Suppression of Glioblastoma Stem Cell Potency and Tumor Growth via LRRK2 Inhibition.

Leucine-rich repeat kinase 2 (LRRK2), a large GTP-regulated serine/threonine kinase, is well-known for its mutations causing late-onset Parkinson's disease. However, the role of LRRK2 in glioblastoma (GBM) carcinogenesis has not yet been fully elucidated. Here, we discovered that LRRK2 was overexpressed in 40% of GBM patients, according to tissue microarray analysis, and high LRRK2 expression correlated with poor prognosis in GBM patients. LRRK2 and stemness factors were highly expressed in various patient-derived GBM stem cells, which are responsible for GBM initiation. Canonical serum-induced differentiation decreased the expression of both LRRK2 and stemness factors. Given that LRRK2 is a key regulator of glioma stem cell (GSC) stemness, we developed DNK72, a novel LRRK2 kinase inhibitor that penetrates the blood-brain barrier. DNK72 binds to the phosphorylation sites of active LRRK2 and dramatically reduced cell proliferation and stemness factors expression in in vitro studies. Orthotopic patient-derived xenograft mouse models demonstrated that LRRK2 inhibition with DNK72 effectively reduced tumor growth and increased survival time. We propose that LRRK2 plays a significant role in regulating the stemness of GSCs and that suppression of LRRK2 kinase activity leads to reduced GBM malignancy and proliferation. In the near future, targeting LRRK2 in patients with high LRRK2-expressing GBM could offer a superior therapeutic strategy and potentially replace current clinical treatment methods.

Comparing axon regeneration in male and female mice after peripheral nerve injury.

Axons in the adult mammalian central nervous system fail to regenerate after injury. By contrast, spontaneous axon regeneration occurs in the peripheral nervous system (PNS) due to a supportive PNS environment and an increase in the intrinsic growth potential induced by injury via cooperative activation of multifaceted biological pathways. This study compared axon regeneration and injury responses in C57BL/6 male and female mice after sciatic nerve crush (SNC) injury. The extent of axon regeneration in vivo was indistinguishable in male and female mice when observed at 3 days after SNC injury, and primary dorsal root ganglion (DRG) neurons from injured, male and female mice extended axons to a similar length. Moreover, the induction of selected regeneration-associated genes (RAGs), such as Atf3, Sprr1a, Gap43, Sox11, Jun, Gadd45a, and Smad1 were comparable in male and female DRGs when assessed by quantitative real-time reverse transcription polymerase chain reaction. Furthermore, the RNA-seq analysis of male and female DRGs revealed that differentially expressed genes (DEGs) in SNC groups compared to sham-operated groups included many common genes associated with neurite outgrowth. However, we also found that a large number of genes in the DEGs were sex dependent, implicating the involvement of distinct gene regulatory network in the two sexes following peripheral nerve injury. In conclusion, we found that male and female mice mounted a comparable axon regeneration response and many RAGs were commonly induced in response to SNC. However, given that many DEGs were sex-dependently expressed, future studies are needed to investigate whether they contribute to peripheral axon regeneration, and if so, to what extent.

Brain injury induces HIF-1α-dependent transcriptional activation of LRRK2 that exacerbates brain damage.

Leucine-rich repeat kinase 2 (LRRK2), originally identified as a causative genetic factor in Parkinson's disease, is now associated with a number of pathologies. Here, we show that brain injury induces a robust expression of endogenous LRRK2 and suggest a role of LRRK2 after injury. We found that various in vitro and in vivo models of traumatic brain injury (TBI) markedly enhanced LRRK2 expression in neurons and also increased the level of hypoxia-inducible factor (HIF)-1α. Luciferase reporter assay and chromatin immunoprecipitation revealed direct binding of HIF-1α in LRRK2 proximal promoter. We also found that HIF-1α-dependent transcriptional induction of LRRK2 exacerbated neuronal cell death following injury. Furthermore, application of G1023, a specific, brain-permeable inhibitor of LRRK2, substantially prevented brain tissue damage, cell death, and inflammatory response and alleviated motor and cognitive defects induced by controlled cortical impact injury. Together, these results suggest HIF-1α-LRRK2 axis as a potential therapeutic target for brain injury.

