Neural Precursor Cell Expressed Research Articles

Fenpropathrin Induces GLT-1 Ubiquitination and Increases IL-6 Secretion through the Mdm2-p53 Pathway.

Human exposure to fenpropathrin, a widely used pesticide, is linked to Parkinson's-like symptoms in the body. However, a specific pathogenic mechanism is still unclear. This study found that fenpropathrin increased the expression of murine double minute 2 (Mdm2) and reduced the expression of p53. Fenpropathrin stimulated the expression of neural precursor cell expressed, developmentally down-regulated 4-like (Nedd4L) and promoted the secretion of the inflammatory cytokine interleukin-6 (IL-6) through the Mdm2-p53 pathway. Nedd4L, a ubiquitin ligase, mediated the ubiquitination degradation of glutamate transporter 1 (GLT-1), resulting in glutamate accumulation and excitotoxicity aggravation. Our findings elucidate part of the pathogenic mechanism of fenpropathrin toxicity and provide scientific evidence to help develop guidance for pesticide control and environmental protection.

Curcumin modulates neurogliogenesis and purinergic receptor expression in neural precursor cells infected with Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis, and its congenital transmission is of paramount concern. During embryonic development, infection with the parasite causes irreversible damage to the still-forming fetus's central nervous system (CNS). In the pathogenesis of neurotoxoplasmosis, purinergic receptors prejudice neuroprotection, neuroinflammation, and activation of microbicide mechanisms against the parasitic vacuole. This study used curcumin as a treatment for neural precursor cells (NPCs) infected with T. gondii. The congenital toxoplasmosis induction consisted of maternal infection with the VEG strain, and NPCs were obtained from the telencephalon of mouse embryos. Curcumin at increasing concentrations was administered in vitro to analyze NPC metabolic activity, cell number, and size, as well as neurogliogenesis, proving to be effective in recovering the size of infected NPCs. Curcumin partially re-established impaired neurogenesis. Purinergic A1, A2A, and P2X7 receptors may be related to neuroprotection, neuroinflammatory control, and activation of mechanisms for inducing the parasite's death. ERK 1/2 was highly expressed in infected cells, while its expression rates decreased after the addition of the treatment, highlighting the possible anti-inflammatory action of curcumin. These findings suggest that curcumin treats neurological perturbations induced by toxoplasmosis.

MiR-4735-3p inhibits cell migration and invasion of gastric cancer by downregulating NEDD9.

Gastric cancer (GC) comprises the 3rd cause of cancer-related death worldwide. Increased expression of neural precursor cell expressed, developmentally downregulated 9 (NEDD9) is commonly observed in GC, however, its underlying molecular mechanism in GC remains unknown. The potential interaction between miR-4735-3p and NEDD9 was predicted by TargetScan 7.1. Expression profiles of miR-4735-3p and NEDD9 were examined between GC tissues and normal tissues by reverse transcription-quantitative (RT-q) PCR. The relationship between miR-4735-3p and NEDD9 was validated by RT-qPCR, western blotting, dual luciferase reporter assay and RNA immunoprecipitation assay. Biological relationship between miR-4735-3p and NEDD9 was evidenced by the cell invasion and cell migration assays. NEDD9 level was negatively associated with miR-4735-3p level in GC tissues. miR-4735-3p suppressed NEDD9 levels in GC cells. NEDD9 was revealed to be a target gene of miR-4735-3p. miR-4735-3p overexpression suppressed cell migration and invasion of GC cells, which were antagonized by overexpression of NEDD9. Moreover, miR-4735-3p mimic decreased the levels of matrix metalloproteinases 2/9, increased the level of E-cadherin, which were reversed by overexpression of NEDD9. Collectively, the present study provided a potential mechanism for the tumor suppressor role of miR-4735-3p in GC by targeting NEDD9.

NEDD9 Mediates the FAK/Src Signaling Pathway to Promote the Adhesion of Human Trabecular Meshwork Cells after Dexamethasone Treatment

