ABSTRACT
During acute viral infections, innate immune cells invade inflamed tissues and face hypoxic areas. Hypoxia-inducible factors (HIFs) adapt cellular responses towards these conditions. We wanted to investigate the effects of a loss of HIF-2α in macrophages during acute Friend murine leukemia retrovirus (FV) infection in C57BL/6 mice using a Cre/loxP system. Remarkably, mice with floxed Hif-2a (Hif-2afl; Hif-2a is also known as Epas1) did not show any signs of FV infection independent of Cre activity. This prevented a detailed analysis of the role of macrophage HIF-2α for FV infection but allowed us to study a model of unexpected FV resistance. Hif-2afl mice showed a significant decrease in the expression of the Atp6v1e2 gene encoding for the E2 subunit of the vacuolar H+-ATPase, which resulted in a decreased acidification of lysosomes and limited virus entry into the cell. These findings highlight that the insertion of loxP sites is not always without functional consequences and has established a phenotype in the floxed Hif-2a mouse, which is not only unexpected, but unwanted and is of relevance for the use of this mouse strain in (at least virus) experiments.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors , Friend murine leukemia virus , Vacuolar Proton-Translocating ATPases , Animals , Mice , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Friend murine leukemia virus/genetics , Lysosomes/metabolism , Macrophages/metabolism , Macrophages/virology , Macrophages/immunology , Mice, Inbred C57BL , Retroviridae Infections/genetics , Retroviridae Infections/metabolism , Retroviridae Infections/virology , Tumor Virus Infections/genetics , Tumor Virus Infections/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , Vacuolar Proton-Translocating ATPases/geneticsABSTRACT
BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive malignant neuroendocrine tumour. There are two subsets of MCC, one related to Merkel cell polyomavirus (MCPyV) and the other to ultraviolet radiation (UVR). MCPyV-positive and MCPyV-negative MCCs have been considered to be different tumours, as the former harbour few DNA mutations and are not related to UVR, and the latter usually arise in sun-exposed areas and may be found in conjunction with other keratinocytic tumours, mostly squamous cell carcinomas. Two viral oncoproteins, large T antigen (LT; coded by MCPyV_gp3) and small T antigen (sT; coded by MCPyV_gp4), promote different carcinogenic pathways. OBJECTIVES: To determine which genes are differentially expressed in MCPyV-positive and MCPyV-negative MCC; to describe the mutational burden and the most frequently mutated genes in both MCC subtypes; and to identify the clinical and molecular factors that may be related to patient survival. METHODS: Ninety-two patients with a diagnosis of MCC were identified from the medical databases of participating centres. To study gene expression, a customized panel of 172 genes was developed. Gene expression profiling was performed with nCounter technology. For mutational studies, a customized panel of 26 genes was designed. Somatic single nucleotide variants (SNVs) were identified following the GATK Best Practices workflow for somatic mutations. RESULTS: The expression of LT enabled the series to be divided into two groups (LT positive, n = 55; LT negative, n = 37). Genes differentially expressed in LT-negative patients were related to epithelial differentiation, especially SOX9, or proliferation and the cell cycle (MYC, CDK6), among others. Congruently, LT displayed lower expression in SOX9-positive patients, and differentially expressed genes in SOX9-positive patients were related to epithelial/squamous differentiation. In LT-positive patients, the mean SNV frequency was 4.3; in LT-negative patients it was 10 (P = 0.03). On multivariate survival analysis, the expression of SNAI1 [hazard ratio (HR) 1.046, 95% confidence interval (CI) 1.007-1.086; P = 0.02] and CDK6 (HR 1.049, 95% CI 1.020-1.080; P = 0.001) were identified as risk factors. CONCLUSIONS: Tumours with weak LT expression tend to co-express genes related to squamous differentiation and the cell cycle, and to have a higher mutational burden. These findings are congruent with those of earlier studies.
Merkel cell carcinoma (MCC) is an aggressive form of skin tumour. There are two subtypes of MCC: one of them is related to a virus called Merkel cell polyomavirus (MCPyV); the other one is related to persistent exposure to sunlight. The aim of this research was to find differences between these subtypes in their molecular behaviour (the genes that are expressed and the mutations that may be found). To do this, we carried out two studies, one to investigate gene expression (the process cells use to convert the instructions in our DNA into a functional product such as a protein) and one to look at gene mutations (changes in the DNA sequence). We found that the tumours that were not related to MCPyV expressed genes related to epithelial differentiation (the process by which unspecialized cells gain features characteristics of epithelial cells, which, among other things, make up the outer surface of the body), which means that the origin of both MCC subtypes may be different. We also found that MCPyV-related tumours had fewer mutations. Our findings are important because they help us to understand the biology of the MCC subtypes and could help with the development of new treatments for people diagnosed with skin tumours.
