519 Paranuclear cytokeratin aggregates help Merkel cell carcinoma evade extrinsic apoptosis

Abstract

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer. Most MCC tumors are virus positive and express Merkel cell polyomavirus oncogenes, whereas virus negative MCC are associated with abundant UV signature mutations. The majority of MCC tumors express CK20. Staining for CK20 and other intermediate filaments in a paranuclear dot pattern is a histological feature used to diagnose MCC. The functional significances of these paranuclear protein aggregates is unknown. We immunostained native MCC tumors with a pan-CK antibody and found areas of paranuclear dot staining in 94% of tumors. Immunofluorescent microscopy (IF) of MCC cell lines showed that paranuclear dot proteins often extend into a lattice-like structure surrounding the nucleus. Electron microscopy revealed that the intermediate filaments in the paranuclear dot did not resided within any cellular organelles but formed a cytoplasmic inclusion surrounding the centrosome. Using IF we confirmed that in 100% of interphase cells the paranuclear dot colocalized with the centrosome. In contrast, mitotic MCC cells showed partial disaggregation of the paranuclear dot and stochastic segregation of dot proteins into daughter cells, suggesting the paranuclear dot is a dynamic structure that reforms with each cell division. Disruption of microtubules with nocodazole resulted in dot disaggregation, demonstrating that microtubules are essential for dot maintenance. In contrast, inhibition of ubiquitin ligation enhanced cytokeratin aggregation. Interestingly, IF staining showed colocalization of the paranuclear dot with Fas Associate Death Domain (FADD). Consistent with functional sequestration of FADD in the paranuclear dot, TNFα and Fas ligand failed to induce apoptosis in dot-positive MCC cell lines. Taken together, the paranuclear dot may allow MCC tumors to evade extrinsic apoptosis by sequestering FADD and disrupting death receptor signaling.