Recent Studies Investigate Pathways Mediating Drug Resistance in Cancer Cells
Since activation of a number of different signaling pathways may promote the cancer phenotype, screening tools that simultaneously assess the phosphorylation state of multiple proteins can be extremely useful. R&D Systems Proteome Profiler Human Phospho-RTK Antibody Array (Catalog # ARY001B), Phospho-MAPK Array (Catalog # ARY002B), and Phospho-Kinase Array (Catalog # ARY003B) allow the phosphorylation of multiple RTKs, or intracellular kinases to be assessed in a single sample of cell lysate (Figure 1). Several recent high profile studies have used the Proteome Profiler Arrays to provide insight into the mechanisms by which some tumors become resistant to specific anticancer drugs.
Receptor tyrosine kinases (RTKs) are transmembrane proteins that initiate signaling pathways required for normal cellular processes such as growth and development. Mutations in RTKs can cause constitutive activation of downstream signaling pathways implicated in the pathogenesis of different forms of cancer. Therefore, monitoring RTK phosphorylation in cancer cells can be useful for the development of treatments.
Scientists at the Dana Farber Cancer Institute and Harvard Medical School utilized the Phospho-RTK Antibody Array to identify phosphorylated RTKs in brain tumor glioblastoma multiforme (GBM) in an effort to determine why therapies targeting single RTKs in these tumors have failed.1 They found that not one, but several RTKs are activated in GBM cell lines (Figure 2A) and in primary GBMs, as well as in other solid tumor cell types such as lung and pancreatic adenocarcinomas. Most phosphorylated RTKs were also activated under conditions of serum starvation and in tumor cell xenografts, indicating that RTK phosphorylation detected in cultured cells was likely not induced by factors present in the cell culture media (Figure 2B). Significantly, this study led to the finding that targeting multiple RTKs simultaneously could suppress the cancer phenotype in glioma cells far more significantly than treatments targeting single RTKs alone.
Other researchers at these institutions used the Phospho-RTK Antibody Array to investigate the mechanisms by which non-small cell lung cancers (NSCLCs) containing EGF R-activating mutations develop resistance to gefitinib, an EGF R kinase inhibitor.2 Engelman et al. showed that ErbB3 and Met (HGF R) are phosphorylated in drug-resistant cells, while phosphorylation of both RTKs is markedly reduced in gefitinib-treated, non-resistant cells (Figure 3). Further research demonstrated that gefitinib resistance in NSCLCs is mediated by MET amplification and the subsequent activation of ErbB3 signaling pathways. These results suggest that Met kinase inhibitors may be useful in combination with other therapies for treating gefitinib-resistant lung cancer tumors with EGF R-activating mutations.2
||Figure 1. R&D Systems Proteome Profiler Phospho-RTK Antibody Array Detects the Phosphorylation State of Multiple Receptors in a Single Sample. Proteome Profiler Antibody Arrays are designed using carefully selected capture antibodies that are spotted in duplicate on nitrocellulose membranes. When these membranes are incubated with experimental samples, capture antibodies printed on the membranes bind to their specific target proteins (Step 1). Capture antibodies on the Phospho-RTK array bind the extracellular domain of both phosphorylated and unphosphorylated RTKs, and an HRP-conjugated pan anti-Phospho-Tyrosine antibody is subsequently used to specifically detect phosphorylated RTKs (Step 2). Proteins are visualized using chemiluminescent detection reagents which produce a signal that is proportional to the amount of analyte bound. The use of Proteome Profiler antibody arrays requires no specialized equipment and eliminates the need to perform multiple immunoprecipitation/Western blot experiments.
||Figure 2. Detection of Activated Receptor Tyrosine Kinases (RTKs) in Glioma Cell Lines using the Proteome Profiler Human Phospho-RTK Antibody Array. A. Stommel et al. assessed the phosphorylation of 42 different receptor tyrosine kinases (RTKs) in whole cell extracts prepared from the indicated glioma cell lines using the Proteome Profiler Human Phospho-RTK Array Kit (Catalog # ARY001B). B. RTK phosphorylation was assessed in whole cell extracts from cultured SF767 or LN340 glioma cells (bottom) or in xenograft tumors derived from these cell lines (top) using the Human Phospho-RTK Array. Activated RTKs are indicated at the bottom. [Figure from Stommel, J.A. et al. (2007) Science 318:287. Reprinted with permission from AAAS.]
||Figure 3. Detection of Phosphorylated RTKs in Gefitinib-Resistant NSCLCs. RTK phosphorylation in control (HCC827) and gefitinib-resistant (HCC827 GR5) non-small cell lung cancers (NSCLCs), in the absence (left) or presence (right) of gefitinib treatment, was determined by Engelman et al. using the Proteome Profiler Human Phospho-RTK Array Kit (Catalog # ARY001B). Spots representing the relative levels of phosphorylated EGF R, ErbB3 and Met (HGF R) are indicated by the arrows. [Figure from Engelman, J.A. et al. (2007) Science 316:1039. Reprinted with permission from AAAS.]
A similar approach was used by Eckstein et al. at the Stiftung Center for Advanced European Studies and Research in Germany to identify mechanisms mediating cisplatin resistance in breast cancer.3 Lysates from engineered cisplatin-resistant MCF-7 cells were incubated with the Phospho-RTK Array and drug resistance was found to be dependent on mechanisms involving EGFR and ErbB2 phosphorylation. Intracellular signaling molecules activated as a result of EGF R or ErbB2 phosphorylation were subsequently assessed using R&D Systems Proteome Profiler Human Phospho-MAPK Antibody Array Kit (Catalog # ARY002B). This analysis revealed that ERK1 (T202/Y204) and Akt1 (S473) are highly phosphorylated in cisplatin-resistant MCF-7 cells. Further investigation demonstrated that the development of cisplatin resistance correlates with increased expression of the EGF R/ErbB ligand, Amphiregulin, which activates PI 3-K/Akt signaling pathways. These findings may open new avenues of research in the pursuit of alternative treatment options for cisplatin-resistant breast cancers.
As these recent studies show, antibody arrays simplify the screening process required to identify proteins or pathways involved in establishing specific cellular phenotypes. By monitoring changes in protein levels and/or post-translational modifications of several proteins simultaneously, molecules of interest can be rapidly identified and targeted for further study. In addition to the Phospho-RTK and Phospho-MAPK Array Kits, arrays are also available for profiling other cancer-related processes such as angiogenesis and apoptosis. Please visit our Proteome Profiler page for more information.
- Stommel, J.A. et al. (2007) Science 318:287.
- Engelman, J.A. et al. (2007) Science 316:1039.
- Eckstein, N. et al. (2008) J. Biol. Chem. 283:739.
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