Misregulation of Nucleoporins 98 and 96 leads to defects in protein synthesis that promote hallmarks of tumorigenesis.

Nucleoporin 98KD (Nup98) is a promiscuous translocation partner in hematological malignancies. Most disease models of Nup98 translocations involve ectopic expression of the fusion protein under study, leaving the endogenous Nup98 loci unperturbed. Overlooked in these approaches is the loss of one copy of normal Nup98 in addition to the loss of Nup96 - a second Nucleoporin encoded within the same mRNA and reading frame as Nup98 - in translocations. Nup98 and Nup96 are also mutated in a number of other cancers, suggesting that their disruption is not limited to blood cancers. We found that reducing Nup98-96 function in Drosophila melanogaster (in which the Nup98-96 shared mRNA and reading frame is conserved) de-regulates the cell cycle. We found evidence of overproliferation in tissues with reduced Nup98-96, counteracted by elevated apoptosis and aberrant signaling associated with chronic wounding. Reducing Nup98-96 function led to defects in protein synthesis that triggered JNK signaling and contributed to hallmarks of tumorigenesis when apoptosis was inhibited. We suggest that partial loss of Nup98-96 function in translocations could de-regulate protein synthesis, leading to signaling that cooperates with other mutations to promote tumorigenesis.

Monitoring Spontaneous Quiescence and Asynchronous Proliferation-Quiescence Decisions in Prostate Cancer Cells.

The proliferation-quiescence decision is a dynamic process that remains incompletely understood. Live-cell imaging with fluorescent cell cycle sensors now allows us to visualize the dynamics of cell cycle transitions and has revealed that proliferation-quiescence decisions can be highly heterogeneous, even among clonal cell lines in culture. Under normal culture conditions, cells often spontaneously enter non-cycling G0 states of varying duration and depth. This also occurs in cancer cells and G0 entry in tumors may underlie tumor dormancy and issues with cancer recurrence. Here we show that a cell cycle indicator previously shown to indicate G0 upon serum starvation, mVenus-p27K-, can also be used to monitor spontaneous quiescence in untransformed and cancer cell lines. We find that the duration of spontaneous quiescence in untransformed and cancer cells is heterogeneous and that a portion of this heterogeneity results from asynchronous proliferation-quiescence decisions in pairs of daughters after mitosis, where one daughter cell enters or remains in temporary quiescence while the other does not. We find that cancer dormancy signals influence both entry into quiescence and asynchronous proliferation-quiescence decisions after mitosis. Finally, we show that spontaneously quiescent prostate cancer cells exhibit altered expression of components of the Hippo pathway and are enriched for the stem cell markers CD133 and CD44. This suggests a hypothesis that dormancy signals could promote cancer recurrence by increasing the proportion of quiescent tumor cells poised for cell cycle re-entry with stem cell characteristics in cancer.

Pulpbow: A Method to Study the Vasculogenic Potential of Mesenchymal Stem Cells from the Dental Pulp.

Understanding how Mesenchymal Stem Cells (MSCs) form blood vessels is critical for creating mechanism-based approaches for the therapeutic use of these cells. In addition, understanding the determinants and factors involved in lineage hierarchy is fundamental to creating accurate and reliable techniques for the study of stem cells in tissue engineering and repair. Dental Pulp Stem Cells (DPSC) from permanent teeth and Stem cells from Human Exfoliated Deciduous teeth (SHED) are particularly interesting sources for tissue engineering as they are easily accessible and expandable. Previously, we have shown that DPSCs and SHEDs can differentiate into endothelial cells and form functional blood vessels through vasculogenesis. Here, we described how we created the "pulpbow" (pulp + rainbow), a multicolor tag experimental model that is stable, permanent, unique to each cell and passed through generations. We used the pulpbow to understand how dental pulp stem cells contributed to blood vessel formation in 3D models in in vitro and ex vivo live cell tracking, and in vivo transplantation assays. Simultaneous tracking of cells during sprout formation revealed that no single multicolor-tagged cell was more prone to vasculogenesis. During this process, there was intense cell motility with minimal proliferation in early time points. In later stages, when the availability of undifferentiated cells around the forming sprout decreased, there was local clonal proliferation mediated by proximity. These results unveiled that the vasculogenesis process mediated by dental pulp stem cells is dynamic and proximity to the sprouting area is critical for cell fate decisions.