Project description:Cervical cancer and a subset of anogenital and head-and-neck carcinomas are caused by persistent infection with high-risk types of the human papillomavirus (hrHPV). Early stages of hrHPV-induced carcinogenesis can be faithfully mimicked in vitro. A major hallmark of hrHPV-transformed cells is their ability to grow anchorage independently, an oncogenic trait known to depend on inactivation of tumour suppressor genes. This study used an in vitro model of hrHPV-induced transformation to delineate in a longitudinal manner to what extent DNA methylation-mediated silencing of tumour suppressive microRNAs (miRNAs) contributed to hrHPV-induced anchorage independence. Genome-wide miRNA expression profiles were yielded from anchorage dependent (n=11) and independent passages (n=19) of 4 hrHPV-immortalised keratinocyte cell lines with and without demethylating treatment (DAC). Unsupervised clustering analysis showed that overall miRNA expression patterns discriminated between anchorage dependent and independent cells. Ten miRNA genes potentially silenced by methylation were selected and validated by bisulfite sequencing and methylation-specific PCR. Hsa-mir-129-2, -137, -935, -3663, -3665, and -4281 showed increased methylation in both HPV-transformed keratinocytes and cervical cancer cell lines compared to primary keratinocytes. Mature miRNAs derived from hsa-mir-129-2, -137, -3663, and -3665 decreased anchorage independence in cervical cancer cell lines. Finally, significantly increased methylation of hsa-mir-129-2, -935, -3663, -3665, and -4281 was observed in cervical (pre)cancerous lesions, underlining the clinical relevance of our findings. In conclusion, methylation-mediated silencing of tumour suppressive miRNAs contributes to the acquisition of anchorage independence, supporting the importance of miRNAs during early stages of carcinogenesis and underlining their potential as both disease markers and therapeutic targets.
Project description:Cervical cancer and a subset of anogenital and head-and-neck carcinomas are caused by persistent infection with high-risk types of the human papillomavirus (hrHPV). Early stages of hrHPV-induced carcinogenesis can be faithfully mimicked in vitro. A major hallmark of hrHPV-transformed cells is their ability to grow anchorage independently, an oncogenic trait known to depend on inactivation of tumour suppressor genes. This study used an in vitro model of hrHPV-induced transformation to delineate in a longitudinal manner to what extent DNA methylation-mediated silencing of tumour suppressive microRNAs (miRNAs) contributed to hrHPV-induced anchorage independence. Genome-wide miRNA expression profiles were yielded from anchorage dependent (n=11) and independent passages (n=19) of 4 hrHPV-immortalised keratinocyte cell lines with and without demethylating treatment (DAC). Unsupervised clustering analysis showed that overall miRNA expression patterns discriminated between anchorage dependent and independent cells. Ten miRNA genes potentially silenced by methylation were selected and validated by bisulfite sequencing and methylation-specific PCR. Hsa-mir-129-2, -137, -935, -3663, -3665, and -4281 showed increased methylation in both HPV-transformed keratinocytes and cervical cancer cell lines compared to primary keratinocytes. Mature miRNAs derived from hsa-mir-129-2, -137, -3663, and -3665 decreased anchorage independence in cervical cancer cell lines. Finally, significantly increased methylation of hsa-mir-129-2, -935, -3663, -3665, and -4281 was observed in cervical (pre)cancerous lesions, underlining the clinical relevance of our findings. In conclusion, methylation-mediated silencing of tumour suppressive miRNAs contributes to the acquisition of anchorage independence, supporting the importance of miRNAs during early stages of carcinogenesis and underlining their potential as both disease markers and therapeutic targets.
