Journal: Oncogene

Article Title: Transduction of the SkBr3 breast carcinoma cell line with the HOXB7 gene induces bFGF expression, increases cell proliferation and reduces growth factor dependence.

doi: 10.1038/sj.onc.1201875

Figure Lengend Snippet: Figure 1 Expression of HOXB7 and bFGF genes in A375 melanoma, SkBr3 mammary carcinoma and HOXB7-transduced SkBr3. (a) RNase protection analysis. Riboprobes (R) and protected fragments (P) are shown on the right, and molecular size (base pairs) markers (MWM) (pGEM4Z HpaII) on the left. b-actin expression is shown as internal control. (b) Northern blot analysis. b2 microglobulin expression is shown as internal control. (c) Western blot analysis of bFGF produced by A375 melanoma, SkBr3 mammary carcinoma and HOXB7-transduced SkBr3 cells

Article Snippet: Intracellular and secreted bFGF were quanti®ed by a human bFGF ELISA from R&D systems (Minneapolis, MN).

Techniques: Expressing, Control, Northern Blot, Western Blot, Produced

Journal: Oncogene

Article Title: Transduction of the SkBr3 breast carcinoma cell line with the HOXB7 gene induces bFGF expression, increases cell proliferation and reduces growth factor dependence.

doi: 10.1038/sj.onc.1201875

Figure Lengend Snippet: Figure 3 (a) Role of bFGF for SkBr3/HOXB7 cell growth in low serum. Growth kinetics of parental and HOXB7-transduced SkBr3 cells maintained in 10% or 1% FCS were compared. Cell growth was monitored over the indicated time intervals by methylene blue inclusion, as indicated in Materials and methods. (b) Eect of exogenous rbFGF on SkBr3 cell growth. Growth curves of cells maintained in 1% FCS plus dierent concentra- tions of rbFGF evaluated by methylene blue inclusion

Article Snippet: Intracellular and secreted bFGF were quanti®ed by a human bFGF ELISA from R&D systems (Minneapolis, MN).

Techniques:

Journal: Oncogene

Article Title: Transduction of the SkBr3 breast carcinoma cell line with the HOXB7 gene induces bFGF expression, increases cell proliferation and reduces growth factor dependence.

doi: 10.1038/sj.onc.1201875

Figure Lengend Snippet: Figure 4 Inhibition of SkBr3/HOXB7 cell proliferation upon treatment with 30 mM of bFGF antisense (a) or of sense (s) oligomers. Evidence of bFGF intracrine loop operating in cells kept in low serum. Mean+s.d. of three separate experiments is shown

Article Snippet: Intracellular and secreted bFGF were quanti®ed by a human bFGF ELISA from R&D systems (Minneapolis, MN).

Techniques: Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Reactivation of Mitogen-activated Protein Kinase (MAPK) Pathway by FGF Receptor 3 (FGFR3)/Ras Mediates Resistance to Vemurafenib in Human B-RAF V600E Mutant Melanoma *

doi: 10.1074/jbc.M112.377218

Figure Lengend Snippet: Enhanced FGFR3 activation in vemurafenib-resistant B-RAF V600E melanoma cells. A, phospho-RTK antibody array analysis. Cell lysates from A375, M14, A375-R1, and M14-R cell lines were incubated on RTK antibody array for 16 h and phosphorylation status was determined as described under “Experimental Procedures.” Each RTK antibody is spotted in duplicate. Supplemental Table S2 describes the list of RTKs and the layout of the antibody array. B, confirmation of phospho-FGFR3 levels by Western blot analysis. Protein levels of total and phosho-FGFR3 were assessed using immunoblotting. C, ELISA analysis of secreted FGF2 in the conditioned media obtained from A375, A375-R1, M14, and M14-R cells. ELISA was performed as described in “Experimental Procedures.”

Article Snippet: Cells were cultured for 48 h in growth medium described above, and the conditioned medium samples (cell free culture supernatant) were analyzed for concentrations of human FGF2 using human FGF2 Quantikine ELISA Kit (R&D Systems).

Techniques: Activation Assay, Ab Array, Incubation, Phospho-proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 1. Involvement of FGF2-FGFR1 axisin Akt activation. (A) The effect of CAF-CM on proliferation of breast cancer (MCF-7, MDA-MB-231, and MDA-MB-468) cells was determined by the MTT assay. Cells were incubated with or without CAF-CM for 72 hours. ***Significantly different be- tween the groups compared (P < 0.001). (B) MDA-MB-231 cells were incubated with CAF-CM for the indicated time periods. Phosphorylation of Akt and STAT3 were detected by Western blot analysis. (C) MDA-MB-231 cells were exposed to CAF-CM with or without FGF-2-neutralizing antibody for 3 hours. Phosphorylation of Akt was detected by Western blot analysis. *,***Significantly different between the groups compared (*P < 0.05; ***P < 0.001). (D) MDA-MB-231 cells were treated with 20 ng/mL of FGF2 for the indicated time periods. The phosphorylation of FRS2α as well as Akt was analyzed by Western blot. (E) RNA-seq data set of TCGA breast invasive carcinoma was downloaded from XenaBrower (https://xenabrowser.net). mRNA expression levels of total 1,097 samples (Illumina HiSeq log [normalized counts + 1]) were prepared by quantile normalization. Pearson cor- relation coefficient was calculated to assess the relationship between FGF2 and FGFR1. (F, G) Correlation of FGFR1 protein expression with FGF2 (F) and Akt (G), based on 105 breast invasive carcinoma protein specimens (TCGA, Pan-Cancer Atlas) from the cBioportal database (www.cbiopor- tal.org). FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; CAFs, cancer-associated fibroblasts; NFs, normal fibroblasts; CM, conditioned medium; ns, not significantly different; FRS2, FGFR substrate 2; TCGA, The Cancer Genome Atlas; CPTAC, the Clinical Proteomic Tumor Analysis Consortium.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Activation Assay, MTT Assay, Incubation, Phospho-proteomics, Western Blot, RNA Sequencing, Expressing

