Characterization of the interactions between Caspase-9 and phosphatases PP1A and PP2A in apoptosis: applications as therapeutic target in cancer | Caracterización de las interacciones entre Caspasa-9 y las fosfatasas PP1A y PP2A en apoptosis: aplicaciones como diana terapéutica en cáncer

[ES] La apoptosis es el principal mecanismo regulado de muerte celular, que tiene un papel crucial no sólo en condiciones de daño celular o estrés, sino también durante el desarrollo normal, la morfogénesis, el mantenimiento de la homeostasis de los tejidos y para un sistema inmune efectivo. Por ello, su alteración está implicada en numerosas patologías, como enfermedades neurodegenerativas, daño isquémico, enfermedades autoinmunes y cáncer. La maquinaria apoptótica intracelular está controlada por una familia de proteasas que presentan cisteína en el sitio activo y procesan a sus proteínas diana a través de residuos específicos de aspartato, denominadas caspasas, que son necesarias para la apoptosis y la respuesta inflamatoria. Las caspasas están reguladas a nivel posttraduccional por eventos de fosforilación/desfosforilación que afectan a la conformación de la proteína diana y/o a su interacción con otras proteínas, así como a su actividad, función, vida media, estabilidad, afinidad de sustrato o localización subcelular. Este mecanismo está controlado por dos grandes familias de proteínas: las proteín kinasas y las proteín fosfatasas. En este trabajo hemos abordado el estudio de las interacciones entre Caspasa- 9 y las fosfatasas PP1 y PP2A, así como la interacción entre Caspasa-9 y el inhibidor 2 de PP2A, SET. Además, hemos estudiado la abundancia relativa de las variantes de empalme alternativo aberrante de Caspasa-9 y PP2Acα en pacientes de CLL y hemos podido aplicar el conocimiento de la interacción Caspasa-9 – PP2Acα para desarrollar un péptido capaz de inducir apoptosis específicamente en células B de CLL y otro tipo de células tumorales, así como en modelos de xenotransplantes de cáncer de mama. Con el objetivo de llevar a cabo ensayos preclínicos para desarrollar este péptido como fármaco, hemos evaluado el uso de nanopartículas como vehículos para transportar este péptido a las céulas diana.

[EN]Apoptosis is the major mechanism of regulated cell death, having a crucial role not only upon cell damage or stress, but also during normal development, morphogenesis, maintenance of tissue homeostasis and for an effective immune system. Therefore, its disturbance is implicated in numerous pathologies, as neurodegenerative disorders, ischemic damage, autoimmune diseases and cancer. The intracellular apoptotic machinery relies on a family of proteases that present cysteine at their active site and cleave their target proteins through specific aspartate residues, termed caspases, which are necessary for apoptosis and inflammatory response. Caspases are regulated at the posttranslational level by phosphorylation/dephosphorylation events that affect the conformation of the target protein and/or its interaction with other proteins, as well as its activity, function, half-life, stability, substrate affinity or subcellular localization. This mechanism is controlled by two big families of proteins: protein kinases and protein phosphatases. In the present work we have given a step further in the characterization of the interaction between procaspase-9 and the Ser/Thr protein phosphatase 1α (PP1cα), confirming that PP1cα binds to both the CARD and the catalytic domain of procaspase-9 with a KD of 10-7M in a transient interaction. We have also described and characterized the direct interaction between Caspase-9 and the catalytic domain of Ser/Thr protein phosphatase 2A (PP2Acα), determining the binding sites and the key residues of the interaction, as mutants that disrupt the association were found. This interaction seems to be also transient, as the KD of the binding is in the micromolar range. Moreover, in this work we have obtained preliminary evidences for a ternary complex formed by Caspase-9, PP1cα and PP2Acα. These findings have allowed us to generate bifunctional peptides fusing the cell-penetrating peptide DPT to Caspase-9 and PP2Acα derived peptides corresponding to the binding sites. DPT-C9h has shown to be very efficient inducing apoptosis in cancer cell lines, primary human cells and primary human breast cancer xenograft models. In addition, DPT-C9h has a specific effect on transformed B cells isolated from chronic lymphocytic leukemia patients without any effect on primary healthy cells, confirming its tumor specificity. Besides, DPT-C9h is also specific for B cells, not inducing any apoptotic effect on T-cells, NK-cells or monocytes. With the aim of facing preclinical trials, this bifunctional peptide has been optimized removing a serum protease cleavage site in the DPT sequence. Furthermore, in the present work the efficiency of mesoporous silica nanoparticles has been evaluated as a tool for drug delivery. In this sense, MCM-41 nanoparticles have provided promising results that suggest the possibility of a up to 10-fold dose reduction in cancer treatment with C9h. Chronic lymphocytic leukemia (CLL) patients and healthy donors were analyzed to study the relative abundance of aberrant spliced variants of Caspase-9 (Caspase-9b) and PP2Acα (PP2Acα2). 40% of CLL patients presented moderately or highly dysregulated Caspase-9b but we couldn’t correlate this data with other parameters of the disease. More importantly, PP2Acα2 was dysregulated in 88% of the patients and it has been associated with Binet B and C stages of the disease. Therefore, we postulate in this work the use of PP2Acα2 overexpression as a biomarker for CLL diagnose. In other part of the study, we have characterized the binding sites between Caspase-9 and SET, suggesting a direct interaction between them, although it couldn’t be characterized in vitro. The bifunctional peptides derived of binding site 1 of Caspase-9 and binding site 1 of SET resulted in a very effective drug to induce apoptosis in breast cancer cells. In sum, the present work has addressed multidisciplinary approaches to better characterize the interactions between Ser/Thr phosphatases PP1 and PP2A and Caspase-9, as well as its association with the PP2A inbitor SET, which has allowed us to get efficient peptide-based drugs for cancer therapy.