Bortezomib suppresses focal adhesion kinase expression via interrupting nuclear factor-kappa B.
OBJECTIVE
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).


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 24h. 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 NFkappaB binding activities were measured by luciferase-reporter method. NFkappaB subunit p65 binding capacity was determined by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis.


RESULTS
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 NFkappaB binding capacity demonstrated by EMSA and ChIP assay suggested that NFkappaB plays an important role in bortezomib suppressing FAK expression.


CONCLUSIONS
These results suggested that FAK is downregulated by bortezomib through a proteasome-dependent NFkappaB inhibitory mechanism. Thus, FAK could be a potential molecular target of bortezomib for therapeutic strategy.
Ko
Bor-Sheng
BS
Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan.
Chang
Tzu-Ching
TC
Chen
Chien-Hung
CH
Liu
Chia-Chia
CC
Kuo
Cheng-Chin
CC
Hsu
Chiun
C
Shen
Ying-Chun
YC
Shen
Tang-Long
TL
Golubovskaya
Vita M
VM
Chang
Chung-Che
CC
Shyue
Song-Kun
SK
Liou
Jun-Yang
JY
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects*; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D006801:Humans; D020287:In Situ Nick-End Labeling; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D061988:Proteasome Inhibitors; D011719:Pyrazines / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000970:Antineoplastic Agents; D001897:Boronic Acids; D004791:Enzyme Inhibitors; D016328:NF-kappa B; D061988:Proteasome Inhibitors; D011719:Pyrazines; D000069286:Bortezomib; D051416:Focal Adhesion Protein-Tyrosine Kinases