Fig. 6: Ectopic overexpression of FOS counteracts the decrease in TGFβ-induced gene activation after p63 depletion.

a, b The effect of ectopic FOS expression on TGFβ- and EGF-induced AP-1 and EGFR pathway components and target genes upon decrease by p63 knockdown. Control or Flag-FOS (F-FOS)-overexpressing MCF10A MII cells were transfected with non-targeting control (siNTC) or p63 siRNA, serum-starved with EGF for 16 h, stimulated with 5 ng/ml TGFβ1 as indicated, or untreated, and analyzed by immnoblotting (a), and qRT-PCR analysis (b). Statistics were calculated using one-way analysis of variance (ANOVA). The data were further analyzed using Dunnett’s multiple comparisons test and compared with the results from control cells transfected with non-targeting control (siNTC) and treated with TGFβ1 (5 ng/ml). Results from three independent experiments are shown as mean ± SD; ns, not significant, *P < 0.05, **P < 0.01, and ***P < 0.001 (c) Schematic representation of the distinct (red, blue) and cooperative (green) contributions of TGFβ, SMADs, EGF, JUN, FOS members and p63 to the combined EGF + TGFβ invasion program analyzed in this study. In the absence of EGF (left panel) or ΔNp63 (middle panel) TGFβ (red pathway) mainly induces TGFβ target genes controlled by SMAD sites, such as MMP2, via activation of membrane-localized TGFβRI (TBRI) and TGFβRII (TBRII) which phosphorylate and activate SMAD2 and SMAD3 to bind to SMAD4. On the other arm ΔNp63 and EGF (blue pathway) enable potent levels of EGF signaling via cell-membrane-localized EGFR, which triggers phosphorylation and activation of, amongst others, MAP kinases, which in the nucleus activate and induce JUN and FOS family members, that bind ΔNp63 and subsequently can auto-regulate their own expression via AP-1 sites, and induce EGFR and HB-EGF (right panel). The combined action of TGFβ and EGF results in enhanced levels of AP-1 and enables and/or potentiates activation of SMAD/AP-1 target genes such as LAMB3, ITGA2 and WNT7 (green).