The Merkel Cell Polyomavirus T Antigens Function as Tumor Promoters in Murine Skin

Merkel cell polyomavirus (MCPyV) causes the majority of human Merkel cell carcinomas (MCC), a rare but highly aggressive form of skin cancer. We recently reported that constitutive expression of MCC tumor-derived MCPyV tumor (T) antigens in the skin of transgenic mice leads to hyperplasia, increased proliferation, and spontaneous epithelial tumor development. We sought to evaluate how the MCPyV T antigens contribute to tumor formation in vivo using a classical, multi-stage model for squamous cell carcinoma development. In this model, two chemical carcinogens, DMBA and TPA, contribute to two distinct phases of carcinogenesis—initiation and promotion, respectively—that are required for tumors to develop. By treating the MCPyV transgenic mice with each chemical carcinogen, we determined how the viral oncogenes contributed to carcinogenesis. We observed that the MCPyV T antigens synergized with the tumor initiator DMBA, but not with the tumor promoter TPA, cause tumors. Therefore, the MCPyV tumor antigens function primarily as tumor promoters, similar to that seen with human papillomavirus (HPV) oncoproteins. These studies provide insight into the role of MCPyV T antigen expression in tumor formation in vivo and contribute to our understanding of how MCPyV may function as a human DNA tumor virus.

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Merkel Cell Polyomavirus Small T Antigen Is Oncogenic in Transgenic Mice

Journal of Investigative Dermatology ◽

10.1038/jid.2014.446 ◽

2015 ◽

Vol 135 (5) ◽

pp. 1415-1424 ◽

Cited By ~ 69

Author(s):

Monique E. Verhaegen ◽

Doris Mangelberger ◽

Paul W. Harms ◽

Tracy D. Vozheiko ◽

Jack W. Weick ◽

...

Keyword(s):

Transgenic Mice ◽

T Antigen ◽

Merkel Cell Polyomavirus ◽

Merkel Cell ◽

Small T Antigen

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Rapid rational drug targeting of Merkel cell polyomavirus (MCV)-positive Merkel cell carcinoma (MCC) using the survivin inhibitor YM155.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.8577 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 8577-8577

Author(s):

Reety Arora ◽

Masahiro Shuda ◽

Anna Guastafierro ◽

Tuna Toptan ◽

Yanis Tolstov ◽

...

Keyword(s):

Xenograft Model ◽

Survivin Expression ◽

T Antigen ◽

Merkel Cell Polyomavirus ◽

Myelogenous Leukemia ◽

Merkel Cell ◽

Rational Drug ◽

Mouse Xenograft Model

8577 Background: MCC is an aggressive, chemoresistant skin cancer causing more deaths each year than chronic myelogenous leukemia. We discovered a new virus, Merkel cell polyomavirus (MCV), clonally integrated into ~80% of primary and metastatic MCC in 2008. To find therapeutic targets for this cancer, we examined cellular genes perturbed by MCV infection. Methods: Digital transcriptome subtraction was used to discover MCV and also to reveal survivin gene (BIRC5) upregulation in virus-positive tumors. MCV T antigen knockdown studies in seven MCC lines and large T (LT) transduction into BJ fibroblasts were used to confirm this. Drug screening was performed in vitro using Cell-Titer Glo assays in a two stage analysis. In vivo screening used an MKL-1 (MCV+) MCC NOD-SCIDg mouse xenograft model with a single three-week treatment round. Results: MCV large T oncoprotein induces survivin transcription through retinoblastoma protein sequestration by the LT LXCXE motif. MCV T antigen knockdown results in nonapoptotic MCC cell death and loss of survivin expression. YM155, a phase II survivin transcription inhibitor, causes MCV+ MCC cell necroptosis associated with autophagy at 1-12 nM EC50. Of 1359 other drugs from LOPAC and NCI Oncology Set II libraries, only bortezomib had in vitro potency comparable to YM155. In MKL-1 xenograft studies, mice were treated with saline, bortezomib or YM155 for three weeks using standard dosings. Bortezomib did not significantly improve mouse survival (33%) over saline (24%) during treatment. In contrast, all YM155-treated mice survived (100%, p<0.001) the 3 week treatment period. Tumors resumed growth once YM155 treatment was stopped suggesting that YM155 is cytostatic in vivo rather than cytotoxic. Conclusions: Survivin expression is induced by MCV LT and is critical to MCV+ MCC survival. A survivin inhibitor, YM155 was nontoxic to mice and cytostatic for MCV+ MCC xenografts. Using genomic technologies, in less than four years, the primary viral cause for most MCC was discovered, new diagnostic tests developed and a promising rational drug candidate identified. A cooperative group trial (E1611) for YM155 and bortezomib in MCC patients is currently planned.

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Artesunate Affects T Antigen Expression and Survival of Virus-Positive Merkel Cell Carcinoma

Cancers ◽

10.3390/cancers12040919 ◽

2020 ◽

Vol 12 (4) ◽

pp. 919 ◽

Cited By ~ 1

Author(s):

Bhavishya Sarma ◽

Christoph Willmes ◽

Laura Angerer ◽

Christian Adam ◽

Jürgen C. Becker ◽

...

