RESUMO
Persistent infection with high-risk human papillomaviruses (hrHPV) can result in the formation of anogenital cancers. As hrHPV proteins E6 and E7 are required for cancer initiation and maintenance, they are ideal targets for immunotherapeutic interventions. Previously, we have described the development of DNA vaccines for the induction of HPV16 E6 and E7 specific T cell immunity. These vaccines consist of 'gene-shuffled' (SH) versions of HPV16 E6 and E7 that were fused to Tetanus Toxin Fragment C domain 1 (TTFC) and were named TTFC-E6SH and TTFC-E7SH. Gene-shuffling was performed to avoid the risk of inducing malignant transformation at the vaccination site. Here, we describe the preclinical safety evaluation of these candidate vaccines by analysis of their transforming capacity in vitro using established murine fibroblasts (NIH 3T3 cells) and primary human foreskin keratinocytes (HFKs). We demonstrate that neither ectopic expression of TTFC-E6SH and TTFC-E7SH alone or in combination enabled NIH 3T3 cells to form colonies in soft agar. In contrast, expression of HPV16 E6WT and E7WT alone or in combination resulted in effective transformation. Similarly, retroviral transduction of HFKs from three independent donors with both TTFC-E6SH and TTFC-E7SH alone or in combination did not show any signs of immortalization. In contrast, the combined expression of E6WT and E7WT induced immortalization in HFKs from all donors. Based on these results we consider it justified to proceed to clinical evaluation of DNA vaccines encoding TTFC-E6SH and TTFC-E7SH in patients with HPV16 associated (pre)malignancies.
Assuntos
Transformação Celular Neoplásica , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Vacinas contra Papillomavirus/efeitos adversos , Proteínas Repressoras/metabolismo , Vacinas de DNA/efeitos adversos , Animais , Células Cultivadas , Embaralhamento de DNA , Expressão Gênica , Humanos , Camundongos , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Vacinas contra Papillomavirus/administração & dosagem , Vacinas contra Papillomavirus/genética , Proteínas Repressoras/genética , Toxoide Tetânico/efeitos adversos , Toxoide Tetânico/genética , Transdução Genética , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/genéticaRESUMO
We previously showed that silencing of TSLC1, recently renamed CADM1, is functionally involved in high-risk HPV-mediated cervical carcinogenesis. CADM1 silencing often results from promoter methylation. Here, we determined the extent of CADM1 promoter methylation in cervical (pre)malignant lesions and its relation to anchorage-independent growth and gene silencing to select a CADM1-based methylation marker for identification of women at risk of cervical cancer. Methylation-specific PCRs targeting three regions within the CADM1 promoter were performed on high-risk HPV-containing cell lines, PBMCs, normal cervical smears, and (pre)malignant lesions. CADM1 protein expression in cervical tissues was analysed by immunohistochemistry. All statistical tests were two-sided. Density of methylation was associated with the degree of anchorage-independent growth and CADM1 gene silencing in vitro. In cervical squamous lesions, methylation frequency and density increased with severity of disease. Dense methylation (defined as >or= 2 methylated regions) increased from 5% in normal cervical samples to 30% in CIN3 lesions and 83% in squamous cell carcinomas (SCCs) and was significantly associated with decreased CADM1 protein expression (p < 0.00005). The frequency of dense methylation was significantly higher in >or= CIN3 compared with
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/genética , Imunoglobulinas/genética , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Proteínas Supressoras de Tumor/genética , Displasia do Colo do Útero/genética , Neoplasias do Colo do Útero/genética , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Estudos de Casos e Controles , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular , Linhagem Celular Transformada , Metilação de DNA , Feminino , Inativação Gênica , Humanos , Imunoglobulinas/análise , Imuno-Histoquímica , Modelos Logísticos , Proteínas de Membrana/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/análise , Neoplasias do Colo do Útero/metabolismo , Displasia do Colo do Útero/metabolismoRESUMO
We aimed to link DNA methylation events occurring in cervical carcinomas to distinct stages of HPV-induced transformation. Methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) analysis of cervical carcinomas revealed promoter methylation of 12 out of 29 tumour suppressor genes analysed, with MGMT being most frequently methylated (92%). Subsequently, consecutive stages of HPV16/18-transfected keratinocytes (n=11), ranging from pre-immortal to anchorage-independent phenotypes, were analysed by MS-MLPA. Whereas no methylation was evident in pre-immortal cells, progression to anchorage independence was associated with an accumulation of frequent methylation events involving five genes, all of which were also methylated in cervical carcinomas. TP73 and ESR1 methylation became manifest in early immortal cells followed by RARbeta and DAPK1 methylation in late immortal passages. Complementary methylation of MGMT was related to anchorage independence. Analysis of nine cervical cancer cell lines, representing the tumorigenic phenotype, revealed in addition to these five genes frequent methylation of CADM1, CDH13 and CHFR. In conclusion, eight recurrent methylation events in cervical carcinomas could be assigned to different stages of HPV-induced transformation. Hence, our in vitro model system provides a valuable tool to further functionally address the epigenetic alterations that are common in cervical carcinomas.
