Jinxia Hu

GOLPH3 promotes glioblastoma cell migration and invasion via the mTOR-YB1 pathway in vitro

The identification of genes involved in carcinogenesis and tumor progression is of great interest, since these genes might be possible as candidates for new tumor targeted therapy strategies. Our previous study shows that Golgi phosphoprotein 3 (GOLPH3) is involved in glioma cell migration and invasion, the critical characteristics of malignant gliomas. In this study, we explored the mechanism of GOLPH3 affecting cell migration and invasion and found that GOLPH3 promotes glioblastoma (GBM) cell migration and invasion via the mammalian target of rapamycin(mTOR)‐Y‐box binding protein‐1 (YB1) pathway in vitro. Both the protein levels of GOLPH3 and YB1 were up‐regulated in human glioma tissues and they exhibited direct correlation with each other. In addition, down‐regulation of GOLPH3 inhibited glioma cell migration and invasion, while over‐expression of GOLPH3 enhanced them. Meanwhile, GOLPH3 down‐regulation led to a significant decrease of YB1 level as well as mTOR activity, both required for glioma cell migration and invasion. On the contrary, YB1 level and mTOR activity increased after GOLPH3 over‐expression. YB1 down‐regulation or mTOR ATP site inhibitor INK128 treatment inhibited cell migration and invasion, similar to the effect of GOLPH3 down‐regulation. Furthermore, over‐expression of GOLPH3 induced glioma cell migration and invasion was blocked by INK128 and YB1 down‐regulation. Taken together, these results show that GOLPH3 promotes glioblastoma cell migration and invasion via the mTOR‐YB1pathway, indicating that GOLPH3‐mTOR‐YB1 pathway might be a new therapeutic target for glioma treatment.

CacyBP/SIP protein is important for the proliferation of human glioma cells

Recently, calcyclin‐binding protein or Siah‐1‐interacting protein (CacyBP/SIP), a component of a novel ubiquitinylation pathway, could regulate the β‐catenin degradation (Fukushima et al., Immunity 2006, 24, 29 – 39). However, the potential role of CacyBP/SIP itself in human glioma cells has not been clarified. Here, we found that CacyBP/SIP was expressed highly in human glioma tissues. Silencing of CacyBP/SIP by short‐hairpin RNA severely suppressed the proliferation of human glioma cell U251, which was at least partly mediated by downregulation of phospho‐Akt (p‐Akt) and phospho‐β‐catenin (p‐β‐catenin) as well as upregulation of p53 and p21. Furthermore, overexpression of CacyBP/SIP obviously promoted the proliferation of human glioma U251, which exhibited the exactly contrary trend in the expression of p‐Akt, p‐β‐catenin, p53, and p21. Taken together, these findings suggest that CacyBP/SIP plays important roles in the proliferation of human glioma cell which might be involved in the development of human glioma.

Plumbagin induces growth inhibition of human glioma cells by downregulating the expression and activity of FOXM1

Plumbagin, a natural quinonoid constituent isolated from the root of medicinal plant Plumbago zeylanica L, has exhibited anti-tumor and anti-proliferative activities in various tumor cell lines as well as in animal tumor models. However, its anticancer effects and the mechanisms underlying its suppression of glioma cell growth have not been elucidated. Oncogenic transcription factor Forkhead Box M1 (FOXM1) has garnered particular interest in recent years as a potential target for the prevention and/or therapeutic intervention in glioma, nevertheless, less information is currently available regarding FOXM1 inhibitor. Here, we reported that plumbagin could effectively inhibit cell proliferation, migration and invasion and induce apoptosis of glioma cells. Cell cycle assay showed that plumbagin induced G2/M arrest. Interestingly, we found that plumbagin decreased the expression of FOXM1 both at mRNA level and protein level. Plumbagin also inhibited the transactivation ability of FOXM1, resulting in down-regulating the expression of FOXM1 downstream target genes, such as cyclin D1, Cdc25B, survivin, and increasing the expression of p21CIP1 and p27KIP1. Most importantly, down-regulation of FOXM1 by siFOXM1 transfection enhanced plumbagin-induced change in viability. On the contrary, over-expression of FOXM1 by cDNA transfection reduced plumbagin-induced glioma cell growth inhibition. These results suggest that plumbagin exhibits its anticancer activity partially by inactivation of FOXM1 signaling pathway in glioma cells. Our findings indicate that plumbagin may be considered as a potential natural FOXM1 inhibitor, which could contribute to the development of new anticancer agent for therapy of gliomas.