Project description:Next to a persistent infection with high-risk human papillomavirus (HPV), molecular changes are required for the development of cervical cancer. To identify which molecular alterations drive carcinogenesis, we performed a comprehensive and longitudinal molecular characterization of HPV-transformed keratinocyte cell lines. Comparative genomic hybridization, mRNA, and miRNA expression analysis of four HPV-containing keratinocyte cell lines at eight different time points was performed. Data was analyzed using unsupervised hierarchical clustering, integrated longitudinal expression analysis, and pathway enrichment analysis. Biological relevance of identified key regulatory genes was evaluated in vitro and dual-luciferase assays were used to confirm predicted miRNA-mRNA interactions. We show that the acquisition of anchorage independence of HPV-containing keratinocyte cell lines is particularly associated with copy number alterations. Approximately one third of differentially expressed mRNAs and miRNAs was directly attributable to copy number alterations. Focal adhesion, TGF-beta signaling, and mTOR signaling pathways were enriched among these genes. PITX2 was identified as key regulator of TGF-beta signaling and inhibited cell growth in vitro, most likely by inducing cell cycle arrest and apoptosis. Predicted miRNA-mRNA interactions miR-221-3p_BRWD3, miR-221-3p_FOS, and miR-138-5p_PLXNB2 were confirmed in vitro. Integrated longitudinal analysis of our HPV-induced carcinogenesis model pinpointed relevant interconnected molecular changes and crucial signaling pathways in HPV-mediated transformation.
Project description:Next to a persistent infection with high-risk human papillomavirus (HPV), molecular changes are required for the development of cervical cancer. To identify which molecular alterations drive carcinogenesis, we performed a comprehensive and longitudinal molecular characterization of HPV-transformed keratinocyte cell lines. Comparative genomic hybridization, mRNA, and miRNA expression analysis of four HPV-containing keratinocyte cell lines at eight different time points was performed. Data was analyzed using unsupervised hierarchical clustering, integrated longitudinal expression analysis, and pathway enrichment analysis. Biological relevance of identified key regulatory genes was evaluated in vitro and dual-luciferase assays were used to confirm predicted miRNA-mRNA interactions. We show that the acquisition of anchorage independence of HPV-containing keratinocyte cell lines is particularly associated with copy number alterations. Approximately one third of differentially expressed mRNAs and miRNAs was directly attributable to copy number alterations. Focal adhesion, TGF-beta signaling, and mTOR signaling pathways were enriched among these genes. PITX2 was identified as key regulator of TGF-beta signaling and inhibited cell growth in vitro, most likely by inducing cell cycle arrest and apoptosis. Predicted miRNA-mRNA interactions miR-221-3p_BRWD3, miR-221-3p_FOS, and miR-138-5p_PLXNB2 were confirmed in vitro. Integrated longitudinal analysis of our HPV-induced carcinogenesis model pinpointed relevant interconnected molecular changes and crucial signaling pathways in HPV-mediated transformation.
Project description:Next to a persistent infection with high-risk human papillomavirus (HPV), molecular changes are required for the development of cervical cancer. To identify which molecular alterations drive carcinogenesis, we performed a comprehensive and longitudinal molecular characterization of HPV-transformed keratinocyte cell lines. Comparative genomic hybridization, mRNA, and miRNA expression analysis of four HPV-containing keratinocyte cell lines at eight different time points was performed. Data was analyzed using unsupervised hierarchical clustering, integrated longitudinal expression analysis, and pathway enrichment analysis. Biological relevance of identified key regulatory genes was evaluated in vitro and dual-luciferase assays were used to confirm predicted miRNA-mRNA interactions. We show that the acquisition of anchorage independence of HPV-containing keratinocyte cell lines is particularly associated with copy number alterations. Approximately one third of differentially expressed mRNAs and miRNAs was directly attributable to copy number alterations. Focal adhesion, TGF-beta signaling, and mTOR signaling pathways were enriched among these genes. PITX2 was identified as key regulator of TGF-beta signaling and inhibited cell growth in vitro, most likely by inducing cell cycle arrest and apoptosis. Predicted miRNA-mRNA interactions miR-221-3p_BRWD3, miR-221-3p_FOS, and miR-138-5p_PLXNB2 were confirmed in vitro. Integrated longitudinal analysis of our HPV-induced carcinogenesis model pinpointed relevant interconnected molecular changes and crucial signaling pathways in HPV-mediated transformation.