Neural Precursor Cell Expressed, Developmentally Down-regulated 9 Research Articles

Combined high NEDD9 expression and E-cadherin loss correlate with poor clinical outcome in gastric cancer.

Neural precursor cell expressed developmentally downregulated 9 (NEDD9) is a member of the Cas family. Previous studies have revealed that NEDD9 coordinates the focal adhesion kinase and Src signaling cascades that are involved in integrin-dependent adhesion and migration, invasion, cell apoptosis and life cycle, and survival, which may play a role in epithelial-mesenchymal transformation. The aim of this study was to analyze the expression of NEDD9 and E-cadherin in gastric cancer (GC) and evaluate their clinical significance. NEDD9 and E-cadherin expression was analyzed with immunohistochemistry using tissue microarray technique in 435 GC patients who underwent gastrectomy. The NEDD9 expression level was defined by the combination score, which was determined by multiplying the staining intensity score and the proportion score (≥5; NEDD9-high, <5; NEDD9-low). E-cadherin loss was defined as a total loss of staining. The clinicopathologic parameters, overall survival, and disease-free survival rates were analyzed according to the NEDD9 and E-cadherin expression status. The combined NEDD9 and E-cadherin expression status correlated with lymphatic invasion (P=0.001), vascular invasion (P=0.020), and T stage (P=0.001). Combined high NEDD9 expression and loss of E-cadherin expression status had a worse overall survival rate (P<0.001) and served as a poor prognostic factor (Hazard ratio 2.49, 95% CI 1.25-5, P=0.01). Immunohistochemical staining for NEDD9 and E-cadherin may function as a candidate prognostic marker for gastric cancer in everyday practice, especially when applied in combination.

NEDD9 scaffolding protein expression as a negative prediction marker in non-small cell lung cancer (NSCLC)

The purpose of the study was to assess the prognostic significance of a scaffolding NEDD9 protein in patients with non-small cell lung cancer (NSCLC). NEDD9 (Neural precursor cell expressed, developmentally down-regulated 9) is a scaffolding protein in many intracellular protein cascades, such as PI3K-AKT-mTOR, Ras-ERK, which play key roles in tumor progression. several studies define the regulatory role of NEDD9 in progression of several solid cancers, including non-small cell lung cancer (NSCLC), however its role as a prediction biomarker is minimally assessed. Materials and methods. In the current study quantitative immunohistochemical (IHC) approach was utilized to characterize NEDD9 expression in a cohort of NSCLC primary tumor samples (n=16), and correlative statistical analysis was performed between NEDD9 expression and a set of patients’ clinical and pathological characteristics. Results of the IHC analysis were validated using broader TCGA RNAseq dataset (n=566). Results. The study demonstrated significant difference between the expression of Nedd9 in stage iii tumors versus stages II and IV (p&lt;0.05). Next, NEDD9 expression level was approximated to high and low (based on median h-score=56.14) and statistical survival analysis revealed a positive correlative trend between the decreased expression of NEDD9 and the decrease in the overall (OS) and progression-free survival (PFS) of the patients. Analysis of TCGA dataset harboring RNA seq data confirmed statistically significant correlation (p=0.05) between low NEDD9 expression and decreased OS. Conclusion. This study suggests that the expression of the scaffolding protein NEDD9, both at the protein and RNA levels, positively correlates with a negative prognosis in NSCLC, suggesting that it can potentially be used as a novel biomarker of disease progression. Further basic and clinical research defining the role of NEDD9 in the progression and metastasis of NSCLC are strongly warranted.

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.

High neural precursor cell-expressed developmentally down-regulated 9 expression is a poor prognostic indicator for osteosarcoma.

