Combining RNA interference and kinase inhibitors against cell signalling components involved in cancer
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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.
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.
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 β-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.
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.
KeywordsEpidermal Growth Factor Receptor Small Molecule Inhibitor Epidermal Growth Factor Receptor Inhibition Small Molecule Compound Epidermal Growth Factor Stimulation
- AP-1 = Activator Protein 1
bla = beta-lactamase, EGFR = Epidermal Growth Factor Receptor, MEK1 = Mitogen Activated Protein Kinase Kinase 1, Phosphate Buffered Saline PBS, PTKs = Protein Tyrosine Kinases, RNAi = RNA interference.
Cellular processes such as proliferation, differentiation, and death are regulated by signal transduction pathways which commonly exert their function through receptor mediated activation. The discovery in 1978 that the v-Src oncogene was a protein kinase led to a "cascade" of research into the role of kinases in cell-signalling pathways, and the subsequent finding that human cancer can result from the activity of nonviral, endogenous oncogenes, a major portion of which code for protein tyrosine kinases (PTKs) [1, 2]. The epidermal growth factor receptor (EGFR) is a tyrosine kinase which acts as a master switch leading to activation of the transcription factor, activator protein-1 (AP-1), and other related pathways. The receptor itself is composed of extracellular, transmembrane, and tyrosine kinase domains. Ligand binding elicits a conformational change of the extracellular domain leading to receptor dimerization and subsequent transphosphorylation of intracellular domain tyrosines. The phosphorylated tyrosines act as binding sites for signal transducers initiating a series of kinase actions resulting in cellular proliferation and differentiation [3, 4, 5]. Aberrant signalling occurring from EGFR results in its conversion into an oncoprotein, and the consequent malfunction of cellular signalling networks leads to the development of cancers and other proliferative diseases. EGFR and its ligands are involved in over 70% of all cancers [[4, 6], and ].
Hidaki, et.al. in the early 1980's discovered the first protein-kinase inhibitors, and established the principle of changing chemical structure to elicit different kinase inhibition specificity . Drug development has followed the lead of the academic community in developing novel inhibitory compounds at points along these disease-related pathways. The protein kinase target class is now the second largest group of drug targets behind G-protein-coupled-receptors . Kinases of the Tyrosine and Serine/Threonine family have been targeted successfully by small-molecule inhibitors and monoclonal antibodies, with many undergoing human clinical trials or successfully launched as therapeutic entities [9, 10, 11, 12, 13].
Acquired resistance to kinase-targeted anticancer therapy has been documented, and most extensively studied with imatinib (Gleevec™), an inhibitor of the aberrant BCR-ABL kinase, in chronic myelogenous leukemia . Resistance has also occurred in EGFR-targeted inhibitor therapy using gefitinib (Iressa™) and erlotinib (Tarceva™). Mutations occurring in the catalytic domain of the receptor have been implicated in this resistance, but cannot account for all resistance seen to these small molecule inhibitors, indicating other mechanisms are involved in the resistance seen to date [15, 16]. Therefore, multiple strategies will be necessary to overcome the observed resistance to these new molecularly targeted therapies, as well as methods to predict their efficacy.
Most kinase inhibitors target the ATP-binding site common to all kinases, and can bind multiple kinases . This generates an inability to predict compound specificity for a particular kinase, and the subsequent need to analyze large numbers of kinases through a screening or profiling approach. Data from these in vitro assays allow the researcher to predict clinical uses for inhibitors and possible offsite target effects. Studies using purified kinase and substrate are dependent on ATP concentration used, and the apparent Km for ATP can differ between kinases. This can lead to problems in the development of small molecule inhibitors based on competition at the ATP-binding site of a kinase, as the ATP concentration in vivo may differ greatly from that used in vitro. In addition, kinase activity studies in a purified setting may use domains of proteins and peptide substrates, which can lead to erroneous interpretation of the true nature of kinase activity and/or inhibition. The in vivo studies using Western blot analysis also can be difficult to interpret due to the need to use a protein preparation from a cellular lysate, and inherent variability when using antibodies for Western blot analysis. Small changes in any step of the protocol could lead to differences in interpretation of the results. For these reasons, and the need for strategies to prevent or overcome resistance formation in malignancies, we have used an in vitro and functional cellular assay approach to study the EGFR/AP-1 signal transduction pathway. AP-1 activation through EGFR was assessed using a β-lactamase reporter gene assay, and served as a model for inhibition of pathway components on a functional cellular level. Kinase profiling using full length EGFR and peptide substrates was used in parallel for confirmation and specificity of inhibition. The knockdown of qualified targets in the EGFR/AP-1 pathway was further studied by immunocytochemistry, allowing for assessment on a cellular protein level. Following qualification of targets in this cancer-related pathway, a functional cellular assay was used to analyze the potential therapeutic benefit of combining RNAi and kinase inhibitors against EGFR and MEK-1 in the AP-1 activation pathway of a human cervical cancer cell line.
