Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model.

Masahiro Shuda,Patrick S Moore,Xuehui Geng,Stephen M Ostrowski,Yoko Shuda,Anna Guastafierro,Yuan Chang,Stefan Lukianov,Stephen M Maricich,Frank J Jenkins,Kord Honda

doi:10.1371/journal.pone.0142329

Abstract

Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSA sT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult Ubc CreERT2; ROSA sT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult Ubc CreERT2; ROSA sT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1 CreERT2/+; ROSA sT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1 CreERT2/+; ROSA sT and Atoh1 CreERT2/+; ROSA sT; p53f lox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.

Highlights

Merkel cell polyomavirus (MCV) is clonally integrated into ~80% of Merkel cell carcinomas (MCC) and is currently the only human polyomavirus known to be oncogenic [1, 2]
To conditionally induce cre-loxP recombination and small T (sT) expression in multiple organs, ROSAsT mice were mated to UbcCreERT2 mice encoding human ubiquitin C promoter-driven Cre recombinase fused to a triple mutant form of the human estrogen receptor activatable by tamoxifen (TMX)
MCV sT transforms immortalized rodent fibroblasts such as Rat1 and NIH3T3 [14] and viral sT expression is required for growth of MCV-positive MCC cancer cell lines [15, 16]

Summary

Introduction

Merkel cell polyomavirus (MCV) is clonally integrated into ~80% of Merkel cell carcinomas (MCC) and is currently the only human polyomavirus known to be oncogenic [1, 2] This virus was the first human pathogen discovered by a nondirected RNAseq method called digital transcriptome subtraction [3], an approach that is commonly used to discover and characterize human viruses [4]. A region called the large T stabilization domain (LSD) binds and inhibits diverse E3 ligase recognition proteins including Fbw, cdc and cdh1 [12, 13]. The latter two proteins are recognition subunits for the anaphase promoting complex/cyclosome (APC/C) and sT expression promotes mitotic prometaphase arrest [13]. Mutations to the MCV sT LSD eliminate MCV sT-induced transformation, and knockdown of either LT or sT initiates cell death or cell cycle arrest of MCV-positive MCC cells [15, 16]

Methods

Results

Conclusion