Single-Molecule RNA Sequencing Reveals IFNγ-Induced Differential Expression of Immune Escape Genes in Merkel Cell Polyomavirus-Positive MCC Cell Lines.

Tatjana Sauerer,Julio Vera,Carola Berking,Adrian Weich,Christopher Lischer,Jan Dörrie

doi:10.3389/fmicb.2021.785662

Abstract

Merkel cell carcinoma (MCC) is a rare and highly aggressive cancer, which is mainly caused by genomic integration of the Merkel cell polyomavirus and subsequent expression of a truncated form of its large T antigen. The resulting primary tumor is known to be immunogenic and under constant pressure to escape immune surveillance. Because interferon gamma (IFNγ), a key player of immune response, is secreted by many immune effector cells and has been shown to exert both anti-tumoral and pro-tumoral effects, we studied the transcriptomic response of MCC cells to IFNγ. In particular, immune modulatory effects that may help the tumor evade immune surveillance were of high interest to our investigation. The effect of IFNγ treatment on the transcriptomic program of three MCC cell lines (WaGa, MKL-1, and MKL-2) was analyzed using single-molecule sequencing via the Oxford Nanopore platform. A significant differential expression of several genes was detected across all three cell lines. Subsequent pathway analysis and manual annotation showed a clear upregulation of genes involved in the immune escape of tumor due to IFNγ treatment. The analysis of selected genes on protein level underlined our sequencing results. These findings contribute to a better understanding of immune escape of MCC and may help in clinical treatment of MCC patients. Furthermore, we demonstrate that single-molecule sequencing can be used to assess characteristics of large eukaryotic transcriptomes and thus contribute to a broader access to sequencing data in the community due to its low cost of entry.

Highlights

Merkel cell carcinoma (MCC) is a rare and frequently metastatic neuroendocrine skin cancer with increasing incidence and high mortality (Fitzgerald et al, 2015; Paulson et al, 2018)
Antibody staining and fluorescence-activated cell sorting (FACS) analysis of all three MCC cell lines were performed after 72-h incubation with IFNγ and as control without IFNγ
Our findings in flow cytometry did mainly match with our transcriptomic data: We observed significantly increased signal transducer and activator of transcription 1 (STAT1) and bone marrow stromal antigen 2 (BST2) level in all three cell lines

Summary

Introduction

Merkel cell carcinoma (MCC) is a rare and frequently metastatic neuroendocrine skin cancer with increasing incidence and high mortality (Fitzgerald et al, 2015; Paulson et al, 2018). The MCPyV DNA is clonally integrated into the MCC genome in a replication-deficient form and expresses a truncated oncoprotein, called the large T (LT) antigen (Shuda et al, 2008). As this protein contains several tumor suppressors targeting motifs such as the retinoblastoma binding site, it promotes cell cycle progression that leads to uncontrolled cell proliferation (Borchert et al, 2014; Hesbacher et al, 2016). MCC is characterized by an increased expression of programmed cell death protein 1 (PD-1), which allows the tumor cells to escape the control of the immune system (Afanasiev et al, 2013). The exact mechanism of immune escape is of high clinical relevance especially regarding immunotherapy resistance; it is not fully understood yet

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