title: Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics.
abstract: BACKGROUND
  The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance.
  
  
  METHODS
  Here we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost.
  
  
  RESULTS
  Using this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches.
  
  
  CONCLUSIONS
  We have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens.
authors: Center for Molecular Therapeutics, Center for Cancer Research, Massachusetts General Hospital, and Harvard Medical School, CNY 149-Rm7308, Thirteenth St. Charlestown, MA 02129, USA. lchen13@partners.org
authors: Li
authors: L
authors: Chen
authors: Lynda
authors: L
authors: Stuart
authors: Toshiro K
authors: TK
authors: Ohsumi
authors: Shawn
authors: S
authors: Burgess
authors: Gaurav K
authors: GK
authors: Varshney
authors: Anahita
authors: A
authors: Dastur
authors: Mark
authors: M
authors: Borowsky
authors: Cyril
authors: C
authors: Benes
authors: Adam
authors: A
authors: Lacy-Hulbert
authors: Emmett V
authors: EV
authors: Schmidt
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D058893:DNA Barcoding, Taxonomic / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods*; D004251:DNA Transposable Elements / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D006801:Humans; D016254:Mutagenesis, Insertional / Q000379:methods; D009369:Neoplasms / Q000188:drug therapy* / Q000235:genetics; D017239:Paclitaxel / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D004251:DNA Transposable Elements; D012333:RNA, Messenger; D017239:Paclitaxel