Facile method to incorporate high-affinity ACAT/SOAT1 inhibitor F12511 into stealth liposome-based nanoparticle and demonstration of its efficacy in blocking cholesteryl ester biosynthesis without overt toxicity in neuronal cell culture.

BACKGROUND: Acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors have been considered as potential therapeutic agents to treat several diseases, including Alzheimer's disease, atherosclerosis, and cancer. While many ACAT inhibitors are readily available, methods to encapsulate them as nanoparticles have not been reported. NEW METHOD: We report a simple method to encapsulate ACAT inhibitors, using the potent hydrophobic ACAT inhibitor F12511 as an example. By mixing DSPE-PEG2000, egg phosphatidylcholine (PC), and F12511 in ethanol, followed by drying, resuspension and sonication in buffer, we show that F12511 can be encapsulated as stealth liposomes at high concentration. RESULTS: We successfully incorporated F12511 into nanoparticles and found that increasing PC in the nanoparticles markedly increased the amount of F12511 incorporated in stealth liposomes. The nanoparticles containing F12511 (Nanoparticle F) exhibit average size of approximately 200 nm and are stable at 4 ºC for at least 6 months. Nanoparticle F is very effective at inhibiting ACAT in human and mouse neuronal and microglial cell lines. Toxicity tests using mouse primary neuronal cells show that F12511 alone or Nanoparticle F added at concentrations from 2 to 10 µM for 24-, 48-, and 72-hours produces minimal, if any, toxicity. COMPARISON WITH EXISTING METHOD(S): Unlike existing methods, the current method is simple, cost effective, and can be expanded to produce tagged liposomes to increase specificity of delivery. This also offers opportunity to embrace water soluble agent(s) within the aqueous compartment of the nanoparticles for potential combinatorial therapy. CONCLUSIONS: This method shows promise for delivery of hydrophobic ACAT inhibitors at high concentration in vivo.

The helix-loop-helix transcriptional regulator Id4 is required for terminal differentiation of luminal epithelial cells in the prostate.

Inhibitor of differentiation 4 (Id4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer. In this study we investigated the effect of loss of Id4 (Id4-/-) on mouse prostate development. Histological analysis was performed on prostates from 25 days, 3 months and 6 months old Id4-/- mice. Expression of Amacr, Ck8, Ck18, Fkbp51, Fkbp52, androgen receptor, Pten, sca-1 and Nkx3.1 was investigated by immunohistochemistry. Results were compared to the prostates from Nkx3.1-/- mice. Id4-/- mice had smaller prostates with fewer and smaller tubules. Subtle PIN like lesions were observed at 6mo. Decreased Nkx3.1 and Pten and increased stem cell marker sca-1, PIN marker Amacr and basal cell marker p63 was observed at all ages. Persistent Ck8 and Ck18 expression suggested that loss of Id4 results in epithelial commitment but not terminal differentiation in spite of active Ar. Loss of Id4 attenuates normal prostate development and promotes hyperplasia/ dysplasia with PIN like lesions. The results suggest that loss of Id4 maintains stem cell phenotype of "luminal committed basal cells", identifying a unique prostate developmental pathway regulated by Id4.

The effect of botulinum toxin on ureteral inflammation.

PURPOSE: The impact of onabotulinum toxin type A (BoNT-A) on bladder afferent nerve pathways and chemosensory functions is an active area of investigation. There may be a role for BoNT-A in disorders of the ureter; however, no histologic studies have assessed the effects of BoNT-A on ureteral tissue. Our objective was to develop an animal model of ureteral inflammation and determine the impact of ureteral BoNT-A instillation on known mechanisms of inflammation. METHODS: The safety and feasibility of a novel animal model of ureteral inflammation was assessed. Through open cystotomy, the effect of ureteral BoNT-A instillation on inflammation was determined through H&E, masson's trichrome, Ki-67 stain, and prostaglandin E (PGE) synthase expression, a known marker of pain and inflammation in ureteral tissue. Urothelial microstructure was assessed using electron microscopy and standard histologic techniques. RESULTS: All experiments were carried to completion, and no systemic signs of botulinum toxicity were seen. BoNT-A exposure was associated with a decrease in PGE synthase expression in a dose-dependent fashion. BoNT-A exposure was not found to impact collagen deposition or cell proliferation. Disruption of tight junctions between urothelial cells was observed under conditions of inflammation. CONCLUSION: We describe the feasibility of a novel in vivo model of ureteral inflammation and report the first histologic study of the effects of BoNT-A on the ureter. Preliminary findings show that BoNT-A attenuates ureteral PGE synthase expression under conditions of inflammation. The application of BoNT-A may provide anti-inflammatory and analgesic effects in the context of ureteral disorders.