Subject(s)
Antigens, Viral, Tumor , Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , SOX9 Transcription Factor , Skin Neoplasms , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Antigens, Viral, Tumor/genetics , Carcinoma, Merkel Cell/virology , Carcinoma, Merkel Cell/genetics , Carcinoma, Merkel Cell/pathology , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Merkel cell polyomavirus/genetics , Merkel cell polyomavirus/isolation & purification , Mutation , Polyomavirus Infections/genetics , Polyomavirus Infections/virology , Skin Neoplasms/virology , Skin Neoplasms/genetics , Skin Neoplasms/pathology , SOX9 Transcription Factor/genetics , Tumor Virus Infections/genetics , Tumor Virus Infections/virologyABSTRACT
BK polyomavirus-associated nephropathy occurs in kidney transplant recipients under immunosuppressive treatment. BK polyomavirus is implicated in cancer development and invasion, and case reports of renal cell carcinoma and urothelial carcinoma possibly associated with BK polyomavirus has been reported. Further, it has been suggested that the immune responses of KT-related diseases could play a role in the pathogenesis and progression of renal cell carcinoma. Thus, we thought to examine the relationship between BK polyomavirus-associated nephropathy and renal cell carcinoma in terms of gene expression. To identify the common and specific immune responses involved in kidney transplantation-related diseases with a specific focus on BK polyomavirus-associated nephropathy, we performed consensus weighted gene co-expression network analysis on gene profile datasets of renal biopsy samples from different institutions. After the identification of gene modules and validation of the obtained network by immunohistochemistry of the marker across kidney transplantation-related diseases, the relationship between prognosis of renal cell carcinoma and modules was assessed. We included the data from 248 patients and identified the 14 gene clusters across the datasets. We revealed that one cluster related to the translation regulating process and DNA damage response was specifically upregulated in BK polyomavirus-associated nephropathy. There was a significant association between the expression value of hub genes of the identified cluster including those related to cGAS-STING pathway and DNA damage response, and the prognosis of renal cell carcinoma. The study suggested the potential link between kidney transplantation-related diseases, especially specific transcriptomic signature of BK polyomavirus associated nephropathy and renal cell carcinoma.
Subject(s)
BK Virus , Carcinoma, Renal Cell , Carcinoma, Transitional Cell , Kidney Diseases , Kidney Neoplasms , Nephritis, Interstitial , Polyomavirus Infections , Tumor Virus Infections , Urinary Bladder Neoplasms , Humans , BK Virus/genetics , Gene Regulatory Networks , Consensus , Urinary Bladder Neoplasms/complications , Kidney Diseases/complications , Polyomavirus Infections/complications , Kidney Neoplasms/genetics , Kidney Neoplasms/complications , Tumor Virus Infections/geneticsABSTRACT
Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma that is frequently divided into Merkel cell polyomavirus negative and positive tumors due their distinct genomic and transcriptomic profiles, and disease outcomes. Although some prognostic factors in MCC are known, tumorigenic pathways, which that explain outcome differences in MCC are not fully understood. We investigated transcriptomes of 110 tissue samples of a formalin-fixed, paraffin-embedded MCC series by RNA sequencing to identify genes showing a bimodal expression pattern and predicting outcome in cancer and that potentially could play a role in tumorigenesis. We discovered 19 genes among which IGHM, IGKC, NCAN, OTOF, and USH2A were associated also with overall survival (all p-values < 0.05). From these genes, NCAN (neurocan) expression was detected in all 144 MCC samples by immunohistochemistry. Increased NCAN expression was associated with presence of Merkel cell polyomavirus DNA (p = 0.001) and viral large T antigen expression in tumor tissue (p = 0.004) and with improved MCC-specific survival (p = 0.027) and overall survival (p = 0.034). We conclude that NCAN expression is common in MCC, and further studies are warranted to investigate its role in MCC tumorigenesis.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Tumor Virus Infections , Humans , Carcinoma, Merkel Cell/metabolism , Neurocan , Skin Neoplasms/pathology , Merkel cell polyomavirus/genetics , Polyomavirus Infections/genetics , Polyomavirus Infections/complications , Tumor Virus Infections/genetics , Tumor Virus Infections/metabolismABSTRACT
