title: Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies.
abstract: Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16(INK4a)-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16(INK4a) leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity. Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma.
authors.affiliation: Institut des Biomolécules Max Mousseron, CNRS, UMR 5247, Université de Montpellier, Faculté de Pharmacie, 15, Av. Charles Flahault, 34093 Montpellier, France.
authors.forename: Camille
authors.initials: C
authors.lastname: Prével
authors.affiliation: Institut des Biomolécules Max Mousseron, CNRS, UMR 5247, Université de Montpellier, Faculté de Pharmacie, 15, Av. Charles Flahault, 34093 Montpellier, France.
authors.forename: Morgan
authors.initials: M
authors.lastname: Pellerano
authors.affiliation: Institut des Biomolécules Max Mousseron, CNRS, UMR 5247, Université de Montpellier, Faculté de Pharmacie, 15, Av. Charles Flahault, 34093 Montpellier, France.
authors.forename: Juan A
authors.initials: JA
authors.lastname: González-Vera
authors.affiliation: Institut des Biomolécules Max Mousseron, CNRS, UMR 5247, Université de Montpellier, Faculté de Pharmacie, 15, Av. Charles Flahault, 34093 Montpellier, France.
authors.forename: Pauline
authors.initials: P
authors.lastname: Henri
authors.affiliation: Institut des Biomolécules Max Mousseron, CNRS, UMR 5247, Université de Montpellier, Faculté de Pharmacie, 15, Av. Charles Flahault, 34093 Montpellier, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Meunier
authors.affiliation: INSERM U823, Institut Albert Bonniot, F-38000 Grenoble, France; Universite Grenoble Alpes, Institut Albert Bonniot, F-38000 Grenoble, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Vollaire
authors.affiliation: INSERM U823, Institut Albert Bonniot, F-38000 Grenoble, France; Universite Grenoble Alpes, Institut Albert Bonniot, F-38000 Grenoble, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Josserand
authors.affiliation: Institut des Biomolécules Max Mousseron, CNRS, UMR 5247, Université de Montpellier, Faculté de Pharmacie, 15, Av. Charles Flahault, 34093 Montpellier, France. Electronic address: may.morris@univ-montp1.fr.
authors.forename: May C
authors.initials: MC
authors.lastname: Morris
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002457:Cell Extracts / Q000737:chemistry; D045744:Cell Line, Tumor; D051358:Cyclin-Dependent Kinase 4 / Q000032:analysis / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D008545:Melanoma / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D012867:Skin / Q000378:metabolism / Q000473:pathology*; D012878:Skin Neoplasms / Q000378:metabolism* / Q000473:pathology; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D002457:Cell Extracts; D005456:Fluorescent Dyes; D010455:Peptides; D051358:Cyclin-Dependent Kinase 4
keywords: CDK4/Cyclin D; Fluorescent biosensor; Kinase activity; Melanoma