title: Crk adaptor proteins act as key signaling integrators for breast tumorigenesis.
abstract: BACKGROUND
  CT10 regulator of kinase (Crk) adaptor proteins (CrkI, CrkII and CrkL) play a role in integrating signals for migration and invasion of highly malignant breast cancer cell lines. This has important implications, as elevated CrkI/II protein levels were observed in a small cohort of breast cancer patients, which identified a potential role for Crk proteins in breast cancer progression. Numerous in vitro studies identified a role for Crk proteins in cell motility, but little is known about how Crk proteins contribute to breast cancer progression in vivo.
  
  
  METHODS
  The clinical significance of Crk proteins in human breast cancer was assessed by analyzing published breast cancer datasets using a gene expression signature that was generated following CrkII over-expression and by examining Crk protein expression in tissue microarrays of breast tumors (n = 254). Stable knockdown of Crk (CrkI/CrkII/CrkL) proteins was accomplished using a short hairpin RNA (shRNA)-mediated approach in two basal breast cancer cell lines, MDA-231 1833TR and SUM1315, where the former have a high affinity to form bone metastases. Both in vitro assays (cell migration, invasion, soft agar growth) and in vivo experiments (intra-cardiac, tibial and mammary fat pad injections) were performed to assess the functional significance of Crk proteins in breast cancer.
  
  
  RESULTS
  A gene signature derived following CrkII over-expression correlated significantly with basal breast cancers and with high grade and poor outcome in general. Moreover, elevated Crk immunostaining on tissue microarrays revealed a significant association with highly proliferative tumors within the basal subtype. RNAi-mediated knockdown of all three Crk proteins in metastatic basal breast cancer cells established a continued requirement for Crk in cell migration and invasion in vitro and metastatic growth in vivo. Furthermore, Crk ablation suppressed anchorage independent growth and in vivo orthotopic tumor growth. This was associated with diminished cell proliferation and was rescued by expression of non-shRNA targeted CrkI/II. Perturbations in tumor progression correlated with altered integrin signaling, including decreased cell spreading, diminished p130Cas phosphorylation, and Cdc42 activation.
  
  
  CONCLUSIONS
  These data highlight the physiological importance of Crk proteins in regulating growth of aggressive basal breast cancer cells and identify Crk-dependent signaling networks as promising therapeutic targets.
authors.affiliation: Department of Biochemistry, McGill University, Rosalind and Morris Goodman Cancer Research Centre, 3655 Promenade Sir William Osler, Montréal, QC H3G 1Y6, Canada.
authors.forename: Kelly E
authors.initials: KE
authors.lastname: Fathers
authors.forename: Emily S
authors.initials: ES
authors.lastname: Bell
authors.forename: Charles V
authors.initials: CV
authors.lastname: Rajadurai
authors.forename: Sean
authors.initials: S
authors.lastname: Cory
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.forename: Anna
authors.initials: A
authors.lastname: Mourskaia
authors.forename: Dongmei
authors.initials: D
authors.lastname: Zuo
authors.forename: Jason
authors.initials: J
authors.lastname: Madore
authors.forename: Anie
authors.initials: A
authors.lastname: Monast
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Mes-Masson
authors.forename: Andree-Anne
authors.initials: AA
authors.lastname: Grosset
authors.forename: Louis
authors.initials: L
authors.lastname: Gaboury
authors.forename: Michael
authors.initials: M
authors.lastname: Hallet
authors.forename: Peter
authors.initials: P
authors.lastname: Siegel
authors.forename: Morag
authors.initials: M
authors.lastname: Park
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D001859:Bone Neoplasms / Q000556:secondary; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic; D050719:Crk-Associated Substrate Protein / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016023:Integrins / Q000378:metabolism; D015674:Mammary Neoplasms, Animal / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D050737:Proto-Oncogene Proteins c-crk / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C493841:BCAR1 protein, human; C082869:CRKL protein; D050719:Crk-Associated Substrate Protein; D016023:Integrins; D009687:Nuclear Proteins; D050737:Proto-Oncogene Proteins c-crk; D034741:RNA, Small Interfering; D020764:cdc42 GTP-Binding Protein