Characterization of Parkinson's disease-related pathogenic TMEM230 mutants.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Although most PD cases are sporadic, 5-10% of them are hereditary and several pathogenic mutations in related genes have been identified. Mutations in TMEM230 were recently identified as a cause of autosomal dominant PD. However, the basic properties of the mutant proteins are not yet known. We examined stability and neurotoxicity, important characteristics of PD pathogenesis-related proteins, of WT TMEM230 and two pathogenic mutants, R78L and PG5ext, in a dopaminergic neuronal cell line. Our study showed that amount of protein expressed in the same vector backbone was R78L > WT > PG5ext. The stabilities of the mutant proteins were similar to each other, but lower than that of the WT. In addition, overexpression of mutants and WT TMEM230 caused similar levels of neurotoxicity upon MPP+ treatment when compared to the cells transfected with an empty vector. Because the proteins encoded by two PD-causing genes, TMEM230 and LRRK2, function in vesicle trafficking, we tested whether they interact. LRRK2 neither interacts with, nor phosphorylates TMEM230. We also investigated the levels of several Rab proteins (Rab1A, 5, 7, 8A and 11) involved in vesicle trafficking after TMEM230 overexpression. However, there was no clear difference of any Rab proteins among cells transfected with an empty vector, TMEM230 WT and mutants-expressing cells, suggesting that TMEM230 does not directly regulate these Rab proteins. Thus, these TMEM230 PG5ext and R78L mutant proteins are not distinctly different from the WT proteins except for their stability. Abbreviations: LRRK2: Leucine-rich repeat kinase 2; PD: Parkinson's disease; AD: Alzheimer's disease; RT-PCR: reverse transcription-polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; FACS: fluorescence-activated cell sorting; PBS: phosphate buffered saline; FBS: fetal bovine serum; PI: propidium iodide.

Inflammatory signals induce the expression of tonicity-responsive enhancer binding protein (TonEBP) in microglia.

Tonicity-responsive enhancer (TonE) binding protein (TonEBP) is known as an osmosensitive transcription factor that regulates cellular homeostasis during states of hypo- and hypertonic stress. In addition to its role in osmoadaptation, growing lines of evidence suggest that TonEBP might have tonicity-independent functions. In particular, a number of studies suggest that inflammatory stimuli induce the expression and activation of TonEBP in peripheral immune cells. However, whether TonEBP is expressed in microglia, resident immune cells of the central nervous system, is unknown. Here we show that inflammatory signals induce the expression of TonEBP in microglia both in vitro and in vitro. In cultured primary microglia, treatment with lipopolysaccharide (LPS), interferon-γ, and interleukin 4 increased the expression of TonEBP. Moreover, we found that stereotaxic injection of LPS into the substantia nigra region of rat brain increased TonEBP expression in OX-42-positive cells. Furthermore, expression of TonEBP was induced in OX-42-positive cells in a rat model of transient middle cerebral artery occlusion. Together these results show that the expression of TonEBP is regulated by inflammatory signals in mammalian brain, suggesting that TonEBP might play a part during neuroinflammation.

Mutant p53-Notch1 Signaling Axis Is Involved in Curcumin-Induced Apoptosis of Breast Cancer Cells.

Notch1 has been reported to be highly expressed in triple-negative and other subtypes of breast cancer. Mutant p53 (R280K) is overexpressed in MDA-MB-231 triple-negative human breast cancer cells. The present study aimed to determine whether the mutant p53 can be a potent transcriptional activator of the Notch1 in MDA-MB-231 cells, and explore the role of this mutant p53-Notch1 axis in curcumin-induced apoptosis. We found that curcumin treatment resulted in an induction of apoptosis in MDA-MB-231 cells, together with downregulation of Notch1 and its downstream target, Hes1. This reduction in Notch1 expression was determined to be due to the decreased activity of endogenous mutant p53. We confirmed the suppressive effect of curcumin on Notch1 transcription by performing a Notch1 promoter-driven reporter assay and identified a putative p53-binding site in the Notch1 promoter by EMSA and chromatin immunoprecipitation analysis. Overexpression of mutant p53 increased Notch1 promoter activity, whereas knockdown of mutant p53 by small interfering RNA suppressed Notch1 expression, leading to the induction of cellular apoptosis. Moreover, curcumin-induced apoptosis was further enhanced by the knockdown of Notch1 or mutant p53, but it was decreased by the overexpression of active Notch1. Taken together, our results demonstrate, for the first time, that Notch1 is a transcriptional target of mutant p53 in breast cancer cells and suggest that the targeting of mutant p53 and/or Notch1 may be combined with a chemotherapeutic strategy to improve the response of breast cancer cells to curcumin.

Curcumin down-regulates visfatin expression and inhibits breast cancer cell invasion.

