Conversion of Sox2-dependent Merkel cell carcinoma to a differentiated neuron-like phenotype by T antigen inhibition

Abstract

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.

Merkel Cell Polyomavirus T Antigens Induce Merkel Cell-Like Differentiation in GLI1-Expressing Epithelial Cells

“…The discrepancy between induction of mRNA and lack of KRT8 protein in immunostaining upon GLI1 expression might be explained by protein levels below the detection limit of the antibody used. Nevertheless, together, these results suggest that GLI1, the executor of the sonic hedgehog pathway, is capable of initiating the first step of MC differentiation via SOX2 induction [6,9]. .…”

“…Subsequent studies revealed that approximately 80% of MCC cases are Merkel cell polyomavirus (MCPyV)-positive, and expression of the two viral T antigens (TA) (small T (sT) and large T antigens (LT)) are considered as the main drivers for carcinogenesis and growth of such tumors [2]. Interestingly, while several candidates, such as epithelial cells, fibroblasts, neuronal progenitors, or B cells, have been proposed, the nature of the cells giving rise to MCC following infection remains unknown [3][4][5][6].…”

High-resolution analysis of Merkel Cell Polyomavirus in Merkel Cell Carcinoma reveals distinct integration patterns and suggests NHEJ and MMBIR as underlying mechanisms

“…We also find that neural cells cluster with the MCC cell lines, albeit more distantly than the cell lines and types mentioned above. Interestingly, a recent study reported reversion of MCC tumor cells to neuron-like cells after T-Antigen knockdown, suggesting neural precursor cells as putative MCC origin [60].…”

LSD1 inhibition induces differentiation and cell death in Merkel cell carcinoma

“…Intriguingly, they found that LSD1 and RCOR2 binding sites overlap with those of ATOH1, a key transcription factor of normal Merkel cell development (Bardot et al , 2013; Park et al , 2020). Other studies found that the inhibition of the T antigens induces cell cycle arrest (Houben et al , 2010) and induces neuronal differentiation when co‐cultured with keratinocytes (Harold et al , 2019). Collectively, these data suggest that T antigen‐mediated transformation relies on LSD1 to suppress differentiation toward a post‐mitotic Merkel cell fate and lock MCC cells in a stem‐like state.…”