Purpose The differential gene expression of neural precursor cell expressed developmentally downregulated 9 (NEDD9) in human trabecular meshwork (HTM) cells after dexamethasone (Dex) treatment was confirmed through gene expression profiling. However, the regulatory mechanism of NEDD9 expression in HTM cells remains unknown. In this study, we investigated NEDD9 expression in HTM cells and gained a better understanding of glucocorticoid-induced glaucoma (GIG) pathophysiology. Methods The Gene Expression Omnibus database and GEO2R tool were used to identify differentially expressed genes in the GSE37474 and GSE124114 datasets, and NEDD9 gene expression was found to be upregulated. Human corneoscleral segments and HTM cells were treated with 100 nM Dex or an equal volume of ethanol (0.01%) for 7 days. NEDD9 expression in TM tissues was evaluated by immunohistochemistry, and NEED9 expression in HTM cells was confirmed by RT-qPCR and western blotting. HTM cell adhesive behaviors were assessed with a cell adhesion detection kit. NEDD9 expression was knocked down with short hairpin RNA in HTM cells, and FAK/Src signaling pathway activation was found to be regulated by NEDD9. Results After 7 days of HTM cell Dex treatment, NEDD9 expression was upregulated to be approximately twice that of control. FAK, Src, phospho-FAK, and phospho-Src expression in Dex-treated HTM cells was markedly increased. Downregulation of NEDD9 expression reduced HTM cell adhesion to the surface of culture wells and simultaneously led to a reduction in FAK, Src, phospho-FAK and phospho-Src expression. Conclusions NEDD9 expression is upregulated in HTM cells after Dex treatment and promotes HTM cell adhesion. These findings underscore the contribution of NEDD9 overexpression to altered HTM cell adhesion during glucocorticoid therapy and may play a key role in GIG pathological progression. Considering the similarity between GIG and primary open-angle glaucoma (POAG), our findings suggest that targeting NEDD9 may be a new therapeutic strategy for POAG patients.

Conditional knockout of leptin receptor in neural stem cells leads to obesity in mice and affects neuronal differentiation in the hypothalamus early after birth

Leptin, secreted by peripheral adipocytes, binds the leptin receptor (Lepr) in the hypothalamus, thereby contributing to the regulation of satiety and body weight. Lepr is expressed in the embryonic brain as early as embryonic day 12.5. However, the function of Lepr in neural precursor cells in the brain has not been resolved. To address this issue, we crossed the Leprflox/flox mice with each of Shh-Cre mice (Shh, sonic hedgehog) and Nestin (Nes)-Cre mice. We found that deletion of Lepr specifically in nestin-expressing cells led to extreme obesity, but the conditional null of Lepr in Shh-expressing cells had no obvious phenotype. Moreover, the level of leptin-activated pSTAT3 decreased in the anterior and central subregions of the arcuate hypothalamus of Shh-Cre; Leprflox/flox mice compared with the controls. By contrast, in Nes-Cre; Leprflox/flox mice, the level of leptin-activated pSTAT3 decreased in all subregions including the anterior, central, and posterior arcuate hypothalamus as well as the dorsomedial, ventromedial, and median eminence of the hypothalamus, revealing that the extensive lack of Lepr in the differentiated neurons of the hypothalamus in the conditional null mice. Notably, conditional deletion of Lepr in nestin-expressing cells enhanced the differentiation of neural precursor cells into neurons and oligodendroglia but inhibited differentiation into astrocytes early in postnatal development of hypothalamus. Our results suggest that Lepr expression in neural precursor cells is essential for maintaining normal body weight as well as the differentiation of neural precursor cells to the neural/glial fate in the hypothalamus shortly after birth.

Comparison of serum NEDD-9, CA 15-3, and CEA levels and PET metabolic parameters in breast cancer patients with 18 F-FDG PET / CT.

OBJECTIVE Analyze the over expression of neural precursor cell expressed developmentally down-regulated protein 9 (NEDD-9) deregulated associated with a poor prognosis in various carcinomas. Our objective was to investigate the relationship between the levels of NEDD-9, CA 15-3, and CEA and PET (SUVmax, MTV40, TLG40) with the clinical parameters of patients with breast cancer (BC). METHODS One hundred and eleven patients (82 BC patients who underwent 18F-FDG PET/CT and 29 healthy controls) were evaluated. SUVmax, MTV, and TLG of the primary tumor were compared with the molecular and histopathological subtypes. 18F-FDG, MTV, and TLG were evaluated based on the clinical data, i.e., nodal involvement, distant metastasis, ER and PR status, Ki-67, serum levels of NEDD-9, CA15-3, and CEA. We compared the NEDD-9 in the BC and healthy control groups. RESULTS The mean ± SD of SUVmax in the 82 patients was 13.0 ± 8.6. A statistically significant relationship (p = 0.022) was found between the molecular subtypes and 18F-FDG uptake. The relationship between 18F-FDG uptake and TLG measured in patients <50 years, ER-PR negativity, and HER2 positivity were statistically significant (p=0.015, 0.007, 0.046, and 0.001, respectively). MTV40, TLG40, and CA 15-3 in metastatic patients were statistically significant (p=0.004, 0.005, and 0.003, respectively). NEDD-9 in the BC group was significantly higher than in the healthy group (p=0.017). There was a positive correlation between SUVmax and Ki67 and CA 15-3; MTV40 and CEA; CA 15-3, CEA, SUVmax, and MTV40; a negative correlation was found between CEA, TLG40, and age. CONCLUSION The use of SUVmax, MTV40, and TLG40 parameters with NEDD-9 and tumor markers has been shown to provide a high diagnostic, predictive, and prognostic value for the management of BC. This is considered to be the basis of interventions focused on the treatment objectives related to NEDD-9.

