Fig 1: Erlotinib increases pYAP and suppresses YAP/TAZ-regulated genes.a Immunoblot of pEGFR (Y1068), EGFR, pYAP (S127), YAP, β-actin in WSU-HN6 and HCC827 cells. Cells were treated with erlotinib at the indicated concentrations for 2 h. b Relative mRNA levels of CTGF, CYR61, and AMOTL2 in WSU-HN6 and HCC827 cells. Cells were treated with erlotinib (1 μM) for 2 h. c The top 15 enriched oncogenic signatures gene sets from RNA-seq data analysis of HCC827 cells. YAP-regulated signatures gene sets are highlighted in red. Cells were treated with vehicle or erlotinib (1 μM) for 24 h. The original name of signature gene sets are listed in supplementary Fig. S5a. d Heat map showing Z-score normalized mRNA expression of representative YAP/TAZ-regulated genes highlighted in orange and yellow. The genes highlighted in green and blue are consistent with the ones previously reported as up- or downregulated by erlotinib treatment31. e Enrichment plots of YAP conserved signatures. f Immunoblot of YAP, TAZ, β-actin in HN6 and HCC827 cells. Cells were transfected with siRNA for control and YAP/TAZ, and incubated for 48 h. g Cell viability. ANOVA with Tukey–Kramer post hoc test and Student’s t-test were used. Mean ± SEM (b, g); ***P <0.001; **P <0.01.
Fig 2: Loss of LATS1/2 confers resistance to erlotinib treatment in cancer cells with EGFR alterations.a Immunoblot of LATS1, LATS2, pYAP (S127), YAP, TAZ, β-actin in WT or LATS1/2 KO HN6 and HCC827 cells. b Relative mRNA levels of CTGF, CYR61, and AMOTL2. Cells were treated with erlotinib (1 μM) for 2 h. c Cell viability. Cells were treated with erlotinib for 3 days. d Schematic of EGFR-mediated YAP/TAZ activation. When EGFR is inactivated, the hydrophobic site of LATS1/2 is phosphorylated and LATS1/2 are active, leading to YAP/TAZ phosphorylation and cytoplasmic retention or degradation. Upon EGF stimulation or EGFR activation by gene amplification, overexpression or mutations, MOB1 is tyrosine phosphorylated and LATS1/2 are dephosphorylated and inactive, resulting in YAP/TAZ nuclear translocation and expression of growth promoting genes regulated by YAP/TAZ. ANOVA with Tukey–Kramer post hoc test were used. Mean ± SEM (b); ***P <0.001; **P <0.01. *versus WT erlotinib non-treated.
Fig 3: EGFR under-phosphorylates YAP/TAZ, induces nuclear translocation of YAP/TAZ and their interaction with TEADs, promoting CTGF/CYR61/AMOTL2 expression.a Immunoblot of EGFR, pEGFR (Y1068), pYAP (S127), YAP, TAZ, pERK1/2 (T202/Y204), ERK1/2, pAKT (S473), AKT, pS6 (S235/236), S6, CTGF, CYR61, β-actin in vector- or EGFR-overexpressing HEK293A cells. Cells were serum starved for 16 h, and treated with EGF (20 ng/ml) for the indicated time. b Relative mRNA levels of CTGF, CYR61, and AMOTL2. c Immunoblot of pEGFR (Y1068), EGFR, pYAP (S127), YAP, TAZ, CTGF, CYR61, β-actin in EGFR-overexpressing HEK293A cells. Cells were transfected with siRNA control and against YAP/TAZ for 24 h, serum starved for 16 h, and treated with EGF (20 ng/ml) for 1 h. d Relative mRNA levels of CTGF, CYR61, and AMOTL2. e Co-immunoprecipitation of YAP and TEAD1. Lysates were immunoprecipitated with control IgG or an antibody against YAP. Immunoblot of TEAD1, YAP, pYAP (S127), pEGFR (Y1068), EGFR, β-actin in EGFR-overexpressing HEK293A cells. Cells were serum stared for 16 h, and treated with EGF (20 ng/ml) for 1 h. f YAP/TAZ localization analyzed by immunofluorescence staining. Cells were serum starved for 16 h, and treated with EGF (20 ng/ml) for 1 h. Scale bars indicate 5 μm. ANOVA with Tukey–Kramer post hoc test was used. Mean ± SEM (b, d); ***P <0.001; **P <0.01; *P <0.05. *versus EGF 0 h (b).
