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Activator protein (AP)-1 transcription factors are essential elements of the pro-oncogenic functions of transforming growth factor-beta (TGF beta)-SMAD signaling. Here we show that in multiple HER2+ and/or EGFR+ breast cancer cell lines these AP-1-dependent tumorigenic properties of TGF beta critically rely on epidermal growth factor receptor (EGFR) activation and expression of the Delta N isoform of transcriptional regulator p63. EGFR and Delta Np63 enabled and/or potentiated the activation of a subset of TGF beta-inducible invasion/migration-associated genes, e.g., ITGA2, LAMB3, and WNT7A/B, and enhanced the recruitment of SMAD2/3 to these genes. The TGF beta- and EGF-induced binding of SMAD2/3 and JUNB to these gene loci was accompanied by p63-SMAD2/3 and p63-JUNB complex formation. p63 and EGFR were also found to strongly potentiate TGF beta induction of AP-1 proteins and, in particular, FOS family members. Ectopic overexpression of FOS could counteract the decrease in TGF beta-induced gene activation after p63 depletion. p63 is also involved in the transcriptional regulation of heparin binding (HB)-EGF and EGFR genes, thereby establishing a self-amplification loop that facilitates and empowers the pro-invasive functions of TGF beta. These cooperative pro-oncogenic functions of EGFR, AP-1, p63, and TGF beta were efficiently inhibited by clinically relevant chemical inhibitors. Our findings may, therefore, be of importance for therapy of patients with breast cancers with an activated EGFR-RAS-RAF pathway.

It is well established that transforming growth factor-beta (TGF beta) switches its function from being a tumor suppressor to a tumor promoter during the course of tumorigenesis, which involves both cell-intrinsic and environment-mediated mechanisms. We are interested in breast cancer cells, in which SMAD mutations are rare and interactions between SMAD and other transcription factors define pro-oncogenic events. Here, we have performed chromatin immunoprecipitation (ChIP)-sequencing analyses which indicate that the genome-wide landscape of SMAD2/3 binding is altered after prolonged TGF beta stimulation. De novo motif analyses of the SMAD2/3 binding regions predict enrichment of binding motifs for activator protein (AP) 1 in addition to SMAD motifs. TGF beta-induced expression of the AP1 component JUNB was required for expression of many late invasion-mediating genes, creating a feed-forward regulatory network. Moreover, we found that several components in the WNT pathway were enriched among the late TGF beta-target genes, including the invasion-inducing WNT7 proteins. Consistently, overexpression of WNT7A or WNT7B enhanced and potentiated TGF beta-induced breast cancer cell invasion, while inhibition of the WNT pathway reduced this process. Our study thereby helps to explain how accumulation of pro-oncogenic stimuli switches and stabilizes TGF beta-induced cellular phenotypes of epithelial cells.

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