title: CRKL plays a pivotal role in tumorigenesis of head and neck squamous cell carcinoma through the regulation of cell adhesion.
abstract: The signaling adapter protein CRK is an indispensable molecule involved in regulating the malignant potential of human cancers. CRK-like (CRKL) is a hematopoietic cell-dominant homologue of CRK that is reported to be phosphorylated by BCR-ABL tyrosine kinase in chronic myelogenous leukemia patients, but its biological function in non-hematopoietic tumors remains unclear. In this study, we explored the tumorigenic role of CRKL in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. Immunoprecipitation analysis of HNSCC cell line, HSC-3 cells, showed that the dominant binding partner for C3G was CRKL, not CRK. To clarify the molecular function of CRKL, we established lentiviral shRNA-mediated CRKL-knockdown HNSCC cell lines. In CRKL-knockdown HSC-3 and HSC-4 cells, cell growth and motility were diminished compared to control cells. Cell adhesion assays showed that cell attachment onto both fibronectin- and collagen-coated dishes was significantly suppressed in CRKL-knockdown HSC-3 cells, while no significant change was observed for poly-l-lysine-coated dishes. Immunofluorescence staining revealed that focal adhesion was reduced in CRKL-knockdown HSC-3 cells. With a pulldown assay, CRKL-knockdown HSC-3 cells showed decreased amounts of active Rap1 compared to control cells. Moreover, in an in vivo assay, tumor formation of CRKL-knockdown HSC-3 cells in nude mice was significantly abrogated. Our results indicate that CRKL regulates HNSCC-cell growth, motility, and integrin-dependent cell adhesion, suggesting that CRKL plays a principal role in HNSCC tumorigenicity.
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638, Japan. h-naka@med.hokudai.ac.jp
authors.forename: Hiroko
authors.initials: H
authors.lastname: Yanagi
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nishihara
authors.forename: Taichi
authors.initials: T
authors.lastname: Kimura
authors.forename: Mishie
authors.initials: M
authors.lastname: Tanino
authors.forename: Teruki
authors.initials: T
authors.lastname: Yanagi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Fukuda
authors.forename: Shinya
authors.initials: S
authors.lastname: Tanaka
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000502:physiology*; D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology*; D002448:Cell Adhesion / Q000235:genetics; D002465:Cell Movement / Q000235:genetics; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D005260:Female; D055785:Gene Knockdown Techniques; D006258:Head and Neck Neoplasms / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D023041:Xenograft Model Antitumor Assays
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D009687:Nuclear Proteins