BackgroundThis study detected neural precursor cell-expressed developmentally down-regulated 9 (NEDD9) expression in osteosarcoma tissue samples. It also examined the association between NEDD9 expression and clinicopathological features of osteosarcoma.MethodsThe prognostic value of NEDD9 has been verified in multiple solid cancers, but not osteosarcoma. In this study, 45 osteosarcoma paraffin-embedded specimens were accumulated and applied based on immunohistochemistry to identify NEDD9 expression. A correlation linking NEDD9 expression, clinicopathological qualities, and its prognostic significance was assessed to verify whether NEDD9 could be a poor prognosis biomarker.ResultsHigh NEDD9 expression compared to its low expression in osteosarcoma tissue was shown in the immunohistochemical assay. The percentage of high and low NEDD9 expression was 62.2% and 37.8% respectively. NEDD9 expression level was significantly associated with metastasis and Enneking stage (P<0.05), while gender, age, tumor size, sites, and histopathological types were not. Univariate analysis showed that metastasis with a hazard ratio (HR) of 2.63 (P=0.003), Enneking stage III (HR =5.44, P=0.009), chondroblastic osteosarcoma (HR =2.59, P=0.014), and high NEDD9 expression (HR =2.79, P=0.002) were prognostic factors contributing to poor overall survival (OS). Moreover, multivariate analysis revealed that nonconventional pathological osteosarcoma (HR =0.17, P=0.010) and NEDD9 expression (HR =6.03, P<0.001) were independent prognostic factors.ConclusionsHigh NEDD9 expression in osteosarcoma independently indicated poor clinical prognosis, indicating the possibility that NEDD9 could be a therapeutic target for osteosarcoma.

NEDD9 promotes invasion and migration of colorectal cancer cell line HCT116 via JNK/EMT.

Neural precursor cell-expressed, developmentally-downregulated 9 (NEDD9) is a multi-domain skeleton protein that serves an important role in the cell signaling process via modulating invasion, metastasis, proliferation and apoptosis of tumor cells. The present study identified that the expression levels of NEDD9 in colorectal cancer were elevated. Therefore, the effect of downregulating the expression of NEDD9 in terms of invasion and migration of colorectal cancer cells was investigated and the role of the JNK pathway in these processes was also investigated. The data revealed that downregulation of NEDD9 and JNK inhibitors suppressed invasion and migration, decreased expression levels of phosphorylated JNK, increased the expression levels of E-cadherin and decreased the expression levels of vimentin. In summary, NEDD9 promotes invasion and migration of colorectal cancer cells via the JNK pathway.

NEDD9 overexpression predicts poor prognosis in solid cancers: a meta-analysis.

Background: The oncogenicity of neural precursor cell-expressed developmentally down-regulated 9 (NEDD9) has been demonstrated in multiple cancer types. However, the prognostic value of NEDD9 in some solid cancers remains controversial. Thus, this meta-analysis was conducted to evaluate the relationship between NEDD9 expression survival rates in solid tumors.Method: Our meta-analysis included studies searched from various search engines with specific inclusion criteria and exclusion criteria. Combined HRs for overall survival (OS) and disease-free survival (DFS) or progression-free survival (PFS) or recurrence-free survival (RFS) or cancer-specific survival (CSS) were assessed using fixed-effects and random-effects models. The source of heterogeneity was identified by subgroup analysis. Additionally, publication bias was assessed using funnel plot and Egger’s regression asymmetry test.Result: Eighteen studies with a total of 2,476 patients were retrieved for analysis. Pooled HRs and 95% CIs were calculated. Both OS (HR=1.82; 95% CI: 1.43–2.31) and DFS/PFS/RFS/CSS (HR=2.54; 95% CI: 1.93–3.33) indicated that NEDD9 overexpression is associated with poor OS in cancer patients with solid tumors.Conclusion: NEDD9 overexpression might be a potential marker to predict prognosis in solid cancer patients.

E-cadherin and NEDD9 expression in primary colorectal cancer, metastatic lymph nodes and liver metastases.