AP-1-bla ME180 CellSensor™cell line
An AP-1 response element (TGACTAA, 7X) was inserted into the MCS of the β-lactamase lentiviral reporter vector using Gateway® technology according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). The virus created from this vector was used to transduce ME180 cells  according to manufacturer's instructions (Invitrogen, Carlsbad, CA). Following a one day incubation, the cells were split and placed in media with Blasticidin (5 ug/ml) for 3–4 days. Cells were then washed with phosphate buffered saline (PBS), refed, and further selected for seven to ten days in media containing Blasticidin. Flow cytometry was used to select for β-lactamase expressing cells according to previously published protocols .
EGF ligand stimulation of AP-1-bla ME180
AP-1-bla ME180 cells were plated in 96 well plates at a density of 20,000–25,000 cells/well in DMEM with 1 mM sodium pyruvate, 25 mM HEPES, 0.1 mM non-essential amino acids (Invitrogen Gibco, Grand Island, NY) plus 1% fetal bovine serum (FBS) and incubated overnight at 37°C with 5% CO2. The following day rhEGF (Calbiochem, San Diego, CA) was diluted in media with 1% FBS at desired concentrations, and cells incubated at 37°C with 5% CO2 for 5 hours. EGF treatment was performed at n = 8.
β-lactamase reporter analysis
β-lactamase reporter gene expression was determined using the LiveBLAzer™ FRET B/G assay kit (Invitrogen Drug Discovery, Madison, WI) according to manufacturer's instructions.
Inhibitor treatment of AP-1-bla ME180
Small molecule inhibitors (Calbiochem, San Diego, CA) were diluted in cell culture media at the desired concentration and preincubated with the cells for 30 minutes at 37°C with 5% CO2. EGF ligand stimulation was performed as indicated above followed by β-lactamase reporter analysis. DMSO was used as the negative control, per its use as the compound reconstitution medium. Final DMSO concentration in the medium was 0.05% for both compounds and negative controls.
RNAi design and transfection of AP-1-bla ME180
dsRNAi Stealth™ oligos were designed against EGFR. The following sequences were used for the oligos:
EGFR: Sense 5' UUA GAU AAG ACU GCU AAG GCA UAG G 3'
Anti-Sense 5' CCU AUG CCU UAG CAG UCU UAU CUA A 3'
AP-1-bla ME180 cells were plated at a concentration of 10,000 cells/well in a 96 well plate and incubated overnight at 37°C with 5% CO2. AP-1-bla ME180 cells were transfected with 50 nM dsRNAi Stealth™ oligos and 2 μg/ml Lipofectamine 2000 according to manufacturer's suggestions (Invitrogen, Carlsbad, CA). mRNA was extracted from transfected cells 24 hours post transfection and EGFR expression levels determined by RT-qPCR using light upon extension (LUX™) primer sets (Invitrogen, Carlsbad, CA) for the target of interest and cyclophilin as a normalization control. Percent knockdown of the targeted message was determined as a ratio of target versus cyclophilin control.
Functional cellular analysis of the effect of RNAi knockdown was studied using the AP-1-bla ME180 CellSensor. The cells were transfected with ds RNAi oligos as above and incubated for various lengths of time prior to EGF stimulation for 5 hours and subsequent quantitation of β-lactamase using the LiveBLAzer FRET B/G assay kit (Invitrogen).
