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

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 receptormediated 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 associated 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.

Gefitinib and Luteolin Cause Growth Arrest of Human Prostate Cancer PC-3 Cells via Inhibition of Cyclin G-Associated Kinase and Induction of miR-630

“…GAK is overexpressed in U2OS osteosarcoma cells, which lack EGFR expression, and knockdown of GAK inhibits their proliferation [31]. Therefore, we hypothesized that luteolin and/or gefitinib may induce apoptotic cell death of U2OS cells as well as PC-3 cells.…”

“…GAK is also overexpressed in KHOS and U2OS osteosarcoma cells; its overexpression is associated with poor prognosis, and siRNA-mediated knockdown of GAK decreases the proliferation of osteosarcoma cells [31], suggesting that GAK plays a pivotal role in the control of the proliferation rate of osteosarcoma cells. Unlike other osteosarcoma cell lines (HOS, MG-63, and KHOS/NP), U2OS cells lack expression of the EGFR, the well-known target of gefitinib; therefore, GAK is a putative bona fide target of gefitinib in these cells.…”

GAK is phosphorylated by c-Src and translocated from the centrosome to chromatin at the end of telophase

“…4 GAK is overexpressed in U2OS osteosarcoma cells and knockdown of GAK inhibits their proliferation. 10 We recently showed that GAK is modified and overexpressed in metastatic cancer cells, and that nuclear GAK is expressed at high levels in surgical specimens from prostate cancer patients with a positive correlation with the Gleason score. 9 These reports and our results here suggest that GAK functions in the regulation of proper M phase progression, in addition to cytoplasmic membrane trafficking.…”

“…9 The GAK level is also augmented in osteosarcoma cells of patients with a poor prognosis, and siRNA-mediated knockdown of GAK decreases the proliferation of osteosarcoma cells. 10 GAK harbors 4 structural domains: an N-terminal Ser/Thr kinase (K) domain, a PTEN-like (T) domain, a clathrin-binding (CB) domain, and a C-terminal J domain. Hampered clathrindependent trafficking caused by GAK knockout in fibroblasts is rescued by expressing the CB and J domains, suggesting that they play pivotal roles in GAK function.…”

A Kinome-Wide siRNA Screen Identifies Multiple Roles for Protein Kinases in Hypoxic Stress Adaptation, Including Roles for IRAK4 and GAK in Protection against Apoptosis in VHL–/– Renal Carcinoma Cells, Despite Activation of the NF-κB Pathway

“…23,24 GAKs' role in vesicle trafficking was described initially, 25 but subsequent studies identified a second role in cytokinesis, 26 and in fact it was this second role that enabled it to be identified in several recent RNAi screens. [27][28][29] On the basis of an siRNA screen, we have extended these characterizations to include a role for GAK in the adaptation of cancer cells to hypoxia and to an effect on cancer cell survival that cannot be suppressed by the activation of NF-κB. Since NF-κB is a potent tumor survival pathway and the object of many cancer therapeutic strategies, 30,31 an inhibitor that can function independently of the NF-κB survival pathway could be an important therapeutic modality.…”