Merkel cell polyomavirus small T antigen promotes pro-glycolytic metabolic perturbations required for transformation
ABSTRACT: Merkel cell polyomavirus (MCPyV) is an etiological agent of Merkel cell carcinoma (MCC), a highly aggressive skin cancer. The MCPyV small tumor antigen (ST) is required for maintenance of MCC and can transform normal cells. To gain insight into cellular perturbations induced by MCPyV ST, we performed transcriptome analysis of normal human fibroblasts with inducible expression of ST. MCPyV ST dynamically alters the cellular transcriptome with increased levels of glycolytic genes, including the monocarboxylate lactate transporter SLC16A1 (MCT1). Extracellular flux analysis revealed increased lactate export reflecting elevated aerobic glycolysis in ST expressing cells. Inhibition of MCT1 activity suppressed the growth of MCC cell lines and impaired MCPyV-dependent transformation of IMR90 cells. Both NF-κB and MYC have been shown to regulate MCT1 expression. While MYC was required for MCT1 induction, MCPyV-induced MCT1 levels decreased following knockdown of the NF-κB subunit RelA, supporting a synergistic activity between MCPyV and MYC in regulating MCT1 levels. Several MCC lines had high levels of MYCL and MYCN but not MYC. Increased levels of MYCL was more effective than MYC or MYCN in increasing extracellular acidification in MCC cells. Our results demonstrate the effects of MCPyV ST on the cellular transcriptome and reveal that transformation is dependent, at least in part, on elevated aerobic glycolysis. Overall design: Expression of MCPyV ST or GFP was induced in IMR90 fibroblasts, and triplicate RNA samples were extracted and sequenced every 8 hours for a total of 96 hours
Project description:Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine tumor with high mortality rates. Merkel cell polyomavirus (MCPyV), identified in the majority of MCC, may drive tumorigenesis via viral T antigens. However, mechanisms underlying pathogenesis in MCPyV-negative MCC remain poorly understood. To nominate genes contributing to pathogenesis of MCPyV-negative MCC, we performed DNA microarray analysis on 30 MCCs. MCPyV status of MCCs was determined by PCR for viral DNA and RNA. 1593 probe-sets were differentially expressed between MCPyV-negative and -positive MCC, with significant differential expression defined as at least 2-fold change in either direction and p-value of ≤ 0.05. MCPyV-negative tumors showed decreased RB1 expression, whereas MCPyV-positive tumors were enriched for immune response genes. Validation studies included immunohistochemistry demonstration of decreased RB protein expression in MCPyV-negative tumors and increased peritumoral CD8+ T lymphocytes surrounding MCPyV-positive tumors. In conclusion, our data suggest that loss of RB1 expression may play an important role in tumorigenesis of MCPyV-negative MCC. Functional and clinical validation studies are needed to determine whether this tumor suppressor pathway represents an avenue for targeted therapy. We used microarrays to characterize global gene expression patterns related to Merkel cell polyomavirus status in Merkel cell carcinoma. Furthermore, we compared Merkel cell carcinoma to less aggressive primary cutaneous carcinomas. We utilized flash-frozen tumor tissue from primary Merkel cell carcinomas, metastatic Merkel cell carcinomas, primary cutaneous squamous cell carcinomas, and basal cell carcinomas. Merkel cell carcinoma cell lines, which represent a pure population of tumor cells, were also included. Merkel cell polyomavirus status was determined at the DNA and RNA level using multiple primers for viral T-antigen and capsid protein sequences. This Series represents two analyses - one with new Samples normalized together, and another with some of the new Samples re-normalized with Samples previously submitted under Series GSE13355. The latter group contain 'renormalized' in the titles.
