TY - JOUR
T1 - Conversion of Sox2-dependent Merkel cell carcinoma to a differentiated neuron-like phenotype by T antigen inhibition
AU - Harold, Alexis
AU - Amako, Yutaka
AU - Hachisuka, Junichi
AU - Bai, Yulong
AU - Li, Meng Yen
AU - Kubat, Linda
AU - Gravemeyer, Jan
AU - Franks, Jonathan
AU - Gibbs, Julia R.
AU - Park, Hyun Jung
AU - Ezhkova, Elena
AU - Becker, Jürgen C.
AU - Shuda, Masahiro
N1 - Funding Information:
ACKNOWLEDGMENTS. We thank Dr. Kathryn Albers, Dr. Paul Cantalupo, Dr. James Pipas, Dr. Patrick Moore, and Dr. Yuan Chang for critical advice and suggestions; and Michelle Savoldy, Navi Baskar, and Korey Henderson for technical assistance. This project used the University of Pittsburgh Health Science Core Research Facilities Genomic Research Core Single Cell RNA sequencing services. M.S. was supported by the Hillman Foundation, Pennsylvania Tobacco Settlement Grant P50CA121973, the University of Pittsburgh Skin Cancer Specialized Program of Research Excellence (SPORE), and National Institutes of Health Cancer Center Support Grant P30 CA047904. E.E. was supported by the Tisch Cancer Institute under P30 Cancer Support Grant and the Irma T. Hirschl Career Scientist Award of the Irma T. Hirschl Trust. J.C.B. was supported by the European Union’s Seventh Framework Program (FP7-HEALTH-2011-two-stage, Immune Modulating Strategies for Treatment of Merkel Cell Carcinoma [IMMOMEC]) under Grant Agreement 277775 (granted to J.C.B.); J.C.B. was supported by the German Cancer Consortium (DKTK L441). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2019 National Academy of Sciences. All rights reserved.
PY - 2019/10/1
Y1 - 2019/10/1
N2 - Viral cancers show oncogene addiction to viral oncoproteins, which are required for survival and proliferation of the dedifferentiated cancer cell. Human Merkel cell carcinomas (MCCs) that harbor a clonally integrated Merkel cell polyomavirus (MCV) genome have low mutation burden and require viral T antigen expression for tumor growth. Here, we showed that MCV+ MCC cells cocultured with keratinocytes undergo neuron-like differentiation with neurite outgrowth, secretory vesicle accumulation, and the generation of sodium-dependent action potentials, hallmarks of a neuronal cell lineage. Cocultured keratinocytes are essential for induction of the neuronal phenotype. Keratinocyte-conditioned medium was insufficient to induce this phenotype. Single-cell RNA sequencing revealed that T antigen knockdown inhibited cell cycle gene expression and reduced expression of key Merkel cell lineage/MCC marker genes, including HES6, SOX2, ATOH1, and KRT20. Of these, T antigen knockdown directly inhibited Sox2 and Atoh1 expression. MCV large T up-regulated Sox2 through its retinoblastoma protein-inhibition domain, which in turn activated Atoh1 expression. The knockdown of Sox2 in MCV+ MCCs mimicked T antigen knockdown by inducing MCC cell growth arrest and neuron-like differentiation. These results show Sox2-dependent conversion of an undifferentiated, aggressive cancer cell to a differentiated neuron-like phenotype and suggest that the ontology of MCC arises from a neuronal cell precursor.
AB - Viral cancers show oncogene addiction to viral oncoproteins, which are required for survival and proliferation of the dedifferentiated cancer cell. Human Merkel cell carcinomas (MCCs) that harbor a clonally integrated Merkel cell polyomavirus (MCV) genome have low mutation burden and require viral T antigen expression for tumor growth. Here, we showed that MCV+ MCC cells cocultured with keratinocytes undergo neuron-like differentiation with neurite outgrowth, secretory vesicle accumulation, and the generation of sodium-dependent action potentials, hallmarks of a neuronal cell lineage. Cocultured keratinocytes are essential for induction of the neuronal phenotype. Keratinocyte-conditioned medium was insufficient to induce this phenotype. Single-cell RNA sequencing revealed that T antigen knockdown inhibited cell cycle gene expression and reduced expression of key Merkel cell lineage/MCC marker genes, including HES6, SOX2, ATOH1, and KRT20. Of these, T antigen knockdown directly inhibited Sox2 and Atoh1 expression. MCV large T up-regulated Sox2 through its retinoblastoma protein-inhibition domain, which in turn activated Atoh1 expression. The knockdown of Sox2 in MCV+ MCCs mimicked T antigen knockdown by inducing MCC cell growth arrest and neuron-like differentiation. These results show Sox2-dependent conversion of an undifferentiated, aggressive cancer cell to a differentiated neuron-like phenotype and suggest that the ontology of MCC arises from a neuronal cell precursor.
KW - Merkel cell carcinoma
KW - Merkel cell polyomavirus
KW - Phenotypic conversion
KW - Sox2 | Atoh1
UR - http://www.scopus.com/inward/record.url?scp=85072784490&partnerID=8YFLogxK
U2 - 10.1073/pnas.1907154116
DO - 10.1073/pnas.1907154116
M3 - Article
C2 - 31527246
AN - SCOPUS:85072784490
VL - 116
SP - 20104
EP - 20114
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 40
ER -