Mst1 regulates glioma cell proliferation via the AKT/mTOR signaling pathway

Mammalian sterile 20-like 1 (Mst1), an upstream serine/threonine-specific protein kinase of the Hippo pathway, is reported to play important roles in tumor suppression and organ size regulation in mammals via regulating cell proliferation and survival. However, whether it is involved in the pathogenesis of malignant gliomas remains poorly understood. Therefore, in the present work, we examined the effect and mechanism of Mst1 on the proliferation and apoptosis of malignant glioma cells. The cell proliferation and growth of glioma cells were examined by EdU incorporation and CCK-8 assay. In addition, the cell apoptosis was assessed by flow cytometry. We found that down-regulation of Mst1 promoted glioma cell proliferation and growth, but inhibited the cell apoptosis. Consistent with this, over-expression of Mst1 inhibited glioma cell proliferation and growth. Interestingly, Mst1 did not affect the phosphorylation of YAP1, the key downstream molecule of Hippo pathway. However, Mst1 was found to bind to AKT in glioma cell and negatively regulated AKT and mTOR activity. Finally, the increased cell proliferation rate induced by Mst1 down-regulation was partially abolished by down-regulation of AKT1. Meanwhile, glioma cell growth inhibition induced by Mst1 over-expression was partially rescued by over-expression of AKT1. Taken together, these findings suggest that Mst1 regulates proliferation of glioma cells via AKT/mTOR signaling pathway.

FRK Inhibits Migration and Invasion of Human Glioma Cells by Promoting N-cadherin/β-catenin Complex Formation

Fyn-related kinase (FRK), a member of Src-related tyrosine kinases, is recently reported to function as a potent tumor suppressor in several cancer types. Our previous study has also shown that FRK over-expression inhibited the migration and invasion of glioma cells. However, the mechanism of FRK effect on glioma cell migration and invasion, a feature of human malignant gliomas, is still not clear. In this study, we found that FRK over-expression increased the protein level of N-cadherin, but not E-cadherin. Meanwhile, FRK over-expression promoted β-catenin translocation to the plasma membrane, where it formed complex with N-cadherin, while decreased β-catenin level in the nuclear fraction. In addition, down-regulation of N-cadherin by siRNA promoted the migration and invasion of glioma U251 and U87 cells and abolished the inhibitory effect of FRK on glioma cell migration and invasion. In summary, these results indicate that FRK inhibits migration and invasion of human glioma cells by promoting N-cadherin/β-catenin complex formation.

Protein kinase D2 promotes the proliferation of glioma cells by regulating Golgi phosphoprotein 3

Protein kinase D2 (PKD2) has been demonstrated to promote tumorigenesis in many types of cancers. However, how PKD2 regulates cancer cell growth is largely unknown. In this study, we found that over-expression of PKD2 promoted glioma cell growth but down-regulation of PKD2 inhibited it. Further investigation indicated that PKD2 down-regulation decreased the protein level of Golgi phosphoprotein 3(GOLPH3) as well as p-AKT level. On the contrary, over-expression of PKD2 increased the protein level of GOLPH3 and p-AKT. In addition, GOLPH3 exhibited similar effect on glioma cell growth to that of PKD2. Importantly, GOLPH3 down-regulation partially abolished glioma cell proliferation induced by PKD2 over-expression, while over-expression of GOLPH3 also partially rescued the inhibition effect of PKD2 down-regulation on glioma cell growth. Interestingly, the level of PKD2 and GOLPH3 significantly increased and was positively correlated in a cohort of glioma patients, as well as in patients from TCGA database. Taken together, these results reveal that PKD2 promotes glioma cell proliferation by regulating GOLPH3 and then AKT activation. Our findings indicate that both PKD2 and GOLPH3 play important roles in the progression of human gliomas and PKD2-GOLPH3-AKT signaling pathway might be a potential glioma therapeutic target.

