RESUMO
Human papillomavirus (HPV)-induced carcinogenesis is critically dependent on the activities of the viral E6 and E7 oncogenes. Here, we demonstrate that expression of the putative tumor suppressor gene B-cell translocation gene-2 (BTG2) is reinduced in HPV16- and HPV18-positive cancer cells on silencing of viral oncogene expression, indicating that BTG2 is repressed by oncogenic HPVs. Inhibition of BTG2 expression was mediated by the HPV E6 oncogene and occurred in a p53-dependent manner. Luciferase reporter gene analyses revealed that BTG2 repression takes place at the transcriptional level and is dependent on the integrity of the major p53-response element within the BTG2 promoter. Ectopic expression of BTG2 acted antiproliferative in cervical cancer cells. Tissue specimens commonly exhibited reduced BTG2 protein levels in HPV-positive high-grade lesions (CIN2/3) and cervical carcinomas, when compared with normal cervical epithelium. These findings identify the antiproliferative BTG2 gene as a novel cellular target blocked by the HPV E6 oncoprotein.
Assuntos
Genes Supressores de Tumor , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Proteínas Imediatamente Precoces/genética , Proliferação de Células , Proteínas de Ligação a DNA/genética , Células HeLa , Papillomavirus Humano 16/patogenicidade , Papillomavirus Humano 18/patogenicidade , Humanos , Proteínas Oncogênicas Virais/genética , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de TumorRESUMO
Specific types of human papillomaviruses (HPVs) cause cervical cancer. The viral E6 oncogene is a critical factor for maintaining the malignant phenotype of HPV-positive tumour cells. By yeast two-hybrid screening of a randomised peptide expression library, we isolated linear short peptides, which specifically bind to the HPV16 E6 oncoprotein. Sequence alignments and mutational analyses of the peptides identified a hitherto undiscovered E6-binding motif. Intracellular expression of a peptide containing the novel E6-binding motif resulted in inhibition of colony formation capacity, specifically of HPV16-positive cancer cells. A solubility-optimised variant of the peptide was created, which binds to HPV16 E6 with high affinity. Its intracellular expression efficiently induced apoptosis in HPV16-positive cancer cells. This was linked to restoration of intracellular p53 activities. Thus, this newly identified E6-binding motif could form a novel basis for the development of rational strategies for the treatment of HPV16-positive preneoplastic and neoplastic lesions.
Assuntos
Proteínas Oncogênicas Virais/metabolismo , Peptídeos/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Proteína 1 Homóloga a Discs-Large , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Marcação In Situ das Extremidades Cortadas , Cinética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/genética , Biblioteca de Peptídeos , Peptídeos/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Transfecção , Proteína Supressora de Tumor p53/metabolismo , Técnicas do Sistema de Duplo-Híbrido , UbiquitinaçãoRESUMO
Specific types of human papillomaviruses (HPVs) cause cervical cancer, the second most common tumor in women worldwide. Both cellular transformation and the maintenance of the oncogenic phenotype of HPV-positive tumor cells are linked to the expression of the viral E6 and E7 oncogenes. To identify downstream cellular target genes for the viral oncogenes, we silenced endogenous E6 and E7 expression in HPV-positive HeLa cells by RNA interference (RNAi). Subsequently, we assessed changes of the cellular transcriptome by genome-wide microarray analysis. We identified 648 genes, which were either downregulated (360 genes) or upregulated (288 genes), upon inhibition of E6/E7 expression. A large fraction of these genes is involved in tumor-relevant processes, such as apoptosis control, cell cycle regulation, or spindle formation. Others may represent novel cellular targets for the HPV oncogenes, such as a large group of C-MYC-associated genes involved in RNA processing and splicing. Comparison with published microarray data revealed a substantial concordance between the genes repressed by RNAi-mediated E6/E7 silencing in HeLa cells and genes reported to be upregulated in HPV-positive cervical cancer biopsies.
