Merkel cell polyomavirus pan-T antigen knockdown reduces cancer cell stemness and promotes neural differentiation independent of RB1
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ABSTRACT: Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC’s oncogenic phenotype, i.e., repression of TAs results in reactivation of the RB pathway and subsequent cell cycle arrest. However, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond inhibition of the RB pathway, revealing the decrease in expression of stem cell-related genes upon panTA-knockdown. Analysis of gene regulatory networks identified members of the E2F family (E2F1, E2F8, TFDP1) as key transcriptional regulators that maintain stem cell properties in TA-expressing MCC cells. Furthermore, minichromosome maintenance (MCM) genes, which encodes DNA-binding license proteins essential for stem cell maintenance, were suppressed upon panTA-knockdown. The decline in stemness occurred simultaneously with neural differentiation, marked by the increased expression of neurogenesis-related genes such as neurexins, BTG2, and MYT1L. This upregulation can be attributed to heightened activity of PBX1 and BPTF, crucial regulators of neurogenesis pathways. The observations in LoKe were confirmed in an additional MCPyV-positive MCC cell line in which RB1 was silenced prior to panTA-knockdown. Moreover, spatially resolved transcriptomics demonstrated reduced TA expression in situ in a part of a MCC tumor characterized by neural differentiation. In summary, TAs are critical for maintaining stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway.
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: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.
Project description:Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high mortality rate. Merkel cell polyomavirus (MCPyV) is the etiology of 80% of MCC cases via expression of the viral oncogenes small T antigen (sT) and truncated large T antigen (tLT). These proteins impair the Rb1-dependent G1/S checkpoint blockade and subvert the host cell epigenome to promote proliferation and evade immune detection. Through whole proteome analysis and proximal interactomics, we identified a tLT-dependent deregulation of DNA damage response (DDR) proteins. We investigated a novel protein proximal interaction between tLT and the histone methyltransferase EHMT2. In this regard, TA knockdown in MCC cells reduced DDR protein levels and increased levels of the DNA damage marker γH2Ax. EHMT2 normally promotes H3K9 methylation and DDR signaling. Given that inhibition of EHMT2 did not result in extensive proteomic changes, we hypothesized that the tLT-EHMT2 interaction could be involved in DDR pathway deregulation. In the presence of tLT, we report that EHMT2 gained double and single-strand break repair proximal interactors. EHMT2 inhibition rescued proliferation in MCC cells depleted for their T antigens, suggesting impaired DDR and/or lack of cell cycle checkpoint efficiency. Combined tLT and EHMT2 inhibition led to altered DDR signaling, evidenced by multiple alterations in signal transduction. Together, our data strongly suggest that tLT hijacks multiple components of the DNA damage machinery to enhance tolerance to DNA damage in MCC cells which could be at the basis of, the remarkable genetic stability of these cancers.
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: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.
Project description:Merkel cell polyomavirus (MCPyV) is the first human polyomavirus etiologically associated with Merkel cell carcinoma (MCC), a rare and aggressive form of skin cancer. Similar to other polyomaviruses, MCPyV encodes early T antigen genes, a viral oncogene required for MCC tumor growth. To identify the unique oncogenic properties of MCPyV, we analysed the gene expression profiles in human spontaneously immortalized keratinocytes (NIKs) expressing the early genes from five distinct human polyomaviruses (PyVs), including MCPyV. A comparison of the gene expression profiles revealed 28 genes specifically deregulated by MCPyV.
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 (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 (MCC) is an aggressive skin cancer with high propensity for metastasis, caused by Merkel cell polyomavirus (MCPyV), or chronic UV-light-exposure. How MCPyV modulates immune responses within the tumor microenvironment and how such are linked to patient outcomes remain unknown partly due to technical barriers to understanding the spatial organization of the tumor microenvironment at single-cell resolution. We interrogated the cellular and transcriptional landscapes of 60 MCC-patients using Co-detection-by-indexing (CODEX) and targeted bulk RNA sequencing. Notably, we identified an enrichment of dysfunctional T cells spatially associating with CXCL9+ myeloid cells at the tumor invasive front as key feature of virus-positive MCC. While MCPyV-positivity and CD8+ T cell infiltration correlated with metastasis-free-survival, responses to immune checkpoint blockade were high regardless of virus-status. Instead, we found an enrichment of central memory T cells within tertiary-lymphoid-structures that associated with response to immune-checkpoint blockade. These findings highlight fundamental differences in the organization of the native tumor microenvironment of virus-positive MCC, that are linked to differential survival outcomes and could be used for personalized treatment strategies.
Project description:Understanding immunotherapy resistance is challenging due to difficulty identifying cancer-specific T cells. Merkel cell carcinoma (MCC) is typically driven by Merkel cell polyomavirus (MCPyV), facilitating identification of cancer-specific T cells across patients. We characterized cancer-specific T cells in 35 MCC patients, including participants in a neoadjuvant anti-PD-1 trial. Higher MCPyV-specific CD8 T-cell frequency in pre-treatment blood (but not tumors) correlated with response (p=0.005). Single-cell transcriptomics revealed MCPyV-specific CD8 T cells in blood had increased stem/memory signatures and decreased exhaustion signatures relative to their intratumoral counterparts. While frequency of peripheral cancer-specific T cells was associated with response to initial PD-(L)1 blockade, longitudinal study of acquired resistance revealed immune evasion by tumor-cell MHC-I downregulation despite cancer-specific T cell expansion to 0.5% in blood. Blood thus appears to be an important reservoir of cancer-specific CD8 T cells and adoptive cell therapies may be particularly effective in patients without such cells.