RESUMO
Precision cancer therapy requires on the one hand detailed knowledge about a tumor's driver oncogenes and on the other hand an effective targeted therapy that specifically inhibits these oncogenes. While the determination of genomic landscape of a tumor has reached a very precise level, the respective therapy options are scarce. The application of small inhibitory (si) RNAs is a promising field of investigation. Here, we present the effective in vivo-treatment of colorectal cancer (CRC) xenograft tumors with antibody-complexed, endoribonuclease-prepared small inhibitory (esi)RNAs. We chose heterogeneous endoribonuclease-prepared siRNA pools (esiRNAs) against the frequently mutated genes PIK3CA and KRAS and coupled them to the anti-EGFR antibody cetuximab, which was internalized specifically into the tumor cells. esiRNA pools have been shown to exhibit superior specificity in target gene knockdown compared to classic siRNAs. We identified a significant decrease in tumor growth upon this treatment due to decreased tumor cell proliferation. The ex vivo-analysis of the xenograft CRC tumors revealed the expected downregulation of the intended direct targets PIK3CA and KRAS on protein level. Moreover, known downstream targets for EGFR signaling such as p-ERK, p-AKT, and c-MYC were decreased as well. We therefore propose the use of antibody-esiRNA complexes as a novel experimental treatment option against key components of the EGFR signaling cascade.
Assuntos
Cetuximab/uso terapêutico , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias Colorretais/terapia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Animais , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Terapia Combinada , Regulação para Baixo , Feminino , Células HT29 , Humanos , Camundongos , Camundongos Nus , Mutação , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Knockdown of genes by RNA interference (RNAi) in vitro requires methods of transfection or transduction, both of which have limited impact in vivo. As a virus-free approach, we chemically coupled cell surface receptors internalizing antibodies to the short interfering RNA (siRNA) carrier peptide protamine using the bispecific cross-linker sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). First, protamine was conjugated amino-terminally to sulfo-SMCC, and then this conjugate was coupled via cysteine residues to the IgG backbone to carry siRNA. This complex can efficiently find, bind and internalize into receptor-positive cells in vitro and in vivo, which can be checked by flow cytometry, fluorescence microscopy and western blotting. This method obtains results similar to those of siRNA targeting molecules engineered by genetic fusions between receptor-binding and siRNA carrier units, with the advantage of using readily available purified proteins without the need for engineering, expression and purification of respective constructs. The procedure for coupling the complex takes â¼ 2 d, and the functional assays take â¼ 2 weeks.
Assuntos
Anticorpos/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Técnicas de Silenciamento de Genes/métodos , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Animais , Anticorpos/química , Anticorpos/imunologia , Linhagem Celular Tumoral , Receptores ErbB/imunologia , Humanos , Maleimidas/química , Camundongos , Protaminas/química , Protaminas/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismoRESUMO
PURPOSE: KRAS mutations are frequent driver mutations in multiple cancers. KRAS mutations also induce anti-EGFR antibody resistance in adenocarcinoma such as colon cancer. The aim of this study was to overcome anti-EGFR antibody resistance by coupling the antibody to KRAS-specific siRNA. EXPERIMENTAL DESIGN: The anti-EGFR antibody was chemically coupled to siRNA. The resulting complex was tested for antibody binding efficiency, serum stability and ability to deliver siRNA to EGFR-expressing cells. Western blotting, viability, apoptosis, and colony formation assays were performed for efficacy evaluation in vitro. Furthermore, therapeutic activity of the antibody-KRAS-siRNA complexes was examined in in vivo xenograft mouse tumor models. RESULTS: Antibody-siRNA complexes were targeted and internalized via the EGFR receptor. Upon internalization, target gene expression was strongly and specifically repressed, followed by a reduced proliferation and viability, and induced apoptosis of the cells in vitro. Clonogenic growth of mutant KRAS-bearing cells was suppressed by KRAS-siRNA-anti-EGFR antibody complexes. In xenograft mouse models, anti-EGFR antibody-KRAS-siRNA complexes significantly slowed tumor growth in anti-EGFR-resistant cells. CONCLUSIONS: The coupling of siRNA against KRAS to anti-EGFR antibodies provides a novel therapy approach for KRAS-mutated EGFR-positive cancer cells in vitro and in vivo. These findings provide an innovative approach for cancer-specific siRNA application and for enhanced therapeutic potential of monoclonal antibody therapy and personalized treatment of cancer entities.