Slc6a3-dependent expression of a CAPS-associated Nlrp3 allele results in progressive behavioral abnormalities and neuroinflammation in aging mice.

BACKGROUND: An association between neuroinflammation and age-related neurologic disorders has been established but the molecular mechanisms and cell types involved have not been thoroughly characterized. Activity of the proinflammatory NLRP3 inflammasome is implicated in Alzheimer's and Parkinson's disease and our recent studies in patients suggest that dopaminergic neurons within the degenerating mesencephalon express NLRP3 throughout the progression of PD. Here, we directly test the impact of enhanced inflammasome activity in mesencephalic neurons by characterizing motor function, tissue integrity, and neuroinflammation in aging mice harboring hyperactivating mutations within the endogenous murine Nlrp3 locus, enabled only in cells expressing the dopaminergic neuron-specific Slc6a3 promoter. METHODS: We compared mice harboring inducible alleles encoding the cryopyrin-associated periodic syndrome activating mutations Nlrp3A350V and Nlrp3L351P inserted into the endogenous mouse Nlrp3 locus. Tissue specific expression was driven by breeding these animals with mice expressing Cre recombinase under the control of the dopaminergic neuron-specific Slc6a3 promoter. The experimental mice, designed to express hyperactive NLRP3 only when the endogenous mouse Nlrp3 promotor is active in dopaminergic neurons, were analyzed throughout 18 months of aging using longitudinal motor function assessments. Biochemical and histologic analyses of mesencephalic tissues were conducted in 1- and 18-month-old animals. RESULTS: We observed progressive and significant deficits in motor function in animals expressing Nlrp3L351P, compared with animals expressing Nlrp3WT and Nlrp3A350V. Age-dependent neuroinflammatory changes in the mesencephalon were noted in all animals. Analysis of GFAP-immunoreactive astrocytes in the substantia nigra revealed a significant increase in astrocyte number in animals expressing Nlrp3L351P compared with Nlrp3WT and Nlrp3A350V. Further analysis of Nlrp3L351P striatal tissues indicated genotype specific gliosis, elevated Il1b expression, and both morphologic and gene expression indicators of proinflammatory A1 astrocytes. CONCLUSIONS: Dopaminergic neurons have the potential to accumulate NLRP3 inflammasome activators with age, including reactive oxygen species, dopamine metabolites, and misfolded proteins. Results indicate the Nlrp3 locus is active in dopaminergic neurons in aging mice, and that the hyperactive Nlrp3L351P allele can drive neuroinflammatory changes in association with progressive behavioral deficits. Findings suggest neuronal NLRP3 inflammasome activity may contribute to neuroinflammation observed during normal aging and the progression of neurologic disorders.

Characterizing the heterogeneity in 5-aminolevulinic acid-induced fluorescence in glioblastoma.

OBJECTIVE: 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is an effective surgical adjunct for the intraoperative identification of tumor tissue during resection of high-grade gliomas. The use of 5-ALA-induced PpIX fluorescence in glioblastoma (GBM) has been shown to double the extent of gross-total resection and 6-month progression-free survival. The heterogeneity of 5-ALA-induced PpIX fluorescence observed during surgery presents a technical and diagnostic challenge when utilizing this tool intraoperatively. While some regions show bright fluorescence after 5-ALA administration, other regions do not, despite that both regions of the tumor may be histopathologically indistinguishable. The authors examined the biological basis of this heterogeneity using computational methods. METHODS: The authors collected both fluorescent and nonfluorescent GBM specimens from a total of 14 patients undergoing surgery and examined their gene expression profiles. RESULTS: In this study, the authors found that the gene expression patterns characterizing fluorescent and nonfluorescent GBM surgical specimens were profoundly different and were associated with distinct cellular functions and different biological pathways. Nonfluorescent tumor tissue tended to resemble the neural subtype of GBM; meanwhile, fluorescent tumor tissue did not exhibit a prominent pattern corresponding to known subtypes of GBM. Consistent with this observation, neural GBM samples from The Cancer Genome Atlas database exhibited a significantly lower fluorescence score than nonneural GBM samples as determined by a fluorescence gene signature developed by the authors. CONCLUSIONS: These results provide a greater understanding regarding the biological basis of differential fluorescence observed intraoperatively and can provide a basis to identify novel strategies to maximize the effectiveness of fluorescence agents.

Glioma Cell Secretion: A Driver of Tumor Progression and a Potential Therapeutic Target.

