RESUMO
Histone deacetylase 2 (HDAC2) is a chromatin modifier involved in epigenetic regulation of cell cycle, apoptosis, and differentiation that is upregulated commonly in human hepatocellular carcinoma (HCC). In this study, we show that specific targeting of this HDAC isoform is sufficient to inhibit HCC progression. siRNA-mediated silencing of HDAC inhibited HCC cell growth by blocking cell-cycle progression and inducing apoptosis. These effects were associated with deregulation of HDAC-regulated genes that control cell cycle, apoptosis, and lipid metabolism, specifically, by upregulation of p27 and acetylated p53 and by downregulation of CDK6 and BCL2. We found that HDAC2 silencing in HCC cells also strongly inhibited PPARγ signaling and other regulators of glycolysis (ChREBPα and GLUT4) and lipogenesis (SREBP1C and FAS), eliciting a marked decrease in fat accumulation. Notably, systemic delivery of HDAC2 siRNA encapsulated in lipid nanoparticles was sufficient to blunt the growth of human HCC in a murine xenograft model. Our findings offer preclinical proof-of-concept for HDAC2 blockade as a systemic therapy for liver cancer.
Assuntos
Carcinoma Hepatocelular/genética , Histona Desacetilase 2/genética , Neoplasias Hepáticas/genética , Isoformas de Proteínas/genética , Animais , Apoptose/genética , Carcinoma Hepatocelular/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 6 Dependente de Ciclina/genética , Progressão da Doença , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/genética , Glicólise/genética , Células Hep G2 , Humanos , Metabolismo dos Lipídeos/genética , Lipogênese/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos SCID , PPAR gama/genética , Antígeno Nuclear de Célula em Proliferação/genética , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Regulação para Cima/genética , Proteína X Associada a bcl-2/genéticaRESUMO
The development of targeted therapeutics for hepatocellular carcinoma (HCC) remains a major challenge. The ubiquitination modulator COP1 regulates p53 activity by ubiquitination and it is frequently overexpressed in human HCC. In this study, we tested the hypothesis that COP1 blockade by short interfering RNA (siRNA)-mediated inhibition could affect the course of HCC progression. The COP1 isoform COP1-1 was selected as the most effective target for siRNAs in terms of growth inhibition and apoptotic induction in several HCC cell lines. Growth inhibition occurred in HCC cells that retained wild-type p53 or expressed mutant p53 (Y220C or R249S), whereas p53-null Hep3B cells were resistant. Microarray expression analysis revealed that the antiproliferative effects of COP1 blockade were driven by a common subset of molecular alterations including a p53-associated functional network. In an orthotopic mouse xenograft model of HCC, systemic delivery of a modified COP1 siRNA by stable nucleic acid-lipid particles suppressed neoplastic growth in liver without unwanted immune responses. Our findings offer a first proof of principle that COP1 can be a promising target for systemic therapy of HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/prevenção & controle , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/prevenção & controle , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma Hepatocelular/genética , Ciclo Celular , Perfilação da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos SCID , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
siRNAs that specifically silence the expression of cancer-related genes offer a therapeutic approach in oncology. However, it remains critical to determine the true mechanism of their therapeutic effects. Here, we describe the preclinical development of chemically modified siRNA targeting the essential cell-cycle proteins polo-like kinase 1 (PLK1) and kinesin spindle protein (KSP) in mice. siRNA formulated in stable nucleic acid lipid particles (SNALP) displayed potent antitumor efficacy in both hepatic and subcutaneous tumor models. This was correlated with target gene silencing following a single intravenous administration that was sufficient to cause extensive mitotic disruption and tumor cell apoptosis. Our siRNA formulations induced no measurable immune response, minimizing the potential for nonspecific effects. Additionally, RNAi-specific mRNA cleavage products were found in tumor cells, and their presence correlated with the duration of target mRNA silencing. Histological biomarkers confirmed that RNAi-mediated gene silencing effectively inhibited the target's biological activity. This report supports an RNAi-mediated mechanism of action for siRNA antitumor effects, suggesting a new methodology for targeting other key genes in cancer development with siRNA-based therapeutics.
Assuntos
Terapia Genética , Neoplasias/genética , Neoplasias/terapia , Interferência de RNA , RNA Interferente Pequeno/genética , Animais , Proteínas de Ciclo Celular/genética , Modelos Animais de Doenças , Duplicação Gênica , Camundongos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Quinase 1 Polo-LikeRESUMO
Short interfering RNAs (siRNAs) that mediate specific gene silencing through RNA interference (RNAi) are widely used to study gene function and are also being developed for therapeutic applications. Many nucleic acids, including double- (dsRNA) and single-stranded RNA (ssRNA), can stimulate innate cytokine responses in mammals. Despite this, few studies have questioned whether siRNA may have a similar effect on the immune system. This could significantly influence the in vivo application of siRNA owing to off-target effects and toxicities associated with immune stimulation. Here we report that synthetic siRNAs formulated in nonviral delivery vehicles can be potent inducers of interferons and inflammatory cytokines both in vivo in mice and in vitro in human blood. The immunostimulatory activity of formulated siRNAs and the associated toxicities are dependent on the nucleotide sequence. We have identified putative immunostimulatory motifs that have allowed the design of siRNAs that can mediate RNAi but induce minimal immune activation.