Integrated Systems and Technologies High-Throughput Time-Resolved FRET Reveals Akt / PKB Activation as a Poor Prognostic Marker in Breast Cancer

Dysregulation of the Akt/PKB pathway has been associated with poor prognosis in several human carcinomas. Current approaches to assess Akt activation rely on intensity-basedmethods, which are limited by the subjectivity of manual scoring and poor specificity. Here, we report the development of a novel assay using amplified, timeresolved F€ orster resonance energy transfer (FRET), which is highly specific and sensitive and can be adapted to any protein. Using this approach to analyze primary breast tissue microarrays, we quantified levels of activated pAkt at a spatial resolution that revealed molecular heterogeneity within tumors. High pAkt status assessed by amplified FRET correlated with worse disease-free survival. Our findings support the use of amplified FRET to determine pAkt status in cancer tissues as candidate biomarker for the identification of high-risk patients. Cancer Res; 74(18); 1–13. 2014 AACR. Introduction Dysregulation of Akt/PKB (protein kinase B) signaling is considered to be a hallmark of many human cancers (1). In cancer, Akt plays a central role in cell proliferation, survival, glucose metabolism, genome stability, and neovascularization (2, 3). Akt also contributes to tumor invasion and metastatic spread by induction of epithelial–mesenchymal transition (4). Breast cancer is the most common malignancy diagnosed in women worldwide (1). In breast cancer, Akt activation is correlated with advanced disease, poor prognosis, reduced patient survival, and resistance to radiotherapy (5, 6). Akt is a member of the AGC family of protein serine/threonine kinases and contains an N-terminal pleckstrin homology (PH) domain, which interacts with PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (7). Various mechanisms contribute to activation of the Akt pathway in human tumors, including disruption of PTEN, upregulation of PI3K and mTOR, as well as mutation of Akt itself (8, 9). Stimulation of the EGFR by EGF leads to activation of Akt in a PI3K-dependent manner (1, 10). Because Akt activation is both an early event in tumor progression and also a characteristic of many advanced carcinomas, Akt activation status (as opposed to Akt expression level) may represent a useful prognostic biomarker as well as a predictive biomarker in both adjuvant and metastatic settings (11). The appreciation of functional molecular heterogeneity of tumors has far reaching implications for the development of personalized medicine and improving tumor biopsy methodologies for predictive biomarkers (12). The assessment of molecular heterogeneity at the protein level has not been extensively reported, in part due to a lack of technologies that can accurately perform this task directly on histologic samples. The accurate quantification of Akt activation status as well as its molecular heterogeneity in patient samples is expected to be highly informative. At present, the detection of endogenous oncoproteins in fixed tumor samples faces several important challenges: (i) the accurate quantification of posttranslational modifications such as phosphorylation; (ii) the simultaneous localization of these oncoprotein epitopes in preserved tissue architecture; and (iii) the acquisition and processing of a large quantity of data in an automated, high-throughput manner. IHC is the most readily available method to assess activation of intracellular proteins such as Akt; however, it is limited by the absence of precise quantification and the lack of a standardized scoring system. In addition, it is a "one-site" assay (including fluorophore-based confocal assays) producing results that can only be obtained frommeasuring the intensity of one label at a time, Cell Biophysics Laboratory, Cancer Research UK, London Research Institute, London, United Kingdom. Centre Emotion, Hôpital de la Piti e Salpetriere, Paris, France. Protein Phosphorylation Laboratory, Cancer Research UK, London Research Institute, London, United Kingdom. Experimental Histopathology Laboratory, Cancer Research UK, London Research Institute, London, United Kingdom. Stanford University School of Medicine, Stanford, California. Cancer Division King's College London, London,UnitedKingdom. Cell Biophysics Laboratory,UnidaddeBiofísica (CSIC-UPV/EHU), Sarriena s/n, Leioa, Spain. Ikerbasque, Basque Foundation for Science, University of the Basque Country, Sarriena s/n, Leioa, Spain. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). S. Veeriah, P. Leboucher, and J. de Naurois contributed equally to this work. Corresponding Author: Banafsh e Larijani, Cell Biophysics Laboratory, Unidad de Biofísica, Ikerbasque, Basque Foundation for Science, University of the Basque Country, Sarriena s/n, 48940 Leioa, Spain. Phone: 34-94-601-8505; Fax: 34-94-601-3360; E-mail: banafshe.larijani@ikerbasque.org doi: 10.1158/0008-5472.CAN-13-3382 2014 American Association for Cancer Research. Cancer Research www.aacrjournals. org OF1 Research. on May 30, 2017. © 2014 American Association for Cancer cancerres.aacrjournals. org Downloaded from Published OnlineFirst June 26, 2014; DOI: 10.1158/0008-5472.CAN-13-3382