Keyword(s):

Cell Carcinoma ◽

Merkel Cell Carcinoma ◽

Antigen Expression ◽

T Antigen ◽

Merkel Cell ◽

T Antigens ◽

Growth And Survival ◽

Proliferative Effect

Merkel cell carcinoma (MCC) is a rare and highly aggressive skin cancer with frequent viral etiology. Indeed, in about 80% of cases, there is an association with Merkel cell polyomavirus (MCPyV); the expression of viral T antigens is crucial for growth of virus-positive tumor cells. Since artesunate—a drug used to treat malaria—has been reported to possess additional anti-tumor as well as anti-viral activity, we sought to evaluate pre-clinically the effect of artesunate on MCC. We found that artesunate repressed growth and survival of MCPyV-positive MCC cells in vitro. This effect was accompanied by reduced large T antigen (LT) expression. Notably, however, it was even more efficient than shRNA-mediated downregulation of LT expression. Interestingly, in one MCC cell line (WaGa), T antigen knockdown rendered cells less sensitive to artesunate, while for two other MCC cell lines, we could not substantiate such a relation. Mechanistically, artesunate predominantly induces ferroptosis in MCPyV-positive MCC cells since known ferroptosis-inhibitors like DFO, BAF-A1, Fer-1 and β-mercaptoethanol reduced artesunate-induced death. Finally, application of artesunate in xenotransplanted mice demonstrated that growth of established MCC tumors can be significantly suppressed in vivo. In conclusion, our results revealed a highly anti-proliferative effect of the approved and generally well-tolerated anti-malaria compound artesunate on MCPyV-positive MCC cells, suggesting its potential usage for MCC therapy.

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Merkel Cell Polyomavirus-Infected Merkel Cell Carcinoma Cells Require Expression of Viral T Antigens

Journal of Virology ◽

10.1128/jvi.02400-09 ◽

2010 ◽

Vol 84 (14) ◽

pp. 7064-7072 ◽

Cited By ~ 278

Author(s):

Roland Houben ◽

Masahiro Shuda ◽

Rita Weinkam ◽

David Schrama ◽

Huichen Feng ◽

...

Keyword(s):

Cell Carcinoma ◽

Cell Lines ◽

Merkel Cell Carcinoma ◽

Annexin V ◽

T Antigen ◽

Merkel Cell Polyomavirus ◽

Merkel Cell ◽

T Antigens ◽

Negative Cell ◽

Exon 1

ABSTRACT Merkel cell carcinoma (MCC) is the most aggressive skin cancer. Recently, it was demonstrated that human Merkel cell polyomavirus (MCV) is clonally integrated in ∼80% of MCC tumors. However, direct evidence for whether oncogenic viral proteins are needed for the maintenance of MCC cells is still missing. To address this question, we knocked down MCV T-antigen (TA) expression in MCV-positive MCC cell lines using three different short hairpin RNA (shRNA)-expressing vectors targeting exon 1 of the TAs. The MCC cell lines used include three newly generated MCV-infected cell lines and one MCV-negative cell line from MCC tumors. Notably, all MCV-positive MCC cell lines underwent growth arrest and/or cell death upon TA knockdown, whereas the proliferation of MCV-negative cell lines remained unaffected. Despite an increase in the number of annexin V-positive, 7-amino-actinomycin D (7-AAD)-negative cells upon TA knockdown, activation of caspases or changes in the expression and phosphorylation of Bcl-2 family members were not consistently detected after TA suppression. Our study provides the first direct experimental evidence that TA expression is necessary for the maintenance of MCV-positive MCC and that MCV is the infectious cause of MCV-positive MCC.

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Merkel cell polyomavirus T antigens promote cell proliferation and inflammatory cytokine gene expression

Journal of General Virology ◽

10.1099/jgv.0.000287 ◽

2015 ◽

Vol 96 (12) ◽

pp. 3532-3544 ◽

Cited By ~ 19

Author(s):

Kathleen F. Richards ◽

Anna Guastafierro ◽

Masahiro Shuda ◽

Tuna Toptan ◽

Patrick S. Moore ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cellular Proliferation ◽

Human Fibroblasts ◽

T Antigen ◽

Merkel Cell Polyomavirus ◽

Cytokine Gene Expression ◽

Merkel Cell ◽

T Antigens ◽

Gene Expression Alterations

Merkel cell polyomavirus (MCV) is clonally integrated in over 80 % of Merkel cell carcinomas and mediates tumour development through the expression of viral oncoproteins, the 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 C-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 tumour-derived LT, tumour-derived LT along with sT, and tumour-derived LT with a mutated Rb interaction domain. Gene expression alterations in the presence of tumour-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 WT tumour-derived LT, while co-expression of sT did not significantly affect these patterns of gene expression. LXCXE-dependent upregulation of cyclin E and CDK2 correlated with increased proliferation in tumour-derived LT-expressing cells. Tumour-derived LT and tumour-derived LT plus sT increased expression of multiple cytokines and chemokines, which resulted in elevated levels of secreted IL-8. We concluded that, in human fibroblasts, the LXCXE motif of tumour-derived LT enhances cellular proliferation and upregulates cell cycle and immune signalling gene transcription.

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