Combining RNA interference and kinase inhibitors against cell signalling components involved in cancer

Abstract

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.

Conclusion: 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.

Figure 1

EGF mediated activation in ME180AP-1 CellSensor. EGF effectively stimulated the EGFR/AP-1 pathway in ME180 cervical cancer cells. Cells expressing β-lactamase were detected using a fluorescent plate reader (Panel A) or under fluorescent microscopy (Panel B). β-lactamase was measured using Invitrogen's LiveBLAzer™ FRET B/G assay. Ratios represent the mean of (± SEM) eight independent data points (n = 8).

Figure 2

Biochemical assay results with kinase inhibitors. Percent inhibition values determined by using the Z'-LYTE technology platform (Invitrogen), and single point concentration testing (1 μM) with two independent data points (n = 2). Small molecule compounds CL387785, PD153035, and AG1478 show strong inhibition of the EGFR, while others tested exhibit none.

Figure 3

Kinase inhibitors block activation at multiple points in EGFR/AP-1 pathway. Inhibitors decrease the response of the AP-1-bla ME180 CellSensor when stimulated with EGF. β-lactamase was measured using Invitrogen's LiveBLAzer™ assay (Panel A). Percent inhibition is determined in comparison to untreated sample and represents the mean of (± SEM) eight independent data points (n = 8). Photomicrographs of cells were obtained under fluorescent microscopy (Panel B).

Figure 4

Potency of individual inhibitors towards kinases in the AP-1 pathway determined in a dose response manner. Inhibitors were used in a dose response manner on the AP-1-bla ME180 CellSensor, and β-lactamase measured using Invitrogen's liveBLAzer™ FRET B/G assay (Panels A and B). IC₅₀ determinations were performed using GraphPad Prism^® and values shown with images of inhibitors at 0.2 μM concentration (Panel B).

Figure 5

Autophosphorylation of EGFR shows similar inhibition profile as the AP-1-bla ME180 functional cellular assay. Immunocytochemistry with phospho-specific antibodies demonstrates the same inhibition profile as the AP-1 activation assay performed using the ME180 CellSensor. Fluorescence was measured from the Alexa fluor 488 and 594 labelled secondary antibodies used for detection (Panel A). Percent inhibition was determined in comparison to untreated sample and represents the mean of (± SEM) eight independent data points (n = 8). Images were obtained from the Alexa fluor 488 labelled secondary antibody under fluorescent microscopy (Panel B).

Figure 6

Knockdown by RNAi qualifies EGFR involvement in ME180 AP-1 CellSensor pathway. AP-1-bla ME180 cells transfected with RNAi targeting EGFR were analyzed by RT-qPCR (Panel A). EGFR mRNA expression is knocked down ~80% as compared to a non-specific control (Med GC). Results are represented as a ratio using cyclophilin as a control. Cells RNAi treated with oligos specific for EGFR show a reduced level of EGFR protein on the cell surface including a loss of autophosphorylation (Panels B and C). RFU represent measurement of the Alexa fluor 488 and 594 labelled secondary antibodies used for detection of the phospho and pan EGFR antibodies and represent the mean of (± SEM) eight independent data points (n = 8). RNAi treated AP-1-bla ME180 cells also exhibit a knockdown of AP-1 activation when EGF stimulated and analyzed at different timepoints (Panel D). Normalized response represents the amount of AP-1 gene activation present when compared with a non-specific control (Med GC), and represent the mean of (± SEM) eight independent data points (n = 8).

Figure 7

Combining RNAi toward EGFR and the kinase inhibitors U0126 and AG1478 increases the potency of the small molecule compounds. Small molecule kinase inhibitors shown to have inhibitory properties in the AP-1 CellSensor assay were used in tandem with RNAi. RNAi targeting the EGFR was kept constant while U0126 targeting MEK-1 kinase (Panel A) and AG1478 targeting EGFR (Panel C) were used in a dose response manner to examine potency effects of the combined treatment. The shift in IC₅₀ indicates the "cocktail" effect seen with the tandem treatment.