Two accepted possible pathways for Merkel cell carcinoma (MCC) pathogenesis include the clonal integration of the Merkel cell polyomavirus (MCPyV) into the neoplastic cells and by UV irradiation. We hypothesize that, in UV etiology, the expression of genes associated with epithelial-mesenchymal transition (EMT) would be higher in MCPyV-negative MCCs. We compared RNA expression in 16 MCPyV-negative with that in 14 MCPyV-positive MCCs in 30 patients using NanoString panel of 760 gene targets as an exploratory method. Subsequently, we confirmed the findings with a publicly available RNA sequencing data set. The NanoString method showed that 29 of 760 genes exhibited significant deregulation. Ten genes (CD44, COL6A3, COL11A1, CXCL8, INHBA, MMP1, NID2, SPP1, THBS1, and THY1) were part of the EMT pathway. The expression of CDH1/E-cadherin, a key EMT gene, and TWIST1, regulator gene of EMT, was higher in MCPyV-negative tumors. To further investigate the expression of EMT genes in MCPyV-negative MCCs, we analyzed publicly available RNA sequencing data of 111 primary MCCs. Differential expression and gene set enrichment analysis of 35 MCPyV-negative versus 76 MCPyV-positive MCCs demonstrated significantly higher expression of EMT-related genes and associated pathways such as Notch signaling, TGF-ß signaling, and Hedgehog signaling, and UV response pathway in MCPyV-negative MCCs. The significance of the EMT pathway in MCPyV-negative MCCs was confirmed independently by a coexpression module analysis. One of the modules (M3) was specifically activated in MCPyV-negative MCCs and showed significant enrichment for genes involved in EMT. A network analysis of module M3 revealed that CDH1/E-cadherin was among the most connected genes (hubs). E-cadherin and LEF1 immunostains demonstrated significantly more frequent expression in MCPvV-negative versus MCPyV-positive tumors (P < .0001). In summary, our study showed that the expression of EMT-associated genes is higher in MCPyV-negative MCC. Because EMT-related proteins can be targeted, the identification of EMT pathways in MCPyV-negative MCCs is of potential therapeutic relevance.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Tumor Virus Infections , Humans , Carcinoma, Merkel Cell/genetics , Carcinoma, Merkel Cell/metabolism , Carcinoma, Merkel Cell/pathology , Skin Neoplasms/metabolism , Merkel cell polyomavirus/genetics , Tumor Virus Infections/complications , Tumor Virus Infections/genetics , Polyomavirus Infections/complications , Polyomavirus Infections/genetics , Epithelial-Mesenchymal Transition/genetics , Hedgehog Proteins , CadherinsABSTRACT
Merkel cell carcinoma (MCC), a rare primary cutaneous neuroendocrine neoplasm, is extremely aggressive and has a higher mortality rate than melanoma. Based on Merkel cell polyomavirus (MCPyV) status and morphology, MCCs are often divided into several distinct subsets: pure MCPyV-positive, pure MCPyV-negative, and combined MCC. MCPyV-positive MCC develops by the clonal integration of viral DNA, whereas MCPyV-negative MCC is induced by frequent ultraviolet (UV)-mediated mutations, that are characterized by a high mutational burden, UV signature mutations, and many mutations in TP53 and retinoblastoma suppressor gene (RB1). Combined MCC consists of an intimate mix of MCC and other cutaneous tumor populations, and is usually MCPyV-negative, with rare exceptions. Based on the existing subsets of MCC, it is speculated that there are at least 4 stages in the natural history of stem cell differentiation: primitive pluripotent stem cells, divergent differentiated stem cells, unidirectional stem cells, and Merkel cells (or epidermal/adnexal cells). In the first stage, MCPyV may integrate into the genome of primitive pluripotent stem cells, driving oncogenesis in pure MCPyV-positive MCC. If MCPyV integration does not occur, the stem cells enter the second stage and acquire the ability to undergo multidirectional neuroendocrine and epidermal (or adnexal) differentiation. At this stage, accumulated UV-mediated mutations may drive the development of combined MCC. In the third stage, the stem cells differentiate into unidirectional neuroendocrine stem cells, UV-mediated mutations can induce carcinogenesis in pure MCPyV-negative MCC. Therefore, it has been speculated that several subsets of MCCs arise from different stages of differentiation of common stem cells.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Tumor Virus Infections , Humans , Carcinoma, Merkel Cell/pathology , Skin Neoplasms/pathology , Cell Transformation, Neoplastic , Carcinogenesis/genetics , Stem Cells , Cell Differentiation , Merkel cell polyomavirus/genetics , Polyomavirus Infections/genetics , Polyomavirus Infections/pathology , Tumor Virus Infections/complications , Tumor Virus Infections/genetics , Tumor Virus Infections/pathologyABSTRACT
OBJECTIVES: We aim to determine molecular differences between Merkel cell polyomavirus (MCPyV)-negative Merkel cell carcinomas (MCCs) and neuroendocrine carcinomas (NECs). METHODS: Our study included 56 MCCs (28 MCPyV negative, 28 MCPyV positive) and 106 NECs (66 small cell NECs, 21 large cell NECs, and 19 poorly differentiated NECs) submitted for clinical molecular testing. RESULTS: APC, MAP3K1, NF1, PIK3CA, RB1, ROS1, and TSC1 mutations, in addition to high tumor mutational burden and UV signature, were frequently noted in MCPyV-negative MCC in comparison to small cell NEC and all NECs analyzed, while KRAS mutations were more frequently noted in large cell NEC and all NECs analyzed. Although not sensitive, the presence of either NF1 or PIK3CA is specific for MCPyV-negative MCC. The frequencies of KEAP1, STK11, and KRAS alterations were significantly higher in large cell NEC. Fusions were detected in 6.25% (6/96) of NECs yet in none of 45 analyzed MCCs. CONCLUSIONS: High tumor mutational burden and UV signature, as well as the presence of NF1 and PIK3CA mutations, are supportive of MCPyV-negative MCC, whereas KEAP1, STK11, and KRAS mutations are supportive of NEC in the appropriate clinical context. Although rare, the presence of a gene fusion is supportive of NEC.