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:MicroRNAs have been implicated in various skin cancers, including melanoma, squamous cell carcinoma, and basal cell carcinoma; however, the expression of microRNAs and their role in Merkel cell carcinoma (MCC) have yet to be explored in depth. To identify microRNAs specific to MCC (MCC-miRs), next-generation sequencing (NGS) of small RNA libraries was performed on different tissue samples including MCCs, other cutaneous tumors, and normal skin. Comparison of the profiles identified several microRNAs upregulated and downregulated in MCC. For validation, their expression was measured via qRT-PCR in a larger group of MCC and in a comparison group of non-MCC cutaneous tumors and normal skin. Eight microRNAs were upregulated in MCC: miR-502-3p, miR-9, miR-7, miR-340, miR-182, miR-190b, miR-873, and miR-183. Three microRNAs were downregulated: miR-3170, miR-125b, and miR-374c. Many of these MCC-miRs, with the miR-183/182/96a cistron in particular, have connections to tumorigenic pathways implicated in MCC pathogenesis. In situ hybridization confirmed that the highly expressed MCC-miR, miR-182, is localized within tumor cells. Furthermore, NGS and qRT-PCR reveals that several of these MCC-miRs are highly expressed in the patient-derived MCC cell line, MS-1. These data indicate that we have identified a set of MCC-miRs with high implications for MCC research. To identify microRNAs specific to Merkel cell carcinoma (MCC) next-generation sequencing (NGS) of small RNA libraries was performed on different tissue samples including MCCs, other cutaneous tumors, and normal skin
Project description:Merkel cell carcinoma (MCC) is a polyomavirus-associated skin cancer that is frequently lethal and lacks established prognostic biomarkers. The purpose of this study was to use gene expression analysis to identify biomarkers that may improve prognostic accuracy and provide insight into MCC biology. Overall design: We profiled 35 tumors from 34 patients. Tumors were macrodissected from surrounding stroma. RNA isolation was performed with RNeasy or Allprep Mini kits (Qiagen, Alameda, CA), and RNA quality assessed by Agilent Bioanalyzer analysis. RNAs were profiled on the Human Rosetta Custom Affymetrix 2.0 chip.
Project description:Purpose: Due to the rarity of Merkel Cell Carcinoma (MCC), prospective clinical trials have not been practical. This study seeks to identify biomarkers to differentiate between patients with a good and poor prognosis. Methods: Patients were stratified into good, moderate or poor prognosis. Merkel cell carcinoma (MCC) patients were stratified into one of three groups based upon status 24 months following treatment. Poor prognosis patients either presented with or progressed to distant metastasis. Patients with favorable prognosis had local disease presentation with no subsequent recurrence or nodal disease at presentation with no progression during follow-up of longer than 24 months. Moderate prognosis had recurrent local disease, development of nodal metastasis, or nodal disease at presentation with no progression during follow-up of fewer than 24 months. Using ArcturusXT Laser Capture Microdissection (Molecular Devices), tumor cells were isolated from the specific areas of interest. The captured tumor tissue was subjected to RNA extraction using the Invitrogen PureLink™ FFPE RNA Isolation Kit. The extracted RNA was analyzed for integrity with the Agilent Bioanalyzer then amplified and hybridized to Affymetrix GeneChip Human Exon 1.0 ST arrays using the NuGEN WT- Ovation™ FFPE System. Results: A total of 191 genes showed significant differential expression (p≤0.05 and 1.5-fold cutoff) between the different between the good and poor prognosis groups. Keratin 20 (KRT20) and Neurofilament protein (NEFM) have been identified in previous studies as proteins of interest in MCC. Our study showed these genes to be significantly upregulated in patients with a poor prognosis. Of interest, phospholipase A2, group X was upregulated in poor responders. Phospholipases liberate arachidonic acid from cellular membranes which can be metabolized to eicosanoids through three major pathways: the cyclooxygenase (COX), the lipoxygenase (LOX) and the cytochrome P450 monooxygenase pathways. This pathway has been implicated in several cancers. Conclusions: The study identified genes with a previous association with MCC as well as some novel genes. These genes provide the basis for further research into possible biomarkers that will enable the differentiation between patients with a good and poor prognosis. Using laser capture microdissection, tumor cells of patients with a good (n=5), moderate (n=3), and poor prognosis (n=7) were isolated from paraffin embedded archival tissue of 15 patients with Merkel cell carcinoma. Affymetrix Human Exon 1.0ST microarrays were utilized to compare gene expression signatures from good and poor prognosis patients.
Project description:Merkel cell carcinoma is a skin cancer associated with the Merkel cell polyomavirus (MCPyV) which is found integrated in the genome of the tumor cells. Here 4 MCPyV-positive tumor cell lines (AlDo, LoKe, BroLi and WoWe) were analyzed by comparative gernomic hybridization to define further genetic alterations. Corresponding peripheral blood lymphocytes (PBL) in case of AlDo and LoKe, purified T cells in case of BroLi and Tumor-derived but virus-negative cells of unknown origin (WoWe1) in case of WoWe served as controls. Overall design: Comparative genomic hybridization to define amplifications and deletions in the MCC genome.
