An intact retinoblastoma protein‐binding site in Merkel cell polyomavirus large T antigen is required for promoting growth of Merkel cell carcinoma cells

Abstract

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer that frequently harbours Merkel cell polyomavirus (MCV) DNA integrated in the genome of the tumor cells. In our study, we elaborate our recent finding that MCV-positive MCC cell lines require the expression of the viral T antigens (TA). Indeed, in a xeno-transplantation model, we prove that TA expression is essential also in an in vivo situation, as knock down of TA leads to tumor regression. Moreover, rescuing TA short hairpin RNA (shRNA)-treated MCV-positive MCC cells by ectopic expression of shRNA-insensitive TAs clearly demonstrates that the observed effect is caused by TA knockdown. Notably, introduction of a mutation in the LTA protein interfering with LTA binding to the retinoblastoma protein (RB) ablated this rescue. The importance of this interaction was further confirmed as LTA-specific knockdown leads to explicit cell growth inhibition. In summary, the presented data demonstrate that established MCV-positive MCC tumors critically depend on TA expression, in particular the LTA and RB interaction, for sustained tumor growth. Consequently, interference with LTA/RB interaction appears as promising strategy to treat MCC.

Rationale For Immune-Based Therapies in Merkel Polyomavirus-Positive and -Negative Merkel Cell Carcinomas

“…MCPyV LT promotes oncogenesis partially through the highly conserved LXCXE motif, which binds to Rb [24]. Rb normally sequesters the transcription factor E2F, however, LT binding to Rb releases E2F resulting in increased expression of cyclin E and CDK2.…”

Retinoblastoma gene mutations detected by whole exome sequencing of Merkel cell carcinoma

“…13,14 Emerging data implicate maintenance and expression of the polyomavirus large T antigen in cell cycle dysregulation and the pathogenesis of viral transformation leading to Merkel cell carcinoma. 12,[15][16][17][18][19][20][21] Although the molecular role of Merkel cell polyomavirus is quickly evolving, the overall biology of Merkel cell carcinoma is poorly understood. Moreover, the presence of polyomavirus alone is not sufficient for carcinogenesis, namely in those Merkel cell carcinoma cases considered as polyomavirusnegative.…”

Survivin downregulation is not required for T antigen knockdown mediated cell growth inhibition in MCV infected merkel cell carcinoma cells

“…To this end, we used an shRNA targeting both LTA and sTA because the only available LTA specific shRNA induces also considerable off target effects. 6 Moreover, TA shRNA induced growth inhibition can be rescued by ectopic expression of shRNA insensitive LTA (data not shown) indicating that in our TA shRNA system sTA knockdown does not affect cellular growth significantly. In accordance with previous published data, knockdown of TA in MCVþ MCC cells resulted in decreased LTA and survivin protein expression (Fig.…”

“…This rescue, however, was abrogated when a point mutation in the LTA protein interfering with LTA binding to the retinoblastoma protein (RB) was introduced. 6 In a recent publication, Arora et al reported increased expression of survivin in MCVþ compared with MCV-MCC cases. 7 Survivin is an inhibitor of apoptosis protein family member that contributes to many human cancers by promoting cell survival and cell division.…”