Cellular secretion is an important mediator of cancer progression. Secreted molecules in glioma are key components of complex autocrine and paracrine pathways that mediate multiple oncogenic pathologies. In this review, we describe tumor cell secretion in high-grade glioma and highlight potential novel therapeutic opportunities. Cancer Res; 78(21); 6031-9. ©2018 AACR.

ID2 promotes survival of glioblastoma cells during metabolic stress by regulating mitochondrial function.

Tumor cells proliferate in cellular environments characterized by a lack of optimal tissue organization resulting oftentimes in compromised cellular metabolism affecting nutrition, respiration, and energetics. The response of tumor cells to adverse environmental conditions is a key feature affecting their pathogenicity. We found that inhibitor of DNA binding 2 (ID2) expression levels significantly correlate with the ability of glioblastoma (GBM)-derived cell lines to survive glucose deprivation. ID2 suppressed mitochondrial oxidative respiration and mitochondrial ATP production by regulating the function of mitochondrial electron transport chain (mETC) complexes, resulting in reduced superoxide and reactive oxygen species (ROS) production from mitochondria. ID2 suppression of ROS production reduced mitochondrial damage and enhanced tumor cell survival during glucose deprivation. Bioinformatics analysis of GBM gene expression data from The Cancer Genome Atlas (TCGA) database revealed that expression of ID2 mRNA is unique among ID gene family members in correlating with the expression of nuclear genes involved in mitochondrial energy metabolism and assembly of mETC. Our data indicate that the expression level of ID2 in GBM cells can predict the sensitivity of GBM-derived tumor cells to decreased glucose levels. Low levels of ID2 expression in human GBM tissues may identify a clinical group in which metabolic targeting of glycolytic pathways can be expected to have the greatest therapeutic efficacy.

Insulin-Mediated Signaling Facilitates Resistance to PDGFR Inhibition in Proneural hPDGFB-Driven Gliomas.

Despite abundant evidence implicating receptor tyrosine kinases (RTK), including the platelet-derived growth factor receptor (PDGFR), in the pathogenesis of glioblastoma (GBM), the clinical use of RTK inhibitors in this disease has been greatly compromised by the rapid emergence of therapeutic resistance. To study the resistance of proneural gliomas that are driven by a PDGFR-regulated pathway to targeted tyrosine kinase inhibitors, we utilized a mouse model of proneural glioma in which mice develop tumors that become resistant to PDGFR inhibition. We found that tumors resistant to PDGFR inhibition required the expression and activation of the insulin receptor (IR)/insulin growth-like factor receptor (IGF1R) for tumor cell proliferation and survival. Cotargeting IR/IGF1R and PDGFR decreased the emergence of resistant clones in vitro Our findings characterize a novel model of glioma recurrence that implicates the IR/IGF1R signaling axis in mediating the development of resistance to PDGFR inhibition and provide evidence that IR/IGF1R signaling is important in the recurrence of the proneural subtype of glioma in which PDGF/PDGFR is most commonly expressed at a high level. Mol Cancer Ther; 16(4); 705-16. ©2017 AACR.

Phosphorylation Regulates Id2 Degradation and Mediates the Proliferation of Neural Precursor Cells.

Inhibitor of DNA binding proteins (Id1-Id4) function to inhibit differentiation and promote proliferation of many different cell types. Among the Id family members, Id2 has been most extensively studied in the central nervous system (CNS). Id2 contributes to cultured neural precursor cell (NPC) proliferation as well as to the proliferation of CNS tumors such as glioblastoma that are likely to arise from NPC-like cells. We identified three phosphorylation sites near the N-terminus of Id2 in NPCs. To interrogate the importance of Id2 phosphorylation, Id2(-/-) NPCs were modified to express wild type (WT) Id2 or an Id2 mutant protein that could not be phosphorylated at the identified sites. We observed that NPCs expressing this mutant lacking phosphorylation near the N-terminus had higher steady-state levels of Id2 when compared to NPCs expressing WT Id2. This elevated level was the result of a longer half-life and reduced proteasome-mediated degradation. Moreover, NPCs expressing constitutively de-phosphorylated Id2 proliferated more rapidly than NPCs expressing WT Id2, a finding consistent with the well-characterized function of Id2 in driving proliferation. Observing that phosphorylation of Id2 modulates the degradation of this important cell-cycle regulator, we sought to identify a phosphatase that would stabilize Id2 enhancing its activity in NPCs and extended our analysis to include human glioblastoma-derived stem cells (GSCs). We found that expression of the phosphatase PP2A altered Id2 levels. Our findings suggest that inhibition of PP2A may be a novel strategy to regulate the proliferation of normal NPCs and malignant GSCs by decreasing Id2 levels. Stem Cells 2016;34:1321-1331.