In Croatia, colorectal cancer mortality rates in males are the third highest in Europe, after Hungary and Slovakia. The results for females rank Croatia in second place after Hungary. According to previous studies, the loss of E-cadherin expression and the higher expression of neural precursor cell-expressed developmentally downregulated 9 (NEDD9) are associated with a worse prognosis. The aim of the present study was to analyze the immunohistochemical expression of NEDD9 and E-cadherin as markers of metastatic potential using a tissue microarray. This retrospective study included 40 previously untreated patients, including 23 males and 17 females with a median age of 64.5 years (range 38–84), with colorectal cancer and synchronous liver metastases that underwent simultaneous colorectal and hepatic resection between January 1st 2006 and December 31st 2013, in the Clinical Hospital Center Sestre Milosrdnice (Zagreb, Croatia). The most frequent tumor stage was T3, while the most frequent nodal stage was N1. Microvascular invasion was present in 37.5% of patients, while perineural invasion was observed in 30% of patients. The immunohistochemical staining index of E-cadherin was highly positive in 87.5% samples of colorectal cancer, 67.7% of lymph nodes and 77.5% of liver metastases. In the primary tumor, highly positive NEDD9 expression was identified in 22.5% of patients. In lymph nodes, it was identified in 35.5% of patients, while in the liver, it was identified in 30% of patients. Significant positive correlations were observed between the percentage of positive lymph nodes and the immunohistochemical staining index of E-cadherin (ρ=0.372; P=0.039) and NEDD9 (ρ=0.451; P=0.011) in lymph nodes. After the conclusion of the study, 55% of the patients succumbed. No significant differences in survival rates were identified regarding the expression of E-cadherin and NEDD9 in the primary tumor, metastatic lymph nodes and liver metastases. Due to the small sample size and the negative results obtained, further research is required to implement these parameters as prognostic factors.

Vimentin and Non-Muscle Myosin IIA are Members of the Neural Precursor Cell Expressed Developmentally Down-Regulated 9 (NEDD9) Interactome in Head and Neck Squamous Cell Carcinoma Cells

Here we demonstrate an interaction between neural precursor cell expressed, developmentally-downregulated 9 (NEDD9) and the cytoskeletal proteins vimentin and non-muscle myosin IIA (NMIIA), based on co-immunoprecipitation and mass spectrometric sequence identification. Vimentin was constitutively phosphorylated at Ser56 but vimentin associated with NEDD9-was not phosphorylated at Ser56. In contrast, NMIIA bound to NEDD9 was phosphorylated on S1943 consistent with its function in invasion and secretion. Treatment of cells with the vimentin-targeting steroidal lactone withaferin A had no effect on vimentin turnover as previously reported, instead causing NEDD9 cleavage and cell death. The NMIIA-selective inhibitor blebbistatin induced cells to form long extensions and attenuated secretion of matrix metalloproteinases (MMPs) 2 and 9. While the site of vimentin interaction on NEDD9 was not defined, NMIIA was found to interact with NEDD9 at its substrate domain. NEDD9 interactions with vimentin and NMIIA are consistent with these proteins having roles in MMP secretion and cell invasion. These findings suggest that a better understanding of NEDD9 signaling is likely to reveal novel therapeutic targets for the prevention of invasion and metastasis.

NEDD9 stimulated MMP9 secretion is required for invadopodia formation in oral squamous cell carcinoma.

Neural precursor cell expressed developmentally downregulated 9 (NEDD9) is a component of the metastatic signatures of melanoma, breast cancer, glioblastoma, lung cancer and head and neck squamous cell carcinoma (HNSCC). Here we tested the efficacy of NEDD9's domains in stimulating matrix metalloproteinase (MMP) secretion and invadopodia formation in cells stably expressing various NEDD9 mutants. Replacement of the 13 YxxP motif substrate domain (SD) tyrosines and the C-terminal Y629 with phenylalanines (F14NEDD9) eliminated tyrosine phosphorylation, MMP9 secretion and loss of invadopodia formation. Mutation of the N-terminal SH3 domain Y12 to glutamic acid (Y12ENEDD9) or phenylalanine (Y12FNEDD9) reduced MMP9 secretion and inhibited invadopodia formation. SH3 domain deletion (∆SH3NEDD9) resulted in the loss of MMP9 secretion and a lack of invadopodia formation. The SH3–SD domain (SSNEDD9) construct exhibited tyrosine phosphorylation and stimulated MMP9 secretion, as did ∆CTNEDD9 which lacked the C-terminus (∆C-terminal; ∆CT). E13NEDD9 expression blocked MMP9 secretion and invadopodia formation. MICAL1 (molecule interacting with Cas-L1) silencing with a short hairpin RNA reduced MMP9 secretion, vimentin and E-cadherin levels while increasing N-cadherin and Rab6 levels, consistent with reduced invasive behavior. These findings indicate that NEDD9 SD phosphorylation and SH3 domain interactions are necessary for increasing MMP9 secretion and invadopodia formation.