Immunocytochemistry for cellular protein knockdown and phosphorylation status of EGFR
AP-1-bla ME180 cells were analyzed for EGFR knockdown at a cellular protein level following RNAi treatment. Cells were transfected with dsRNAi oligos as in the functional cellular studies above and incubated for ~60 hrs. The cells were then fixed with 4% Paraformaldehyde for 30 minutes at 22°C, followed by permeabilization in PBS + 0.25% Triton X-100 for 5 mins at 22°C. Blocking of antigen binding sites was performed with PBS + 2% FBS for 2 hours at 22°C with rocking. Cells were washed twice with PBS + 2% FBS, then incubated with rabbit anti-phospho-EGFR (Tyr1086) or mouse anti-EGFR (31G7) primary antibody (Invitrogen Zymed, San Francisco, CA) at 1:100 concentration and 1:25 concentration respectively in PBS + 1% FBS for 1 hour at 22°C with rocking. Following primary antibody incubation, the cells were washed twice with PBS + 1% FBS at 22°C with rocking. The secondary antibodies goat anti-rabbit IgG (H+L) Alexa Fluor® 488 and goat anti-mouse IgG (H+L) Alexa Fluor® 594 (Invitrogen Molecular Probes, Eugene, OR) were used at 1 μg/mL concentration in PBS + 1% FBS, and incubated at 22°C with rocking. Cells were washed three times with PBS + 1% FBS followed by addition of PBS + 10% glycerol for assay and storage. A Zeiss Axiovert 25CFL microscope with a FITC filter set (excitation D480/30X, emission D535/40 M, Chroma Technology Corporation, Rockingham VT) for Alexa 488, and a Texas Red filter set (excitation D560/40X, emission D630/60 M, Chroma Technology Corporation, Rockingham VT) for Alexa 594 was used for imaging of cells. Quantitation of knockdown was done using a Tecan Safire® instrument using excitation wavelength of 485/7.5 nm and emission wavelength of 510/7.5 nm for the Alexa 488 labelled secondary antibody and excitation wavelength of 590/7.5 nm and emission wavelength of 615/7.5 nm for the Alexa 594 labelled secondary antibody.
Immunocytochemistry for EGFR phosphorylation following kinase inhibitor treatment
AP-1-bla ME180 cells were treated with kinase inhibitors as outlined above. Immunocytochemistry using appropriate phospho-specific antibodies was performed as in the RNAi experiments to analyze EGFR autophosphorylation.
Combining RNAi and kinase inhibitors on the AP-1-bla ME180 CellSensor
AP-1-bla ME180 cells were treated with 50 nM RNAi against EGFR as in experiments outlined above. Only one-half of a 96 well plate was treated, while the other half was mock treated with Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) alone. At 60 hours the cells were treated with a dilution series of AG1478 or U0126 as outlined above. Cells were then stimulated with EGF and the resulting β-lactamase readout was analyzed using GraphPad Prism® to determine IC50 values. Controls for RNAi transfection (Med GC; 40–50% GC nucleotide content), EGFR knockdown (EGFR RNAi), and β-Lactamase knockdown (β-Lac) were used to set standards for transfection, single treatment, and maximal effects respectively.
Kinase profiling of compounds used in functional cellular assays
Small molecule compounds used in the cellular assays were profiled in the SelectScreen™ Kinase Profiling Service (Invitrogen Drug Discovery Solutions, Madison, WI). This service utilizes the Z'-Lyte technology assay platform  where the biochemical assays were performed at a final concentration of 1 μM compound in 0.1% DMSO and an ATP concentration of Km, app for the EGFR protein. The assays were analyzed on a Tecan Safire2® detection instrument. The percent inhibition values were calculated using XL fit 4.0 by ID-BS.
ME180 EGFR/AP-1 signal transduction pathway is responsive to EGF stimulation
EGFR/AP-1 signal transduction can be inhibited at multiple points in the pathway
The commercial availability of known small molecule kinase inhibitors allowed for the study of their potency in our cellular system. Prior to analyzing efficacy in a functional cellular assay, we tested their inhibition of EGFR in a biochemical assay using Z'-LYTE, a FRET-based platform.
Cellular phosphorylation of EGFR on a cellular protein level matches reporter gene results
RNAi qualifies EGFR as a target for AP-1 pathway inhibition
Combining EGFR RNAi with AG1478 or U0126 increases the potency of the kinase inhibitors against AP-1 activation
Molecular targeted therapies against signalling pathway components involved in cancer have arrived in the clinic, and drug discovery efforts are increasing in this evolving area [[3, 9, 11, 13, 24], and ]. The growth of screening for small molecule compounds which act as kinase inhibitors has led to their becoming the second most targeted group of druggable entities after G-protein-coupled receptors .