The Role of Neural Precursor Cell Expressed Developmentally Down-Regulated Protein 4.1- Mediated Insulin-Like Growth Factor-1R? Ubiquitination in the Pathogenesis of Alzheimer?s disease

To study the role of Neural precursor cell expressed developmentally down-regulated protein 4.1 on ubiquitination of central Insulin-like growth factor 1β pathway in Alzheimer’s Disease, and to clarify the molecular mechanism of Neural precursor cell expressed developmentally down-regulated protein 4.1 and Insulin-like growth factor 1β for central insulin signalling pathway damage. Mouse hippocampal neurons cells were isolated from Intracerebral haemorrhage mice. 30 μM or 300 μM S-Nitroso-l-cysteine were incubated with hippocampal neurons cells for 4 h. pCMV-Neural precursor cell expressed developmentally down-regulated protein 4.1 or siR- Neural precursor cell expressed developmentally down-regulated protein 4.1 were synthesized and transfected into S-Nitroso-l-cysteine treated hippocampal neurons cells. Cycloheximide and MG132 were incubated with hippocampal neuron cells after S-Nitroso-l-cysteine stimulation. Insulin resistant brain state model were constructed by streptozotocin injection. Western blot were used to test the expression of neural precursor cell expressed developmentally down-regulated protein 4.1, Insulin-like growth factor-1Rβ, Protein kinase B and p- Protein kinase B, Glycogen synthase kinase 3β and p- Glycogen synthase kinase 3β, Tau protein and p-Tau protein in vitro and in vivo. Coimmunoprecipitation were used to analyse Neural precursor cell expressed developmentally downregulated protein 4.1 and Insulin-like growth factor-1Rβ. Neural precursor cell expressed developmentally down-regulated protein 4.1 in the hippocampus of Tg2576 mice increased significantly compared to the control mice (p<0.05), while the expression of Insulin-like growth factor-1Rβ decreased significantly compared to the control mice (p<0.05). S-Nitroso-l-cysteine stimulated hippocampal neuron cells to increase the ubiquitination of Insulin-like growth factor-1Rβ (p<0.05) and decrease the expression of Insulin-like growth factor-1Rβ (p<0.05). Down-regulation of Neural precursor cell expressed developmentally downregulated protein 4.1 can decreased the ubiquitination of Insulin-like growth factor-1Rβ significantly (p<0.05), and rescued the S-Nitroso-l-cysteine inhibition effect on expression of Insulin-like growth factor-1Rβ. Over-expression Neural precursor cell expressed developmentally down-regulated protein 4.1 could increase the ubiquitination level of Insulin-like growth factor-1Rβ (p<0.05). MG132 and cyclohexide both can reverse the degradation of Insulin-like growth factor-1Rβ induced by S-Nitroso-l-cysteine. The phosphorylation degree of Protein kinase B and Glycogen synthase kinase 3β in neurons cells treated with S-Nitroso-l-cysteine decreased significantly (p<0.05), while Tau phosphorylation level in hippocampal neuron cells treated with Insulin-like growth factor-1 increased significantly (p<0.05). S-Nitroso-lcysteine can improve Tau protein phosphorylation (p<0.05) in neurons cells. C1060 site is necessary for its ubiquitination and degradation. Insulin-like growth factor-1Rβ were significantly lower (p<0.05) in the Streptozotocin model while Neural precursor cell expressed developmentally down-regulated protein 4.1 and Aβ were significantly higher (p<0.05) in the Streptozotocin model. p- Protein kinase B/Protein kinase B and p- Glycogen synthase kinase 3β/Glycogen synthase kinase 3β in the Streptozotocin dementia rats were significantly lower (p<0.05), and p-Tau/Tau were significantly increased (p<0.05). Abnormal increasing neural precursor cell expressed developmentally down-regulated protein 4.1 in brain injury tissue induced the rising ubiquitination of Insulin-like growth factor-1Rβ and rising of Aβ amount and Tau protein phosphorylation. C1060 site in Insulin-like growth factor-1Rβ was necessary for the Neural precursor cell expressed developmentally down-regulated protein 4.1 of ubiquitination and degradation of Insulin-like growth factor-1Rβ. In addition, Neural precursor cell expressed developmentally down- regulated protein 4.1 affects Insulin-like growth factor-1 signal pathway in brain, such as inhibiting Protein kinase B and Glycogen synthase kinase 3β phosphorylation, promoting Tau protein phosphorylation, and promoting β-amyloid secretions. The Role of Neural Precursor Cell Expressed Developmentally Down-Regulated Protein 4.1- Mediated Insulin-Like Growth Factor-1Rβ Ubiquitination in the Pathogenesis of Alzheimer’s disease

Sox9 overexpression exerts multiple stage-dependent effects on mouse spinal cord development.