Differential genotypical expression of a NEDD9 in normal and tumor tissues: a possible pharmacological target

Background: Neural precursor cell expressed developmentally down regulated-9 (NEDD9) is a E, D, D scaffolding metastatic marker protein in multiple cancer types. Generally, the expression occurs during the embryonic development and depletes in adults. Expression of NEDD9 in adults leads to the progression of tumor which is sufficient for the cellular invasion. Elevated behavior of the gene mediates metastatic movement which includes protease dependent neovessel formation, invasion and migration of tumor cells from the site of origin to the distant tissues.Methods: The current study involves the screening and elucidation of differential expression of NEDD9 in normal and tumor subtypes with various tissues of mice by immunohistochemistry.Results: The validating approaches in the study, low expression of NEDD9 was observed in the normal tissues and predominance in the tumor subsets.Conclusions: The experimental analysis proven that NEDD9 expression is merely associated with tumor progression and the molecular mechanism of NEDD9 is restricted in the establishment of metastatic cascade. NEDD9 association in tumor prognosis which helps in the emergence of diagnostic and therapeutic approaches.

Expression of three proteins in endometrioid adenocarcinoma and their significance in clinical nursing, diagnosis and treatment.

This study aimed to investigate the role of NEDD9 (neural precursor cell expressed developmentally down-regulated 9), BCAR1/P130CAS (BCAR1/P130 Crk-associated substrate) and paxillin in predicting the prognosis of endometrioid adenocarcinoma (EA), so as to guild the nursing of EA. A total of 65 patients who visited Affiliated Hongqi Hospital of Mudanjiang Medical University between June 2015 and June 2017 were enrolled. They underwent gynecological surgery and had their EA confirmed by pathology, and they were assigned to the EA group. All EA tissues were sampled and archived in paraffin blocks. In addition, 40 specimens of atypical endometrial hyperplasia (EAH) (the EAH group) and 40 specimens of normal proliferative endometria with benign uterine fibroids (the EN group) were selected as controls. The protein levels of NEDD9, BCAR1/P130CAS, and paxillin in each group were then detected by immunohistochemical staining. The expression of the three proteins in the EA group and EAH group was significantly higher than that in the EN group, and their expression was significantly correlated with the clinical stage, histological grade and lymph node metastasis of EA. In addition, the expression of NEDD9, BCAR1/P130CAS, and paxillin in the EA group was positively correlated with each other. BCAR1/P130CAS and paxillin interact with NEDD9 to participate in the growth and migration of EA cells. Therefore, their proteins can be used as biomarkers for the diagnosis, treatment, and prognosis of EA.

Progesterone and calcitriol reduce invasive potential of endometrial cancer cells by targeting ARF6, NEDD9 and MT1-MMP.