Modulation of kinase activity can be accomplished by strategies other than inhibition of phosphorylation activity through the blocking of ATP binding. Such methods include disruption of protein-protein interactions and the knockdown or downregulation of kinase gene expression by antisense or RNA interference approaches [16, 24]. The need for multiple approaches for therapies targeting kinases can be seen in the reports of resistance towards the recently launched kinase inhibitors gefitinib (Iressa™) and imatinib (Gleevec™), which inhibit the EGFR and Bcr-Abl kinase respectively [14, 27]. Strategies implemented for overcoming or preventing this resistance have included chemical modifications of the inhibitor compound using a rational drug design strategy to increase the potency against the targeted kinase. The ATP binding site of kinases has proven to be a "hot spot" for kinase mutations and includes a "gatekeeper" region shown to be difficult to block with inhibitors and their new more potent derivatives. Recent work has been published on inhibitors targeting the substrate binding site of the Bcr-Abl kinase , which seem to inhibit wild-type and all imatinib-resistant kinase domain mutations, including the "gatekeeper" mutation . These approaches have proven successful for imatinib and will undoubtedly work in the near term for other small molecule inhibitors, but experience suggests the possibility of further mutations leading to increased resistance [[15, 16], and ].
Blockade of one kinase alone might not be sufficient to achieve needed pathway inhibition, and so targeting of multiple kinases could be more promising in terms of efficacy and prevention of resistance. This type of combinatorial therapeutic approach has become the standard for HIV treatment to maximize potency, minimize toxicity, and diminish the risk for resistance development . Targeted compounds could be used together or in combination with toxic agents such as chemotherapy or ionizing radiation, as well as with other novel agents. This approach has proven successful in recent studies using chemotherapeutic agents or ionizing radiation in combination with kinase inhibitors [[31, 32, 33] and ]. However, given the rapidly growing number of inhibitory agents and an exponential number of possible combinations, it will not be possible to test all such groupings in a clinical trial setting [16, 24]. The need for predictive preclinical models allowing for the choice of which studies to advance through the drug discovery process led to the experiments outlined in this paper with RNAi and the kinase inhibitors U0126 and AG1478.
First, in order to establish a functional cellular model for investigation of the EGFR/AP-1 pathway we built a stable cell line responsive to EGF stimulation. Kinase inhibitors and RNAi analysis established components that were involved in the functional response of the EGFR/AP-1 pathway that could be inhibited by molecularly targeted agents.
Biochemical analysis of EGFR inhibition using the same small molecule inhibitors further supported our cellular results and demonstrated the importance of complimentary approaches when analyzing signal transduction pathways. Inhibitors such as U0126 inhibit the functional response of the AP-1 pathway as shown in the cellular reporter readout, but work at a level independent of EGFR inhibition determined by in vitro assays.
RNAi analysis and immunocytochemistry further demonstrated the essential role EGFR plays at the beginning of the AP-1 activation cascade. Showing that inhibition or knockdown of the receptor leads to the functional results observed in the cellular assay model, led us to analyze combinatorial effects of using RNAi and kinase inhibitors in tandem. When the targeted agents were used together, an increased potency of the inhibitors was observed. This finding demonstrates the use of a cellular reporter system to predict the cocktail effects of a growing number of targeted agents against cell signalling components involved in cancer.
Our results demonstrate the essential role for EGFR in AP-1 activation when analyzed using an ME180 cervical cancer cell line. Confirmation of "druggable" targets was shown using the parallel approach of known kinase inhibitors and RNA interference. An immunocytochemistry approach using phospho- and pan-specific EGFR antibodies strengthened the argument for the use of the AP-1-bla ME180 cell line as a viable model for the analysis of a combinatorial targeted agent approach to cancer-related pathways. Results obtained combining RNAi toward EGFR and small molecule inhibitors of MEK-1 and EGFR indicate a beneficial effect on EGFR/AP-1 pathway inhibition. This cellular model approach could lead to further studies combining kinase inhibitors with other targeted agents, and suggests possible implications for screening of compound libraries to uncover novel pathway inhibitors.
Thanks to Greg Parker for figure preparation. Andy Kopp, Tom Zielinski, Jessica Honer, Heidi Braun, Jeff Beauchaine, Dave Lasky, Leah Aston, and Jenny Fronczak at Invitrogen-Madison, and Kristin Wiederholt and Mason Brooks at Invitrogen-Carlsbad, for research support. Also thanks to Brian Pollok, Kurt Vogel, Tammy Turek-Etienne, and Peter Welch for critical reading of the manuscript.
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