Transduction-induced overexpression of Merkel cell T antigens in human hair follicles induces formation of pathological cell clusters with Merkel cell carcinoma-like phenotype.

Abstract

To the editor, Merkel cell carcinoma (MCC) is a rare skin cancer, induced by the Merkel cell polyomavirus (MCPyV)1 in 80% of cases (S1 in Appendix S1). Following genomic integration of MCPyV, expression of the MCPyV T antigens (TA), small T (sT) and large T (LT), is regarded as a crucial oncogenic determinant. sT and LT are both required for proliferation of MCPyV-positive tumour cells while only sT has been demonstrated to induce tumour formation.1 The cell identity in which MCC-inducing MCPyV integration occurs is still unknown.1 Even though MCC tumour cells are phenotypically similar to Merkel cells (MCs), it is unlikely that MCs constitute the cells of origin of MCCs due to their postmitotic status and poor MCPyV infectability.1 Importantly, Nguyen et al.2 recently demonstrated that MC formation in hairy mice skin arises from epithelial Sox9+ progenitors of the hair follicle (HF) and requires Sonic Hedgehog (Shh) pathway activation. Recently, we analysed a combined tumour comprising an MCPyV-positive MCC and a trichoblastoma (benign epithelial tumour displaying hair follicle and MC differentiation; S2 in Appendix S1).3 By applying massive parallel sequencing, we demonstrated that MCPyV integration in a trichoblastoma cell gave rise to MCPyV-positive MCC, providing evidence of the epithelial origin of this MCC (S3 in Appendix S1).3, 4 After confirming SOX9 expression and SHH activation in trichoblastoma cells,3, 4 we hypothesized that MC progenitors of the HF might constitute a cell of origin of MCPyV-positive MCC. To probe our hypothesis, we aimed in the current pilot study to evaluate whether TA overexpression in human scalp HF might lead to MCC-like tumour formation. We first assessed the functional impact of ectopic expression of the MCPyV-TAs in human anagen scalp HFs by transducing the HF cells with a lentiviral vector encoding sT and truncated LT (Supplementary Material and Methods in Appendix S1). HFs transduced with an empty lentiviral vector served as controls, and analysis was performed after 7 days of organ culture (S4 in Appendix S1). Following this procedure, strong nuclear LT expression was evidenced by immunohistochemistry in the inner root sheath and in the infundibular region of TA-transduced HFs, whereas no significant staining was observed in controls (Figure S2). Since only transient HF transfections were previously described (S5 in Appendix S1), these results suggest that lentiviral transduction of HFs is possible in principle, which would expand the range of mechanistic studies in human HF organ culture (S6 in Appendix S1). Interestingly, morphological analysis of TA-transduced HFs at day 7 revealed several cell clusters detached from the HF structure, composed of highly mitotic cells with a morphology similar to MCC cells5: elevated nucleocytoplasmic ratio and dusty chromatin, while no clusters were detected prior to day 7 or in control HFs (Figure S1; Table S1). Of note, in one case, we observed similar cluster formation within the hair follicle structure, suggesting an epithelial origin of these clusters (Figure S3). Consequently, MCC marker expression was investigated by immunohistochemistry.5 This revealed high LT expression in the cell clusters while Keratin 8 or 20 appeared to be absent, strong positivity for SATB2—a highly specific MCC marker was observed in these cells (S7–S9 in Appendix S1) (Figure S1; Table S1) and was completely absent in the controls. In addition, diffuse EPCAM expression (S10–S11 in Appendix S1), and positivity of some cells for synaptophysin and chromogranin was detected, while none of these MCC markers was observed in the controls. Proliferation index (Ki67 staining) was higher than 50% (Figure S4). This suggested induction of an MCC-like phenotype in TA-expressing cells. In conclusion, although this pilot study has important limitations (eg no proof of immortalization and no precise identification of the lentivirus-transduced HF cells), it suggests that MCPyV-TA-positive MCC-like cells may originate from epithelial cells present in HFs, an important finding for the development of MCC transgenic mice model.1 We thank Dr Sonja Hesbacher, Dr Patricia Berthon, Pr Serge Guyétant, Pr Mahtab Samimi, Pr Antoine Touzéand Pr Boris Laure for their help and assistance. We thank the foundation ARC and la ligue contre le cancer, the IZKF (B-343) and the German Research Foundation SCHR 1178/3-1. The authors declare no conflict of interest. TK, JC, RP, RH, DS have made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data; and have been involved in drafting the manuscript or revising it critically for important intellectual content. TK, JC, RP, RH, DS have given final approval of the version to be published. Each author should have participated sufficiently in the work to take public responsibility for appropriate portions of the content; and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. No data are available. Figure S1. T Antigen transduction in hair follicles led to the formation of cell clusters displaying MCC-like features Figure S2. Impact of ectopic T antigen expression in human hair follicles. Figure S3. Formation of a MCC-like cell cluster in the hair follicle structure. Figure S4. Ki67 expression in a T antigen-transduced hair follicle and in a MCC-like cell cluster. Table S1. Morphologic and immunohistochemical characterization of T antigen-expressing hair follicles and controls. Appendix S1. Supplementary methods: Hair follicle culture; Lentivirus production; Immunohistochemistry. Appendix S1. Supplementary references. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.