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Cyclin G–Associated Kinase Is Necessary for Osteosarcoma Cell Proliferation and Receptor Trafficking

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Cyclin G–Associated Kinase Is Necessary for Osteosarcoma Cell Proliferation and Receptor Trafficking Published OnlineFirst September 23, 2010; DOI: 10.1158/1535-7163.MCT-10-0637 Molecular Cancer Preclinical Development Therapeutics Cyclin G–Associated Kinase Is Necessary for Osteosarcoma Cell Proliferation and Receptor Trafficking Michiro Susa1,2, Edwin Choy2, Xianzhe Liu1,2, Joseph Schwab1,2, Francis J. Hornicek1,2, Henry Mankin2, and Zhenfeng Duan1,2 Abstract Osteosarcoma is the most frequent primary malignant bone tumor among the children. The advent of neoadjuvant chemotherapy significantly improved the prognosis of patients with osteosarcoma in the 1980s, but it has since plateaued in the past decades. Recently, one of the most researched areas in sarcoma treatment is tyrosine kinases. Here, we describe research on a serine/threonine kinase, cyclin G–associated kinase (GAK), which has not been reported in osteosarcoma previously. In this study, a lentiviral based human shRNA library was utilized to screen for kinases in KHOS and U-2OS osteosarcoma cells. The expression of GAK was examined in osteosarcoma and the effect on cell proliferation was analyzed by GAK siRNA knockdown. The level of GAK expression and its correlation to prognosis was analyzed in osteosarcoma tissue microarray. The effect of GAK depletion on insulin-like growth factor and epidermal growth factor receptor– mediated signal transduction was analyzed by Western blot. We observed that GAK was overexpressed in both osteosarcoma cell lines and tissue samples when compared with human osteoblasts. GAK knockdown by siRNA decreased cell proliferation in both drug-sensitive and multidrug-resistant osteosarcoma cell lines. Immunohistochemistry of osteosarcoma tissue microarray revealed that overexpression of GAK was asso- ciated with poor prognosis. Finally, knockdown of GAK resulted in alterations of receptor trafficking and several downstream proteins. In conclusion, our results suggest that osteosarcoma cell proliferation and survival are dependent on GAK. These findings may lead to the development of new therapeutic options for osteosarcoma. Mol Cancer Ther; 9(12); 3342–50. Ó2010 AACR. Introduction osteosarcoma has yet to be elucidated. Nevertheless, targeting kinases has tremendous potential as shown in Osteosarcoma has the highest prevalence (approxi- the treatment of gastrointestinal stromal tumors treated mately 60%) among patients with primary malignant bone with imatinib mesylate (Gleevec; Novartis Pharmaceu- tumors. Each year, there are more than 800 new patients, ticalsCorp;ref.2). younger than 20 years, with a diagnosis of osteosarcoma in Cyclin G–associated kinase (GAK), also known as the United States (1). Major progress has been made in the auxillin 2, is a 160-kDa serine/threonin protein kinase treatment of patients with osteosarcoma because of the that was cloned from rats and humans in 1997 (3, 4). It is use of chemotherapy, leading to an improved overall composed from N-terminal kinase domain that phos- survival rate of 65%. However, 30% to 40% of the patients phorylates the m-subunits of adaptor protein 1 and 2, with osteosarcoma still experience recurrence or metas- clathrin-binding domain, and a C-terminal J-domain that tasis despite the improved multimodality therapy. Many interacts with heat shock cognate 70 (Hsc 70). Although it will develop resistance to multiple types of chemotherapy shares 43% homology to auxillin, which is expressed only after prolonged periods of treatments. in the neuronal cells (5), GAK is expressed ubiquitously The importance of kinases has been established in various organs. GAK is known to assemble clathrin in many cancers, but their diversity of functions in into baskets during endocytosis and support Hsc 70 in uncoating clathrin-coated vesicles (CCV; refs. 6, 7). In yeast and Caenorhabditis elegans, auxillin depletion has Authors' Affiliations: 1Department of Orthopaedic Surgery, and 2Sarcoma been reported to cause accumulation of CCVs, impair Biology Laboratory, Center for Sarcoma and Connective Tissue Oncology, cargo delivery to the vacuoles, and slow down cell Massachusetts General Hospital, Boston, MA growth (8–10). GAK was identified in our lentiviral Corresponding Author: Zhenfeng Duan, Center for Sarcoma and Con- nective Tissue Oncology, Massachusetts General Hospital, 55 Fruit St. shRNA screen as being critical for osteosarcoma cell Jackson 1115, Boston, MA 02114. Phone: (617) 724-3144; Fax: (617) 724- proliferation. Because GAK has important functions as 7396. E-mail: [email protected] a regulator of receptor tyrosine kinase trafficking, which doi: 10.1158/1535-7163.MCT-10-0637 is situated at the upstream of many kinases