Using Antibody Arrays for Assessing the Integration of Multiple Signaling Pathways and Off-Target Inhibitors

Greta Wegner, Erin Eleria, Jackie Ernst, & Amy James

THE PROBLEM: Time-consuming Western blots

• Assessing phosphorylation changes in multiple interrelated pathways
• Performing pharmacological screens in a timely manner
• Identifying off-target pharmacological effects

THE SOLUTION: Proteome Profiler™ Phospho-Antibody Arrays

• Easier to perform than a Western blot
• Uncover new relationships between signaling pathways
• Identify off-target pharmacological responses that can confound results and waste time
• Collect data from the array membrane in the same manner as an immunoblot

INTRODUCTION

Kinases are enzymes that regulate protein activity by transferring phosphate groups to serine, threonine, or tyrosine residues in a wide range of substrates. They impact most cellular activities, and much effort is placed on studying how they affect such processes as cell proliferation, survival, differentiation, and motility. Tight regulation of kinase activity is crucial for maintaining cellular homeostasis, and aberrant kinase activity can directly contribute to cellular transformation. Consequently, kinases are often targets for drug development.

Common experiments associated with drug discovery include screening pharmacological candidates for efficacy or off-target activity. Also of interest to researchers is assessing the interrelationship of multiple signaling pathways. Traditional single analyte assays, such as Western blot, do not have the throughput necessary for these types of experiments.

MATERIALS & METHODS

Proteome Profiler Array Assay Principle

Arrays are composed of capture and control antibodies spotted in duplicate on nitrocellulose membranes. Cell lysates were diluted, mixed with a cocktail of biotinylated detection antibodies, and incubated overnight with the Proteome Profiler Phospho-Kinase Array (Catalog # ARY003B). Streptavidin-HRP and chemiluminescent detection reagents were applied to the membrane, and the signal produced at each capture spot corresponded to the amount of phosphorylated protein bound. The data was collected using x-ray film and analyzed using image analysis software.

Additional Kits and Reagents

Recombinant Human EGF was from R&D Systems (Catalog # 236-EG). Inhibitors were from Tocris, an R&D Systems Company: AS 605240 (Catalog # 3578), LY 294002 (Catalog # 1130), PI 103 (Catalog # 2930), PD 0325901 (Catalog # 4192), PD 98059 (Catalog # 1213), SL 327 (Catalog # 1969), U0126 (Catalog # 1144). ELISA development kits were from R&D Systems: Phospho-CREB (S133) DuoSet® IC ELISA (Catalog # DYC2510), Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) DuoSet IC ELISA (Catalog # DYC1018B). Antibodies were from R&D Systems: Anti-Human/Mouse/Rat Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) Affinity-Purified Polyclonal Antibody (Catalog # AF1018), Anti-Human/Mouse/Rat Total ERK1/ERK2 Monoclonal Antibody (Catalog # MAB15761).

RESULTS

Measuring Efficacy and Off-Target Responses to PI 3-Kinase Inhibitors

Protein phosphorylation profiles of A549 human lung adenocarcinoma cells were obtained to assess their response to EGF and several PI 3-Kinase Inhibitors. The data revealed a range of signaling molecules that were phosphorylated in response to EGF treatment, some of which are highlighted in array images (Figure 1A). Histogram profiles were generated for select analytes to look more closely at the effects of inhibitors (Figure 1B). The phosphorylation of CREB appeared to be unaffected, while the phosphorylation of the PI 3-Kinase effector Akt was suppressed by all inhibitors. In this context, the commonly used inhibitor LY 294002 was least effective. We also found that EGF R phosphorylation increased in response to two of the inhibitors. This type of observation could prove important to data interpretation or when assessing mechanisms of resistance, and might be missed if single analyte assays were used.

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Figure 1. Induction and Inhibition of Kinase Phosphorylation in Lung Adenocarcinoma Cells. The A549 human lung adenocarcinoma cell line was untreated, treated with 100 ng/mL EGF alone for 15 minutes, or treated with EGF following a 3 hour pretreatment with 5 μM of the PI 3-Kinase inhibitors AS 605240, LY 294002, or PI-103. Images of the Proteome Profiler Human Phospho-Kinase membrane array (A) and corresponding histogram profiles of select analytes are shown (B). CREB phosphorylation was unaffected, while LY 294002 was the least effective inhibitor of Akt phosphorylation. An increase in the phosphorylation of EGF R was observed in the presence the inhibitors AS 60524 or PI 103.

