kinase expression during murine mammary development. Dev. Biol. 219:259–276. - PubMed
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