Id2 mediates oligodendrocyte precursor cell maturation arrest and is tumorigenic in a PDGF-rich microenvironment.

Maturation defects occurring in adult tissue progenitor cells have the potential to contribute to tumor development; however, there is little experimental evidence implicating this cellular mechanism in the pathogenesis of solid tumors. Inhibitor of DNA-binding 2 (Id2) is a transcription factor known to regulate the proliferation and differentiation of primitive stem and progenitor cells. Id2 is derepressed in adult tissue neural stem cells (NSC) lacking the tumor suppressor Tp53 and modulates their proliferation. Constitutive expression of Id2 in differentiating NSCs resulted in maturation-resistant oligodendroglial precursor cells (OPC), a cell population implicated in the initiation of glioma. Mechanistically, Id2 overexpression was associated with inhibition of the Notch effector Hey1, a bHLH transcription factor that we here characterize as a direct transcriptional repressor of the oligodendroglial lineage determinant Olig2. Orthotopic inoculation of NSCs with enhanced Id2 expression into brains of mice engineered to express platelet-derived growth factor in the central nervous system resulted in glioma. These data implicate a mechanism of altered NSC differentiation in glioma development and characterize a novel mouse model that reflects key characteristics of the recently described proneural subtype of glioblastoma multiforme. Such findings support the emerging concept that the cellular and molecular characteristics of tumor cells are linked to the transformation of distinct subsets of adult tissue progenitors.

p53 directly represses Id2 to inhibit the proliferation of neural progenitor cells.

Neural progenitor cells (NPCs) have the capacity to proliferate and give rise to all major central nervous system cell types and represent a possible cell of origin in gliomagenesis. Deletion of the tumor suppressor gene Tp53 (p53) results in increased proliferation and self-renewal of NPCs and is a common genetic mutation found in glioma. We have identified inhibitor of DNA binding 2 (Id2) as a novel target gene directly repressed by p53 to maintain normal NPC proliferation. p53((-/-)) NPCs express elevated levels of Id2 and suppression of Id2 expression is sufficient to inhibit the increased proliferation and self-renewal which results from p53 loss. Elevated expression of Id2 in wild-type NPCs phenocopies the behavior of p53((-/-)) NPCs by enhancing NPC proliferation and self-renewal. Interestingly, p53 directly binds to a conserved site within the Id2 promoter to mediate these effects. Finally, we have identified elevated Id2 expression in glioma cell lines with mutated p53 and demonstrated that constitutive expression of Id2 plays a key role in the proliferation of glioma stem-like cells. These findings indicate that Id2 functions as a proproliferative gene that antagonizes p53-mediated cell cycle regulation in NPCs and may contribute to the malignant proliferation of glioma-derived tumor stem cells.

A novel mechanism of transcriptional repression of p27kip1 through Notch/HRT2 signaling in vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) proliferation occurs in vascular obstructive events such as atherosclerosis and restenosis. We previously showed that Notch receptors are induced in smooth muscle cells during vascular remodeling. Our goal was to determine the mechanisms employed by Notch signaling to regulate proliferation. Activation of Notch1 and Notch4 induced the VSMC-selective target genes HRT1 and HRT2, promoted cell cycle transit in smooth muscle cells, and led to loss of density-dependent growth inhibition. This was associated with a reduction in levels of the cyclin-dependent kinase inhibitor (cdk) p27(kip1). Over-expression of p27(kip1) resulted in a dose-dependent rescue of the Notch-induced phenotype and exit from the cell cycle. In addition, HRT2 expression was sufficient to promote S-phase entry, and we demonstrate that HRT2 interacts directly with the p27(kip1) promoter to repress transcription. Transcriptional repression occurred within the approximately 774 bp minimal p27(kip1) promoter region and mutational analysis demonstrated that repression is largely dependent on a conserved class-C domain. Our data show that Notch signaling acts to promote a proliferative phenotype in VSMC by modulation of the G1/S-phase checkpoint. In addition, we define a novel mechanism by which the Notch effector, HRT2, interacts directly with the class-C domain of the p27(kip1) promoter, repressing its expression. These studies identify a novel transcriptional target of HRT2, and show that Notch effectors directly control cell cycle regulatory components. We suggest that this mechanism is relevant to hyperproliferative states in VSMC seen during vascular remodeling and repair.