Hugl-1 inhibits glioma cell growth in intracranial model

Drosophila lethal (2) giant larvae (lgl) has been reported as a tumor suppressor and could regulate the Drosophila hippo signaling. Human giant larvae-1(Hugl-1), one human homologue of Drosophila lgl, also has been reported to be involved in the development of some human cancers. However, whether Hugl-1 is associated with the pathogenesis of malignant gliomas remains poorly understood. In the present work, we examined the effect of Hugl-1 on glioma cell growth both in vitro and in vivo. Firstly, we found that Hugl-1 protein levels decreased in the human glioma tissues, suggesting that Hugl-1 is involved in glioma progression. Unfortunately, either stably or transiently over-expressing Hugl-1 did not affect glioma cell proliferation in vitro. In addition, Hugl-1 over-expression did not regulate hippo signaling pathway. Interestingly, over-expression of Hugl-1 not only inhibited gliomagenesis but also markedly inhibited cell proliferation and promoted the apoptosis of U251 cells in an orthotopic model of nude mice. Taken together, this study provides the evidence that Hugl-1 inhibits glioma cell growth in intracranial model of nude mice, suggesting that Hugl-1 might be a potential tumor target for glioma therapy.

Inhibiting geranylgeranyltransferase I activity decreases spine density in central nervous system

Geranylgeranyltransferase I (GGT), a protein prenyltransferase, is responsible for the posttranslational lipidation of Rho GTPases, such as Rac, Rho and Cdc42, all of which play an important role in neuronal synaptogenesis. We previously demonstrated that GGT promotes dendritic morphogenesis in cultured hippocampal neurons and cerebellar slices. We report here that inhibiting GGT activity decreases basal‐ and activity‐dependent changes in spine density as well as in learning and memory ability of mice in vivo. We found that KCl‐ or bicuculline‐induced dendritic spine density increases was abolished by specific GGT inhibitor GGTi‐2147 treatment in cultured hippocampal neurons. GGTi‐2147 lateral ventricular injection reduced GGT activity and membrane association of Rac and decreased the density of dendritic spines in the mouse hippocampus, frontal cortex and cerebellum. GGTi‐2147 administration also impaired learning and memory ability of mice. More importantly, mice exposed to environmental enrichment (EE) showed increased spine density and learning and memory ability, which were significantly reversed by GGTi‐2147 administration. These data demonstrate that inhibiting GGT activity prevents both basal‐ and activity‐dependent changes in spine density in central nervous system both in vitro and in vivo. Manipulating GGT activity may be a promising strategy for the therapies of neurodevelopmental disorders, such as autism, depression, and schizophrenia.

β-Catenin is involved in Bex2 down-regulation induced glioma cell invasion/migration inhibition

Previously, we found that brain expressed X-linked gene 2 (Bex2) regulates the invasion/migration ability of glioma cells. However, the mechanism of this effect remains unknown. In current study, we reported that Bex2 down-regulation inhibited glioma cell migration and invasion by decreasing the nucleus and cytoplasm protein level of β-catenin. We found that the protein levels of Bex2 and β-catenin were up-regulated and showed direct correlation in glioma tissues. Bex2 down-regulation significantly decreased β-catenin protein levels but not its mRNA levels. Furthermore, the decreased protein level of β-catenin was located in the nucleus and cytoplasm but not in the cell membrane. Further study found that the effects of Bex2 down-regulation on the invasion and migration of glioma cell could be reversed by β-catenin over-expression. Taken together, Bex2 affects the invasion and migration ability of glioma cells by regulating β-catenin.

Over‐expression of Rap2a inhibits glioma migration and invasion by down‐regulating p‐AKT

Ras‐oncogenic pathway contributes to the pathogenesis of various tumours in humans, in which mutations of three canonical genes including H‐Ras, N‐Ras and K‐Ras are the most common events. Dysregulation of Ras signalling can be tumourigenic, especially gliomas of the central nervous system. Rap proteins are members of the small GTPase superfamily that involved in many biological processes. However, it remains largely unclear as to whether and how Rap proteins are involved in the development of multiple gliomas. We found that the levels of the protein Rap2a and the activity of Rap2a (GTP‐Rap2a) were weakly expressed in glioma tissues. Overexpressed Rap2a significantly inhibited the migration and invasion of glioma cells with an increase of GTP‐Rap2a. Overexpression of the dominant‐active (DA‐Rap2a), but not the dominant‐negative (DN‐Rap2a) form of Rap2a, also similarly inhibited the migration and invasion of glioma cells by reducing the phosphorylation level of AKT. In contrast, downregulation of Rap2a promoted glioma migration and invasion, and raised the phosphorylation level of AKT, whereas these effects were inhibited by PI3K‐specific inhibitor, LY294002. Thus unlike the other family members of Ras, Rab2a probably serves as a tumour suppressor in the pathogenesis of glioma.