Abstract 3502: Proximity ligation assays reveal protein-protein interactions associated with oncogenic signaling and drug sensitivity.
Authors: Smith Matthew A. , Hall Richard , Haake Scott , Li Jiannong , Haura Eric B. 
Published: August 2014

Abstract
Predictive biomarkers are critical to optimize the use of receptor tyrosine kinase (RTK) inhibitors for cancer therapy. Because multi-protein complexes are critical to maintain oncogenic signaling, we hypothesized that assays that measure protein-protein interactions could identify patients with RTK activity and predict RTK inhibitor response. We developed in situ proximity ligation assays (PLA) to assess the degree of active EGFR signaling in both non-small cell lung cancer (NSCLC) cell lines and formalin-fixed paraffin-embedded (FFPE) patient specimens with the goal of establishing PLA assays as biomarkers of erlotinib sensitivity.
Using in situ PLA, we evaluated EGFR association with its signaling adaptors, GRB2 and SHC1. By confocal microscopy we observed increased PLA signals in EGFR mutant NSCLC cell lines (PC9, H1650, HCC4006, HCC827) relative to EGFR wildtype (H23, H1299, H322, A549), indicative of constitutively active signaling complexes in mutant cell lines. PLA signal intensity was independent of EGFR, GRB2 and SHC1 protein expression levels, but did correlate with co-IP and pEGFR immunoblot results. Erlotinib dose-dependently reduced PLA signals in PC9 cells. H1650 xenografts exhibited high PLA signals that were reduced by erlotinib, corresponding with tumor shrinkage. Conversely, low PLA signals were observed in xenografts from EGFR wildtype cell lines (H322, H157). In human xenografts, PLA signals were significantly higher in EGFR mutant xenografts and reduced by erlotinib. In human FFPE specimens, PLA signals were absent in normal non-epithelial tissues and low in epithelial portions. FFPE lung tumor specimens (N = 221) revealed a wide range of PLA signal intensities (scored as 0+ to 3+), which localized to cytokeratin-positive regions. High PLA signals were found in approximately 25% of specimens across multiple histological classifications, including adenocarcinoma, squamous and large cell carcinoma. Furthermore, specimens from metastatic brain lesions were highly enriched for high PLA signal (p&lt;.0001). Automated Quantitative Analysis (AQUA) of EGFR expression revealed multiple examples of EGFR expression-PLA signal discordance - we observed both high PLA signal (&gt;2+) in specimens with relatively low EGFR protein expression and low PLA signal (&lt;1+) in specimens with relatively high EGFR protein expression. Additionally, PLA signal was observed in both EGFR mutant and wildtype specimens, indicating that PLA provides additional information beyond immunohistochemistry and mutational analysis. Our results establish PLA as a powerful technique to detect signaling activity by monitoring protein complexes in clinical specimens. PLA-based approaches will likely have utility in biomarker studies for other receptor tyrosine kinase systems and their respective inhibitors.
Citation Format: Matthew A. Smith, Richard Hall, Scott Haake, Jiannong Li, Eric B. Haura. Proximity ligation assays reveal protein-protein interactions associated with oncogenic signaling and drug sensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3502. doi:10.1158/1538-7445.AM2013-3502