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Background: p63 is a transcription factor with intrinsic pioneer factor activity and pleiotropic functions. Transforming growth factor beta (TGF beta) signaling via activation and cooperative action of canonical, SMAD, and non-canonical, MAP-kinase (MAPK) pathways, elicits both anti- and pro-tumorigenic properties, including cell stemness and invasiveness. TGF beta activates the Delta Np63 transcriptional program in cancer cells; however, the link between TGF beta and p63 in unmasking the epigenetic landscape during tumor progression allowing chromatin accessibility and gene transcription, is not yet reported.

Methods: Small molecule inhibitors, including protein kinase inhibitors and RNA-silencing, provided loss of function analyses. Sphere formation assays in cancer cells, chromatin immunoprecipitation and mRNA expression assays were utilized in order to gain mechanistic evidence. Mass spectrometry analysis coupled to co-immunoprecipitation assays revealed novel p63 interactors and their involvement in p63-dependent transcription.

Results: The sphere-forming capacity of breast cancer cells was enhanced upon TGF beta stimulation and significantly decreased upon Delta Np63 depletion. Activation of TGF beta signaling via p38 MAPK signaling induced Delta Np63 phosphorylation at Ser 66/68 resulting in stabilized Delta Np63 protein with enhanced DNA binding properties. TGF beta stimulation altered the ratio of H3K27ac and H3K27me3 histone modification marks, pointing towards higher H3K27ac and increased p300 acetyltransferase recruitment to chromatin. By silencing the expression of Delta Np63, the TGF beta effect on chromatin remodeling was abrogated. Inhibition of H3K27me3, revealed the important role of TGF beta as the upstream signal for guiding Delta Np63 to the TGF beta/SMAD gene loci, as well as the indispensable role of Delta Np63 in recruiting histone modifying enzymes, such as p300, to these genomic regions, regulating chromatin accessibility and gene transcription. Mechanistically, TGF beta through SMAD activation induced dissociation of Delta Np63 from NURD or NCOR/SMRT histone deacetylation complexes, while promoted the assembly of Delta Np63-p300 complexes, affecting the levels of histone acetylation and the outcome of Delta Np63-dependent transcription.

Conclusions: Delta Np63, phosphorylated and recruited by TGF beta to the TGF beta/SMAD/Delta Np63 gene loci, promotes chromatin accessibility and transcription of target genes related to stemness and cell invasion.

Transforming growth factor-β (TGF-β) exerts its cellular effects via binding to type I and type II kinase-associated receptors, whereby SMAD-dependent and SMAD-independent pathways are activated; amongst the latter is the Rho pathway with the downstream effectors Rho-associated coiled-coiled kinases 1 (ROCK1) and ROCK2. In the present study, we investigated whether ROCK1 and ROCK2 regulate TGF-β-SMAD signaling in breast cancer cells. We found that knockdown of ROCK2 or treatment with a highly selective ROCK2 kinase inhibitor (KD025) suppresses TGF-β-SMAD signaling, which was opposite to the effect of knockdown of ROCK1. Moreover, we demonstrate that overexpression of ROCK1 inhibits TGF-β-induced CAGA12-luc reporter expression, whereas a kinase-dead ROCK1 mutant or a ROCK inhibitor GSK42928A reversed this effect. Overexpression of ROCK2 enhanced TGF-β-induced CAGA12-luc reporter activity, while a kinase-dead ROCK2 mutant and KD025 reversed this effect. These observations suggest that the kinase activities of ROCK isoforms are needed for their inhibitory or stimulatory effects on TGF-β-SMAD signaling. In addition, we found that ROCK1 and ROCK2 have different subcellular localizations, and that SMAD3 interacts with ROCK1, but not with ROCK2. Furthermore, ROCK1 depletion in MDA-MB-231 cells promoted cell proliferation and reduced cell invasion, whereas, in contrast, ROCK2 depletion reduced cell proliferation and promoted cell invasion in vitro. Thus, our observations support the notion that the two ROCK isoforms have opposite effects on TGF-β-SMAD signaling.

In the present study, we performed a CRISPR-Cas9 screen in the human triple-negative breast cancer cell line MDA-MB-231, using a CAGA12-GFP-reporter, to identify cellular factors with the ability to enhance or suppress TGF-β-SMAD signaling, we performed a CRISPR-Cas9 genetic screen in the human triple-negative breast cancer cell line MDA-MB-231, using a SMAD3/4-dependent CAGA12-transcriptional reporter. We found that the chromodomain-helicase-DNA-binding protein 4 (CHD4), a subunit of the nucleosome remodeling and deacetylation (NuRD) complex, suppresses TGF-β-SMAD signaling. CHD4 is highly expressed in all subtypes of breast cancers and we found that CHD4 reduced TGF-β-induced SMAD3/4-driven CAGA12-luciferase transcriptional reporter activity in several types of breast cancer cell lines. We also found that CHD4 was mainly localized in the nucleus, and that it interacted with SMAD3, and less so with SMAD2, but not with SMAD4, in a ligand-dependent manner. 

Transforming growth factor-β (TGFβ)-SMAD signaling induces both tumor-suppressive and tumor-promoting pathways; the latter is critically dependent on the induction of activator protein (AP)-1 transcription factors, but the mechanisms regulating this pathway are not yet fully understood. In the present study, we show that integrin-αvβ5 (ITGAV-ITGB5) enhances the AP-1-dependent tumorigenic properties of TGFβ-SMAD signaling in breast cancer cells. Mechanistically, ITGAV-ITGB5 facilitates the activation of the mitogen activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway, which stimulates the expression of AP-1 transcription factor components, especially FOS members, thereby enhancing the expression of a subset of TGFβ-inducible migration/invasion-associated genes, such as lamilin subunit b3 (LAMB3), WNT7B, matrix metalloproteinase (MMP)9, and interleukin (IL)11. Importantly, ITGAV-ITGB5 was necessary for TGFβ-induced migration/invasion but did not affect tumor suppressive pathways induced by TGFβ, such as induction of CDKNA1, encoding the cell cycle inhibitor p21, and suppression of the proto-oncogene MYC. Our observations provide insight into the mechanisms involved in the activation of pro-tumorigenic signals by TGFβ in breast cancer, which will be important for the development of selective TGFb inhibitors, specifically perturbing the pro-tumorigenic effects of TGFb.