Research ArticleCancer

Annotation of human cancers with EGFR signaling–associated protein complexes using proximity ligation assays

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Science Signaling  13 Jan 2015:
Vol. 8, Issue 359, pp. ra4
DOI: 10.1126/scisignal.2005906

Visualizing Protein Complexes in Clinical Samples

Aberrant activation of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR), plays a role in cancer initiation, progression, and acquired drug resistance. Genetic analysis may not always reveal aberrant activity of receptor tyrosine kinase signaling; thus, detecting active signaling through these kinases in clinical samples should improve prognostication and personalization of therapies. Diverse cancers, for example, those found in the lung, colon, or head and neck, can have aberrant activation of EGFR signaling, and EGFR-targeted therapies are used to treat these diseases. Smith et al. developed a proximity ligation assay (PLA) to detect the interaction between EGFR and the requisite signaling adaptor GRB2 (growth factor receptor–bound protein 2) in common clinical preparations. EGFR:GRB2 PLA recapitulated traditional readouts of active EGFR signaling in cultured cells, in a panel of tumor xenografts in mice derived from primary patient samples, and in samples from three large cohorts of human patients. Moreover, EGFR:GRB2 PLA detected increased EGFR signaling in tumors with wild-type EGFR, which would have been undetectable by genetic analysis, and was predictive of therapeutic response to EGFR inhibitors in both mice and humans. Thus, using PLA to detect signaling-associated protein complexes has the potential to improve current diagnostic measures.


Strategies to measure functional signaling-associated protein complexes have the potential to augment current molecular biomarker assays, such as genotyping and expression profiling, used to annotate diseases. Aberrant activation of epidermal growth factor receptor (EGFR) signaling contributes to diverse cancers. We used a proximity ligation assay (PLA) to detect EGFR in a complex with growth factor receptor–bound protein 2 (GRB2), the major signaling adaptor for EGFR. We used multiple lung cancer cell lines to develop and characterize EGFR:GRB2 PLA and correlated this assay with established biochemical measures of EGFR signaling. In a panel of patient-derived xenografts in mice, the intensity of EGFR:GRB2 PLA correlated with the reduction in tumor size in response to the EGFR inhibitor cetuximab. In tumor biopsies from three cohorts of lung cancer patients, positive EGFR:GRB2 PLA was observed in patients with and without EGFR mutations, and the intensity of EGFR:GRB2 PLA was predictive of overall survival in an EGFR inhibitor–treated cohort. Thus, we established the feasibility of using PLA to measure EGFR signaling–associated protein complexes in patient-based materials, suggesting the potential for similar assays for a broader array of receptor tyrosine kinases and other key signaling molecules.

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