Project description:When using cell lines to study cancer, phenotypic similarity to the original tumor is paramount. Yet, little has been done to characterize how closely Merkel cell carcinoma (MCC) cell lines model native tumors. To determine their similarity to MCC tumor samples, we characterized MCC cell lines via gene expression microarrays. Using whole transcriptome gene expression signatures and a computational bioinformatic approach, we identified significant differences between variant cell lines (UISO, MCC13, and MCC26) and fresh frozen MCC tumors. Conversely, the classic WaGa and Mkl-1 cell lines more closely represented the global transcriptome of MCC tumors. When compared to publicly available cancer lines, WaGa and Mkl-1 cells were similar to other neuroendocrine tumors, but the variant cell lines were not. WaGa and Mkl-1 cells grown as xenografts in mice had histological and immunophenotypical features consistent with MCC, while UISO xenograft tumors were atypical for MCC. Spectral karyotyping and short tandem repeat analysis of the UISO cells matched the original cell line's description, ruling out contamination. Our results validate the use of transcriptome analysis to assess the cancer cell line representativeness and indicate that UISO, MCC13, and MCC26 cell lines are not representative of MCC tumors, whereas WaGa and Mkl-1 more closely model MCC. RNA was extracted from MCC cell lines and MCC and SCLC tumor samples and hybridized to Affymetrix microarrays for transcriptome profiling.
Project description:Merkel cell carcinoma (MCC) is a polyomavirus-associated skin cancer that is frequently lethal and lacks established prognostic biomarkers. The purpose of this study was to use gene expression analysis to identify biomarkers that may improve prognostic accuracy and provide insight into MCC biology. We profiled 35 tumors from 34 patients. Tumors were macrodissected from surrounding stroma. RNA isolation was performed with RNeasy or Allprep Mini kits (Qiagen, Alameda, CA), and RNA quality assessed by Agilent Bioanalyzer analysis. RNAs were profiled on the Human Rosetta Custom Affymetrix 2.0 chip.
Project description:The net transcriptome of a cancer cell is defined by relative levels of transcription factors, activators, suppressors and co-factors. These in turn are controlled by epigenetic, genetic and metabolic restraints. Here we used RNA-Seq, ChIP-qPCR, and ChIP-Seq to determine the impact of MYCN protein levels on p53 and MYCN mediated transcription in neuroblastoma, a p53 wild-type neural crest derived pediatric malignancy. Of note, about 25% of neuroblastoma is MYCN amplified and expresses 10-100 fold higher levels of MYCN protein than non-amplified tumors. We hypothesized that p53 functions would be globally altered by extreme MYCN levels and this could help to explain aggressive biology and poor long term survival which distinguishes MYCN-amplified neuroblastoma. In fact, we found that in the context of high MYCN levels, activation of p53 results in marked changes in transcription of specific p53 and MYCN target loci regulating apoptosis, DNA repair and cell cycle progression. Co-immunoprecipitation of endogenous and GST-fused proteins, as well as ChIP-qPCR confirms formation of p53/MYCN complexes and enrichment of both transcription factors at active loci. This p53/MYC interaction is independent of MYC/MAX complexes. Together, these data demonstrate MYCN to be a direct co-factor modulating p53 transcriptional output in MYCN amplified cancer. Overall design: To explore the MYCN binding at genomic locations under MYCN low and MYCN high conditions
Project description:Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. Using a MYC target gene signature that predicts poor neuroblastoma prognosis we identified the histone chaperone, FAcilitates Chromatin Transcription (FACT), as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small molecule Curaxin compound, CBL0137, markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with chemotherapy in standard use by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN amplified neuroblastoma cells and a treatment strategy for MYCN-driven neuroblastoma Overall design: BE(2)C neuroblastoma cells were transfected with either control non-targeting siRNA's or siRNA's directed against MYCN. 36 hours post-transfection, RNA was isolated for hybridisation on Affymetrix Human Gene 2.0 ST arrays.