Combining RNA interference and kinase inhibitors against cell signalling components involved in cancer.
BACKGROUND
The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including oncogenic transformation. The tyrosine kinases of the epidermal growth factor receptor (EGFR) constitute the beginning of one signal transduction cascade leading to AP-1 activation and are known to control cell proliferation and differentiation. Drug discovery efforts targeting this receptor and other pathway components have centred on monoclonal antibodies and small molecule inhibitors. Resistance to such inhibitors has already been observed, guiding the prediction of their use in combination therapies with other targeted agents such as RNA interference (RNAi). This study examines the use of RNAi and kinase inhibitors for qualification of components involved in the EGFR/AP-1 pathway of ME180 cells, and their inhibitory effects when evaluated individually or in tandem against multiple components of this important disease-related pathway.


METHODS
AP-1 activation was assessed using an ME180 cell line stably transfected with a beta-lactamase reporter gene under the control of AP-1 response element following epidermal growth factor (EGF) stimulation. Immunocytochemistry allowed for further quantification of small molecule inhibition on a cellular protein level. RNAi and RT-qPCR experiments were performed to assess the amount of knockdown on an mRNA level, and immunocytochemistry was used to reveal cellular protein levels for the targeted pathway components.


RESULTS
Increased potency of kinase inhibitors was shown by combining RNAi directed towards EGFR and small molecule inhibitors acting at proximal or distal points in the pathway. After cellular stimulation with EGF and analysis at the level of AP-1 activation using a beta-lactamase reporter gene, a 10-12 fold shift or 2.5-3 fold shift toward greater potency in the IC50 was observed for EGFR and MEK-1 inhibitors, respectively, in the presence of RNAi targeting EGFR.


CONCLUSIONS
EGFR pathway components were qualified as targets for inhibition of AP-1 activation using RNAi and small molecule inhibitors. The combination of these two targeted agents was shown to increase the efficacy of EGFR and MEK-1 kinase inhibitors, leading to possible implications for overcoming or preventing drug resistance, lowering effective drug doses, and providing new strategies for interrogating cellular signalling pathways.
O'Grady
Michael
M
Invitrogen Corporation, Madison, WI 53719, USA. michael.ogrady@invitrogen.com
Raha
Debasish
D
Hanson
Bonnie J
BJ
Bunting
Michaeline
M
Hanson
George T
GT
D000911:Antibodies, Monoclonal / Q000737:chemistry; D002070:Butadienes / Q000494:pharmacology; D002454:Cell Differentiation; D049109:Cell Proliferation; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D007150:Immunohistochemistry; D020128:Inhibitory Concentration 50; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation; D011799:Quinazolines; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000494:pharmacology; D014162:Transfection; D020032:Tyrphostins / Q000494:pharmacology; D001618:beta-Lactamases / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002070:Butadienes; D004791:Enzyme Inhibitors; D009570:Nitriles; D016376:Oligonucleotides, Antisense; D011799:Quinazolines; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D020032:Tyrphostins; C113580:U 0126; C101044:RTKI cpd; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D048369:MAP Kinase Kinase 1; D001618:beta-Lactamases