title: A pooled shRNA screen for regulators of primary mammary stem and progenitor cells identifies roles for Asap1 and Prox1.
abstract: BACKGROUND
  The molecular regulators that orchestrate stem cell renewal, proliferation and differentiation along the mammary epithelial hierarchy remain poorly understood. Here we have performed a large-scale pooled RNAi screen in primary mouse mammary stem cell (MaSC)-enriched basal cells using 1295 shRNAs against genes principally involved in transcriptional regulation.
  
  
  METHODS
  MaSC-enriched basal cells transduced with lentivirus pools carrying shRNAs were maintained as non-adherent mammospheres, a system known to support stem and progenitor cells. Integrated shRNAs that altered culture kinetics were identified by next generation sequencing as relative frequency changes over time. RNA-seq-based expression profiling coupled with in vitro progenitor and in vivo transplantation assays was used to confirm a role for candidate genes in mammary stem and/or progenitor cells.
  
  
  RESULTS
  Utilizing a mammosphere-based assay, the screen identified several candidate regulators. Although some genes had been previously implicated in mammary gland development, the vast majority of genes uncovered have no known function within the mammary gland. RNA-seq analysis of freshly purified primary mammary epithelial populations and short-term cultured mammospheres was used to confirm the expression of candidate regulators. Two genes, Asap1 and Prox1, respectively implicated in breast cancer metastasis and progenitor cell function in other systems, were selected for further analysis as their roles in the normal mammary gland were unknown. Both Prox1 and Asap1 were shown to act as negative regulators of progenitor activity in vitro, and Asap1 knock-down led to a marked increase in repopulating activity in vivo, implying a role in stem cell activity.
  
  
  CONCLUSIONS
  This study has revealed a number of novel genes that influence the activity or survival of mammary stem and/or progenitor cells. Amongst these, we demonstrate that Prox1 and Asap1 behave as negative regulators of mammary stem/progenitor function. Both of these genes have also been implicated in oncogenesis. Our findings provide proof of principle for the use of short-term cultured primary MaSC/basal cells in functional RNAi screens.
authors: ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. sheridan@wehi.EDU.AU.|Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. sheridan@wehi.EDU.AU.|Department of Medical Biology, The University of Melbourne, Parkville, VIC, 3010, Australia. sheridan@wehi.EDU.AU.
authors: Julie M
authors: JM
authors: Sheridan
authors: Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. mritchie@wehi.edu.au.|School of Mathematics and Statistics, The University of Melbourne, Parkville, VIC, 3010, Australia. mritchie@wehi.edu.au.
authors: Matthew E
authors: ME
authors: Ritchie
authors: ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. best@wehi.edu.au.|Department of Medical Biology, The University of Melbourne, Parkville, VIC, 3010, Australia. best@wehi.edu.au.
authors: Sarah A
authors: SA
authors: Best
authors: ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. kun_153@hotmail.com.
authors: Kun
authors: K
authors: Jiang
authors: ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. mclennan@wehi.edu.au.
authors: Tamara J
authors: TJ
authors: Beck
authors: ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. vaillant@wehi.edu.au.|Department of Medical Biology, The University of Melbourne, Parkville, VIC, 3010, Australia. vaillant@wehi.edu.au.
authors: François
authors: F
authors: Vaillant
authors: ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. liu.k@wehi.EDU.AU.
authors: Kevin
authors: K
authors: Liu
authors: Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. rdickins@wehi.EDU.AU.|Department of Medical Biology, The University of Melbourne, Parkville, VIC, 3010, Australia. rdickins@wehi.EDU.AU.
authors: Ross A
authors: RA
authors: Dickins
authors: Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. smyth@wehi.edu.au.|School of Mathematics and Statistics, The University of Melbourne, Parkville, VIC, 3010, Australia. smyth@wehi.edu.au.
authors: Gordon K
authors: GK
authors: Smyth
authors: ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. lindeman@wehi.edu.au.|Department of Medicine, The University of Melbourne, Parkville, VIC, 3010, Australia. lindeman@wehi.edu.au.|Department of Medical Oncology, The Royal Melbourne Hospital, Grattan Street, Parkville, VIC, 3050, Australia. lindeman@wehi.edu.au.
authors: Geoffrey J
authors: GJ
authors: Lindeman
authors: ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. visvader@wehi.edu.au.|Department of Medical Biology, The University of Melbourne, Parkville, VIC, 3010, Australia. visvader@wehi.edu.au.
authors: Jane E
authors: JE
authors: Visvader
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D000818:Animals; D002452:Cell Count; D002454:Cell Differentiation / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D059014:High-Throughput Nucleotide Sequencing; D018398:Homeodomain Proteins / Q000235:genetics*; D016130:Immunophenotyping; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D034741:RNA, Small Interfering / Q000235:genetics*; D015203:Reproducibility of Results; D013234:Stem Cells / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D000098551:Prospero-Related Homeobox 1 Protein
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C485546:Asap1 protein, mouse; D018398:Homeodomain Proteins; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D000098551:Prospero-Related Homeobox 1 Protein