Previously, we have demonstrated that progesterone and calcitriol synergistically inhibit growth of endometrial and ovarian cancer by enhancing apoptosis and causing cell cycle arrest. Metastasis is the main reason of mortality in cancer patients. Activation of ADP-Ribosylation Factor 6 (ARF6), Neural Precursor cell expressed Developmentally Downregulated 9 (NEDD9), and Membrane-Type-1 Matrix Metalloproteinase (MT1-MMP) have been implicated in promoting tumor growth and metastasis. We examined the effects of progesterone, calcitriol and progesterone-calcitriol combination on metastasis promoting proteins in endometrial cancer. Expression of ARF6, NEDD9, and MT1-MMP was enhanced in advanced-stage endometrial tumors and in cancer cell lines compared to normal tissues and immortalized EM-E6/E7-TERT endometrial epithelial cells. Knockdown of these proteins significantly inhibited the invasiveness of the cancer cells. The expression levels of all three proteins was reduced with progesterone and progesterone-calcitriol combination treatment, whereas calcitriol alone showed no effect on their expression but moderately decreased MT1-MMP activity. Fluorescence microscopy showed membrane expression of MT1-MMP in vehicle and calcitriol-treated endometrial cancer cells. However, progesterone and calcitriol-progesterone combination treatment revealed MT1-MMP in the cytoplasm. Furthermore, progesterone and calcitriol reduced the activity of MT1-MMP, MMP-9, and MMP-2. In addition, invadopodia regulatory proteins were attenuated in both progesterone and progesterone-calcitriol combination treated cells as well as in MT1-MMP knockdown cells. Thus, targeting the aberrant MT1-MMP signaling with progesterone-calcitriol may be a novel approach to impede MT1-MMP mediated cancer dissemination and may have therapeutic benefits for endometrial cancer patients.

Overexpression of NEDD9 promotes cell invasion and metastasis in hepatocellular carcinoma

Neural precursor cell expressed, developmentally downregulated9 (NEDD9), is a focal adhesion scaffold protein which has been associated with metastasis in several cancers. Recent study found that NEDD9 expression was upregulated in HCC. However, the precise function of NEDD9 in HCC is still unclear. In the present study, we demonstrated that high NEDD9 expression was associated with the invasiveness of HCC in clinical samples. Moreover, by gain-and-loss function studies, we revealed that silencing of NEDD9 expression inhibited cancer cells proliferation, migration and invasion, while upregulated expression of NEDD9 promoted invasion and metastasis of HCC cells in vitro and in vivo. Further studies revealed that NEDD9 inversely regulated E-cadherin in HCC cells and HCC tissues, which indicated that NEDD9 might promotes the invasion and metastasis of HCC cells through the downregulation of E-cadherin, possibly by inducing EMT. On the whole, our findings thus indicate that NEDD9 may serve as a metastasis-promoting gene and potential therapeutic target for the treatment of hepatocellular carcinoma.

MiR-125a-5p upregulation suppresses the proliferation and induces the cell apoptosis of lung adenocarcinoma by targeting NEDD9.

MicroRNAs (miRNAs) are critical translational regulators that act as oncogenes or tumor-suppressor genes. qRT-PCR assay results showed that the expression levels of miR-125a-5p are lower in lung adenocarcinoma(AD) tissues than expression levels in adjacent non-neoplastic tissues. This relative expression was found to be significantly associated with lymph node metastases. Cell growth, apoptosis, caspase activity and Transwell invasion assay results showed that in two lung adenocarcinoma cell lines transfected with a miR-125a-5p mimic, proliferation and invasion rates were found to be significantly reduced, whereas the apoptosis rate of the miR-125a-5p mimic group was enhanced. Subsequent western blotting and luciferase reporter assays showed that miR-125a-5p is able to bind to putative binding sites within the mRNA 3'untranslated region (UTR) of neural precursor cell expressed, developmentally downregulated9 (NEDD9). Our findings suggest that miR-125a-5p may serve as a therapeutic agent for lung adenocarcinoma through its major target, NEDD9.

