Inherited duplications of PPP2R3B predispose to nevi and melanoma via a C21orf91-driven proliferative phenotype.

PURPOSE: Much of the heredity of melanoma remains unexplained. We sought predisposing germline copy-number variants using a rare disease approach. METHODS: Whole-genome copy-number findings in patients with melanoma predisposition syndrome congenital melanocytic nevus were extrapolated to a sporadic melanoma cohort. Functional effects of duplications in PPP2R3B were investigated using immunohistochemistry, transcriptomics, and stable inducible cellular models, themselves characterized using RNAseq, quantitative real-time polymerase chain reaction (qRT-PCR), reverse phase protein arrays, immunoblotting, RNA interference, immunocytochemistry, proliferation, and migration assays. RESULTS: We identify here a previously unreported genetic susceptibility to melanoma and melanocytic nevi, familial duplications of gene PPP2R3B. This encodes PR70, a regulatory unit of critical phosphatase PP2A. Duplications increase expression of PR70 in human nevus, and increased expression in melanoma tissue correlates with survival via a nonimmunological mechanism. PPP2R3B overexpression induces pigment cell switching toward proliferation and away from migration. Importantly, this is independent of the known microphthalmia-associated transcription factor (MITF)-controlled switch, instead driven by C21orf91. Finally, C21orf91 is demonstrated to be downstream of MITF as well as PR70. CONCLUSION: This work confirms the power of a rare disease approach, identifying a previously unreported copy-number change predisposing to melanocytic neoplasia, and discovers C21orf91 as a potentially targetable hub in the control of phenotype switching.

Accounting for photophysical processes and specific signal intensity changes in fluorescence-detected sedimentation velocity.

Fluorescence detected sedimentation velocity (FDS-SV) has emerged as a powerful technique for the study of high-affinity protein interactions, with hydrodynamic resolution exceeding that of diffusion-based techniques, and with sufficient sensitivity for binding studies at low picomolar concentrations. For the detailed quantitative analysis of the observed sedimentation boundaries, it is necessary to adjust the conventional sedimentation models to the FDS data structure. A key consideration is the change in the macromolecular fluorescence intensity during the course of the experiment, caused by slow drifts of the excitation laser power, and/or by photophysical processes. In the present work, we demonstrate that FDS-SV data have inherently a reference for the time-dependent macromolecular signal intensity, resting on a geometric link between radial boundary migration and plateau signal. We show how this new time-domain can be exploited to study molecules exhibiting photobleaching and photoactivation. This expands the application of FDS-SV to proteins tagged with photoswitchable fluorescent proteins, organic dyes, or nanoparticles, such as those recently introduced for subdiffraction microscopy and enables FDS-SV studies of their interactions and size distributions. At the same time, we find that conventional fluorophores undergo minimal photobleaching under standard illumination in the FDS. These findings support the application of a high laser power density for the detection, which we demonstrate can further increase the signal quality.

Functional analysis of encapsulated hepatic progenitor cells.

A major challenge in developing therapies based on progenitor or stem cell populations (from sources other than bone marrow) involves developing a mode to deliver these cells in a manner that optimizes their viability, engraftment, proliferation, and differentiation. We have previously isolated a hepatic progenitor cell (HPC) population from adult liver tissue that differentiates into hepatic and biliary cell subtypes. We postulated that, using electrostatic encapsulation, we could reproducibly generate an ex vivo environment for the HPCs. We also theorized that this approach would foster cellular viability and function of the progenitor cell population. Using this encapsulation process, we consistently produced beads with uniform diameters between 200 and 700 microm. In vitro analysis of the encapsulated beads demonstrated extended periods of viability and function based on albumin production, urea metabolism, and glycogen storage. In conclusion, HPC encapsulation fosters the subsequent differentiation of HPCs into functional cells while maintaining their viability in long-term culture. These results demonstrate the efficacy of this method using somatic-derived progenitor cell populations and pave the way for clinical therapies.