The high-mobility-group (HMG)-domain protein Sox9 is one of few transcription factors implicated in gliogenesis in the vertebrate central nervous system. To further study the role of Sox9 in early spinal cord development, we generated a mouse that allows expression of Sox9 in a temporally and spatially controlled manner. Using this mouse, we show that premature Sox9 expression in neural precursor cells disrupted the neuroepithelium of the ventricular zone. Sox9 also compromised development and survival of neuronal precursors and neurons. Additionally, we observed in these mice substantial increases in oligodendroglial and astroglial cells. Reversing the normal order of appearance of essential transcriptional regulators during oligodendrogenesis, Sox10 preceded Olig2. Our study reinforces the notion that Sox9 has a strong gliogenic activity. It also argues that Sox9 expression has to be tightly controlled to prevent negative effects on early spinal cord structure and neuronal development.

Dexmedetomidine exerts dual effects on human annulus fibrosus chondrocytes depending on the oxidative stress status.

Dexmedetomidine (Dex) is an anesthetic widely used in lumbar discectomy, but its effect on chondrocytes remains unclear. Dex is speculated to promote cartilage degeneration by activating α-2 adrenergic receptor. However, the antioxidative and anti-inflammatory effects of Dex implied the potential chondrocyte protective effect under stress conditions. The present study aimed to determine the effect of Dex on chondrocytes under non-stress and stress conditions. Chondrocytes were isolated from human annulus fibrosus (AF) tissues and oxidative stress was induced by treatment with 1 mM hydrogen peroxide (H2O2). Chondrocytes were treated with Dex alone or in combination with H2O2. Treatment with Dex alone decreased mRNA expression of COL2A1 and increased that of MMP-3 and MMP-13, thus contributing to cartilage degeneration. However, Dex prevented H2O2-induced death and degeneration of chondrocytes partly by enhancing antioxidant capacity. Mechanistically, Dex attenuated H2O2-mediated activation of NF-κB and NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), both of which play key roles in inflammation and inflammatory damage. Dex inactivated NLRP3 through the suppression of NF-κB and JNK signals. Co-treatment with Dex and H2O2 increased protein level of XIAP (X-linked inhibitor-of-apoptosis, an anti-apoptosis protein), compared with H2O2 treatment alone. H2O2 treatment increased the expression of neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) that is a ubiquitin ligase targeting XIAP. However, Dex decreased the amount of NEDD4 adhering to XIAP, thus protecting XIAP protein from NEDD4-mediated ubiquitination and degradation. Given that surgery inevitably causes oxidative stress and inflammation, the protective effect of Dex on chondrocytes during oxidative stress is noteworthy and warrants further study.

WNT signaling represses astrogliogenesis via Ngn2-dependent direct suppression of astrocyte gene expression.

Neural progenitor cells (NPCs) are sequentially specified into neurons and glia during the development of central nervous system. WNT/β-catenin signaling is known to regulate the balance between the proliferation and differentiation of NPCs during neurogenesis. However, the function of WNT/β-catenin signaling during gliogenesis remains poorly defined. Here, we report that activation of WNT/β-catenin signaling disrupts astrogliogenesis in the developing spinal cord. Conversely, inhibition of WNT/β-catenin signaling leads to precocious astrogliogenesis. Further analysis reveals that activation of WNT/β-catenin pathway results in a dramatic increase of neurogenin 2 (Ngn2) expression in transgenic mice, and knockdown of Ngn2 expression in neural precursor cells can reverse the inhibitory effect of WNT/β-catenin on astrocytic differentiation. Moreover, Ngn2 can directly bind to the promoters of several astrocyte specific genes and suppress their expression independent of STATs activity. Together, our studies provide the first in vivo evidence that WNT/β-catenin signaling inhibits early astrogliogenesis via an Ngn2-dependent transcriptional repression mechanism.

Immunohistochemical expression of NEDD9, E-cadherin and γ-catenin and their prognostic significance in pancreatic ductal adenocarcinoma (PDAC).

