Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Effects of FGFR1 and FGFR4 on lung squamous carcinoma cell lines. See also Supplementary Figure S1. Growth curves in 10% FBS (a) and soft agar assays (b) of FGFR4-overexpressing lung squamous carcinoma cell lines. (c) Western blot analysis of the activation of FGFR-related signalling pathways in FGFR4-overexpressing lung squamous carcinoma cell lines compared to empty-vector-expressing cell lines after stimulation with FBS. Growth curves in 0.5% FBS (d) and soft agar assays (e) of FGFR1- and FGFR4-silenced H520 cells (lung squamous cell carcinoma). (f) Western blot analysis of the activation of FGFR-related signalling pathways in FGFR1- and FGFR4-silenced H520 cells. All experiments were reproduced a minimum of three times in the laboratory, and three technical replicates were obtained for each experiment. For growth curves and western blots, a representative figure/image is shown. On the growth curves, the means and standard deviations of the technical replicates are shown. In the soft agar assays, all values were normalised to the empty vector control, and the mean and standard deviation of all the normalised replicates are presented. Silencing of either gene was performed using two different shRNAs, referred to as “a” and “b”. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). ADC = Adenocarcinoma, SCC = Squamous cell carcinoma, I = Immortalised, KRAS = KRAS-mutated, EGFR = EGFR-mutated, ALK = ALK translocation bearer, TN = “Triple negative” (referring to the absence of alterations in KRAS, EGFR and ALK), EV = empty vector control, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, scramble = scrambled shRNA control, shFGFR1 = FGFR1 shRNA, shFGFR4 = FGFR4 shRNA, FBS = foetal bovine serum. Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Western Blot, Activation Assay, Plasmid Preparation, Expressing, Control, Standard Deviation, MANN-WHITNEY, Translocation Assay, shRNA, Molecular Weight

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Effects of FGFR1 and FGFR4 on non-squamous lung cell lines. See also Supplementary Figure S2. Growth curves in 10% FBS (a) and soft agar assays (b) of the FGFR1- or FGFR4-overexpressing lung adenocarcinoma cell lines H2009 and H3122. (c) Western blot analysis of the activation of FGFR-related signalling pathways in FGFR1-overexpressing H2009 and H3122 cells compared to empty-vector-expressing cells. Growth curves in 10% FBS (d) and soft agar assays (e) of FGFR1- and FGFR4-silenced A549 cells (adenocarcinoma cell line). (f) Western blot analysis of the activation of FGFR-related signalling pathways in FGFR1- and FGFR4-silenced A549 cells. All experiments were reproduced a minimum of three times in the laboratory, and three technical replicates were obtained for each experiment. For growth curves and western blots, a representative figure/image is shown. On the growth curves, the means and standard deviations of the technical replicates are shown. In the soft agar assays, all values were normalised to the empty vector control, and the mean and standard deviation of all the normalised replicates are presented. For western blots, cells were serum-starved for five hours prior to protein extraction. For the serum-stimulated conditions, serum-starved cells were incubated in serum-containing complete medium for 15 min before protein extraction. Silencing of either gene was performed using two different shRNAs, referred to as “a” and “b”. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). EV = empty vector control, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, scramble = scrambled shRNA control, shFGFR1 = FGFR1 shRNA, shFGFR4 = FGFR4 shRNA, FBS = foetal bovine serum. Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Western Blot, Activation Assay, Plasmid Preparation, Expressing, Control, Standard Deviation, Protein Extraction, Incubation, MANN-WHITNEY, shRNA, Molecular Weight

