Journal: Cell & Bioscience

Article Title: Oncogenic activation revealed by FGFR2 genetic alterations in intrahepatic cholangiocarcinomas

doi: 10.1186/s13578-023-01156-7

Figure Lengend Snippet: Study design. 474 ICCs were included in this study. Among 474 cases, 290 ICCs were analyzed both by FISH and NGS testing (DNA-based and RNA-based) for FGFR2 genetic alterations, and the remaining 184 cases were only analyzed by FISH. In total, there were thirty patients with FGFR2 fusion/translocation and four patients with FGFR2 genetic mutations in our cohort. Further, we selected 1534 ICC patients in 10 studies from public databases and found 38 FGFR2 genetic alterations excluding fusions in 36 patients. In addition, we also collected 31 cases with FGFR2 in-frame deletions and 65 cases with FGFR2 site mutations across multiple cancers. Finally, we depicted the comutation plot of these FGFR2 translocation/fusion, in-frame deletion and site mutation cases

Article Snippet: RBEwere infected with recombinant lentiviruses expressing different FGFR2 fusion mutants, then were distributed into 96-well plates with indicated concentrations of BGJ398 (#T1975, TargetMol, USA).

Techniques: Next-Generation Sequencing, Translocation Assay, Mutagenesis

Journal: Cell & Bioscience

Article Title: Oncogenic activation revealed by FGFR2 genetic alterations in intrahepatic cholangiocarcinomas

doi: 10.1186/s13578-023-01156-7

Figure Lengend Snippet: FGFR2 lesion spectrum in a large cohort of ICC and pan-cancer patients. In a total of 290 ICCs, five FGFR2 genetic alterations (except for translocation/fusion) were found, the types of genomic alterations were color coded and the length of the bar represents the frequency of mutations ( A ). In total of 1534 ICCs from ten studies, 38 FGFR2 genetic alterations excluding fusions were found and the types of genomic alterations were color coded ( B ). A consortium of 91,129 multiple tumors were collected to identify FGFR2 in-frame deletions. In total, eleven tumor types that carried FGFR2 genetic short in frame deletions were identified with ICC at the highest frequency (0.62%, 7/1122), notched rectangles represent the frame deletion represented twice, the percentages represent the frequency of the mutation ( C ). Pan-cancer study of 9999 cases from public database was analyzed to evaluate the prevalence of FGFR2 site mutations, there were 70 site mutations in 65 patients were found, and in ICC the prevalence was 1.08% ( D ). Other tumors that may show scattered FGFR2 point mutations but are not listed in the figure include: cancer of unknown (1/120, A511T), uterine corpus endometrial carcinoma (1/61, S252W), extrahepatic cholangiocarcinoma (1/351, R255W), gallbladder carcinoma (1/240, N441S), gastric cancer (1/866, K399Q), ovarian cancer(1/261, S252W) and urothelial carcinoma (1/96,Q259L)

Article Snippet: RBEwere infected with recombinant lentiviruses expressing different FGFR2 fusion mutants, then were distributed into 96-well plates with indicated concentrations of BGJ398 (#T1975, TargetMol, USA).

Techniques: Translocation Assay, Mutagenesis

Journal: Cell & Bioscience

Article Title: Oncogenic activation revealed by FGFR2 genetic alterations in intrahepatic cholangiocarcinomas

doi: 10.1186/s13578-023-01156-7

Figure Lengend Snippet: Histopathological features of FGFR2 genetic alteration ICCs. FGFR2 gene fusion/translocation ICCs were enriched for specific subtypes in small duct cholangiocarcinoma (CLC) with specific immunological features showing MUC5A negativity, MUC6 negativity or sporadic positivity, and CD56 positivity. HE and IHC staining are presented at 200×, the scale represents 100 µm ( A ). ICCs with FGFR2 site mutations, in-frame deletion and frame-shift deletion presented large duct (LD) or small duct (SD) in histological subtypes. HE staining are presented at 200×, the scale represents 100 µm ( B )

Article Snippet: RBEwere infected with recombinant lentiviruses expressing different FGFR2 fusion mutants, then were distributed into 96-well plates with indicated concentrations of BGJ398 (#T1975, TargetMol, USA).

Techniques: Translocation Assay, Immunohistochemistry, Staining

Journal: Cell & Bioscience

Article Title: Oncogenic activation revealed by FGFR2 genetic alterations in intrahepatic cholangiocarcinomas

doi: 10.1186/s13578-023-01156-7

Figure Lengend Snippet: Underlying co-mutation features of different types of FGFR2 genetic alterations. Comutation plot of seventeen FGFR2 translocation/fusion cases ( A ), thirty-one FGFR2 in-frame deletion cases ( B ) and sixty-five FGFR2 site mutation cases ( C )

Article Snippet: RBEwere infected with recombinant lentiviruses expressing different FGFR2 fusion mutants, then were distributed into 96-well plates with indicated concentrations of BGJ398 (#T1975, TargetMol, USA).