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 2. Role of FGFR1 in Akt phosphorylation and breast cancer cell growth and progression. (A) MDA-MB-231 cells were transfected with scrambled or FGFR1 si-RNA for 24 hours. Cells were then incubated with 20 ng/mL of FGF2 for 15 minutes to measure phosphorylated FRS2α. (B) Mice were subjected to xenograft co-injecting with fibroblasts and MDA-MB-231 breast cancer cells. A complex collagen network was detected in H&E-stained tumors by an intense pink and in Masson’s trichrome stain by a blue stain (arrows). Stromal compartment was also detected by α-SMA immunostaining. Magnification, x100. Bars, 100 μm. (C) Phosphorylated Akt in the xenograft tumors was determined by Western blot analysis. *Sig- nificantly different between the groups compared (P < 0.05). (D) Enrichment plots of hallmark gene sets in the high FGFR1-expressing group. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; FRS2, FGFR substrate 2; α-SMA, alpha-smooth muscle actin; CONT, control; EMT, epithelial- mesenchymal transition.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Phospho-proteomics, Transfection, Incubation, Staining, Immunostaining, Western Blot, Expressing, Control

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 3. The involvement of FGF2-induced ROS generation in nuclear localization of FGFR1. (A) MDA-MB-231 cells were co-cultured with NFs or CAFs for 24 hours. MDA-MB-231 (5 x 10 3 cells) and NFs or CAFs (5 x 10 3 cells) were mixed prior to seeding and incubated for 24 hours. Immunocytochemical analysis was performed using anti-FGFR1 antibody. Cells were then stained with DAPI for detection of nuclei. Magnification, x100. Bars, 200 μm. (B) MDA-MB-231 cells were incubated with FGF2 for 1 hour. Immunocytochemical analysis was performed using anti-FGFR1 antibody. Cells were then stained with PI for detection of nuclei. Magnification, x100. Bars, 200 μm. (C) MDA-MB-231 cells were treated with 20 ng/ mL of FGF2 for 1 hour, followed by Western blot analysis of FGFR1 in cytosolic and nuclear extracts. Lamin B was used as a nuclear marker. *Sig- nificantly different between the groups compared (P < 0.05). (D, E) MDA-MD-231 cells were incubated with CAF-CM or FGF2 for 3 hours and 1 hour, respectively. After staining with DCF-DA for 30 minutes, fluorescent microscopic (D) or flow cytometric (E) analysis was performed to detect intracellu- lar ROS accumulation. Magnification, x40. (F) After pretreatment with NAC for 3 hours, cells were exposed to FGF2 for additional 1 hour. Nuclear ex- tracts were subjected to Western blot analysis to detect the presence of FGFR1 and Nrf2 in the nucleus. **Significantly different between the groups compared (P < 0.01). (G) MDA-MB-231 cells were exposed to FGF2 (20 ng/mL) for 1 hour. Cell lysates were subjected to immunoprecipitation using CBP antibody for 16 hours followed by immunoblotting with. FGFR1 or Nrf2 antibody. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; ROS, reactive oxygen species; CAFs, cancer-associated fibroblasts; CM, conditioned medium; NFs, normal fibroblasts; DAPI, 4′,6-diamidino-2-phenylindole; PI, propidium iodide; CONT, cotrol; DCF-DA, 2’,7’-dichlorodihydrofluorescein diacetate; NAC, N-acetylcysteine; CBP, CREB-binding protein.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Cell Culture, Incubation, Staining, Western Blot, Marker, Immunoprecipitation, Binding Assay

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 4. Possible association between nuclear FGFR1 and Nrf2. (A) TNBC patient cohorts were validated based on the mean expression value of the indicated single genes (FGFR1 or NFE2L2) or as a signature of two genes together and patient survival was analyzed (n = 255). (B, C) MDA- MB-231 cells were transfected with scrambled or Nrf2 si-RNA for 24 hours. Cells were then incubated with 20 ng/mL of FGF2 for 3 hours. The mRNA (B) and protein (C) expression of cyclin D1 was assessed by RT-PCR and Western blot analyses, respectively. The expression of cyclin D1 was mea- sured by RT-PCR (B) and Western blot (C) analyses. (D) In tumor microenvironment, fibroblasts are activated to form CAFs, which secrete FGF2. CAF-derived FGF2 could induces nuclear translocation as well as de novo synthesis of FGFR1, ultimately contributing to cancer cell proliferation, mi- gration and tumor growth. While membrane bound FGFR1 may translocate to nucleus as a complex with FGF2 which has nuclear localization signal (NLS), the complex is likely rather to stimulate the intracellular signaling via FRS2α, which induces transcription of FGFR-1 gene. On the other hand, newly synthesized FGFR-1 is speculated to enter the nucleus as a complex with a cargo protein harboring NLS. FGFR-1 is translocated to the inner nuclear membrane through the nuclear pore complexes (NPCs), which is regulated by importin β. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; TNBC, triple negative breast cancer; HR, hazard ratio; CAFs, cancer-associated fibroblasts; ER, endoplasmic reticulum; FRS2, FGFR substrate 2; CBP, CREB-binding protein.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Expressing, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Derivative Assay, Translocation Assay, Membrane, Synthesized, Binding Assay