Project description:Merkel cells are epidermal mechanoreceptor cells responsible for the perception of gentle touch. Merkel cell carcinoma (MCC) is a rare and highly aggressive skin cancer. Although MCC histologically resembles Merkel cells, the cell of origin for MCC is unknown. MCC frequently contains integrated Merkel cell polyomavirus (MCPyV), a small DNA tumor virus with widespread prevalence. Whether MCPyV can transform Merkel cells is unknown. Here, we describe the isolation and long-term expansion of human Merkel cells from neonatal foreskin. We validated the expression of several Merkel cell-related factors by RNASeq, and assessed the ultrastructure by electron microscopy. Culture of Merkel cell preparations on an artificial basement membrane promoted the formation of structures containing both Merkel and non-Merkel cell populations. To determine whether Merkel cells were susceptible to transformation, we expressed tumor-derived MCPyV T antigens and additional oncogenes. We were unable to demonstrate tumorigenesis in immunodeficient mice, but were able to detect T antigen expression from excised cells weeks after implantation. These results highlight that foreskin-isolated Merkel cells can be propagated extensively, sustain expression of MCPyV T antigens, but are not susceptible to transformation by MCPyV, suggesting that Merkel cells from non-glabrous skin may not be a cell of origin for MCC.
Project description:We performed miRNA expression profiling in a series of human Merkel Cell carcinoma samples using a microarray approach. Significant differentially expressed miRNAs among groups were identified using SAM analysis. Agilent microarray platform containing 723 human miRNAs was used to determine miRNA expression profiles in 16 human Merkel cell carcinoma (MCC) samples. To validate the microarray platform, the expression levels of selected miRNAs were evaluated using qRT-PCR.
Project description:We performed miRNA expression profiling in a series of human Merkel Cell carcinoma samples using a microarray approach. Significant differentially expressed miRNAs among groups were identified using SAM analysis.
Project description:Merkel cell polyomavirus (MCPyV) small tumor antigen (ST), truncated large tumor antigen (TLT), early region (ER), and GFP control inducible vectors were introduced into IMR90 human lung embryonic fibrobrasts. After induction, cells were extracted and sequenced in triplicate every 4 hours for the first 24 hours, then every 8 hours until 48 hours. The purpose of this experiment was to identify the dynamic host response to MCPyV oncogenes, using both differential expression analysis and transcriptional network reconstruction, and to provide a systems-level understanding of the initial steps toward Merkel cell carcinoma.
Project description:Merkel cell polyomavirus (MCV) is clonally integrated in over 80% of Merkel cell carcinomas and mediates tumor development through the expression of viral oncoproteins large T (LT) and small T antigens (sT). Viral integration is associated with signature mutations in the T antigen locus that result in deletions of carboxy-terminal replicative functions of the LT antigen. Despite these truncations, the LT LxCxE retinoblastoma (Rb) pocket protein family-binding domain is retained, and the entire sT isoform is maintained intact. To investigate the ability of MCV oncoproteins to regulate host gene expression, we performed microarray analysis on cells stably expressing tumor-derived LT, tumor-derived LT along with sT, and tumor-derived LT with a mutated Rb interaction motif. Gene expression alterations in the presence of tumor-derived LT could be classified into three main groups: genes that are involved in the cell cycle (specifically the G1/S transition), genes involved in DNA replication, and genes involved in cellular movement. The LxCxE mutant LT largely reversed gene expression alterations detected with the wild type LT, while co-expression of sT did not significantly affect these patterns of gene expression. LxCxE-dependent upregulation of cyclin E and CDK2 correlates with increased proliferation in tumor-derived LT expressing cells. We conclude that in hTERT-immortalized human fibroblasts the LXCXE motif of tumor-derived LT enhances cellular proliferation and upregulates cell cycle and immune signaling gene transcription. RNA was isolated from BJ-hTERT cell lines and hybridized to an Illumina chip for global gene expression analysis
Project description:Merkel cell polyomavirus (MCPyV) is linked to Merkel cell carcinoma (MCC), a rare and aggressive skin cancer. This study investigated the influence of MCPyV T antigens on the host genome using transcriptomics and epigenomics. Results revealed a role for the small Tumor (sT) antigen in subverting type I interferon response and immune evasion, contributing to persistent infection and tumor progression. These findings enhance our understanding of MCPyV pathogenesis and may inform new therapeutic strategies.
Project description:Merkel cell carcinoma is supposed to be derived from Merkel cells after infection by Merkel cell polyomavirus (MCV) and other poorly known events. A transcriptional profiling with cDNA microarrays was performed on cells from MCV+ Merkel cell carcinomas and isolated normal Merkel cells. This microarray revealed numerous significantly upregulated genes and down-regulated genes. The extensive list of genes identified in these experiments provides a large body of potentially valuable information of Merkel cell carcinoma carcinogenesis and could represent a source of potential targets for cancer therapy. Two-conditions experiment, MCV vs Normal Merkel Cell. Biological replicates : 4 MCV (Cy5), 1 control = pool of Normal Merkel cells from 3 liftings
Project description:Merkel cell polyomavirus (MCPyV) is linked to Merkel cell carcinoma (MCC), a rare and aggressive skin cancer. This study investigated the influence of MCPyV T antigens on the host genome using transcriptomics and epigenomics. Results revealed a role for the small Tumor (sT) antigen in subverting type I interferon response and immune evasion, contributing to persistent infection and tumor progression. These findings enhance our understanding of MCPyV pathogenesis and may inform new therapeutic strategies.
Project description:Merkel cell polyomavirus (MCV) is clonally integrated in over 80% of Merkel cell carcinomas and mediates tumor development through the expression of viral oncoproteins large T (LT) and small T antigens (sT). Viral integration is associated with signature mutations in the T antigen locus that result in deletions of carboxy-terminal replicative functions of the LT antigen. Despite these truncations, the LT LxCxE retinoblastoma (Rb) pocket protein family-binding domain is retained, and the entire sT isoform is maintained intact. To investigate the ability of MCV oncoproteins to regulate host gene expression, we performed microarray analysis on cells stably expressing tumor-derived LT, tumor-derived LT along with sT, and tumor-derived LT with a mutated Rb interaction motif. Gene expression alterations in the presence of tumor-derived LT could be classified into three main groups: genes that are involved in the cell cycle (specifically the G1/S transition), genes involved in DNA replication, and genes involved in cellular movement. The LxCxE mutant LT largely reversed gene expression alterations detected with the wild type LT, while co-expression of sT did not significantly affect these patterns of gene expression. LxCxE-dependent upregulation of cyclin E and CDK2 correlates with increased proliferation in tumor-derived LT expressing cells. We conclude that in hTERT-immortalized human fibroblasts the LXCXE motif of tumor-derived LT enhances cellular proliferation and upregulates cell cycle and immune signaling gene transcription.
Project description:Merkel cell carcinoma is supposed to be derived from Merkel cells after infection by Merkel cell polyomavirus (MCPyV) and other poorly known events. A transcriptional profiling with cDNA microarrays was performed on cells from MCPyV(+) Merkel cell carcinomas and isolated normal Merkel cells. This microarray revealed numerous significantly upregulated genes and downregulated genes. The extensive list of genes identified in these experiments provides a large body of potentially valuable information of Merkel cell carcinoma carcinogenesis and could represent a source of potential targets for cancer therapy.