Bortezomib suppresses focal adhesion kinase expression via interrupting nuclear factor-kappa B Bor-Sheng Ko a,b,1 , Tzu-Ching Chang a,1 , Chien-Hung Chen a , Chia-Chia Liu a , Cheng-Chin Kuo a , Chiun Hsu c , Ying-Chun Shen c , Tang-Long Shen d , Vita M. Golubovskaya e,2 , Chung-Che Chang f , Song-Kun Shyue g, ⁎, Jun-Yang Liou a, ⁎ a Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan b Department of Internal Medicine, National Taiwan University Hospital, Taipei 100, Taiwan c Department of Oncology, National Taiwan University Hospital, Taipei 100, Taiwan d Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 10617, Taiwan e Department of Surgery, School of Medicine, UF Shands Cancer Center, Gainesville, FL 32610, USA f The Methodist Hospital Research Institutes, Department of Pathology, Weill Medical College of Cornell University, Houston, TX 77030, USA g Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan abstract article info Article history: Received 8 July 2009 Accepted 4 December 2009 Keywords: Bortezomib Proteasome FAK NFκB p53 Aims: Bortezomib is a potent proteasome inhibitor currently used to treat various malignancies with promising results. To explore the role of bortezomib in reducing cancer cell migration and inducing apoptosis, we evaluated the effects of bortezomib on the expression of focal adhesion kinase (FAK). Main methods: Various types of cancer cells including lung cancer A549, H1299; a breast cancer MCF7; a hepatocellular carcinoma Huh7, and a tongue squamous cell carcinoma SCC-25 were treated with different concentrations of bortezomib or MG-132 as indicated for 24 h. Protein and mRNA levels were determined by Western blotting and real-time PCR. Apoptosis was analyzed by caspase 3 cleavage and activity. FAK promoter and NFκB binding activities were measured by luciferase-reporter method. NFκB subunit p65 binding capacity was determined by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis. Key findings: Both bortezomib and another proteasome inhibitor, MG-132, significantly reduced FAK expression, suppressed cancer cell migration and increased cell apoptosis. Results of real-time PCR and promoter activity assay revealed that bortezomib decreased FAK expression through transcriptional inactivation. Results of FAK promoter activity and ChIP assays in A549 and H1299 cells indicated that bortezomib suppressed FAK activity through a p53-independent pathway. Furthermore, reduction of NFκB binding capacity demonstrated by EMSA and ChIP assay suggested that NFκB plays an important role in bortezomib suppressing FAK expression. Significance: These results suggested that FAK is downregulated by bortezomib through a proteasome- dependent NFκB inhibitory mechanism. Thus, FAK could be a potential molecular target of bortezomib for therapeutic strategy. © 2009 Elsevier Inc. All rights reserved. Introduction Focal adhesion kinase (FAK) was originally identified as a protein tyrosine kinase that localizes to cell focal adhesion contacts and adhesion sites (McLean et al. 2005; van Nimwegen and van de Water 2007; Wozniak et al. 2004). FAK interacts with a cluster of transmem- brane integrins and plays a prominent role in regulating integrin- mediated signaling (Toutant et al. 2002). Activated FAK recruits proteins to form a complex that regulates multiple signaling downstream pathways implicated in various cellular processes, including cell adhesion, motility, division, proliferation, and survival. Downregulation of FAK inhibits cell spreading and the response to exogenous stimuli of migration (Ilic et al. 1995; Owen et al. 1999; Sieg et al. 2000), whereas overexpression of FAK enhances cell motility, and reconstituting FAK-deficient cells with wild-type FAK restores cell migration (Richardson et al. 1997; Taylor et al. 2001). Attenuation or inhibition of FAK expression is associated with loss of cell adhesion and anoikis, which results in induction of tumor-cell and fibroblast apoptosis (Frisch et al. 1996; Hungerford et al. 1996; Xu et al. 1996). Moreover, a large Life Sciences 86 (2010) 199–206 ⁎ Corresponding authors. Liou is to be contacted at Tel.: + 886 37 246 166x38309; fax: +886 37 587 408. Shyue, Tel.: +886 2 26523962. E-mail addresses: skshyue@ibms.sinica.edu.tw (S.-K. Shyue), jliou@nhri.org.tw (J.-Y. Liou). 1 Bor-Sheng Ko and Tzu-Ching Chang contributed equally to this work. 2 Current address: Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.12.003 Contents lists available at ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie