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BACKGROUND: Tumor resistance is a frequent cause of therapy failure and remains a major challenge for the long-term management of colorectal cancer (CRC). The aim of this study was to determine the implication of the tight junctional protein claudin 1 (CLDN1) in the acquired resistance to chemotherapy. METHODS: Immunohistochemistry was used to determine CLDN1 expression in post-chemotherapy liver metastases from 58 CRC patients. The effects of oxaliplatin on membrane CLDN1 expression were evaluated by flow cytometry, immunofluorescence and western blotting experiments in vitro and in vivo. Phosphoproteome analyses, proximity ligation and luciferase reporter assays were used to unravel the mechanism of CLDN1 induction. RNAseq experiments were performed on oxaliplatin-resistant cell lines to investigate the role of CLDN1 in chemoresistance. The "one-two punch" sequential combination of oxaliplatin followed by an anti-CLDN1 antibody-drug conjugate (ADC) was tested in both CRC cell lines and murine models. RESULTS: We found a significant correlation between CLDN1 expression level and histologic response to chemotherapy, CLDN1 expression being the highest in resistant metastatic residual cells of patients showing minor responses. Moreover, in both murine xenograft model and CRC cell lines, CLDN1 expression was upregulated after exposure to conventional chemotherapies used in CRC treatment. CLDN1 overexpression was, at least in part, functionally related to the activation of the MAPKp38/GSK3ß/Wnt/ß-catenin pathway. Overexpression of CLDN1 was also observed in oxaliplatin-resistant CRC cell lines and was associated with resistance to apoptosis, suggesting an anti-apoptotic role for CLDN1. Finally, we demonstrated that the sequential treatment with oxaliplatin followed by an anti-CLDN1 ADC displayed a synergistic effect in vitro and in in vivo. CONCLUSION: Our study identifies CLDN1 as a new biomarker of acquired resistance to chemotherapy in CRC patients and suggests that a "one-two punch" approach targeting chemotherapy-induced CLDN1 expression may represent a therapeutic opportunity to circumvent resistance and to improve the outcome of patients with advanced CRC.
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Accumulation of PrP(Sc), an abnormal form of cellular prion protein (PrP), in the brain of animals and humans leads to fatal neurodegenerative disorders known as prion diseases. Limited protease digestion of PrP(Sc) produces a truncated form called PrP(27-30) that retains prion infectivity and is the main marker of disease targeted in most diagnostic tests. In the search for new anti-prion molecules, drug-screening assays on prion-infected murine cells have been oriented toward decreasing levels of PrP(27-30). In contrast, we screened for drugs promoting multimers of PrP(27-30), illustrating a possible stabilization of mouse PrP(Sc) species, because recent studies aiming to characterize the conformational stability of various prion strains showed that stable recombinant amyloids produced more stable prion strain, leading to longest incubation time. We identified a family of thienyl pyrimidine derivatives that induce SDS-resistant dimers and trimers of PrP(27-30). Bioassays performed on mice brain homogenates treated with these compounds showed that these thienyl pyrimidine derivatives diminished prion infectivity in vivo. Oligomeric-induced activity by thienyl pyrimidine compounds is a promising approach not only to understanding the pathogenesis of prions but also for prion diagnostics. This approach could be extended to other neurodegenerative "prionopathies," such as Alzheimer's, Huntington, or Parkinson's diseases.
Assuntos
Encéfalo/efeitos dos fármacos , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Doenças Priônicas/patologia , Pirimidinas/farmacologia , Anilidas/farmacologia , Anilidas/uso terapêutico , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Endopeptidase K/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Camundongos , Modelos Moleculares , Neuroblastoma/patologia , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas PrPC/análise , Doenças Priônicas/tratamento farmacológico , Conformação Proteica/efeitos dos fármacos , Pirimidinas/uso terapêutico , Silício , Estatísticas não Paramétricas , Fatores de Tempo , Transfecção/métodosRESUMO
BACKGROUND: Prion diseases are characterized by the accumulation in the central nervous system of an abnormally folded isoform of the prion protein, named PrP(Sc). Aggregation of PrP(Sc) into oligomers and fibrils is critically involved in the pathogenesis of prion diseases. Oligomers are supposed to be the key neurotoxic agents in prion disease, so modulation of prion aggregation pathways with small molecules can be a valuable strategy for studying prion pathogenicity and for developing new diagnostic and therapeutic approaches. We previously identified thienyl pyrimidine compounds that induce SDS-resistant PrP(Sc) (rSDS-PrP(Sc)) oligomers in prion-infected samples. RESULTS: Due to the low effective doses of the thienyl pyrimidine hits, we synthesized a quaterthiophene-bis-triazine compound, called MR100 to better evaluate their diagnostic and therapeutic potentials. This molecule exhibits a powerful activity inducing rSDS-PrP(Sc) oligomers at nanomolar concentrations in prion-infected cells. Fluorescence interaction studies of MR100 with mouse PrP fibrils showed substantial modification of the spectrum, and the interaction was confirmed in vitro by production of rSDS-oligomer species upon incubation of MR100 with fibrils in SDS-PAGE gel. We further explored whether MR100 compound has a potential to be used in the diagnosis of prion diseases. Our results showed that: (i) MR100 can detect rSDS-oligomers in prion-infected brain homogenates of various species, including human samples from CJD patients; (ii) A protocol, called "Rapid Centrifugation Assay" (RCA), was developed based on MR100 property of inducing rSDS-PrP(Sc) oligomers only in prion-infected samples, and avoiding the protease digestion step. RCA allows the detection of both PK-sensitive and PK-resistant PrP(Sc) species in rodents samples but also from patients with different CJD forms (sporadic and new variant); (iii) A correlation could be established between the amount of rSDS-PrP(Sc) oligomers revealed by MR100 and the duration of the symptomatic phase of the disease in CJD patients; and (iv) Bioassay experiments showed that MR100 can trap prion infectivity more efficiently than P30 drug. CONCLUSIONS: MR100 is a powerful tool not only for studying the prion aggregation pathways regarding oligomeric and sPrP(Sc) species, but also for developing alternative methods for the detection of prion-infected samples. Considering our bioassay results, MR100 is a promising molecule for the development of prion decontamination approaches.
Assuntos
Encéfalo/metabolismo , Proteínas PrPC/metabolismo , Doenças Priônicas/diagnóstico , Doenças Priônicas/metabolismo , Príons , Pirimidinas/metabolismo , Animais , Western Blotting/métodos , Modelos Animais de Doenças , Corantes Fluorescentes/metabolismo , Humanos , CamundongosRESUMO
Transmissible spongiform encephalopathies (TSEs), also called prion diseases, are fatal, infectious, genetic or sporadic neurodegenerative disorders of humans and animals. In humans, TSEs are represented by Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome, Fatal Familial Insomnia and Kuru. In animals, the most prominent prion diseases are scrapie of sheep and goats, bovine spongiform encephalopathy (BSE) of cattle and chronic wasting disease (CWD) of deer and elk. A critical event in prion diseases is the accumulation in the central nervous system (CNS) of the abnormally folded PrP(Sc) protein that is the protease-resistant isoform of a normal cellular protein encoded by the host and called PrP(C). PrP(Sc) (also known as rPrP(Sc) or PrP27-30) represents the main marker of prion diseases and is routinely used in the reference method for the diagnosis of prion diseases. Most of the therapeutic strategies developed so far aimed at identifying compounds that diminish the levels of PrP(Sc), with variable success when tested in vivo. In this review, we present an alternative approach in which small molecules that induce PrP(Sc) oligomers are identified. By using virtual and cellular screenings, we found several thienyl pyrimidine compounds that trigger PrP(Sc) oligomerization and trap prion infectivity.