Downregulation of NEDD9 by apigenin suppresses migration, invasion, and metastasis of colorectal cancer cells

Apigenin is a natural flavonoid which possesses multiple anti-cancer properties such as anti-proliferation, anti-inflammation, and anti-metastasis in many types of cancers including colorectal cancer. Neural precursor cell expressed developmentally downregulated 9 (NEDD9) is a multi-domain scaffolding protein of the Cas family which has been shown to correlate with cancer metastasis and progression. The present study investigates the role of NEDD9 in apigenin-inhibited cell migration, invasion, and metastasis of colorectal adenocarcinoma DLD1 and SW480 cells. The results show that knockdown of NEDD9 inhibited cell migration, invasion, and metastasis and that overexpression of NEDD9 promoted cell migration and invasion of DLD1 cells and SW4890 cells. Apigenin treatment attenuated NEDD9 expression at protein level, resulting in reduced phosphorylations of FAK, Src, and Akt, leading to inhibition on cell migration, invasion, and metastasis of both DLD1 and SW480 cells. The present study has demonstrated that apigenin inhibits cell migration, invasion, and metastasis through NEDD9/Src/Akt cascade in colorectal cancer cells. NEDD9 may function as a biomarker for evaluation of cancer aggressiveness and for selection of therapeutic drugs against cancer progression.

Mice null for NEDD9 (HEF1) display extensive hippocampal dendritic spine loss and cognitive impairment

NEDD9 (neural precursor cell expressed, developmentally down-regulated 9) is a member of the CAS (Crk-associated substrate) family of scaffolding proteins that regulate cell adhesion and migration. A Nedd9 knock-out/lacZ knock-in mouse (Nedd9−/−) was developed in order to study Nedd9 expression and function in the nervous system. Herein we show that NEDD9 is expressed in the adult brain and is prominently expressed in the hippocampus. Behavioral testing uncovered functional deficits in Nedd9−/− mice. In the Morris water maze test, Nedd9−/− mice showed deficits in both the ability to learn the task as well as in their ability to recall the platform location. There was no change in the gross morphology of the hippocampus, and stereological analysis of BrdU-labeled newly formed hippocampal cells suggested that this defect is not secondary to altered neurogenesis. However, analysis of the hippocampus revealed extensive loss of dendritic spine density in both the dentate gyrus (DG) and CA1 regions. Spine loss occurred equally across all branch orders and regions of the dendrite. Analysis of spine density in Nedd9−/− mice at 1.5, 6 and 10 months revealed an age-dependent spine loss. This work shows that NEDD9 is required for the maintenance of dendritic spines in the hippocampus, and suggests it could play a role in learning and memory.

Prognostic roles of serum NEDD9 levels in patients with gastric cancer.

e15103 Background: Neural precursor cell-expressed, developmentally downregulated 9 (NEDD9), a member of Crk-associated substrate (CAS) family, is highly expressed in multiple cancer types and invo...

Abstract PR09: Genetic interaction of NEDD9 with melanoma-prone oncogenes and tumor suppressor genes

