Figure 3. Cetuximab inactivates YAP.

(A) Correlation between gene expression changes induced by cetuximab (20 mg/kg, intraperitoneal injection twice a week for 6 weeks) in CRC PDXs and those induced by YAP knockout (YAPΔ/Δ) in the normal mouse intestine (35). Scatter plot of gene expression changes (left) and Pearson’s correlationof differentially regulated genes (right) are shown. Cetux, cetuximab. (B) GSEA plot showing the regulation of YAP-inducible target genes (36) in PDXs from mice treated with cetuximab. NES, normalized enrichment score; FDR, false discovery rate. (C) Graphs show qualitative morphometric assessment of YAP total and nuclear abundance in PDXs from the reference collection after treatment with cetuximab, compared with vehicle-treated counterparts. Representative images of YAP staining in PDXs from the reference collection after treatment with vehicle (until tumors reached an average volume of 1500 mm³) or cetuximab (for 6 weeks) are shown. Scale bar, 50 μm. (D) Measurement of YAP transcriptional activity in DiFi and HCA46 cell lines after treatment for 48h with vehicle (DMSO), cetuximab (20 μg/ml), selumetinib (1 μM), or dactolisib (250 nM). Luciferase activity was normalized against reporter plasmid concentrations as determined by DNA qPCR and expressed as arbitrary units (A.U.). Three independent experiments were performed in biological quadruplicates (n = 12). The plots show means ± SD. Statistical analysis by one-way ANOVA followed by Benjamini, Krieger and Yekutieli FDR correction. (E) Effect of doxycycline-regulated YAP activation, with or without concomitant EGFR inhibition, in established DiFi xenografts. (Left) Tumor volume changes after 3 weeks of treatment with cetuximab (20 mg/kg, intraperitoneal injection twice a week), doxycycline (50 mg/kg, daily oral gavage), or both. (Right) Tumor volume changes after 4 weeks from treatment suspension. n = 7 animals per each treatment arm. Statistical analysis by one-way ANOVA followed by Benjamini, Krieger and Yekutieli FDR correction (left) or two-tailed Wilcoxon test (right). Control, doxycycline vehicle + cetuximab vehicle; Doxy, doxycycline; Combo, doxycycline + cetuximab.