Obesity is frequently associated with breast cancer. Such associations are possibly mediated by adipokines. Visfatin, an adipokine, has recently been shown to be related to the development and progression of breast cancer. Therefore, the down-regulation of visfatin may be a novel strategy for breast cancer therapy. Curcumin has anticancer activities by modulating multiple signaling pathways and genes. The purpose of this study was to investigate whether visfatin gene expression is affected by curcumin in human breast cancer cells and to characterize the functional role of visfatin in breast cancer. We found that the mRNA and protein levels of visfatin were down-regulated by curcumin in MDA-MB-231, MDA-MB-468, and MCF-7 breast cancer cells, along with decreased activity of constitutive nuclear factor (NF)-κB. We confirmed the repressive effect of curcumin on visfatin transcription by performing a visfatin promoter-driven reporter assay and identified two putative NF-κB-binding sites on visfatin promoter that are important for this effect. EMSA and chromatin immunoprecipitation analysis indicated the binding of p65 to the visfatin promoter, which was effectively blocked by curcumin. Enforced expression of p65 protein increased visfatin promoter activity, whereas blocking NF-κB signaling suppressed visfatin gene expression. Visfatin could enhance the invasion of MDA-MB-231 cells and also attenuate curcumin-induced inhibition of cell invasion; on the other hand, visfatin knockdown by small interfering RNA led to the reduction of cell invasion. Our data demonstrate, for the first time, that curcumin down-regulates visfatin gene expression in human breast cancer cells by a mechanism that is, at least in part, NF-κB dependent and suggest that visfatin may contribute to breast cancer cell invasion and link obesity to breast cancer development and progression.

Notch1 mediates visfatin-induced FGF-2 up-regulation and endothelial angiogenesis.

AIMS: Our aims were to determine the role of Notch1 in mediating visfatin-induced angiogenesis and to explore potential target genes involved. METHODS AND RESULTS: Inhibition of Notch signalling attenuated visfatin-induced angiogenesis in vitro, ex vivo, and in vivo. Visfatin increased γ-secretase activity, Notch1 cleavage and activation, and Hes1 gene induction. Visfatin also stimulated fibroblast growth factor-2 (FGF-2) gene expression in a Notch1-dependent manner. Enforced expression of active Notch1 intracellular domain increased FGF-2 protein levels and stimulated endothelial tube formation, whereas blocking Notch1 signalling or knockdown of Notch1 by small interfering RNA suppressed visfatin-induced FGF-2 up-regulation and angiogenesis. Reporter analysis of FGF-2 promoter revealed the presence of CSL (CBF-1, suppressor of hairless, LAG-1)-binding site, and chromatin immunoprecipitation analysis demonstrated the binding of Notch1-CSL complex to this site in response to visfatin. CONCLUSION: Our data provide the first example of Notch1-dependent endothelial FGF-2 induction by visfatin and of Notch1 activation in visfatin-stimulated endothelial angiogenesis, suggesting that the signalling axis of visfatin/Notch1/angiogenic factors like FGF-2 might be a valuable target for pathological angiogenesis.

Visfatin through STAT3 activation enhances IL-6 expression that promotes endothelial angiogenesis.

Signal transducer and activator of transcription 3 (STAT3) acts as a mediator and biomarker in endothelial activation. We have recently shown that a novel adipokine visfatin promotes endothelial angiogenesis. The present study was to determine whether visfatin affects STAT3 activity and to explore the potential target gene(s). Here, we found that visfatin induced the activation of STAT3, as characterized by increased tyrosine phosphorylation, nuclear translocation, and DNA-binding activity in human endothelial cells. In addition, visfatin significantly upregulated mRNA and protein levels of endothelial interleukin-6 (IL-6), which was blocked by a specific inhibitor of STAT3 signaling and by the transfection of siRNA specific for STAT3. Furthermore, visfatin-induced angiogenesis was reduced by the inhibition of STAT3 signaling or neutralization of IL-6 function, as measured by tube formation, rat aortic ring assay, and mouse Matrigel plug assay. Taken together, our results provide the first example of STAT3-dependent endothelial IL-6 induction by visfatin and of the role of IL-6 in mediating visfatin-induced angiogenesis.

Neuromedin B induces angiogenesis via activation of ERK and Akt in endothelial cells.

Neuromedin B (NMB) is one of the bombesin-like peptides in mammals. Recently, bombesin-like peptides have been characterized as growth factors in highly vascularized tumors. In this study, we report that NMB potently stimulates in vivo neovascularization in a mouse Matrigel plug and the sprouting of endothelial cells ex vivo in rat aortic rings. In addition, NMB increases the migration and tube formation in human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with NMB activates the extracellular signal-regulated kinase 1/2 (ERK(1/2)), Akt, and endothelial nitric oxide synthase (eNOS) and increases the level of NO production in a dose- and time-dependent manner. Furthermore, ERK activation and angiogenic sprouting in response to NMB are significantly blocked by the MEK inhibitor. Inhibition of phosphatidylinositol 3-kinase (PI3K) suppresses the NMB-stimulated tubular formation of HUVECs, along with reduction in the phosphorylation of Akt and eNOS. Taken together, these results indicate that NMB is a novel angiogenic peptide, and its angiogenic activity is mediated by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent pathways. This study suggests that NMB may play important roles in mediating a variety of pathophysiological angiogenesis.