Oncogenic signaling in the adult Drosophila prostate-like accessory gland leads to activation of a conserved pro-tumorigenic program, in the absence of proliferation.

Drosophila models for tumorigenesis and metastasis have revealed conserved mechanisms of signaling that are also involved in mammalian cancer. Many of these models use the proliferating tissues of the larval stages of Drosophila development, when tissues are highly mitotically active, or stem cells are abundant. Fewer Drosophila tumorigenesis models use adult animals to initiate tumor formation when many tissues are largely terminally differentiated and postmitotic. The Drosophila accessory glands are prostate-like tissues and a model for some aspects of prostate tumorigenesis using this tissue has been explored. In this model, oncogenic signaling was induced during the proliferative stage of accessory gland development, raising the question of how oncogenic activity would impact the terminally differentiated and postmitotic adult tissue. Here, we show that oncogenic signaling in the adult Drosophila accessory gland leads to activation of a conserved pro-tumorigenic program, similar to that observed in mitotic larval tissues, but in the absence of proliferation. Oncogenic signaling in the adult postmitotic gland leads to tissue hyperplasia with nuclear anaplasia and aneuploidy through endoreduplication, which increases polyploidy and occasionally results in non-mitotic neoplastic-like extrusions. We compare gene expression changes in our Drosophila model with that of endocycling prostate cancer cells induced by chemotherapy, which potentially mediate tumor recurrence after treatment. Similar signaling pathways are activated in the Drosophila gland and endocycling cancer cells, suggesting the adult accessory glands provide a useful model for aspects of prostate cancer progression that do not involve cellular proliferation.

Initiation of wound healing is regulated by the convergence of mechanical and epigenetic cues.

Wound healing in the skin is a complex physiological process that is a product of a cell state transition from homeostasis to repair. Mechanical cues are increasingly being recognized as important regulators of cellular reprogramming, but the mechanism by which it is translated to changes in gene expression and ultimately cellular behavior remains largely a mystery. To probe the molecular underpinnings of this phenomenon further, we used the down-regulation of caspase-8 as a biomarker of a cell entering the wound healing program. We found that the wound-induced release of tension within the epidermis leads to the alteration of gene expression via the nuclear translocation of the DNA methyltransferase 3A (DNMT3a). This enzyme then methylates promoters of genes that are known to be down-regulated in response to wound stimuli as well as potentially novel players in the repair program. Overall, these findings illuminate the convergence of mechanical and epigenetic signaling modules that are important regulators of the transcriptome landscape required to initiate the tissue repair process in the differentiated layers of the epidermis.

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.

Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data.

Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95pdz3) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations.

PLETHORA Genes Control Regeneration by a Two-Step Mechanism.

Regeneration, a remarkable example of developmental plasticity displayed by both plants and animals, involves successive developmental events driven in response to environmental cues. Despite decades of study on the ability of the plant tissues to regenerate a complete fertile shoot system after inductive cues, the mechanisms by which cells acquire pluripotency and subsequently regenerate complete organs remain unknown. Here, we show that three PLETHORA (PLT) genes, PLT3, PLT5, and PLT7, regulate de novo shoot regeneration in Arabidopsis by controlling two distinct developmental events. Cumulative loss of function of these three genes causes the intermediate cell mass, callus, to be incompetent to form shoot progenitors, whereas induction of PLT5 or PLT7 can render shoot regeneration hormone-independent. We further show that PLT3, PLT5, and PLT7 establish pluripotency by activating root stem cell regulators PLT1 and PLT2, as reconstitution of either PLT1 or PLT2 in the plt3; plt5-2; plt7 mutant re-established the competence to regenerate shoot progenitor cells but did not lead to the completion of shoot regeneration. PLT3, PLT5, and PLT7 additionally regulate and require the shoot-promoting factor CUP-SHAPED COTYLEDON2 (CUC2) to complete the shoot-formation program. Our findings uncouple the acquisition of competence to regenerate shoot progenitor cells from completion of shoot formation, indicating a two-step mechanism of de novo shoot regeneration that operates in all tested plant tissues irrespective of their origin. Our studies reveal intermediate developmental phases of regeneration and provide a deeper understanding into the mechanistic basis of regeneration.

Competence and regulatory interactions during regeneration in plants.

The ability to regenerate is widely exploited by multitudes of organisms ranging from unicellular bacteria to multicellular plants for their propagation and repair. But the levels of competence for regeneration vary from species to species. While variety of living cells of a plant display regeneration ability, only a few set of cells maintain their stemness in mammals. This highly pliable nature of plant cells in-terms of regeneration can be attributed to their high developmental plasticity. De novo organ initiation can be relatively easily achieved in plants by proper hormonal regulations. Elevated levels of plant hormone auxin induces the formation of proliferating mass of pluripotent cells called callus, which predominantly express lateral root meristem markers and hence is having an identity similar to lateral root primordia. Organ formation can be induced from the callus by modulating the ratio of hormones. An alternative for de novo organogenesis is by the forced expression of plant specific transcription factors. The mechanisms by which plant cells attain competence for regeneration on hormonal treatment or forced expression remain largely elusive. Recent studies have provided some insight into how the epigenetic modifications in plants affect this competence. In this review we discuss the present understanding of regenerative biology in plants and scrutinize the future prospectives of this topic. While discussing about the regeneration in the sporophyte of angiosperms which is well studied, here we outline the regenerative biology of the gametophytic phase and discuss about various strategies of regeneration that have evolved in the domain of life so that a common consensus on the entire process of regeneration can be made.