Measuring Efficacy and Off-Target Responses to MEK Inhibitors

The T47D human ductal breast epithelial cell line was used to generate an EGF-induced phosphorylation profile and to assess the effects of several MEK inhibitors. Select analytes are highlighted in array images (Figure 2A). A histogram profile was generated showing the effects on ERK1/2, Akt, STAT3, and c-Jun phosphorylation (Figure 2B). The results indicated varied levels of efficacy and possible cross-talk between traditional pathways. For instance, PD 0325901 was the most potent inhibitor of ERK1/2 phosphorylation, while T47D cells displayed resistance to the MEK inhibitors PD98059, U0126, and SL372. In addition, we noted potential off-target inhibitor effects. These included an increase in phospho-Akt in the presence of 3 of the 4 inhibitors, and decreased STAT3 and c-Jun phosphorylation in the presence of PD 98089. Again the array revealed inhibitor-related changes in phosphorylation that could be biologically relevant but may have been missed using single analyte assays.

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Figure 2. Induction and Inhibition of Kinase Phosphorylation in Breast Cancer Cells. The T47D human ductal breast epithelial cell line was untreated, treated with 100 ng/mL EGF for 15 minutes, or EGF following a 2 hour pretreatment with the MEK inhibitors 10 μM PD 0325901, 20 μM PD 98059, 10 μM SL 327, or 10 μM U0126. Images of Proteome Profiler Human Phospho-Kinase membrane arrays (A) and the corresponding histogram profiles are shown for select analytes (B). PD 0325901 was the most effective suppressor of ERK1/2, while PD 98089 resulted in decreased phosphorylation of STAT3 and c-Jun. Increases in Akt phosphorylation were also observed in response to certain MEK inhibitors.

Comparing Array Results to Western Blot and ELISA Data

The results obtained from array experiments can provide direction for the design of hypothesis-driven single analyte assays used to support or expand on array data. For instance, in TD47 cells the efficacy of MEK inhibitors on phospho-ERK1/2 was tested with similar results using Western blot (Figure 3A), or using phospho-ELISA or phospho-array (Figure 3B). Phospho-CREB, an ERK1/2 effector, also shows a similar phosphorylation pattern using phospho-array or phospho-ELISA (Figure 3C). In all instances, PD 0325901 was the most effective inhibitor.

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Figure 3. Comparing Array Data to Single Analyte ELISA and Western Blot in a MEK Inhibitor Screen. The T47D human ductal breast epithelial cell line was untreated, treated with 100 ng/mL EGF for 15 minutes or treated with the MEK inhibitors 10 µM PD 0325901, 20 µM PD 98059, 10 µM SL 327, or 10 µM U0126 for 2 hours, followed by treatment with EGF. Phosphorylation of ERK1/2 was measured using Western blot (A). Similar results are shown in histogram profiles comparing the array data to ELISA for Phospho-ERK1/2 (B) or Phospho-CREB (C).

Expanding on these results, concentration-response experiments were performed using PD 0325901. Measuring EGF-induced phosphorylation of ERK1/2 using Western blot (Figure 4A), or phospho-ELISA or phospho-Array all gave comparable results (Figure 4B).

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Figure 4. MEK Inhibitor PD 0325901 Concentration-Response Measurements. The T47D human ductal breast epithelial cell line was treated with different concentrations of PD 0325901 for 2 hours, followed by treatment with100 ng/mL EGF for 5 minutes. (A) ERK1/2 phosphorylation was measured using Western blot. (B) Histogram profiles obtained from the array were comparable to those obtained by Phospho-ELISA and Western blot.

CONCLUSIONS

The Phospho-Kinase Array is an economical and time-saving alternative to traditional methods, such as Western blot, for pharmacological screens to assess efficacy and/or off-target effects. The arrays also provide a much better picture of the overall signaling response by revealing phosphorylation changes in multiple interrelated pathways. This information could prove important for data interpretation or when assessing compensation mechanisms associated with tumor resistance. Array assays are easy to perform. In addition, the data is collected from the array membrane using chemiluminescence in the same manner as an immunoblot and no additional specialized equipment is necessary. The arrays are sufficiently sensitive to measure changes in phosphorylation caused by both ligand and inhibitor treatment, and in follow-up experiments results are comparable to both Western blot and ELISA.

For research use only. Not for use in diagnostic procedures.

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