Nuclear localization signals are short amino acid sequences that target proteins for nuclear import. In this manuscript, we have generated a chimeric tri-functional peptide composed of a cell penetrating peptide (CPP), a nuclear localization sequence and an interfering peptide blocking the interaction between TEAD and YAP, two transcription factors involved in the Hippo signalling pathway, whose deregulation is related to several types of cancer. We have validated the cell penetration and nuclear localization by flow cytometry and fluorescence microscopy and shown that the new generated peptide displays an apoptotic effect in tumor cell lines thanks to the specific nuclear delivery of the cargo, which targets a protein/protein interaction in the nucleus. In addition, the peptide has an anti-tumoral effect in vivo in xenograft models of breast cancer. The chimeric peptide designed in the current study shows encouraging prospects for developing nuclear anti- neoplastic drugs.
Apoptotic signaling pathways are altered in numerous pathologies such as cancer. In this scenario, caspase-9/PP2Acα interaction constitutes a key target with pharmacological interest to re-establish apoptosis in tumor cells. Very recently, a short peptide (C9h) known to disrupt caspase-9/PP2Acα interaction with subsequent apoptosis induction was described. Here, we prepared two sets of mesoporous silica nanoparticles loaded with safranin O (S2) or with C9h peptide (S4) and functionalized with ϵ-polylysine as capping unit. Aqueous suspensions of both nanoparticles showed negligible cargo release whereas in the presence of pronase, a marked delivery of safranin O or C9h was observed. Confocal microscopy studies carried out with HeLa cells indicated that both materials were internalized and were able to release their entrapped cargos. Besides, a marked decrease in HeLa cell viability (ca. 50 %) was observed when treated with C9h-loaded S4 nanoparticles. Moreover, S4 provides peptide protection from degradation additionally allowing for a dose reduction to observe an apoptotic effect when compared with C9h alone or in combination with a cell-penetrating peptide (i.e., Mut3DPT-C9h). Flow cytometry studies, by means of Annexin V-FITC staining, showed the activation of apoptotic pathways in HeLa as a consequence of S4 internalization, release of C9h peptide and disruption of caspase-9/PP2Acα interaction.
BackgroundDisruption of alternative splicing in apoptotic factors has been associated to chronic lymphocytic leukemia among other cancers and hematological malignancies. The proapoptotic proteins Caspase-9 and PP2Acα are functionally related in a direct interaction, which constitutes a promising target for cancer therapy. Both proteins present aberrant mRNA splicing variants that are antiapoptotic (Caspase-9b) and catalytically inactive (PP2Acα2), respectively.ResultsIn this work we have analyzed the relative abundance of the aberrant spliced forms Caspase-9b and PP2Acα2 in several cell lines and chronic lymphocytic leukemia patients and correlated it with several parameters of the disease. Despite 40 % of the patients presented Caspase-9b dysregulation, there was no direct association between alterations in Caspase-9b relative abundance and the parameters analyzed in medical records. More importantly, PP2Acα2 dysregulation was observed in 88 % of CLL patients and was related with advanced stages of the malignancy.ConclusionsCaspase-9b dysregulation seemed to be associated with the disease, although the differences between healthy donors and CLL patients were not statistically significant. However, PP2Acα2 dysregulation was significantly different between healthy donors and CLL patients and correlated with Binet B and C stages; therefore, we propose the use of PP2Acα2 dysregulation as a potential biomarker for advanced stages of chronic lymphocytic leukemia.
Introduction. The interaction between intracellular caspase-9 and PP2A proteins is critical to apoptosis. We designed PEP-010, a Cell Penetrating & Interfering Peptide, which specifically disrupts the interaction between caspase-9 and PP2A. We evaluated in vitro and in vivo its therapeutic properties to demonstrate its potential as an innovative approach to breast cancers treatment. Material and Methods. In vitro evaluation was done by apoptosis (Annexin V) and cell viability (MTT) measurements on cancer cell lines from different origins. In vivo efficacy studies were conducted on patient-derived xenograft (PDX) mice models of triple-negative breast cancer (TNBC) and hormone-positive HER2-negative breast adenocarcinoma (BC). Pharmacokinetic and biodistribution studies were conducted on mice after administration by iv and ip route. Preliminary tolerance studies were done on mice and rats after repeated administration of PEP-010. Results. First, we demonstrated that PEP-010 is able to penetrate into tumor cells and induces caspase-9-dependent apoptosis in several tumor cell types. Moreover, we demonstrated that PEP-010 specifically induces the death of cancer cells (CLL) without harm to healthy cells.After PEP-010 treatment, we observed a significant tumor growth inhibition in PDX mice models of TNBC, compared to the untreated control. In BC PDX mice models, we observed also a significant tumor growth inhibition with complete response after PEP-010 treatment. We also improved the stability and the pharmacokinetic parameters of PEP-010. Point mutations on a protease cleavage site clearly improved peptide stability while keeping the functional activity. Biodistribution studies demonstrated that the optimized peptide is able to reach the targeted tumor and accumulate there at higher concentration than the former peptide. Pharmacokinetic studies in mice administered with PEP-010 by iv and ip route were conducted as well as in vitro study of plasmatic clearance of PEP-010 in different species. We finally performed formulation studies to improve the solubility of PEP-010 and confirmed the efficacy of the drug product in a BC PDX mice model.Preliminary tolerance studies were done on mice and rats after repeated administration of PEP-010 without demonstrating any signs of toxicity. Moreover, no immunogenic response has be observed after repeated administration of PEP-010 in mice. Conclusion. Using PEP-010, a CP&IP blocking caspase-9/PP2A interaction, we have demonstrated that this peptide has an interesting in vitro and in vivo therapeutic effect. PEP-010 constitutes a new innovative therapeutic approach for the treatment of human breast cancers. PEP-010 will enter GLP toxicity studies shortly in order to prepare the first in human clinical trial. Citation Format: Sophie Lebel-Binay, Fariba Nemati, Leticia Dominguez-Berrocal, Justine Fleury, Adnan Naguez, Didier Decaudin, Angelita Rebollo. PEP-010, a cell penetrating & interfering peptide as a new therapeutic approach in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3904.
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