Extensive research is being conducted to identify novel diagnostic, predictive and prognostic biomarkers for pancreatic ductal adenocarcinoma (PDAC), as only a few markers have been routinely used so far with limited success. Our aim was to assess the expression of neural precursor cell expressed developmentally down-regulated protein 9 (NEDD9), E-cadherin, and γ-catenin in PDAC in relation to clinicopathological parameters and patient survival. We also investigated if there is a correlation of NEDD9 expression with E-cadherin or γ-catenin. The protein expression was determined by immunohistochemistry in 61 PDAC and 61 samples of normal pancreatic tissue. The log rank test and Kaplan-Meier survival curve were used for survival analysis. E-cadherin and γ-catenin expressions were reduced in PDAC, and completely retained in normal pancreatic tissue. Expression of NEDD9 was significantly increased in PDAC (strong expression in 78.7% of cases and moderate in 21.3%) and reduced in normal pancreatic tissue (strong positivity in 45.9% of cases, moderate in 31.1%, and weak in 23%). There was a positive correlation between reduced E-cadherin and γ-catenin expression in PDAC (p = 0.015). The loss or reduced expression of E-cadherin had a negative impact on patient survival (p = 0.020). A negative correlation between E-cadherin expression and tumor grade was also observed (p = 0.011). Decreased E-cadherin expression was more common in male patients with PDAC (81.3% vs. 60% for females, p = 0.005). γ-catenin and NEDD9 expressions were not statistically correlated with tumor stage and grade, gender, nor with patient survival. Our results support the role of NEDD9, E-cadherin and γ-catenin proteins in PDAC, but further research should clarify in detail their mechanism of action in pancreatic cancer.

Expression of Nedd9 in Transbronchial Biopsies of Lung Adenocarcinoma.

SUMMARY – Lung cancer as the major cause of cancer mortality worldwide includes several histologic subtypes evolving from numerous genetic and epigenetic changes emerging in alveolar, bronchiolar and bronchial epithelium. The majority of cases are diagnosed in advanced stage (i.e. stages IIIB and IV), often with scanty amount of tissue in transbronchial biopsies or cytology specimens. The aim of the present study was to investigate the expression of the scaffolding protein neural precursor cell expressed, developmentally downregulated 9 (NEDD9) in small biopsies of lung adenocarcinoma. The expression of NEDD9 was analyzed immunohistochemically in 71 formalin-fixed and paraffin-embedded transbronchial biopsy specimens of primary lung adenocarcinoma. Nuclear and cytoplasmic NEDD9 expression was detected indicating activation of the epithelial-mesenchymal transition process. Direct relationship between the expression of NEDD9 and survival was not demonstrated.

Correlation of NEDD4-1 and PTEN expression with the invasive capacity of pituitary adenomas.

The aim of the present study was to correlate the expression of neural precursor cell expressed developmentally downregulated 4-1 (NEDD4-1) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) with the invasive capacity of pituitary adenomas. A total of 50 pituitary adenoma tissues and 10 normal pituitary tissues were divided into the invasive group (26 cases), the non-invasive group (24 cases) and the normal group (10 cases). The expression of NEDD4-1 and PTEN was determined by immunohistochemistry. NEDD4-1 was revealed to be located in the nucleus and cytoplasm, whereas PTEN was only located in the cytoplasm. Furthermore, expression of NEDD4-1 was higher in pituitary adenomas compared with normal pituitary tissues (P<0.05), and higher in the invasive group compared with the non-invasive group, whereas the opposite trend was observed for PTEN. There was a strong negative correlation between NEDD4-1 and PTEN expression, indicating a dependency between the two and an association with invasiveness. In conclusion, NEDD4-1 may serve as a diagnostic and prognostic factor, and as a novel therapeutic target, in pituitary adenomas.

DENEDDYLASE1 deconjugates NEDD8 from non-cullin protein substrates in Arabidopsis thaliana.

The evolutionarily conserved 8-kD protein NEDD8 (NEURAL PRECURSOR CELL EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED8) belongs to the family of ubiquitin-like modifiers. Like ubiquitin, NEDD8 is conjugated to and deconjugated from target proteins. Many targets and functions of ubiquitylation have been described; by contrast, few targets of NEDD8 have been identified. In plants as well as in non-plant organisms, the cullin subunits of cullin-RING E3 ligases are NEDD8 conjugates with a demonstrated functional role for the NEDD8 modification. The existence of other non-cullin NEDD8 targets has generally been questioned. NEDD8 is translated as a precursor protein and proteolytic processing exposes a C-terminal glycine required for NEDD8 conjugation. In animals and yeast, DENEDDYLASE1 (DEN1) processes NEDD8. Here, we show that mutants of a DEN1 homolog from Arabidopsis thaliana have no detectable defects in NEDD8 processing but do accumulate a broad range of NEDD8 conjugates; this provides direct evidence for the existence of non-cullin NEDD8 conjugates. We further identify AUXIN RESISTANT1 (AXR1), a subunit of the heterodimeric NEDD8 E1 activating enzyme, as a NEDD8-modified protein in den1 mutants and wild type and provide evidence that AXR1 function may be compromised in the absence of DEN1 activity. Thus, in plants, neddylation may serve as a regulatory mechanism for cullin and non-cullin proteins.