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Effects of N-cadherin on the pro-oncogenic role of FGFR1 and FGFR4. See also Supplementary Figures S3 and S4. (a) Western blots of N-cadherin and E-cadherin protein expression in our lung cell line panel. To assess the expression of these proteins in the 18 cell lines, different blots were performed in parallel with an internal reference sample and the assembled images are shown. 10% FBS growth curves (b) and soft agar assays (c) of H2009 and H3122 cells overexpressing N-cadherin and either FGFR1 or FGFR4. (d) Western blot analysis of the activation of FGFR-related signalling pathways in these cell lines. All experiments were reproduced a minimum of three times in the laboratory and three technical replicates were obtained for each experiment. For growth curves and western blots, a representative figure/image is shown. On the growth curves, the means and standard deviations of the technical replicates are shown. In the soft agar assays, all values were normalised to the empty vector control, and the mean and standard deviation of all the normalised replicates are presented. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001) non-SCC = non-Squamous, SCC = Squamous cell carcinoma, I = Immortalised, KRAS = KRAS-mutated, EGFR = EGFR-mutated, ALK = ALK translocation bearer, TN = “Triple negative” (referring to the absence of alterations in KRAS, EGFR and ALK), EV1 = empty vector 1, EV2 = empty vector 2, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, CDH2 = N -cadherin-overexpressing. Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Western Blot, Expressing, Activation Assay, Plasmid Preparation, Control, Standard Deviation, MANN-WHITNEY, Translocation Assay, Molecular Weight

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Effects of N-cadherin on the pro-oncogenic role of FGFR1 and FGFR4 and the interaction of N-cadherin with FGFR1 and FGFR4. See also Supplementary Figure S4. (a) 0.5% FBS growth curves for FGFR1-overexpressing and N-cadherin-silenced (left) or FGFR4-overexpressing and N-cadherin-silenced (right) NL20 cells. (b) Soft agar assays of FGFR-overexpressing and N-cadherin-silenced NL20 cells. (c) Western blot analysis of the activation of FGFR-related signalling pathways in these cell lines. (d) Xenograft tumour volumes of the FGFR1, FGFR4 and N-cadherin interaction models in the immortalised NL20 cell line. (e) Proximity ligation assays (PLA) to assess the physical interaction of N-cadherin with FGFR1 (upper panel) or FGFR4 (lower panel). The interactions detected were quantified and normalised by cell number for each condition. As controls to distinguish signal from noise, interactions were quantified in the single antibody conditions (labelled as “N-cadherin”, “FGFR1” and “FGFR4”). To assess the interaction between the two proteins, both antibodies were used, either N-cadherin + FGFR1 (upper panel) or N-cadherin + FGFR4 (lower panel), in the condition labelled as “combo”. Representative images and quantifications are shown. (f) Co-immunoprecipitation of N-cadherin with FGFR1 and with FGFR4 in the H520 cell line. (g) Kaplan-Meier curves of overall survival (OS) for the entire NSCLC patient cohort ( N = 109). Patients were grouped based on FGFR1 and N-cadherin expression levels or on FGFR4 and N-cadherin expression levels. (h) OS curve of patients in the cohort with high expression of FGFR1 and/or FGFR4 stratified by N-cadherin expression levels. In each analysis, for the FGFR1 and N-cadherin genes, the cut-off point was the median mRNA expression value for that variable. For FGFR4, the cut-off point was the first-quartile mRNA expression value in the TCGA adenocarcinoma cohort. The Kaplan-Meier method was used for survival analyses of the clinical data and cell line xenograft experiments, with a Cox proportional hazards model used to adjust for explanatory variables. A log Rank analysis was used to analyse differences in survival between groups. To obtain the hazard ratio values, the Cox proportional hazards model was used. All in vitro experiments were reproduced a minimum of three times in the laboratory, and three technical replicates were obtained for each experiment. For growth curves and western blots, a representative figure/image is shown. On the growth curves, the means and standard deviations of the technical replicates are shown. In the soft agar assays, all values were normalised to the empty vector control, and the mean and standard deviation of all the normalised replicates are presented. N-cadherin silencing was performed using two different shRNAs. Results generated with the alternative shRNA are shown in Supplementary Figure S3 c-e. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). EV1 = empty vector 1, EV2 = empty vector 2, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, CDH2 = N -cadherin-overexpressing, scramble = scrambled shRNA control, shCDH2 = silenced with N-cadherin shRNA. Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Western Blot, Activation Assay, Ligation, Immunoprecipitation, Expressing, In Vitro, Plasmid Preparation, Control, Standard Deviation, Generated, shRNA, MANN-WHITNEY, Molecular Weight

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: RNAseq analysis of TCGA lung adenocarcinoma and squamous cell carcinoma datasets. (a) Differential gene expression analysis in FGFR1/4high-CDH2high ( n = 145) versus FGFR1/4high-CDH2low ( n = 94) patients. The gene dataset was filtered by discarding genes whose expression was dependant on CDH2 high/low status irrespective of FGFR1 and/or FGFR4 expression (FGFR1/4low-CDH2high patients, n = 21). Parameters were set up as logFC>1, B >0. (b) Query of defined gene expression signature against Gene Ontology. The results shown here are based in whole or in part upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/ .