Abstract Mouse models have proven their utility in establishing causal roles and addressing genetic interactions of candidate alterations in tumorigenesis. Previously, our cross-species comparison of human and mouse melanoma genomes led to the identification of NEDD9 (Neural precursor cell Expressed, Developmentally Down-regulated 9) as a novel melanoma metastasis gene that is up-regulated in 35~50% of metastatic melanomas. We have shown that NEDD9 enhances proliferation and drives invasion in vitro and lung seeding in vivo of melanoma cells. In this study, to explore the biological roles of NEDD9 in melanoma tumorigenesis, we have generated tet-inducible NEDD9 mouse model that carries a doxycycline-responsive, melanocyte-targeted NEDD9. Two independent transgenic founder lines (line A and D) were obtained and genetic interaction of NEDD9 with melanoma-prone oncogenes and tumor suppressor genes have been tested. Melanoma displays frequent activation of NRAS and BRAF and inactivation of CDKN2A (INK4A/ARF) and PTEN. First, we asked whether NEDD9 cooperates with RAS/RAF/MAPK pathway activation by crossing Nedd9 alleles onto the non-metastatic Tyr-RAS* Ink4a/Arf-/- melanoma model to establish a colony of compound mutant mice designated as “iNEDD9” (e.g. Tyr-HRAS*;Tyr-rtTA/Tet-NEDD9;Ink4a/Arf-/-). These mice developed spontaneous cutaneous nodular type melanomas and ocular melanomas indistinguishable from the ones developed in Tyr-RAS* Ink4a/Arf-/- model. In these mice, NEDD9 expression in melanocytes via doxycycline administration led to decreased tumor latency (Median latency: 88 (NEDD9 ON) vs. 120 (NEDD9 OFF) days (p=0.041, log-rank test)) and increased tumor initiation without signs of metastasis. Median melanoma-free survival of iNEDD9 mice was 111 (line A, p=0.009, log-rank test) and 114.5 (line D, p=0.032) days, lower than 166 days observed in control mice without NEDD9. This data supports the cooperation of NEDD9 with RAS/RAF/MAPK pathway activation in melanoma tumorigenesis. Secondly, we addressed whether NEDD9 can synergize with loss of PTEN and INK4a/ARF in vitro and in vivo. We observed that NEDD9 cooperated with PTEN loss in Ink4a/Arf-/- melanocytes without activating RAS or RAF mutation and enhanced proliferation, anchorage independent growth, and invasion in vitro and tumor growth in nude mice. NEDD9 expression correlated with increased phosphorylation of SFKs (Src family kinases), AKT2, and STAT3. To analyze the biological consequence of NEDD9 expression in vivo, we generated a mouse model based on loss of PTEN and CDKN2A (INK4A/ARF) with or without NEDD9 up-regulation designated as “NIP” (e.g. Tyr-CreERT2;Tyr-rtTA/Tet-NEDD9;cInk4a/ArfL/L;cPtenL/L). These mice developed cutaneous and ocular melanomas following tamoxifen treatment, which are under characterization. This study will generate a body of knowledge for the in vivo roles of NEDD9 in melanoma tumorigenesis and invaluable melanoma models driven by genetic lesions observed in human melanoma patients. Since most of the current mouse melanoma models are driven by activating mutations in NRAS or BRAF genes, our model may provide a novel opportunity to understand melanomas without these mutations. This abstract is also presented as Poster B04. Citation Format: Hyeran Sung, Jong Woo Lee, Gretchen Alicea, Ji-Hyun Lee, Jane Messina, Scott Granter, Lynda Chin, Minjung Kim. Genetic interaction of NEDD9 with melanoma-prone oncogenes and tumor suppressor genes. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR09.

NEDD9 Regulates Actin Dynamics through Cortactin Deacetylation in an AURKA/HDAC6–Dependent Manner

The prometastatic protein NEDD9 (neural precursor cell expressed, developmentally downregulated 9) is highly expressed in many cancers and is required for mesenchymal individual cell migration and progression to the invasive stage. Nevertheless, the molecular mechanisms of NEDD9-driven migration and the downstream targets effecting metastasis are not well defined. In the current study, knockdown of NEDD9 in highly metastatic tumor cells drastically reduces their migratory capacity due to disruption of actin dynamics at the leading edge. Specifically, NEDD9 deficiency leads to a decrease in the persistence and stability of lamellipodial protrusions similar to knockdown of cortactin (CTTN). Mechanistically, it was shown that NEDD9 binds to and regulates acetylation of CTTN in an Aurora A kinase (AURKA)/HDAC6-dependent manner. The knockdown of NEDD9 or AURKA results in an increase in the amount of acetylated CTTN and a decrease in the binding of CTTN to F-actin. Overexpression of the deacetylation mimicking (9KR) mutant of CTTN is sufficient to restore actin dynamics at the leading edge and migration proficiency of the tumor cells. Inhibition of AURKA and HDAC6 activity by alisertib and Tubastatin A in xenograft models of breast cancer leads to a decrease in the number of pulmonary metastases. Collectively, these findings identify CTTN as the key downstream component of NEDD9-driven migration and metastatic phenotypes. This study provides a mechanistic platform for therapeutic interventions based on AURKA and HDAC6 inhibition for patients with metastatic breast cancer to prevent and/or eradicate metastases.

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.