Abstract 2003: CPE-delta-N promotes metastasis by regulating Nedd9 and Yap1 expression

Abstract Cancer mortality is often from metastatic disease rather than the direct effect of the primary tumor. Identification of metastasis inducing genes may offer valuable mechanistic insight for guiding specific therapeutic strategies. We report here that the carboxypeptidase E gene (CPE) is alternatively spliced in human tumors to yield an N-terminal truncated protein (CPE-DELTA-N) that drives metastasis. CPE-DELTA-N mRNA was elevated in human metastatic colon, breast and HCC cell lines. Suppression of CPE-DELTA-N expression in these cell lines by si-RNA significantly inhibited their growth migration and invasion. To confirm these observations in vivo, an orthotopic nude mouse model was established. The mice implanted with a tumor derived from HCC cells transfected with si- CPE-DELTA-N RNA in the liver did not show tumor growth or metastasis, compared to scrambled controls. In HCC cytosolic CPE-DELTA-N protein was translocated to the nucleus and upregulated the expression of neural precursor cell expressed, developmentally downregulated gene 9 (Nedd9), through interaction with histone deacetylase (HDAC) 1/2. Inhibition of HDAC activity by the HDAC inhibitors suppressed expression of NEDD9, without effecting CPE-DELTA-N expression. The enhanced invasive phenotype of HCC cells stably transfected with CPE-DELTA-N was suppressed when Nedd9 was silenced by si-RNA. Furthermore CPE-DELTA-N protein up-regulation also increased Yes Associated Protein 1 (YAP1), Carcinoembryonic Antigen-related Cell Adhesion Molecule 5 (ceacam5) and Carbonyl Reductase 1(cbr1) and silencing CPE-DELTA-N with siRNA suppressed YAP1, CEACAM5 and CBR1 protein expression. cDNA Microarray studies of HCC cells overexpressing CPE-DELTA-N showed elevated expression of 27 genes associated with metastasis, while 30 genes associated with tumor suppressor function, which included insulin-like growth factor binding protein 5 and 3 (igfbp5 and igfbp3) and h19 were down-regulated. These data were further confirmed by qRTPCR.. In clinical studies, CPE-DELTA-N mRNA quantification in 99 patients with primary tumor (T) and normal surrounding tissue (N) established a T/N cut off value which predicted future intra-hepatic metastasis with high sensitivity and specificity, independent of cancer stage. Additionally, prospective study on 46 resected PHEO/PGLs having very high CPE-DELTA-N mRNA copy numbers accurately predicted future metastasis or recurrence in patients diagnosed with benign tumors at time of surgery, versus low copy numbers in post-surgery, disease-free patients. Additionally, CPE-DELTA-N is a diagnostic biomarker for metastasis in papillary thyroid carcinoma and colorectal cancer. Thus CPE-DELTA-N induces tumor metastasis and is a powerful biomarker for predicting future metastasis and recurrence in HCC and PHEO/PGL patients, superior to histopathological diagnosis. Citation Format: Saravana R.K Murthy, Terence K. Lee, Niamh X. Cawley, Stephen M. Hewitt, Peng Y. Loh. CPE-delta-N promotes metastasis by regulating Nedd9 and Yap1 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2003. doi:10.1158/1538-7445.AM2014-2003

NEDD9 overexpression correlates with poor prognosis in gastric cancer

In this study, the expression of neural precursor cell expressed developmentally downregulated 9 (NEDD9) in benign and malignant gastric tissues was investigated, and the significance of NEDD9 in gastric cancer prognosis was explored. Immunohistochemistry was used to detect NEDD9 expression in gastric cancer, nontumor gastric, and normal gastric tissues. The relationship between NEDD9 expression in gastric cancer tissues and the clinicopathologic factors was examined using the Mann-Whitney U test. The two factors between NEDD9 expression and tumor node metastasis (TNM) stage in gastric cancer patients were analyzed by Spearman rank correlation analysis. The Kaplan-Meier method and log-rank test were used to compare the overall survival of NEDD9 negative, weak positive expression, and strong positive expression group. NEDD9 expression rates were significantly higher (P < 0.001) in gastric cancer tissues (162 out of 187, 86.6 %) compared with normal (2 out of 11, 18.2 %) and nontumor (11 out of 58, 19.0 %) gastric tissues. The upregulated NEDD9 expression in gastric cancer tissue was significantly correlated with high preoperative CEA level (P = 0.044), poor differentiation (P = 0.007), tissue invasion (P = 0.015), present lymph node metastasis (P < 0.001), and high TNM stage (P < 0.001). NEDD9 expression was positively correlated with clinical TNM stage. Advancing clinical TNM stage corresponded with higher NEDD9 expression (r s = 0.289, P < 0.001). The overall 5-year survival of gastric cancer patients with strong positive NEDD9 expression was significantly shorter compared with the survival of NEDD9 negative and weakly positive expression group. NEDD9 may be used as a biomarker in the clinical setting to predict the prognosis of gastric cancer patients.