Subject(s)
Carcinoma, Merkel Cell , Carcinoma, Neuroendocrine , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Tumor Virus Infections , Humans , Skin Neoplasms/pathology , Kelch-Like ECH-Associated Protein 1/genetics , Merkel cell polyomavirus/genetics , Tumor Virus Infections/genetics , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins p21(ras)/genetics , NF-E2-Related Factor 2/genetics , Proto-Oncogene Proteins/genetics , Carcinoma, Neuroendocrine/genetics , High-Throughput Nucleotide Sequencing , Class I Phosphatidylinositol 3-Kinases/geneticsABSTRACT
Merkel cell carcinoma is a rare, aggressive skin tumor initiated by polyomavirus integration or UV light DNA damage. In New Zealand, there is a propensity toward the UV-driven form (31 of 107, 29% virus positive). Using archival formalin-fixed, paraffin-embedded tissues, we report targeted DNA sequencing covering 246 cancer genes on 71 tumor tissues and 38 nonmalignant tissues from 37 individuals, with 33 of 37 being negative for the virus. Somatic variants of New Zealand virus-negative Merkel cell carcinomas partially overlapped with those reported overseas, including TP53 variants in all tumors and RB1, LRP1B, NOTCH1, and EPHA3/7 variants each found in over half of the cohort. Variants in genes not analyzed or reported in previous studies were also found. Cataloging variants in TP53 and RB1 from published datasets revealed a broad distribution across these genes. Chr 1p gain and Chr 3p loss were identified in around 50% of New Zealand virus-negative Merkel cell carcinomas, and RB1 loss of heterozygosity was found in 90% of cases. Copy number variants accumulate in most metastases. Virus-negative Merkel cell carcinomas have complex combinations of somatic DNA-sequence variants and copy number variants. They likely carry the small genomic changes permissive for metastasis from early tumor development; however, chromosomal alterations may contribute to driving metastatic progression.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Tumor Virus Infections , Humans , Carcinoma, Merkel Cell/pathology , Mutation , Skin Neoplasms/genetics , Oncogenes , Chromosome Aberrations , Merkel cell polyomavirus/genetics , Polyomavirus Infections/genetics , Tumor Virus Infections/geneticsABSTRACT
Senescent cells accumulate in the host during the aging process and are associated with age-related pathogeneses, including cancer. Although persistent senescence seems to contribute to many aspects of cellular pathways and homeostasis, the role of senescence in virus-induced human cancer is not well understood. Merkel cell carcinoma (MCC) is an aggressive skin cancer induced by a life-long human infection of Merkel cell polyomavirus (MCPyV). Here, we show that MCPyV large T (LT) antigen expression in human skin fibroblasts causes a novel nucleolar stress response, followed by p21-dependent senescence and senescence-associated secretory phenotypes (SASPs), which are required for MCPyV genome maintenance. Senolytic and navitoclax treatments result in decreased senescence and MCPyV genome levels, suggesting a potential therapeutic for MCC prevention. Our results uncover the mechanism of a host stress response regulating human polyomavirus genome maintenance in viral persistency, which may lead to targeted intervention for MCC.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Tumor Virus Infections , Humans , Polyomavirus Infections/genetics , Tumor Virus Infections/genetics , Tumor Virus Infections/pathology , Antigens, Viral, Tumor/genetics , Merkel cell polyomavirus/genetics , Merkel cell polyomavirus/metabolism , Carcinoma, Merkel Cell/genetics , Carcinoma, Merkel Cell/pathology , Skin Neoplasms/pathology , Cellular Senescence , Genome, ViralABSTRACT
BackgroundMerkel cell carcinoma (MCC) is an aggressive neuroendocrine (NE) skin cancer caused by severe UV-induced mutations or expression of Merkel cell polyomavirus (MCPyV) large and small T antigens (LT and ST). Despite deep genetic differences between MCPyV-positive and -negative subtypes, current clinical diagnostic markers are indistinguishable, and the expression profile of MCC tumors is, to our knowledge, unexplored.MethodsHere, we leveraged bulk and single-cell RNA-Seq of patient-derived tumor biopsies and cell lines to explore the underlying transcriptional environment of MCC.ResultsStrikingly, MCC samples could be separated into transcriptional subtypes that were independent of MCPyV status. Instead, we observed an inverse correlation between a NE gene signature and the Hippo pathway transcription factors Yes1-associated transcriptional regulator (YAP1) and WW domain-containing transcriptional regulator 1 (WWTR1). This inverse correlation was broadly present at the transcript and protein levels in the tumor biopsies as well as in established and patient-derived cell lines. Mechanistically, expression of YAP1 or WWTR1 in a MCPyV-positive MCC cell line induced cell-cycle arrest at least in part through TEA domain-dependent (TEAD-dependent) transcriptional repression of MCPyV LT.ConclusionThese findings identify what we believe to be a previously unrecognized heterogeneity in NE gene expression within MCC and support a model of YAP1/WWTR1 silencing as essential for the development of MCPyV-positive MCC.FundingUS Public Health Service grants R35CA232128, P01CA203655, and P30CA06516.