Oncogenic transformation of mammary epithelial cells by transforming growth factor beta independent of mammary stem cell regulation.

BACKGROUND: Transforming growth factor beta (TGFß) is transiently increased in the mammary gland during involution and by radiation. While TGFß normally has a tumour suppressor role, prolonged exposure to TGFß can induce an oncogenic epithelial to mesenchymal transition (EMT) program in permissive cells and initiate the generation of cancer stem cells. Our objective is to mimic the transient exposure to TGFß during involution to determine the persistent effects on premalignant mammary epithelium. METHOD: CDßGeo cells, a transplantable mouse mammary epithelial cell line, were treated in vitro for 14 days with TGFß (5 ng/ml). The cells were passaged for an additional 14 days in media without TGFß and then assessed for markers of EMT and transformation. RESULTS: The 14-day exposure to TGFß induced EMT and transdifferentiation in vitro that persists after withdrawal of TGFß. TGFß-treated cells are highly tumorigenic in vivo, producing invasive solid de-differentiated tumours (100%; latency 6.7 weeks) compared to control (43%; latency 32.7 weeks). Although the TGFß-treated cells have initiated a persistent EMT program, the stem cell population was unchanged relative to the controls. The gene expression profiles of TGFß-treated cells demonstrate de-differentiation with decreases in the expression of genes that define luminal, basal and stem cells. Additionally, the gene expression profiles demonstrate increases in markers of EMT, growth factor signalling, TGFß2 and changes in extra cellular matrix. CONCLUSION: This model demonstrates full oncogenic EMT without an increase in stem cells, serving to separate EMT markers from stem cell markers.

p53 and ΔNp63α Coregulate the Transcriptional and Cellular Response to TGFß and BMP Signals.

UNLABELLED: The TGFß superfamily regulates a broad range of cellular processes, including proliferation, cell-fate specification, differentiation, and migration. Molecular mechanisms underlying this high degree of pleiotropy and cell-type specificity are not well understood. The TGFß family is composed of two branches: (i) TGFßs, activins, and nodals, which signal through SMAD2/3, and (ii) bone morphogenetic proteins (BMP), which signal through SMAD1/5/8. SMADs have weak DNA-binding affinity and rely on coactivators and corepressors to specify their transcriptional outputs. This report reveals that p53 and ΔNp63α act as transcriptional partners for SMAD proteins and thereby influence cellular responses to TGFß and BMPs. Suppression of p53 or overexpression of ΔNp63α synergistically enhance BMP-induced transcription. Mechanistically, p53 and ΔNp63α physically interact with SMAD1/5/8 proteins and co-occupy the promoter region of inhibitor of differentiation (ID2), a prosurvival BMP target gene. Demonstrating further convergence of these pathways, TGFß-induced canonical BMP regulated transcription in a ΔNp63α- and p53-dependent manner. Furthermore, bioinformatic analyses revealed that SMAD2/3 and ΔNp63α coregulate a significant number of transcripts involved in the regulation of epithelial-to-mesenchymal transition. Thus, p53 and ΔNp63α are transcriptional partners for a subset of TGFß- and BMP-regulated SMAD target genes in the mammary epithelium. Collectively, these results establish an integrated gene network of SMADs, p53, and ΔNp63α that contribute to EMT and metastasis. IMPLICATIONS: This study identifies aberrant BMP activation as a result of p53 mutation or ΔNp63α expression.

ΔNp63α-mediated activation of bone morphogenetic protein signaling governs stem cell activity and plasticity in normal and malignant mammary epithelial cells.

Genetic analysis of TP63 indicates that ΔNp63 isoforms are required for preservation of regenerative stasis within diverse epithelial tissues. In squamous carcinomas, TP63 is commonly amplified, and ΔNp63α confers a potent survival advantage. Genome-wide occupancy studies show that ΔNp63 promotes bidirectional target gene regulation by binding more than 5,000 sites throughout the genome; however, the subset of targets mediating discreet activities of TP63 remains unclear. We report that ΔNp63α activates bone morphogenic proteins (BMP) signaling by inducing the expression of BMP7. Immunohistochemical analysis indicates that hyperactivation of BMP signaling is common in human breast cancers, most notably in the basal molecular subtype, as well as in several mouse models of breast cancer. Suppression of BMP signaling in vitro with LDN193189, a small-molecule inhibitor of BMP type I receptor kinases, represses clonogenicity and diminishes the cancer stem cell-enriched ALDH1(+) population. Importantly, LDN193189 blocks reconstitution of mixed ALDH1(+)/ALDH1(-) cultures indicating that BMP signaling may govern aspects of cellular plasticity within tumor hierarchies. These results show that BMP signaling enables reversion of committed populations to a stem-like state, potentially supporting progression and maintenance of tumorigenesis. Treatment of a mouse model of breast cancer with LDN193189 caused reduced expression of markers associated with epithelial-to-mesenchymal transition (EMT). Furthermore, in vivo limiting dilution analysis assays revealed that LDN193189 treatment suppressed tumor-initiating capacity and increased tumor latency. These studies support a model in which ΔNp63α-mediated activation of BMP signaling governs epithelial cell plasticity, EMT, and tumorigenicity during breast cancer initiation and progression.

Phosphorylation of ΔNp63α via a novel TGFß/ALK5 signaling mechanism mediates the anti-clonogenic effects of TGFß.

Genetic analysis of TP63 implicates ΔNp63 isoforms in preservation of replicative capacity and cellular lifespan within adult stem cells. ΔNp63α is also an oncogene and survival factor that mediates therapeutic resistance in squamous carcinomas. These diverse activities are the result of genetic and functional interactions between TP63 and an array of morphogenic and morphostatic signals that govern tissue and tumor stasis, mitotic polarity, and cell fate; however the cellular signals that account for specific functions of TP63 are incompletely understood. To address this we sought to identify signaling pathways that regulate expression, stability or activity of ΔNp63α. An siRNA-based screen of the human kinome identified the Type 1 TGFß receptor, ALK5, as the kinase required for phosphorylation of ΔNp63α at Serine 66/68 (S66/68). This activity is TGFß-dependent and sensitive to either ALK5-directed siRNA or the ALK5 kinase inhibitor A83-01. Mechanistic studies support a model in which ALK5 is proteolytically cleaved at the internal juxtamembrane region resulting in the translocation of the C-terminal ALK5-intracellular kinase domain (ALK5(IKD)). In this study, we demonstrate that ALK5-mediated phosphorylation of ΔNp63α is required for the anti-clonogenic effects of TGFΒ and ectopic expression of ALK5(IKD) mimics these effects. Finally, we present evidence that ultraviolet irradiation-mediated phosphorylation of ΔNp63α is sensitive to ALK5 inhibitors. These findings identify a non-canonical TGFß-signaling pathway that mediates the anti-clonogenic effects of TGFß and the effects of cellular stress via ΔNp63α phosphorylation.