ML3 Is a NEDD8- and Ubiquitin-Modified Protein

NEDD8 (NEURAL PRECURSOR CELL-EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED PROTEIN8) is an evolutionarily conserved 8-kD protein that is closely related to ubiquitin and that can be conjugated like ubiquitin to specific lysine residues of target proteins in eukaryotes. In contrast to ubiquitin, for which a broad range of substrate proteins are known, only a very limited number of NEDD8 target proteins have been identified to date. Best understood, and also evolutionarily conserved, is the NEDD8 modification (neddylation) of cullins, core subunits of the cullin-RING-type E3 ubiquitin ligases that promote the polyubiquitylation of degradation targets in eukaryotes. Here, we show that Myeloid differentiation factor-2-related lipid-recognition domain protein ML3 is an NEDD8- as well as ubiquitin-modified protein in Arabidopsis (Arabidopsis thaliana) and examine the functional role of ML3 in the plant cell. Our analysis indicates that ML3 resides in the vacuole as well as in endoplasmic reticulum (ER) bodies. ER bodies are Brassicales-specific ER-derived organelles and, similar to other ER body proteins, ML3 orthologs can only be identified in this order of flowering plants. ML3 gene expression is promoted by wounding as well as by the phytohormone jasmonic acid and repressed by ethylene, signals that are known to induce and repress ER body formation, respectively. Furthermore, ML3 protein abundance is dependent on NAI1, a master regulator of ER body formation in Arabidopsis. The regulation of ML3 expression and the localization of ML3 in ER bodies and the vacuole is in agreement with a demonstrated importance of ML3 in the defense to herbivore attack. Here, we extend the spectrum of ML3 biological functions by demonstrating a role in the response to microbial pathogens.

Abstract 3877: An N-terminal truncated carboxypeptidase E splice isoform induces metastasis by activating nedd9, ceacam5, cbr1 and other metastasis inducing genes.

Abstract Cancer mortality is often from metastatic disease rather than the direct effect of the primary tumor. Identification of metastasis inducing genes may offer valuable mechanistic insight for guiding specific therapeutic strategies. We report here that the carboxypeptidase E gene (CPE) is alternatively spliced in human tumors to yield an N-terminal truncated protein (CPE-DELTA-N) that drives metastasis. CPE-DELTA-N mRNA was elevated in human metastatic colon, breast and HCC cell lines. Suppression of CPE-DELTA-N expression in these cell lines by si-RNA significantly inhibited their growth migration and invasion. To confirm these observations in vivo, an orthotopic nude mouse model was established. The mice implanted with a tumor derived from HCC cells transfected with si- CPE-DELTA-N RNA in the liver did not show tumor growth or metastasis, compared to scrambled controls. In HCC cytosolic CPE-DELTA-N protein was translocated to the nucleus and upregulated the expression of neural precursor cell expressed, developmentally downregulated gene 9 (Nedd9), through interaction with histone deacetylase (HDAC) 1/2. Inhibition of HDAC activity by the HDAC inhibitors suppressed expression of NEDD9, without effecting CPE-DELTA-N expression. The enhanced invasive phenotype of HCC cells stably transfected with CPE-DELTA-N was suppressed when Nedd9 was silenced by si-RNA. cDNA Microarray studies of HCC cells overexpressing CPE-DELTA-N showed elevated expression of 27 genes associated with metastasis such as Nedd9, claudin 2 (cldn2), carcinoembryonic antigen-related cell adhesion molecule 5 (ceacam5) and carbonyl reductase 1(cbr1), while 30 genes associated with tumor suppressor function, which included insulin-like growth factor binding protein 5 and 3 (igfbp5 and igfbp3) and h19 were down-regulated. These data were further confirmed by qRTPCR. Suppression of CPE-DELTA-N expression in HT1080 cell line by si-RNA significantly down regulated ceacam5 and cbr1. In clinical studies, CPE-DELTA-N mRNA quantification in 99 patients with primary tumor (T) and normal surrounding tissue (N) established a T/N cut off value which predicted future intra-hepatic metastasis with high sensitivity and specificity, independent of cancer stage. Additionally, prospective study on 16 resected PHEO/PGLs having very high CPE-DELTA-N mRNA copy numbers accurately predicted future metastasis or recurrence in patients diagnosed with benign tumors at time of surgery, versus low copy numbers in post-surgery, disease-free patients. Additionally, CPE-DELTA-N is a diagnostic biomarker for metastasis in papillary thyroid carcinoma and colorectal cancer. Thus CPE-DELTA-N induces tumor metastasis and is a powerful biomarker for predicting future metastasis and recurrence in HCC and PHEO/PGL patients, superior to histopathological diagnosis. Citation Format: Saravana R. Murthy, Terence K. Lee, Niamh X. Cawley, Stephen M. Hewitt, Karel Pacak, Peng Y. Loh. An N-terminal truncated carboxypeptidase E splice isoform induces metastasis by activating nedd9, ceacam5, cbr1 and other metastasis inducing genes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3877. doi:10.1158/1538-7445.AM2013-3877