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Tumor Virus Infections , Humans , Carcinoma, Merkel Cell/genetics , Carcinoma, Merkel Cell/pathology , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Merkel cell polyomavirus/genetics , Intracellular Signaling Peptides and Proteins , Cell Line , Polyomavirus Infections/genetics , Tumor Virus Infections/genetics , Transcriptional Coactivator with PDZ-Binding Motif ProteinsABSTRACT
BACKGROUND: Cytochrome P450 3A5 (CYP3A5) includes two active genotypes, namely CYP3A5*1/*1 or *1/*3 with the fast metabolic activity and CYP3A5*3/*3 with slow metabolic. We retrospectively analyzed the correlation between CYP3A5 gene polymorphism and the susceptibility to the BK virus (BKV) infection in renal transplant recipients. METHODS: According to the inclusion/ exclusion criteria, we selected 134 recipients who received kidney transplantation at the Renmin Hospital of Wuhan University from January 2019 to December 2019. Based on the pre-operative CYP3A5 sequencing results, 134 recipients were divided into two groups: those expressing the fast metabolic CYP3A5*1/*1 or *1/*3 genotype; and, those expressing slow metabolic CYP3A5*3/*3 genotype. These two recipient groups were then analyzed for the BKV infection rate with different metabolic types to establish the potential relationship between CYP3A5 gene polymorphism and BKV infection. RESULTS: The overall incidence of BKV viruria was 37.3%, whereas BKV viremia was 4.5% among all 134 recipients. The fast metabolism group had 9.1% incidence of BKV viremia and 49.1% incidence of BKV viruria. In contrast, the slow metabolism group had only 1.3%incidence of BKV viremia (P = 0.031) with only 29.1% BKV viruria (P = 0.011). The incidence of low levels of urinary BKV in the fast metabolism group was higher than that in the slow metabolism group (P = 0.005), while no significant statistical difference in the incidence of high levels of urinary BKV and high and low levels of blood BKV. CONCLUSION: After kidney transplantation, CYP3A5 gene polymorphism of recipients present a certain relationship with the occurrence of BKV infection, which may be of value for the prediction and prevention of BKV infection.
Subject(s)
Cytochrome P-450 CYP3A , Kidney Transplantation , Polyomavirus Infections , Tumor Virus Infections , Humans , BK Virus , Cytochrome P-450 CYP3A/genetics , Kidney Diseases/surgery , Kidney Transplantation/adverse effects , Polymorphism, Genetic , Polyomavirus Infections/epidemiology , Polyomavirus Infections/genetics , Retrospective Studies , Transplant Recipients , Tumor Virus Infections/epidemiology , Tumor Virus Infections/genetics , Viremia/epidemiology , Viremia/geneticsABSTRACT
The germinal center (GC) plays a central role in the generation of antigen-specific B cells and antibodies. Tight regulation of the GC is essential due to the inherent risks of tumorigenesis and autoimmunity posed by inappropriate GC B cell processes. Gammaherpesviruses such as Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) utilize numerous armaments to drive infected naïve B cells, independent of antigen, through GC reactions to expand the latently infected B cell population and establish a stable latency reservoir. We previously demonstrated that the MHV68 microRNA (miRNA) mghv-miR-M1-7-5p represses host EWSR1 (Ewing sarcoma breakpoint region 1) to promote B cell infection. EWSR1 is a transcription and splicing regulator that is recognized for its involvement as a fusion protein in Ewing sarcoma. A function for EWSR1 in B cell responses has not been previously reported. Here, we demonstrate that 1) B cell-specific deletion of EWSR1 had no effect on generation of mature B cell subsets or basal immunoglobulin levels in naïve mice, 2) repression or ablation of EWSR1 in B cells promoted expansion of MHV68 latently infected GC B cells, and 3) B cell-specific deletion of EWSR1 during a normal immune response to nonviral antigen resulted in significantly elevated numbers of antigen-specific GC B cells, plasma cells, and circulating antibodies. Notably, EWSR1 deficiency did not affect the proliferation or survival of GC B cells but instead resulted in the generation of increased numbers of precursor GC B cells. Cumulatively, these findings demonstrate that EWSR1 is a negative regulator of B cell responses.
