title: Co-deleting Pten with Rb in retinal progenitor cells in mice results in fully penetrant bilateral retinoblastomas.
abstract: BACKGROUND
  Rb1 is the most frequently mutated gene in the pediatric cancer retinoblastoma, and its loss causes E2F transcription factors to induce proliferation related genes. However, high E2F levels following pRB loss also induce apoptosis-promoting genes as a safeguard mechanism to suppress emergent tumors. Although p53 accumulation and apoptosis induction is believed to be a primary mechanism to eliminate cells with excess E2F activity, p53 deletion doesn't suppress RB/E2F induced apoptosis in vivo in the retina. This prompted us to test the PTEN/PI3K/AKT signaling pathway on RB/E2F apoptosis suppression in vivo, to ascertain if the PI3K pathway may provide a potential avenue for retinoblastoma therapy.
  
  
  METHODS
  We developed a mouse model in which Rb1 and Pten were conditionally deleted from retinal progenitor cells using Chx10-Cre, whereas Rbl1 (p107) was constitutively deleted. Pathway components were also tested individually by in vivo electroporation into newborn retinas for an effect on apoptosis and tumor initiation. Mouse retinal tissues were analyzed by immunohistochemistry (IHC) for proliferation, apoptosis, and pathway activation. ShRNAs were used in vitro to assess effects on apoptosis and gene expression.
  
  
  RESULTS
  Co-deleting Pten with Rb1 and Rbl1 in mouse retinal progenitor cells (RPCs) causes fully penetrant bilateral retinoblastomas by 30 days and strongly suppresses Rb/E2F-induced apoptosis. In vivo electroporation of constitutively active (ca)-Pik3ca, ca-Akt, or dominant-negative (dn)-Foxo1 into apoptosis prone newborn murine retina with deleted Rb/p107 eliminate Rb/E2F induced apoptosis and induce retinoblastoma emergence. Retinal deletion of Pten activates p-AKT and p-FOXO1 signaling in incipient retinoblastoma. An unbiased shRNA screen focusing on Akt phosphorylation targets identified FOXOs as critical mediators of Rb/E2F induced apoptosis and expression of Bim and p73 pro-apoptotic genes.
  
  
  CONCLUSIONS
  These data indicate that we defined a key molecular trigger involving E2F/FOXO functioning to control retinal progenitor cell homeostasis and retinoblastoma tumor initiation. We anticipate that our findings could provide contextual understanding of the proliferation of other progenitor cells, considering the high frequency of co-altered signaling from RB/E2F and PTEN/PI3K/AKT pathways in a wide variety of normal and malignant settings.
authors.affiliation: Department of Pediatrics, University of Minnesota, Minneapolis, MN, 55455, USA. cxie@umn.edu.
authors.forename: Chencheng
authors.initials: C
authors.lastname: Xie
authors.affiliation: Department of Pediatrics, University of Minnesota, Minneapolis, MN, 55455, USA. luxxx245@umn.edu.
authors.forename: Huarui
authors.initials: H
authors.lastname: Lu
authors.affiliation: Department of Surgery, University of Minnesota, Minneapolis, MN, 55455, USA. nomur013@umn.edu.
authors.forename: Alice
authors.initials: A
authors.lastname: Nomura
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, 55455, USA. ehanse@umn.edu.
authors.forename: Eric Allan
authors.initials: EA
authors.lastname: Hanse
authors.affiliation: BioNet, Academic Health Center, University of Minnesota, Minneapolis, MN, 55455, USA. cforster@umn.edu.
authors.forename: Colleen Lynn
authors.initials: CL
authors.lastname: Forster
authors.affiliation: Department of Veterinary Population Medicine, University of Minnesota, St Paul, MN, 55108, USA. park0463@umn.edu.
authors.forename: Josh Berken
authors.initials: JB
authors.lastname: Parker
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, 55455, USA. linde013@umn.edu.
authors.forename: Michael Andrew
authors.initials: MA
authors.lastname: Linden
authors.affiliation: Department of Pediatrics, University of Minnesota, Minneapolis, MN, 55455, USA. kara0195@umn.edu.
authors.forename: Chris
authors.initials: C
authors.lastname: Karasch
authors.affiliation: Department of Pediatrics, University of Minnesota, Minneapolis, MN, 55455, USA. halls026@umn.edu.
authors.forename: Timothy Curtis
authors.initials: TC
authors.lastname: Hallstrom
mesh_terms: D000818:Animals; D017209:Apoptosis; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D058534:Class I Phosphatidylinositol 3-Kinases; D004195:Disease Models, Animal; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000378:metabolism; D017353:Gene Deletion; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D051059:PTEN Phosphohydrolase / Q000235:genetics* / Q000378:metabolism; D019683:Penetrance; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012160:Retina / Q000166:cytology; D012175:Retinoblastoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016160:Retinoblastoma Protein / Q000235:genetics* / Q000378:metabolism; D050720:Retinoblastoma-Like Protein p107 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013234:Stem Cells / Q000378:metabolism*
chemical_list: D058534:Class I Phosphatidylinositol 3-Kinases; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D016160:Retinoblastoma Protein; D050720:Retinoblastoma-Like Protein p107; C467200:Foxo1 protein, mouse; C546639:Pik3ca protein, mouse