Abstract 5327: An N-terminal truncated carboxypeptidase E splice isoform induces metastasis by activating nedd9 and other metastasis inducing genes

Abstract Cancer mortality is often from metastatic disease rather than the direct effect of the primary tumor. Identification of metastasis inducing genes may offer valuable mechanistic insight for guiding specific therapeutic strategies. We report here that the carboxypeptidase E gene (CPE) is alternatively spliced in human tumors to yield an N-terminal truncated protein (CPE-ΔN) that drives metastasis. CPE-ΔN mRNA was elevated in human metastatic colon, breast and HCC cell lines. Suppression of CPE-ΔN expression in these cell lines by si-RNA significantly inhibited their growth and invasion. To confirm these observations in vivo, an orthotopic nude mouse model was established. The mice implanted with a tumor derived from HCC cells transfected with si- CPE-ΔN RNA in the liver did not show tumor growth or metastasis, compared to scrambled controls. In HCC cytosolic CPE-ΔN protein was translocated to the nucleus and upregulated the expression of neural precursor cell expressed, developmentally downregulated gene 9 (Nedd9), through interaction with histone deacetylase (HDAC) 1/2. Inhibition of HDAC activity by the HDAC inhibitors suppressed expression of NEDD9, without effecting CPE-ΔN expression. The enhanced invasive phenotype of HCC cells stably transfected with CPE-ΔN was suppressed when Nedd9 was silenced by si-RNA. cDNA Microarray studies of HCC cells overexpressing CPE-ΔN showed elevated expression of 27 genes associated with metastasis such as Nedd9, claudin 2 (cldn2), carcinoembryonic antigen-related cell adhesion molecule 5 (ceacam5), matrix metallopeptidase 1 (mmp1), plasminogen activator (plat) and inositol 1,4,5-trisphosphate 3-kinase A (itpka), while 30 genes associated with tumor suppressor function, which included insulin-like growth factor binding protein 5 and 3 (igfbp5 and igfbp3) and h19 were down-regulated. These data were further confirmed by qRTPCR. Robust increase in the levels of protranscriptional acetylated histone H3 and H4 in these cells suggest epigenetic regulation of downstream genes by CPE-ΔN. In clinical studies, CPE-ΔN mRNA quantification in 99 patients with primary tumor (T) and normal surrounding tissue (N) established a T/N cut off value which predicted future intra-hepatic metastasis with high sensitivity and specificity, independent of cancer stage. Additionally, prospective study on 16 resected PHEO/PGLs having very high CPE-ΔN mRNA copy numbers accurately predicted future metastasis or recurrence in patients diagnosed with benign tumors at time of surgery, versus low copy numbers in post surgery, disease-free patients. Thus CPE-ΔN induces tumor metastasis and is a powerful biomarker for predicting future metastasis and recurrence in HCC and PHEO/PGL patients, superior to histopathological diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5327. doi:1538-7445.AM2012-5327

In Vitro Characterization of Trophic Factor Expression in Neural Precursor Cells

In cellular transplantation strategies for repairing the injured central nervous system, interactions between transplanted neural precursor cells (NPCs) and host tissue remain incompletely understood. Although trophins may contribute to the benefits observed, little research has explored this possibility. Candidate trophic factors were identified, and primers were designed for these genes. Template RNA was isolated from 3 NPC sources, and also from bone marrow stromal cells (BMSCs) and embryonic fibroblasts as comparative controls. Quantitative polymerase chain reaction was performed to determine the effect of cell source, passaging, cellular differentiation, and environmental changes on trophin factor expression in NPCs. Results were analyzed with multivariate statistical analyses. NPCs, BMSCs, and fibroblasts each expressed trophic factors in unique patterns. Trophic factor expression was similar among NPCs whether harvested from rat or mouse, brain or spinal cord, or their time in culture. The expression of neurotrophin NT-3, NT-4/5, glial-derived neurotrophic factor, and insulin-like growth factor-1 decreased with time in culture. Induced differentiation of NPCs led to a marked and statistically significant increase in the expression of trophic factors. Culture conditions and environmental changes were also associated with significant changes in trophin expression. These results suggest that trophins could contribute to the benefits associated with transplantation of NPCs as well as BMSCs. Trophic factor expression changes with NPC differentiation and environmental conditions, which could have important implications with regard to their behavior after in vivo transplantation.