Subject(s)
B-Lymphocytes , Gammaherpesvirinae , Germinal Center , Herpesviridae Infections , MicroRNAs , RNA-Binding Protein EWS , Tumor Virus Infections , Animals , B-Lymphocytes/immunology , B-Lymphocytes/virology , Gammaherpesvirinae/genetics , Gammaherpesvirinae/physiology , Gene Deletion , Germinal Center/immunology , Germinal Center/virology , Herpesviridae Infections/genetics , Herpesviridae Infections/immunology , Herpesviridae Infections/virology , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , RNA-Binding Protein EWS/genetics , RNA-Binding Protein EWS/metabolism , Tumor Virus Infections/genetics , Tumor Virus Infections/immunology , Tumor Virus Infections/virology , Virus LatencyABSTRACT
BACKGROUND: BK polyoma virus (BKPyV) associated nephropathy (BKPyVAN) is a major cause of kidney graft loss in renal transplant patients. Interferons (IFNs) are an important innate immune response against viral infections and genetic polymorphisms of the IFN-pathways can affect susceptibility and mortality during viral infection. Here, we investigated whether the dinucleotide polymorphism rs368234815 (ΔG/TT) in the IFNL4 gene contributed to BKPyV reactivation or BKPyVAN after living-donor kidney transplantation. METHODS: This retrospective case-control study determines the prevalence of IFNL4 variants in a Caucasian population of living-donor kidney transplant recipients and donors and explores its association with BKPyV infection and BKPyVAN development. We included 28 recipients with BKPyV reactivation, 10 of which developed BKPyVAN and 30 BKPyV negative controls. Targeted sequencing of the IFNL4 gene from both recipients and their respective donors was performed. RESULTS: We found IFNL4 rs368234815 ΔG allele frequencies of 41.7% in BKPyV negative and 39.3% in BKPyV positive recipients (P = .85), and 41.7% and 40.4% (P>.99) in their respective donors. IFNL4 rs368234815 ΔG allele frequencies in BKPyVAN developing recipients and their respective donors were 50% and 43.7% (P = .60 and P>.99). CONCLUSIONS: Our results indicate that the IFNL4 rs368234815 ΔG allele is not associated with BKPyV reactivation, nor the manifestation of BKPyVAN.
Subject(s)
BK Virus , Kidney Transplantation , Polyomavirus Infections , Tumor Virus Infections , Case-Control Studies , Humans , Interleukins , Kidney Transplantation/adverse effects , Kidney Transplantation/methods , Living Donors , Polymorphism, Genetic , Polyomavirus Infections/epidemiology , Polyomavirus Infections/genetics , Retrospective Studies , Transplant Recipients , Tumor Virus Infections/complications , Tumor Virus Infections/geneticsABSTRACT
Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer that frequently carries an integrated Merkel cell polyomavirus (MCPyV) genome and expresses viral transforming antigens (TAgs). MCC tumor cells also express signature genes detected in skin-resident, postmitotic Merkel cells, including atonal bHLH transcription factor 1 (ATOH1), which is required for Merkel cell development from epidermal progenitors. We now report the use of in vivo cellular reprogramming, using ATOH1, to drive MCC development from murine epidermis. We generated mice that conditionally expressed MCPyV TAgs and ATOH1 in epidermal cells, yielding microscopic collections of proliferating MCC-like cells arising from hair follicles. Immunostaining of these nascent tumors revealed p53 accumulation and apoptosis, and targeted deletion of transformation related protein 53 (Trp53) led to development of gross skin tumors with classic MCC histology and marker expression. Global transcriptome analysis confirmed the close similarity of mouse and human MCCs, and hierarchical clustering showed conserved upregulation of signature genes. Our data establish that expression of MCPyV TAgs in ATOH1-reprogrammed epidermal cells and their neuroendocrine progeny initiates hair follicle-derived MCC tumorigenesis in adult mice. Moreover, progression to full-blown MCC in this model requires loss of p53, mimicking the functional inhibition of p53 reported in human MCPyV-positive MCCs.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Tumor Virus Infections , Animals , Antigens, Viral , Antigens, Viral, Tumor/genetics , Antigens, Viral, Tumor/metabolism , Carcinoma, Merkel Cell/genetics , Carcinoma, Merkel Cell/metabolism , Carcinoma, Merkel Cell/pathology , Cellular Reprogramming , Merkel cell polyomavirus/genetics , Mice , Polyomavirus Infections/genetics , Polyomavirus Infections/pathology , Skin Neoplasms/pathology , Tumor Virus Infections/genetics , Tumor Virus Infections/pathologyABSTRACT
Merkel cell carcinoma (MCC) is a neuroendocrine tumor either induced by integration of the Merkel cell polyomavirus into the cell genome or by accumulation of UV-light-associated mutations (VP-MCC and UV-MCC). Whether VP- and UV-MCC have the same or different cellular origins is unclear; with mesenchymal or epidermal origins discussed. DNA-methylation patterns have a proven utility in determining cellular origins of cancers. Therefore, we used this approach to uncover evidence regarding the cell of origin of classical VP- and UV-MCC cell lines, i.e., cell lines with a neuroendocrine growth pattern (n = 9 and n = 4, respectively). Surprisingly, we observed high global similarities in the DNA-methylation of UV- and VP-MCC cell lines. CpGs of lower methylation in VP-MCC cell lines were associated with neuroendocrine marker genes such as SOX2 and INSM1, or linked to binding sites of EZH2 and SUZ12 of the polycomb repressive complex 2, i.e., genes with an impact on carcinogenesis and differentiation of neuroendocrine cancers. Thus, the observed differences appear to be rooted in viral compared to mutation-driven carcinogenesis rather than distinct cells of origin. To test this hypothesis, we used principal component analysis, to compare DNA-methylation data from different epithelial and non-epithelial neuroendocrine cancers and established a scoring model for epithelial and neuroendocrine characteristics. Subsequently, we applied this scoring model to the DNA-methylation data of the VP- and UV-MCC cell lines, revealing that both clearly scored as epithelial cancers. In summary, our comprehensive analysis of DNA-methylation suggests a common epithelial origin of UV- and VP-MCC cell lines.