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Gene Expression, Expressing, Generated

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Predictive potential of N-cadherin expression for anti-FGFR therapy in vitro . See also Supplementary Figure S5. Treatment for 72 h with AZD4547 or BGJ398 at a concentration of 0.5 or 1 µM was applied to cells with high endogenous expression of FGFR1 and/or FGFR4, with high or low endogenous expression of N-cadherin (a) and to cells either exogenously expressing FGFR1 or FGFR4, alone or in combination with N-cadherin, or to cells with high endogenous expression of the three genes with N-cadherin downregulation (b). All experiments were reproduced a minimum of three times in the laboratory. For growth curves, a representative figure is shown and the mean and standard deviation for the technical replicates are indicated. N-cadherin silencing was performed using two different shRNAs to avoid off-target effects. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). EV1 = empty vector 1, EV2 = empty vector 2, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, CDH2 = N -cadherin-overexpressing, scramble = scrambled shRNA control, shCDH2 = silenced with N-cadherin shRNA.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Expressing, In Vitro, Concentration Assay, Standard Deviation, MANN-WHITNEY, Plasmid Preparation, shRNA, Control

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: FGFR efficacy in N-cadherin, FGFR1 and/or FGFR4 co-expressing patient-derived xenografts (PDXs). See also Supplementary Figure S6. (a) Western blot showing FGFR1, FGFR4 and N-cadherin protein expression in five different lung PDXs. AZD4547 treatment of low (b) and high (c) N-cadherin-expressing PDXs. (d) Results of the PDX treatments in terms of tumour versus control volume (T/C), and complete regressions. T/C values are expressed as percentages. (e) Graph showing the median variation in tumour volume from the initial volume, for every model, calculated as the increase or decrease in volume and expressed as a percentage. (f) Western blot showing the effects of AZD4547 treatment on FGFR-related signalling pathways in one low-N-cadherin-expressing (TP13) and one high-N-cadherin-expressing (TP114) adenocarcinoma PDX. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Expressing, Derivative Assay, Western Blot, Control, MANN-WHITNEY, Molecular Weight

Journal: Nature Communications

Article Title: Alveolar cell fate selection and lifelong maintenance of AT2 cells by FGF signaling

