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      Time resolved amplified FRET identifies protein kinase B activation state as a marker for poor prognosis in clear cell renal cell carcinoma

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          Abstract

          Purpose

          Clear cell Renal Cell Carcinomas (ccRCC), the largest group of renal tumours, are resistant to classical therapies. The determination of the functional state of actionable biomarkers for the assessment of these adenocarcinomas is essential. The dysregulation of the oncoprotein, PKB/Akt has been linked with poor prognoses in human cancers.

          Material & methods

          We analysed the status of the PKB/Akt pathway in a representative tumour tissue microarray obtained from the primary tumours and their metastases in 60 ccRCC with long term follow up. We sought to define the evolution of this pathway from the primary tumour to the metastatic event and to know the impact of its functional state in tumour aggressiveness and patient survival. Two-site time resolved amplified FRET (A-FRET) was utilised for assessing the activation state of PKB/Akt and this was compared to conventional immunohistochemistry measurements.

          Results

          Activation state of PKB/Akt in primary tumours defined by A-FRET correlated with poorer overall survival (hazard ratio 0.228; p = 0.002). Whereas, increased protein expression of phosphoPKB/Akt, identified using classical immunohistochemistry, yielded no significant difference (hazard ratio 1.390; p = 0.548).

          Conclusions

          Quantitative determination of PKB/Akt activation in ccRCC primary tumours alongside other diagnostics tools could prove key in taking oncologists closer to an efficient personalised therapy in ccRCC patients.

          General significance

          The quantitative imaging technology based on Amplified-FRET can rapidly analyse protein activation states and molecular interactions. It could be used for prognosis and assess drug function during the early cycles of chemotherapy. It enables evaluation of clinical efficiency of personalised cancer treatment.

          Highlights

          • Time Resolved Amplified FRET (A-FRET), has been used to quantitatively assess PKB/Akt activation states in ccRCC.

          • Increased activation state of PKB/Akt in primary tumours was shown to correlate with poorer prognoses.

          • Companion diagnostic tools such as A-FRET will prove valuable for assessing prognostic models and for predicting the response to systemic therapy.

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          Most cited references10

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          An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer.

          Phosphatidylinositol 3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy-based sequencing and reverse-phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT, and PTEN mutations and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor-positive (34.5%) and HER2-positive (22.7%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor-positive cancers. Unlike AKT1 mutations that were absent from cell lines, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant effect on outcome after adjuvant tamoxifen therapy in 157 hormone receptor-positive breast cancer patients. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines. PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss rendered cells significantly more sensitive to growth inhibition by the PI3K inhibitor LY294002 than did PIK3CA mutations. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration present may have implications for the selection of PI3K-targeted therapies in hormone receptor-positive breast cancer.
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            A transforming mutation in the pleckstrin homology domain of AKT1 in cancer.

            Although AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a central member of possibly the most frequently activated proliferation and survival pathway in cancer, mutation of AKT1 has not been widely reported. Here we report the identification of a somatic mutation in human breast, colorectal and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket of AKT1. Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates AKT1 by means of pathological localization to the plasma membrane, stimulates downstream signalling, transforms cells and induces leukaemia in mice. This mechanism indicates a direct role of AKT1 in human cancer, and adds to the known genetic alterations that promote oncogenesis through the phosphatidylinositol-3-OH kinase/AKT pathway. Furthermore, the E17K substitution decreases the sensitivity to an allosteric kinase inhibitor, so this mutation may have important clinical utility for AKT drug development.
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              Activation of AKT kinases in cancer: implications for therapeutic targeting.

              The AKT1, AKT2, and AKT3 kinases have emerged as critical mediators of signal transduction pathways downstream of activated tyrosine kinases and phosphatidylinositol 3-kinase. An ever-increasing list of AKT substrates has precisely defined the multiple functions of this kinase family in normal physiology and disease states. Cellular processes regulated by AKT include cell proliferation and survival, cell size and response to nutrient availability, intermediary metabolism, angiogenesis, and tissue invasion. All these processes represent hallmarks of cancer, and a burgeoning literature has defined the importance of AKT alterations in human cancer and experimental models of tumorigenesis, continuing the legacy represented by the original identification of v-Akt as the transforming oncogene of a murine retrovirus. Many oncoproteins and tumor suppressors intersect in the AKT pathway, finely regulating cellular functions at the interface of signal transduction and classical metabolic regulation. This careful balance is altered in human cancer by a variety of activating and inactivating mechanisms that target both AKT and interrelated proteins. Reprogramming of this altered circuitry by pharmacologic modulation of the AKT pathway represents a powerful strategy for rational cancer therapy. In this review, we summarize a large body of data, from many types of cancer, indicating that AKT activation is one of the most common molecular alterations in human malignancy. We also review mechanisms of activation of AKT kinases, examples of therapeutic modulation of the AKT pathway in animal models, and the current status of efforts to target molecular components of the AKT pathway for cancer therapy and, possibly, cancer prevention.
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                Author and article information

                Contributors
                Journal
                BBA Clin
                BBA Clin
                BBA Clinical
                Elsevier
                2214-6474
                13 October 2017
                December 2017
                13 October 2017
                : 8
                : 97-102
                Affiliations
                [a ]Cell Biophysics Laboratory, Ikerbasque, Basque Foundation for Science, FASTBASE SOLUTIONS Ltd, Research Centre for Experimental Marine Biology and Biotechnology (PiE) & Biofísika Institute (UPV/EHU, CSIC), University of the Basque Country, Spain
                [b ]Institut du Cerveau et de la Moelle épinière, Hôpital de la Pitié-Salpêtrière, Paris, France
                [c ]Basque Centre for Applied Mathematics, Bilbao, Bizkaia, Spain
                [d ]Department of Pathology B, Complejo Hospitalario de Navarra, Pamplona, Spain
                [e ]Department of Pharmacy and Pharmacology, University of Bath, UK
                [f ]Protein Phosphorylation Laboratory, The Francis Crick Institute, London, UK and Division of Cancer Studies, King's College London, London, UK
                [g ]Department of Pathology, Cruces University Hospital, Biocruces Research Institute, University of the Basque Country (UPV/EHU), Barakaldo, Spain
                Author notes
                Article
                S2214-6474(17)30036-3
                10.1016/j.bbacli.2017.10.002
                5744020
                29296589
                c29caba3-b372-43f3-bfa6-b9d1475e805f
                © 2017 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 10 August 2017
                : 11 October 2017
                : 11 October 2017
                Categories
                Regular Article

                clear cell renal cell carcinoma,protein kinase b (pkb/akt),amplified fret,prognosis,fret-flim,biomarker activation

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