title: Structure-function studies of the bHLH phosphorylation domain of TWIST1 in prostate cancer cells.
abstract: The TWIST1 gene has diverse roles in development and pathologic diseases such as cancer. TWIST1 is a dimeric basic helix-loop-helix (bHLH) transcription factor existing as TWIST1-TWIST1 or TWIST1-E12/47. TWIST1 partner choice and DNA binding can be influenced during development by phosphorylation of Thr125 and Ser127 of the Thr-Gln-Ser (TQS) motif within the bHLH of TWIST1. The significance of these TWIST1 phosphorylation sites for metastasis is unknown. We created stable isogenic prostate cancer cell lines overexpressing TWIST1 wild-type, phospho-mutants, and tethered versions. We assessed these isogenic lines using assays that mimic stages of cancer metastasis. In vitro assays suggested the phospho-mimetic Twist1-DQD mutation could confer cellular properties associated with pro-metastatic behavior. The hypo-phosphorylation mimic Twist1-AQA mutation displayed reduced pro-metastatic activity compared to wild-type TWIST1 in vitro, suggesting that phosphorylation of the TWIST1 TQS motif was necessary for pro-metastatic functions. In vivo analysis demonstrates that the Twist1-AQA mutation exhibits reduced capacity to contribute to metastasis, whereas the expression of the Twist1-DQD mutation exhibits proficient metastatic potential. Tethered TWIST1-E12 heterodimers phenocopied the Twist1-DQD mutation for many in vitro assays, suggesting that TWIST1 phosphorylation may result in heterodimerization in prostate cancer cells. Lastly, the dual phosphatidylinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor BEZ235 strongly attenuated TWIST1-induced migration that was dependent on the TQS motif. TWIST1 TQS phosphorylation state determines the intensity of TWIST1-induced pro-metastatic ability in prostate cancer cells, which may be partly explained mechanistically by TWIST1 dimeric partner choice.
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Rajendra P
authors.initials: RP
authors.lastname: Gajula
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Sivarajan T
authors.initials: ST
authors.lastname: Chettiar
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Russell D
authors.initials: RD
authors.lastname: Williams
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Katriana
authors.initials: K
authors.lastname: Nugent
authors.affiliation: The Russell H. Morgan Department of Radiology and Radiological Science, Division of Cancer Imaging Research, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA; In Vivo Cellular and Molecular Imaging Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kato
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Hailun
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Reem
authors.initials: R
authors.lastname: Malek
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Cellular and Molecular Medicine Program, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Kekoa
authors.initials: K
authors.lastname: Taparra
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Cades
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Anvesh
authors.initials: A
authors.lastname: Annadanam
authors.affiliation: Department of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, USA.
authors.forename: A-Rum
authors.initials: AR
authors.lastname: Yoon
authors.affiliation: Department of Oncology, Division of Biostatistics and Bioinformatics, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Elana
authors.initials: E
authors.lastname: Fertig
authors.affiliation: Department of Pediatrics, Riley Heart Research Center, Indiana University School of Medicine, Indianapolis, IN, USA.
authors.forename: Beth A
authors.initials: BA
authors.lastname: Firulli
authors.affiliation: Department of Medicine, Division of Hematology-Oncology, Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Lucia
authors.initials: L
authors.lastname: Mazzacurati
authors.affiliation: Department of Medicine, Division of Hematology-Oncology, Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Timothy F
authors.initials: TF
authors.lastname: Burns
authors.affiliation: Department of Pediatrics, Riley Heart Research Center, Indiana University School of Medicine, Indianapolis, IN, USA.
authors.forename: Anthony B
authors.initials: AB
authors.lastname: Firulli
authors.affiliation: In Vivo Cellular and Molecular Imaging Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, USA; Physical Sciences in Oncology Center, Johns Hopkins University, Baltimore, MD, USA; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Steven S
authors.initials: SS
authors.lastname: An
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA; In Vivo Cellular and Molecular Imaging Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Cellular and Molecular Medicine Program, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Urology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Electronic address: tranp@jhmi.edu.
authors.forename: Phuoc T
authors.initials: PT
authors.lastname: Tran
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D016000:Cluster Analysis; D004195:Disease Models, Animal; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics; D020869:Gene Expression Profiling; D064593:Heterografts; D006801:Humans; D008297:Male; D009154:Mutation; D009362:Neoplasm Metastasis; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D054730:Protein Interaction Domains and Motifs; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D055503:Protein Multimerization; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D059467:Transcriptome; D051879:Twist-Related Protein 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D051879:Twist-Related Protein 1; C103173:TWIST1 protein, human