doi: 10.1038/s41467-022-34059-1

Figure Lengend Snippet: a (Left) Time course showing the proportion of AT2 (Muc1 + ) cells labeled with GFP in control ( LyzM-Cre; Rosa26-mTmG Fgfr2 fl/+ ) or Fgfr2 deleted ( LyzM-Cre; Rosa26-mTmG; Fgfr2 fl/fl ) lungs in postnatal life (control, extension of Fig. , mean values ± SD). No further accumulation of GFP pos AT2 occurs in Fgfr2 deleted lungs (dashed line) after PN20, despite continued LyzM-Cre activity (AT2 labeling) apparent in control (solid line). n = 3955 AT2 (3 animals/genotype). (Right) Time course showing identities of GFP pos cells following Fgfr2 loss (extension of Fig. ). After a rapid increase in AT1 due to AT2 reprogramming in the juvenile period (through PN20), no further accumulation of GFP pos AT1 or AT2 occurs. n = 300 GFP pos cells scored (3 animals/condition and timepoint, mean ± SD). b Close-up of the alveolar region in adult (PN60) LyzM-Cre;Rosa26-mTmG;Fgfr2 fl/fl mouse stained for apoptosis marker cleaved Caspase-3 (cCaspase-3). Note GFP pos AT2 deleted for Fgfr2 undergoing apoptosis (asterisk) but not unlabeled control AT2 nearby (arrowhead). Bar, 10 µm. c – f MASTR system for the conditional, complete deletion of Fgfr2 in AT2. c AAV-Sftpc-Flp virus (top) expresses Flp from Sftpc promoter. MASTR allele (bottom), following Flp-mediated deletion (at frt, triangles) of Pgk-neo-pA cassette, constitutively expresses GFP-Cre fusion. d Scheme. Two weeks after AAV-Sftpc-Flp instillation into lungs of adult Fgfr2 fl/fl ;Rosa26 mTmG/MASTR mice to induce constitutive GFP-Cre and complete deletion of Fgfr2 fl/fl in AT2, lungs were immunostained (GFP, Muc1, Fgfr2, cCaspase-3). e Alveolar regions from Fgfr2 fl/+ control (top) or Fgfr2 fl/fl (bottom) mice treated as in ( d ). Note the reduction in GFP pos AT2 (Muc1 pos ) (bottom). Bar, 25 µm. f Quantification of AT2 loss in ( e ). n = 890 Muc1 pos cells scored/condition (three experiments, mean ± SD). g , h Quantification ( h ) of Fgfr2 immunostaining ( n = 100 GFP pos cells scored/condition, three experiments, mean ± SD). Nearly all (96%) GFP pos AT2 remaining after MASTR-mediated Fgfr2 fl/fl deletion ( e , f ) have not fully lost Fgfr2. Micrograph ( g ) shows an example of rare GFP pos Fgfr2 neg AT2, which were all rounded and extruded into the lumen. Bar, 10 µm. i , j Quantification ( j ) of cCaspase-3 immunostaining ( n = 470 GFP pos cells scored/condition, mean ± SD). Note that 36% of GFP pos AT2 cells remaining after MASTR-mediated Fgfr2 fl/fl deletion were cCaspase-3 pos , indicating apoptosis initiated (micrograph, i ). Bar, 10 µm. k – n Acute inhibition of Fgfr2 signaling by FIIN-1. k Scheme for co-instillation of FIIN-1 and fluorescent marker WGA-405 into lungs of adult Sftpc CreER/+ ;Rosa26 mTmG/+ mice tamoxifen(Tam)-treated to label AT2 then analyzed over 2 weeks. Bar, 200 µm. l Lung regions exposed to FIIN-1 (WGA-labeled, left) and shown merged (right) with mTmG fluorescence (AT2 lineage GFP-labeled, other cells tdTomato (mT)-labeled). Bar, 250 µm. m WGA-labeled alveolar regions exposed 2 h to the vehicle (top) or FIIN-1 (bottom). FIIN-1-exposed AT2 cells (GFP pos ) undergo apoptosis (cCaspase-3 pos , arrowheads). Bar, 20 µm. n Kinetics of AT2 apoptosis in ( m ). In vehicle control, <2.5% of AT2 were cCaspase-3 pos ( n = 650 cells scored/condition, three experiments, mean ± SD). No conversion of labeled AT2 to AT1 was detected during this period ( n = 390 cells scored/condition, three experiments) (see also Suppl. Fig. ). o – q Effect of acute Fgfr2 inhibition in AT2 cells by Fgfr2iiib-Fc protein. o Scheme. After Tam-induction of adult Sftpc CreER/+ ;Rosa26 mTmG/+ mice to GFP-label AT2, Fgfr2iiib-Fc (or vehicle) and WGA-405 were co-instilled and lungs subsequently stained for cCaspase-3. p WGA-labeled alveolar regions from vehicle control (top) and Fgfr2iiib-Fc (bottom) instilled lungs. Bar, 25 µm. q Quantification of p . n , cells scored, six experiments (mean ± SD). *** p = 4.3 × 10 −4 for ( f ), ** p = 3.4 × 10 −3 for ( j ), and *** p = 2.5 × 10 −4 for ( q ) (Student’s two-sided t -test) (panels f , j , q ).

Article Snippet: The ORF encoding Fgfr2iiib (Origene; MC221076), the IRES sequence from pLVX-IRES-Puro (Clontech; 632186), and the sfGFP (superfolder GFP) coding sequence from pBAD-sfGFP (Addgene; 54519) were PCR-amplified with adapter sequences (IDT) and assembled (NEB; E5520) into the lentiviral backbone pTetOne (Takara; 631018) linearized by EcoRI/BamHI digestion (NEB) to generate pTetOne-Fgfr2-IRES-sfGFP (Fig. ).

Techniques: Labeling, Control, Activity Assay, Staining, Marker, Virus, Immunostaining, Inhibition, Fluorescence