Abstract
During reproductive life, the mammary epithelium undergoes consecutive cycles of proliferation, differentiation and apoptosis. Doing so relies on the retained proliferative capacity, prolonged lifespan and developmental potency of mammary stem cells (MaSCs). ΔNp63α, the predominant TP63 isoform in mammary epithelia, is robustly expressed in MaSCs and is required for preservation of self-renewing capacity in diverse epithelial structures. However, the mechanism(s) underlying subversion of this activity during forfeiture of self-renewing capacity are poorly understood. MicroRNAs (miRNAs) govern critical cellular functions including stem cell maintenance, development, cell cycle regulation and differentiation by disrupting translation of target mRNAs. Data presented here indicate that expression of miR203, a miRNA that targets ΔNp63α and ΔNp63β is activated during luminal epithelial differentiation and that this pattern is observed in the murine mammary hierarchy. In addition, we present evidence that the transcription factor Zeb1 represses miR203 expression, thus enhancing ΔNp63α protein levels. Furthermore, ectopic miR203 suppresses ΔNp63α expression, proliferation and colony formation. The anti-clonogenic effects mediated by miR203 require suppression of ΔNp63α. In addition, ectopic miR203 promotes mesenchymal-to-epithelial transition and disrupts activities associated with epithelial stem cells. These studies support a model in which induction of miR203 mediates forfeiture of self-renewing capacity via suppression of ΔNp63α and may also have anti-tumorigenic activity through its reduction of EMT and cancer stem cell populations.
Highlights
Abundant evidence implicates DNp63 isoforms in preservation of self-renewal and tissue stasis in diverse epithelial structures, including the mammary gland
MiR203 directly targets sequences within exon 15 of TP63 that encode the 3’UTR of a and b isoforms.[22]. This finding coupled to the fact that DNp63a is required for mammary stem cells (MaSCs) preservation suggests that increased expression of miR203 may promote differentiation in the mammary regenerative hierarchy
To test this, enriched fractions of MaSCs (Lin À /CD24 þ /CD29high/CD61 þ ), luminal progenitors (Lin À /CD24 þ /CD29low/CD61 þ ) and mature luminal epithelia (Lin À /CD24 þ /CD29low/CD61 À ) were isolated (Figure 1a) and analyzed for expression of miR203. Cytokeratin profiling of these fractions revealed that Lin À / CD24 þ /CD29low/CD61 À fractions were enriched for the luminal epithelial cytokeratins KRT18 and KRT19 (Supplementary Figure S1a), whereas Lin À /CD24 þ / CD29high was highly enriched for expression of basal epithelial markers, KRT14 and KRT5 (Supplementary Figure S1b)
Summary
Abundant evidence implicates DNp63 isoforms in preservation of self-renewal and tissue stasis in diverse epithelial structures, including the mammary gland. Targeted ablation of DNp63 isoforms leads to broad-spectrum epithelial hypoplasias characterized by stem cell depletion and ablated regenerative kinetics.[10,11,12] the precise mechanisms by which DNp63a preserves self-renewing capacity are not fully understood, substantial evidence indicates that it potently inhibits cellular senescence.[13] In addition, haploinsufficiency of TP63 confers a premature aging phenotype associated with a sharp increase in cellular senescence.[13,14,15] In basal breast cancers and head and neck squamous cell carcinomas, DNp63a acts as a pro-survival factor and a mediator of chemoresistance that actively represses expression of pro-apoptotic effectors.[16,17] These studies provide compelling evidence that DNp63a is critical for preservation of replicative capacity, Received 30.12.12; accepted 16.1.13; Edited by E Candi prolonged life span and survival that are characteristic of adult and cancer stem cells They further suggest that specific mechanisms exist to subvert these activities during lineage commitment and cellular differentiation. These studies suggest that miR203 is a potent stem cell regulator and has a significant role in the acquisition of luminal epithelial cell fate through the forfeiture of MaSC activity in the normal mammary gland and breast cancer stem cells
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