that Ó2010 American Association for Cancer Research. have been reported to play key regulatory roles in the 3342 Mol Cancer Ther; 9(12) December 2010 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst September 23, 2010; DOI: 10.1158/1535-7163.MCT-10-0637 Cyclin G–Associated Kinase and Osteosarcoma development of cancer, it was our aim to further examine in accordance with the policies of the institutional review its function in osteosarcoma cell lines. board of the hospital. All diagnoses were confirmed by In osteosarcoma, receptor tyrosine kinases such as light microscopy and immunohistochemistry. insulin-like growth factor 1 receptor (IGF-IR; refs. 11–15) and epidermal growth factor receptor (EGFR; refs. Lentiviral human kinase shRNA library screen 16–19) have been implicated in malignant growth and using MISSION LentiExpress human kinases invasion. When ligands bind to each respective receptor, shRNA library they are activated by autophosphorylation, internalized, A total of 673 human kinases were analyzed for at least in part through clathrin-mediated endocytosis, their effects on osteosarcoma cell growth, using MISSION and sorted to early and late endosomes. Activation of LentiExpress Human Kinases shRNA Library (Sigma) as signal transduction pathways and consequent sub- described previously (20). cellular reactions are mediated through the subcellular compartmentalization of each receptor tyrosine kinases. Synthetic GAK siRNA and transfection It is important to note that osteosarcoma is characterized Two different GAK siRNAs and 1 nonspecific by heterogeneity that has numerous genetic changes. siRNA that has no significant homology to any known Although there are several clinical trials (www. genesequencesfrommouse,rat,orhumanwere clinicaltrials.gov) currently undergoing that utilize var- purchased from Ambion. The siRNA sequences target- ious drugs to block the IGF-IR and EGFR signaling ing GAK (GenBank accession No. NM_005255.2) corre- cascade, it may be logical to do a sequential or combi- sponded to coding regions (1: sense 50-GUCCGUCGC- natorial application of several kinase inhibitors for the UAAUUAUGCAtt-30,antisense50-UGCAUAAUUAG- treatment of osteosarcoma. Therefore, it may be useful CGACGGACtg-30;2:sense50-CACCAGAAAUCAUA- to identify new kinases associated with osteosarcoma GACUUtt-30,antisense50-AAGUCUAUGAUUUCU- to develop appropriate treatment options for diverse GGUGtt-30)oftheGAK gene. The siRNA oligonucleo- patients with osteosarcoma. tides were dissolved in nuclease-free water at a con- In this study, we analyzed the expression of GAK and centration of 100 mmol/L and kept at À20Cuntilthe its function in osteosarcoma cell lines. In addition, we following transfection experiments. Transfections were hypothesized that GAK might have a role as a prognostic performed with Lipofectamine RNAiMAX according to indicator for osteosarcoma. These data may contribute to the manufacturer’s instruction (Invitrogen). Mixtures the growing information of kinases for clinical utility in (100 nmol/L) of siRNA, transfection agent, and medium the treatment of osteosarcoma. were subsequently diluted with the same medium and applied at the designated concentration to each well. Materials and Methods The medium was replaced with RPMI 1640 supplemen- ted with 10% FBS 24 hours after each transfection. Cell culture and tissues The human osteosarcoma cell line U-2OS was obtained Immunofluorescence microscopy from American Type Culture Collection. Dr Efstathios Osteosarcoma cells were grown with the addition of S. Gonos (National Hellenic Research Foundation) kindly either 100 nmol/L of GAK siRNA1 or 100 nmol/L of provided the human osteosarcoma cell line KHOS and nonspecific siRNA on 8-well Lab-Tek chamber slides the multidrug-resistant cell lines KHOSR2 and U-2OSR2. (Thermo Fisher Scientific, Rochester, NY) for 24 hours All cell lines were cultured in RPMI 1640 medium sup- and fixed in 3.7% buffered paraformaldehyde. Immunos- plemented with 10% FBS, 100 U/mL of penicillin, and tainings were done using antibodies against GAK (1:50 100 mg/mL of streptomycin (all obtained from Invitro- dilution; sc-7864; Santa Cruz Biotechnology) and actin gen). Cells were incubated at 37 Cin5%CO2, 95% air (1:200 dilution; Sigma-Aldrich) overnight. After washing, atmosphere, and passaged when near-confluent mono- the cells were incubated with Alexa Fluor secondary anti- layers were achieved using trypsin-EDTA solution. Resis- bodies (Invitrogen). To counterstain the nuclei, the cells tant cell lines were continuously cultured in 0.1 mmol/L were incubated with PBS containing 1 mg/mL of Hoechst of doxorubicin. Doxorubicin was obtained as unused 33342 (Invitrogen) for 15 minutes. Stained cells were then residual clinical material from the Massachusetts General visualized on a Nikon Eclipse Ti-U fluorescence micro- Hospital.
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