Lara Sicouri1, Federica Pisati1, Salvatore Pece2,3, Francesco Blasi*,1 and Elena Longobardi*,1,4
1IFOM (FIRC Institute of Molecular Oncology), Milan, 2Department of Experimental Oncology, European Institute of Oncology, Milan, 3Department of Oncology and Hemato-Oncology, University of Milan, Milan, and 4Section of Pharmacology, Department of Neuroscience, University of Naples Federico II, Naples, Italy
Prep1 (pKnox1) is a homeodomain transcription factor essential for in utero and post-natal development and an oncosuppressor gene in human and adult mice. We have analyzed its role in the development of the mouse mammary gland. We used Prep1i/i hypomorphic and Prep1F/F-Ker5CRE crosses to analyze the role of Prep1 in vivo in adult mouse mammary gland development. We also cultured mammary gland stem/progenitor cells in mammospheres to perform biochemical studies. Prep1 was expressed in mammary gland progenitors and fully differentiated mammary gland cells. Using different Prep1-deficient mouse models we show that in vivo Prep1 contributes to mammary gland branching since the branching efficiency of the mammary gland in Prep1-deleted or Prep1 hypomorphic mice was largely reduced. In-vitro, Prep1 sustained functions of the mammary stem/progenitor compartment. Prep1-deficient mammary stem/progenitor cells showed reduced ability to form mammospheres; they were not able to branch in a 3D assay, and exhibited reduced expression of Snail1, Snail2 and vimentin. The branching phenotype associates with increased Tp53-dependent apoptosis and inability to properly activate signals involved in branching morphogenesis. Finally, Prep1 formed complexes with Snail2, a transcription factor essential in branching morphogenesis, and its absence destabilizes and promotes Snail2 proteasome-mediated degradation. We conclude that Prep1 is required for normal adult mammary gland development, in particular at its branching morphogenesis step. By binding Snail2, Prep1 protects it from the proteasomal degradation.