Subject(s)
Carcinoma, Merkel Cell/genetics , DNA Methylation/genetics , High-Throughput Screening Assays/methods , Tumor Virus Infections/genetics , HumansABSTRACT
Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine carcinoma that is classified as Merkel cell polyomavirus-positive (virus positive [VP]) or Merkel cell polyomavirus-negative (virus negative [VN]). Epigenetic changes, such as DNA methylation, can alter gene expression and influence cancer progression. However, patterns of DNA methylation and the therapeutic efficacy of hypomethylating agents have not been fully explored in MCC. We characterized genome-wide DNA methylation in 16 MCC cell lines from both molecular subclasses in comparison with other cancer types and found that the overall profile of MCC is similar to that of small-cell lung carcinoma. Comparison of VP MCC with VN MCC revealed 2,260 differentially methylated positions. The hypomethylating agent decitabine upregulated the expression of antigen-presenting machinery in MCC cell lines and stimulated membrane expression of HLA-A in VP and VN MCC xenograft tumors. Decitabine also induced prominent caspase- and large T antigenâindependent cell death in VP MCC, whereas VN MCC cell lines displayed decreased proliferation without increased cell death. In mouse xenografts, decitabine significantly decreased the size of VP tumors but not that of VN tumors. Our findings indicate that viral status predicts genomic methylation patterns in MCC and that decitabine may be therapeutically effective against MCC through antiproliferative effects, cell death, and increased immune recognition.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Tumor Virus Infections , Animals , Carcinoma, Merkel Cell/drug therapy , Carcinoma, Merkel Cell/genetics , Carcinoma, Merkel Cell/pathology , DNA Methylation , Decitabine/pharmacology , Decitabine/therapeutic use , Humans , Merkel cell polyomavirus/genetics , Mice , Skin Neoplasms/drug therapy , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Tumor Virus Infections/geneticsABSTRACT
Colorectal cancer is ranked to have high mortality among most malignancies worldwide. In the adult population, the seroprevalence rates of the John Cunningham virus (JCV) range from 70% to 90%. Recently the association for JCV in many malignant tumours have been reported worldwide, including colonic and rectum cancers. Interleukin-1ß (IL-1ß) can promote tumour growth where it is abundant in the tumour microenvironment, and its up-regulation is considered a poor prognostic feature in different types of solid tumours, including colon malignancies. One hundred tissue biopsies belonged to 50 patients with colorectal cancers and 30 benign colonic tumour patients, and 20 colorectal control tissues were enrolled in this study. JCV was detected via chromogenic in situ hybridization (CISH), while IL-1 beta was detected by immunohistochemistry (IHC). The recorded data showed that 21 out of 50 (42%) tissue samples with colorectal carcinoma showed positive CISH reactions for JCV DNA in this study. The benign colorectal tumours group revealed positive signals in 2 out of 30 tissues representing 6.7% of this group. Lastly, no control tissues showed positive signals for the JCV -CISH test. The positive signals of IL-1 Beta-IHC detection were found in 26 out of 50 (52 %) colorectal carcinoma tissues, while in the benign colorectal tumour was 43.3% (13 out of 30) and in AHC was 20% (4 out of 20 tissues). The high rates of JCV infection in this group of Iraqi patients with colonic adenocarcinoma in concordance with IL-1 Beta expression could play an essential role in the development and progression of these malignant tumours along with benign colonic tumours. To analyze the concordant expression of IL-1 beta gene and JCV in issues from a group of Iraqi patients with colonic adenocarcinomas.
Subject(s)
Colorectal Neoplasms , Interleukin-1beta , JC Virus , Tumor Virus Infections , Adult , Humans , Colorectal Neoplasms/epidemiology , Colorectal Neoplasms/virology , Iraq/epidemiology , JC Virus/genetics , JC Virus/metabolism , Seroepidemiologic Studies , Tumor Microenvironment , Tumor Virus Infections/epidemiology , Tumor Virus Infections/geneticsABSTRACT
BACKGROUND: Viral infections are prevalent in human cancers and they have great diagnostic and theranostic values in clinical practice. Recently, their potential of shaping the tumor immune microenvironment (TIME) has been related to the immunotherapy of human cancers. However, the landscape of viral expressions and immune status in human cancers remains incompletely understood. METHODS: We developed a next-generation sequencing (NGS)-based pipeline to detect viral sequences from the whole transcriptome and used machine learning algorithms to classify different TIME subtypes. RESULTS: We revealed a pan-cancer landscape of viral expressions in human cancers where 9 types of viruses were detected in 744 tumors of 25 cancer types. Viral infections showed different tissue tendencies and expression levels. Multi-omics analyses further revealed their distinct impacts on genomic, transcriptomic and immune responses. Epstein-Barr virus (EBV)-infected stomach adenocarcinoma (STAD) and Human Papillomavirus (HPV)-infected head and neck squamous cell carcinoma (HNSC) showed decreased genomic variations, significantly altered gene expressions, and effectively triggered anti-viral immune responses. We identified three TIME subtypes, in which the "Immune-Stimulation" subtype might be the promising candidate for immunotherapy. EBV-infected STAD and HPV-infected HNSC showed a higher frequency of the "Immune-Stimulation" subtype. Finally, we constructed the eVIIS pipeline to simultaneously evaluate viral infection and immune status in external datasets. CONCLUSIONS: Viral infections are prevalent in human cancers and have distinct influences on hosts. EBV and HPV infections combined with the TIME subtype could be promising biomarkers of immunotherapy in STAD and HNSC, respectively. The eVIIS pipeline could be a practical tool to facilitate clinical practice and relevant studies.
