Sudhir, Sweta

Background/Objectives: CSCs are critical drivers of the tumor and stem cell phenotypes of glioblastoma (GBM) cells. Chromatin modifications play a fundamental role in driving a GBM CSC phenotype. The goal of this study is to further our understanding of how stem cell-driving events control changes in chromatin architecture that contribute to the tumor-propagating phenotype of GBM. Methods: We utilized computational analyses to identify a subset of clinically relevant genes that were predicted to be repressed in a Polycomb repressive complex 2 (PRC2)-dependent manner in GBM upon induction of stem cell-driving events. These associations were validated in patient-derived GBM neurosphere models using state-of-the-art molecular techniques to express, silence, and measure microRNA (miRNA) and gene expression changes. Advanced Poly(β-amino ester) nanoparticle formulations (PBAEs) were used to deliver miRNAs in vivo to orthotopic human GBM tumor models. Results: We show that glioma stem cell (GSC) formation and tumor propagation involve the crosstalk between multiple epigenetic mechanisms, resulting in the repression of the miRNAs that regulate PRC2 function and histone H3 lysine 27 tri-methylation (H3K27me3). We also identified miR-217-5p as an EZH2 regulator repressed in GSCs and showed that miR-217-5p reconstitution using advanced nanoparticle formulations re-activates the PRC2-repressed genes, inhibits GSC formation, impairs tumor growth, and enhances the effects of ionizing radiation in an orthotopic model of GBM. Conclusions: These findings suggest that inhibiting PRC2 function by targeting EZH2 with miR-217-5p advanced nanoparticle formulations could have a therapeutic benefit in GBM.

Over 40 years of controversy surround the nuclear localization of actin. Now, it is well understood that actin localization to the nucleus is highly regulated, suggesting actin has critical nuclear functions. Studies have demonstrated that nuclear actin has roles in regulating transcription, chromatin remodeling, nuclear organization, and DNA damage repair. However, much remains unknown about nuclear actin, including its structure in the nucleus and its functions during development. Here we present our novel finding that Drosophila oogenesis, or follicle development, is a model for studying the structure, regulation, and function of nuclear actin. Follicles are composed of roughly 1000 somatic follicle cells and 16 germline cells, including 15 nurse or support cells and a single oocyte, and progress through a series of 14 morphological stages, from the germanium to Stage 14. During Stages 5–9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Known regulators of actin nucleocytoplasmic transport, Cofilin and Profilin, also localize to the nuclei during follicle development. Further, germline expression of GFP‐tagged Actin results in nuclear actin rod formation in nurse cells during Stages 5–9. These nuclear actin rods are static, label with both phalloidin and Cofilin. These findings suggest that nuclear actin is tightly regulated during follicle development. As we have previously determined that Fascin, an actin bundling protein, localizes to nurse cell nuclei, we postulated that Fascin may be a novel regulator of nuclear actin. Overexpression of Fascin enhances the frequency and length of GFP‐Actin rods, and Fascin colocalizes with these rods. Additionally, the loss of Fascin reduces, while the overexpression of Fascin increases, the frequency of nurse cells with high levels of endogenous nuclear actin. However, genetic manipulation of Fascin does not alter the overall nuclear level of actin within the ovary. Thus, these data suggest Fascin regulates the ability of specific cells to accumulate nuclear actin. The loss of Fascin reduces nuclear Cofilin suggesting that Fascin positively regulates nuclear actin through Cofilin, a key factor in the nuclear import of actin. Indeed, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. In conclusion, we have established Drosophila oogenesis as a new system to uncover the physiologically important functions and regulation of nuclear actin. Further, we have identified Fascin as a novel regulator of nuclear actin. The nuclear localization of Fascin and Actin is highly conserved among systems, indicating these findings are applicable far beyond Drosophila follicle development. Support or Funding Information This project is supported by grants by the National Science Foundation MCB‐1158527, National Institutes of Health R01GM116885 and Carver Trust Medical Research Initiative Grant. D.J.K. is partially supported by NINDS T32NS045549.