Techniques: Mutagenesis, Translocation Assay

Journal: Cell & Bioscience

Article Title: Oncogenic activation revealed by FGFR2 genetic alterations in intrahepatic cholangiocarcinomas

doi: 10.1186/s13578-023-01156-7

Figure Lengend Snippet: Various oncogenic activities and diverse responses to FGFR-selective small molecule kinase inhibitor (SMKI) among FGFR2 mutants. Proliferation activities of AML12 cells expressing MCS, FGFR2 and different FGFR2 mutants are shown ( A ). Representative images of transwell migration assay and average numbers of migrated AML12 cells expressing FGFR2 and different FGFR2 mutants are shown ( B ), the scale represents 100 µm. Representative images of invasion assay and average number of invasive AML12 cells expressing FGFR2 and various FGFR2 mutants are shown ( C ). Cellular skeleton staining revealed the morphological changes in AML12 cells when expressing Lenti-CMV-MCS control virus (MCS), FGFR2 and different FGFR2 mutants ( D ). Evaluation the sensitivity of RBE cells with different FGFR2 mutants to BGJ398 ( E )

Article Snippet: RBEwere infected with recombinant lentiviruses expressing different FGFR2 fusion mutants, then were distributed into 96-well plates with indicated concentrations of BGJ398 (#T1975, TargetMol, USA).

Techniques: Expressing, Transwell Migration Assay, Invasion Assay, Staining, Control, Virus

Journal: Cell & Bioscience

Article Title: Oncogenic activation revealed by FGFR2 genetic alterations in intrahepatic cholangiocarcinomas

doi: 10.1186/s13578-023-01156-7

Figure Lengend Snippet: Frameshift mutation resulting in premature stop codons is also a potential therapeutic target. Proliferation activities of AML12 cells expression Lenti-CMV-MCS control virus (MCS), FGFR2, FGFR2-BICC1 fusion and FGFR2 I548Wfs*8 mutant are shown ( A ). Representative images of transwell migration and average numbers of migrated cells expressing above three clones ( B ), the scale represents 100 µm. Representative images of invasion and average colonies of invasion cells expressing above three clones in AML12 cells are shown ( C ). Capability of FGFR2 I548Wfs*8 rendering RBE cells sensitive to BGJ398 ( D )

Article Snippet: RBEwere infected with recombinant lentiviruses expressing different FGFR2 fusion mutants, then were distributed into 96-well plates with indicated concentrations of BGJ398 (#T1975, TargetMol, USA).

Techniques: Mutagenesis, Expressing, Control, Virus, Migration, Clone Assay

Journal: PLoS ONE

Article Title: Receptor Tyrosine Kinases Activate Canonical WNT/β-Catenin Signaling via MAP Kinase/LRP6 Pathway and Direct β-Catenin Phosphorylation

doi: 10.1371/journal.pone.0035826

Figure Lengend Snippet: (A) RCS cells were transfected with wt FGFR3 or activating FGFR3 mutants (N540K, G380R, R248C, Y373C, K650M, K650E), and analyzed for the indicated molecules by WB 48 hours later. The levels of ERK phosphorylation vary among the tested mutants, reflecting the different strength of FGFR3 activation by each particular mutation . K508M - kinase inactive FGFR3 mutant. GFP and empty vectors serve as transfection controls. (B) LRP6 phosphorylation at Thr1572 caused by highly activating FGFR3 mutants R248C and K650E. (C) Cells were transfected with the indicated FGFR3 vectors together with Topflash reporter vectors, treated with WNT3a and analyzed for luciferase activity. Data represent an average from three transfections (each measured twice), with the indicated standard deviations. A logarithmic scale of the y -axis is necessary to express the massive Topflash activation in WNT3a-treated cells expressing activating FGFR3 mutants (* p <0.001; Student’s t -test; compared to wt FGFR3). Results are representative of four experiments. (D) Cells were transfected with wt FGFR2 or activating FGFR2 mutants (S252W, P253R, C342R, C342Y, Y375C), and analyzed for the indicated molecules by WB. Note the significant ERK and LRP6 phosphorylation caused by C342R, C342Y and Y375C mutants, which correlates with increased basal (E; upper graph) and WNT3a-induced (E; lower graph) β-catenin activity, evidenced by Topflash experiment. Results are representative for three experiments (* p <0.001; Student’s t -test; compared to wt FGFR2).

Article Snippet: For the recombinant RTK kinase assays, the reactions were carried-out with 400 ng of recombinant FGFR2, FGFR3, TRKA or EGFR (SignalChem, Richmond, Canada) and 400 ng of recombinant β-catenin (Abcam, Cambridge, MA, USA) in 50 μl of kinase buffer in the presence of 50 μM ATP for 60 minutes at 30°C.

Techniques: Transfection, Phospho-proteomics, Activation Assay, Mutagenesis, Luciferase, Activity Assay, Expressing

Journal: PLoS ONE

Article Title: Receptor Tyrosine Kinases Activate Canonical WNT/β-Catenin Signaling via MAP Kinase/LRP6 Pathway and Direct β-Catenin Phosphorylation

doi: 10.1371/journal.pone.0035826

Figure Lengend Snippet: (A) RCS cells were treated for indicated times with FGF2 (10 ng/ml) in the presence of heparin (1 µg/ml), and analyzed for β-catenin phosphorylation at Tyr142 by WB (arrow). (B) HEK293 cells were transfected with wt FGFR2 or its activating mutant Y375C, and analyzed for indicated molecules 48 hours later. Note the increased β-catenin phosphorylation at Tyr142 (arrow). (C) Active recombinant FGFR3, FGFR2, TRKA and EGFR were subjected to a cell-free kinase assay with recombinant β-catenin as a substrate. Samples with ATP or kinase omitted serve as controls for kinase reaction.

Article Snippet: For the recombinant RTK kinase assays, the reactions were carried-out with 400 ng of recombinant FGFR2, FGFR3, TRKA or EGFR (SignalChem, Richmond, Canada) and 400 ng of recombinant β-catenin (Abcam, Cambridge, MA, USA) in 50 μl of kinase buffer in the presence of 50 μM ATP for 60 minutes at 30°C.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Recombinant, Kinase Assay