Subject(s)
Immunotherapy , Machine Learning , Neoplasms , Oncogenic Viruses , Tumor Microenvironment , Tumor Virus Infections , Biomarkers, Tumor/genetics , Biomarkers, Tumor/immunology , DNA, Viral/genetics , Epstein-Barr Virus Infections , Genetic Variation , Genome, Viral , Head and Neck Neoplasms/immunology , Head and Neck Neoplasms/therapy , Head and Neck Neoplasms/virology , Herpesvirus 4, Human/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Kaplan-Meier Estimate , Leukocytes/classification , Leukocytes/cytology , Mutation , Neoplasms/immunology , Neoplasms/therapy , Neoplasms/virology , Oncogenic Viruses/genetics , Oncogenic Viruses/immunology , Papillomaviridae/genetics , Papillomavirus Infections , RNA-Seq , Squamous Cell Carcinoma of Head and Neck/immunology , Squamous Cell Carcinoma of Head and Neck/virology , Stomach Neoplasms/immunology , Stomach Neoplasms/therapy , Stomach Neoplasms/virology , Support Vector Machine , Transcriptome , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology , Tumor Virus Infections/genetics , Tumor Virus Infections/immunologyABSTRACT
The human BK polyomavirus (BKPyV) is latent in the kidneys of most adults, but can be reactivated in immunosuppressed states, such as following renal transplantation. If left unchecked, BK polyomavirus nephropathy (PyVAN) and possible graft loss may result from viral destruction of tubular epithelial cells and interstitial fibrosis. When coupled with regular post-transplant screening, immunosuppression reduction has been effective in limiting BKPyV viremia and the development of PyVAN. Antiviral drugs that are safe and effective in combating BKPyV have not been identified but would be a benefit in complementing or replacing immunosuppression reduction. The present study explores inhibition of the host DNA damage response (DDR) as an antiviral strategy. Immunohistochemical and immunofluorescent analyses of PyVAN biopsies provide evidence for stimulation of a DDR in vivo. DDR pathways were also stimulated in vitro following BKPyV infection of low-passage human renal proximal tubule epithelial cells. The role of Chk1, a protein kinase known to be involved in the replication stress-induced DDR, was examined by inhibition with the small molecule LY2603618 and by siRNA-mediated knockdown. Inhibition of Chk1 resulted in decreased replication of BKPyV DNA and viral spread. Activation of mitotic pathways was associated with the reduction in BKPyV replication. Chk1 inhibitors that are found to be safe and effective in clinical trials for cancer should also be evaluated for antiviral activity against BKPyV.
Subject(s)
BK Virus/genetics , Checkpoint Kinase 1/metabolism , Polyomavirus Infections/drug therapy , BK Virus/pathogenicity , Cells, Cultured , Checkpoint Kinase 1/physiology , DNA Damage/physiology , DNA Repair/physiology , Humans , Kidney/pathology , Kidney/virology , Kidney Transplantation , Phenylurea Compounds/pharmacology , Polyomavirus Infections/genetics , Polyomavirus Infections/immunology , Pyrazines/pharmacology , Tumor Virus Infections/drug therapy , Tumor Virus Infections/genetics , Virus Replication/drug effects , Virus Replication/physiologyABSTRACT
Though viruses have their own genomes, many depend on the nuclear environment of their hosts for replication and survival. A substantial body of work has therefore been devoted to understanding how viral and eukaryotic genomes interact. Recent advances in chromosome conformation capture technologies have provided unprecedented opportunities to visualize how mammalian genomes are organized and, by extension, how packaging of nuclear DNA impacts cellular processes. Recent studies have indicated that some viruses, upon entry into host cell nuclei, produce factors that alter host chromatin topology, and thus, impact the 3D organization of the host genome. Additionally, a variety of distinct viruses utilize host genome architectural factors to advance various aspects of their life cycles. Indeed, human gammaherpesviruses, known for establishing long-term reservoirs of latent infection in B lymphocytes, utilize 3D principles of genome folding to package their DNA and establish latency in host cells. This manipulation of host epigenetic machinery by latent viral genomes is etiologically linked to the onset of B cell oncogenesis. Small DNA viruses, by contrast, are tethered to distinct cellular sites that support virus expression and replication. Here, we briefly review the recent findings on how viruses and host genomes spatially communicate, and how this impacts virus-induced pathology.