Fig 4: EGFR stimulation leads to MOB1 phosphorylation and LATS1/2 inactivation, independently of MST1/2.a Immunoprecipitation of LATS1. Lysates were immunoprecipitated with control IgG or an antibody against LATS1. Immunoblot of pLATS1 (T1079), LATS1, pEGFR (Y1068), EGFR, β-actin in EGFR-overexpressing HEK293A cells. Cells were serum stared for 16 h, and treated with EGF (20 ng/ml) for 1 h. b In vitro kinase assay of LATS1 against YAP. Lysates were immunoprecipitated with control IgG or an antibody against LATS1, then applied for in vitro kinase reaction with GST-YAP protein. Cells were serum starved for 16 h, and treated with EGF (20 ng/ml) or FBS (10%) as positive control for 1 h. Immunoblot of pYAP (S127), GST, LATS1, pEGFR (Y1068), EGFR, β-actin. Arrow indicates non-specific band. c Immunoblot of LATS1, LATS2, pEGFR (Y1068), EGFR, pYAP (S127), YAP, TAZ, β-actin in WT, or LATS1/2 KO HEK293A cells. WT or LATS1/2 KO HEK293A cells were transfected with EGFR plasmid and incubated for 24 h, serum starved for 16 h, and treated with EGF (20 ng/ml) for 1 h. d Immunoprecipitation of Myc-MST1, FLAG-SAV1, FLAG-LATS1, HA-MOB1, and Myc-YAP. Lysates were immunoprecipitated with control IgG or antibodies against each Tag. Immunoblot of pY, Tag, pEGFR (Y1068), EGFR, β-actin. EGFR-overexpressing HEK293A cells were transfected with the Hippo-components and YAP plasmid, incubated for 24 h, serum starved for 16 h, and treated with EGF (20 ng/ml) for 1 h. e In vitro kinase assay of EGFR and GST-MOB1. In vitro kinase reaction was performed with recombinant EGFR, GST-MOB1 protein, and ATP. Immunoblot for pY and GST. f Co-immunoprecipitation of HA-MOB1 and LATS1. Lysates were immunoprecipitated with control IgG or an antibody against HA-tag. Immunoblot of LATS1, HA, pEGFR (Y1068), EGFR, β-actin. EGFR-overexpressing HEK293A cells were transfected with HA-MOB1 plasmid and incubated for 24 h, serum starved for 16 h, and treated with EGF (20 ng/ml) for 1 h. g Immunoblot of Myc-tag, pMST1/2 (T183/T180), pMOB1 (T35), MOB1, pEGFR (Y1068), EGFR, β-actin. EGFR-overexpressing HEK293A cells were transfected with vector or Myc-MST1 plasmid and incubated for 24 h, serum starved for 16 h, and treated with EGF (20 ng/ml) for 1 h. Asterisks indicate non-specific bands.
Fig 5: EGFR activates YAP/TAZ in HNSCC, independently of PI3K.a Immunoblot of pEGFR (Y1068), EGFR, pYAP (S127), YAP, pERK1/2 (T202/Y204), ERK1/2, pAKT (S473), AKT, pS6 (S235/236), S6, CTGF, CYR61, β-actin in CAL33, CAL27, and WSU-HN6. Right panel showing the status of FAT1 gene alterations. b Relative mRNA levels of CTGF, CYR61, and AMOTL2 in CAL33, CAL27, and WSU-HN6 cells. c GSEA analysis of RNA-seq data in CCLE using the C6 oncogenic gene sets, spiked with several previously published YAP-regulated gene sets. NES, normalized enrichment score; NOM, nominal; FDR, false discovery rate. d Immunoblot of pEGFR (Y1068), EGFR, pYAP (S127), YAP, TAZ, pERK1/2 (T202/Y204), ERK1/2, pAKT (S473), AKT, pS6 (S235/236), S6, CTGF, CYR61, β-actin in CAL27 cells. Cells were serum starved for 16 h, and treated with EGF (20 ng/ml) for the indicated time. e Relative mRNA levels of CTGF, CYR61, and AMOTL2 in CAL27 cells. f Immunoblot of pEGFR (Y1068), EGFR, pAKT (S473), AKT, pYAP (S127), YAP, TAZ, CTGF, CYR61, β-actin in CAL27 cells stably overexpressing empty vector or PIK3CA H1047R. Cells were serum starved for 16 h, and treated with EGF (20 ng/ml) for 1 hr. g Immunoblot of pEGFR (Y1068), EGFR, pAKT (S473), AKT, pYAP (S127), YAP, TAZ, CTGF, CYR61, β-actin in CAL27 cells. Cells were serum starved for 16 h, and pretreated with BYL719 (1 μM) for 1 h and followed by EGF treatment (20 ng/ml) for 1 h. ANOVA with Tukey–Kramer post hoc test was used. Mean ± SEM (b, e); ***P < 0.001; **P < 0.01; *P < 0.05. *versus CAL33 (b) and versus EGF 0 h (e).
Supplier Page from Abcam for Recombinant Human YAP1 protein (GST tag N-Terminus)