Profiling the effects of MEK inhibition on kinase phosphorylation and protease secretion using antibody arrays

Greta Wegner, Erin Eleria, Wen-Chieh Liao, Dave Finkel, and Amy James

ABSTRACT

The frequent deregulation of ERK signaling in tumors has led to the development of MEK inhibitors, which have proven to be clinically relevant to malignancies harboring BRAF mutations. Because proliferation and invasion are intertwined aspects of metastasis, antibody arrays were used to measure the effect of multiple MEK inhibitors on 43 kinases and 34 proteases. Treatment of SK-BR-3 cells with MEK inhibitors resulted in decreased ERK1/2 (T202/Y204, T185/Y187) and STAT3 (S727) phosphorylation. Increased p38 phosphorylation was observed following treatment with multiple inhibitors, but a decrease was observed in response to PD 0325901. These differential effects demonstrate the utility of arrays for parsing out crosstalk between signaling pathways. To assess the effects of inhibitors across multiple cancer cell lines, changes in ERK phosphorylation were examined using ELISA. High ERK activation was observed in both RTK-driven and RAF/RAS mutation-containing cells, but did not correlate with sensitivity to MEK inhibition and was instead determined to be inhibitor dependent. Protease profiles were also impacted by MEK inhibition, with reduced MMP secretion detected by antibody arrays and ELISAs in inhibitor treated cells.

MATERIALS & METHODS

Proteome Profiler^® Array Assay Principle

RESULTS

Induction and inhibition of kinase phosphorylation in breast cancer cells

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Figure 2. The SK-BR-3 human breast cancer cell line was untreated or treated with 10 μM PD 0325901, 20 μM PD 98059, 10 μM SL 327, or 10 μM U0126 for 2 hours. Images of Proteome Profiler Human Phospho-Kinase membrane arrays and the corresponding densitometry histogram profiles are shown.

MAP Kinase inhibition in cancer cell lines by MEK inhibitors

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Figure 3. Eight cell lines were untreated or treated with 10 μM PD 0325901, 20 μM PD 98059, 10 μM SL 327, or 10 μM U0126 for 2 hours, followed by 20 ng/mL EGF or 100 nM PMA treatment. Phosphorylated ERK1 (T202/Y204) and ERK2 (T185/Y187) were quantified with DuoSet® IC ELISA.

Dose-dependent inhibition of MAP Kinase phosphorylation

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Figure 4. The COLO 205 human colorectal adenocarcinoma cell line and the SK-Mel-28 human malignant melanoma cell line were treated with SL 327 and PD 98059 over a range of concentrations to determine the dose response of ERK1/2 phosphorylation. Cells were treated with inhibitors for 2 hours. Phosphorylated ERK1 (T202/Y204) and ERK2 (T185/Y187) were quantified with DuoSet IC ELISA.

Induction and inhibition of protease secretion by breast cancer and colorectal adenocarcinoma cells

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Figure 5. (A, B) The SK-BR-3 human breast cancer cell line was either untreated or treated with 20 ng/mL EGF without and with a variety of MEK inhibitors for 18 hours. Cell supernatants were harvested and assayed for protease secretion using the Proteome Profiler Human Protease Array or Quantikine® ELISA. (A) Images of the Proteome Profiler Human Protease Arrays incubated with untreated or treated cell culture supernatants. (B) MMP-9 secretion measured using the Proteome Profiler Arrays (pixel density) or Quantikine ELISA show comparable results. (C) COLO 205 cells were either untreated or treated with 100 nM PMA without or with 20 μM PD 98059 or 10 μM U0126 for 24 hours. Histogram profiles of array pixel densities and concentrations of MMP-7 determined by Quantikine ELISA are comparable.

CONCLUSIONS

Multianalyte arrays used in coordination with single-analyte ELISAs are an economical and convenient approach to quickly screen the effects of inhibitors across large sample sets. This method enables the optimization of treatment conditions across multiple cell lines and concentration ranges in a single experiment, which is more time-effective than multiple rounds of Western blotting. Clinically, resistance to MEK inhibitors has been observed in BRAF V600 melanomas with RTK- and RAS-driven mutations indicating the need for combination therapies that target multiple signaling pathways. As inhibitors are screened for efficacy and off-target effects, both individually and in combination, arrays and ELISAs may be used as tools to increase throughput.

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

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