title: Patient Mutation Directed shRNA Screen Uncovers Novel Bladder Tumor Growth Suppressors.
abstract: Next-generation sequencing (NGS) of human bladder cancer has revealed many gene alterations compared with normal tissue, with most being predicted to be "loss of function." However, given the high number of alterations, evaluating the functional impact of each is impractical. Here, we develop and use a high-throughput, in vivo strategy to determine which alterations are loss of function in tumor growth suppressors. Genes reported as altered by NGS in bladder cancer patients were bioinformatically processed by MutationTaster and MutationAssessor, with 283 predicted as loss of function. An shRNA lentiviral library targeting these genes was transduced into T24 cells, a nontumorigenic human bladder cancer cell line, followed by injection into mice. Tumors that arose were sequenced and the dominant shRNA constructs were found to target IQGAP1, SAMD9L, PCIF1, MED1, and KATNAL1 genes. In vitro validation experiments revealed that shRNA molecules directed at IQGAP1 showed the most profound increase in anchorage-independent growth of T24 cells. The clinical relevance of IQGAP1 as a tumor growth suppressor is supported by the finding that its expression is lower in bladder cancer compared with benign patient urothelium in multiple independent datasets. Lower IQGAP1 protein expression associated with higher tumor grade and decreased patient survival. Finally, depletion of IQGAP1 leads to increased TGFBR2 with TGFβ signaling, explaining in part how reduced IQGAP1 promotes tumor growth. These findings suggest IQGAP1 is a bladder tumor growth suppressor that works via modulating TGFβ signaling and is a potentially clinically useful biomarker.
  
  
  CONCLUSIONS
  This study used gene mutation information from patient-derived bladder tumor specimens to inform the development of a screen used to identify novel tumor growth suppressors. This included identification of the protein IQGAP1 as a potent bladder cancer growth suppressor.
authors.affiliation: Departments of Surgery (Urology) and Pharmacology, University of Colorado, Aurora, Colorado.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hensel
authors.affiliation: Departments of Surgery (Urology) and Pharmacology, University of Colorado, Aurora, Colorado.
authors.forename: Jason E
authors.initials: JE
authors.lastname: Duex
authors.affiliation: Departments of Surgery (Urology) and Pharmacology, University of Colorado, Aurora, Colorado.
authors.forename: Charles
authors.initials: C
authors.lastname: Owens
authors.affiliation: Department of Mathematics and Computer Science, Eastern Connecticut State University, Willimantic, Connecticut.
authors.forename: Garrett M
authors.initials: GM
authors.lastname: Dancik
authors.affiliation: Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado, Aurora, Colorado.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Edwards
authors.affiliation: Department of Pathology, University of Virginia, Charlottesville, Virginia.
authors.forename: Henry F
authors.initials: HF
authors.lastname: Frierson
authors.affiliation: Departments of Surgery (Urology) and Pharmacology, University of Colorado, Aurora, Colorado. University of Colorado Comprehensive Cancer Center, Aurora, Colorado. dan.theodorescu@ucdenver.edu.
authors.forename: Dan
authors.initials: D
authors.lastname: Theodorescu
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D039301:Computers, Molecular; D018572:Disease-Free Survival; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D005820:Genetic Testing / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D051379:Mice; D009154:Mutation; D009361:Neoplasm Invasiveness; D011379:Prognosis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000378:metabolism; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D020703:ras GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C101203:IQ motif containing GTPase activating protein 1; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; D020703:ras GTPase-Activating Proteins; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II