NEDD9 Protein Research Articles

NEDD9 Restrains dsDNA Damage Response during Non-Small Cell Lung Cancer (NSCLC) Progression.

Simple SummaryAltered DNA damage response (DDR) contributes to numerous processes during the progression of tumors, such as genomic instability, the emergence of neoantigens, aberrations in cell-cell signaling, and acquired tumor resistance to DNA damaging agents, such as platinating agents and irradiation. This study describes a novel role for the scaffolding protein NEDD9 in regulating DDR signaling and characterizes its effects on sensitivity to DNA damaging therapies in a non-small cell lung cancer (NSCLC) setting. Our data demonstrate that NEDD9 depletion is capable of upregulating ATM-CHK2 signaling, shifting NEDD9 depleted cells towards a more mesenchymal phenotype and elevated sensitivity to UV irradiation. Immunohistochemical analysis of the cohort of human NSCLC samples revealed an association between reduced NEDD9 protein expression and a decrease in overall (OS) survival of NSCLC patients.DNA damaging modalities are the backbone of treatments for non-small cell lung cancer (NSCLC). Alterations in DNA damage response (DDR) in tumor cells commonly contribute to emerging resistance to platinating agents, other targeted therapies, and radiation. The goal of this study is to identify the previously unreported role of NEDD9 scaffolding protein in controlling DDR processes and sensitivity to DNA damaging therapies. Using a siRNA-mediated approach to deplete NEDD9 in a group of human and murine KRAS/TP53-mutant NSCLC cell lines, coupled with a set of cell viability and clonogenic assays, flow cytometry analysis, and Western blotting, we evaluated the effects of NEDD9 silencing on cellular proliferation, DDR and epithelial-to-mesenchymal transition (EMT) signaling, cell cycle, and sensitivity to cisplatin and UV irradiation. Using publicly available NSCLC datasets (TCGA) and an independent cohort of primary NSCLC tumors, subsequent in silico and immunohistochemical (IHC) analyses were performed to assess relevant changes in NEDD9 RNA and protein expression across different stages of NSCLC. The results of our study demonstrate that NEDD9 depletion is associated with the increased tumorigenic capacity of NSCLC cells. These phenotypes were accompanied by significantly upregulated ATM-CHK2 signaling, shifting towards a more mesenchymal phenotype in NEDD9 depleted cells and elevated sensitivity to UV-irradiation. IHC analyses revealed an association between reduced NEDD9 protein expression and a decrease in overall (OS) and progression-free survival (PFS) of the NSCLC patients. These data, for the first time, identified NEDD9 as a negative regulator of ATM kinase activity and related DDR signaling in numerous KRAS/TP53 mutated NSCLC, with its effects on the regulation of DDR-dependent EMT signaling, sensitivity to DNA damaging modalities in tumor cells, and the survival of the patients.

Abstract 12577: Pulmonary Endothelial Sulfatase-1 is Regulated by NEDD9 and Increased in Pulmonary Arterial Hypertension

Introduction: Oxidation of the scaffolding protein NEDD9 by aldosterone (ALDO) increases NEDD9 bioactivity in human pulmonary artery endothelial cells (HPAECs), leading to pulmonary arterial hypertension (PAH). In tumor cells, oxidizing conditions alter Sulfatase-1 (SULF-1) activity. In turn, SULF-1 catalyzes 6-O-sulfate removal from heparan sulfate proteoglycans that releases cytokines implicated in the pathogenesis of PAH. Hypothesis: NEDD9 regulates SULF-1 expression in HPAECs, which is associated with pulmonary arterial remodeling in PAH. Methods: HPAECs were treated with ALDO (10 -7 mol/L) for 24 hr and then untransfected (Un-Tx) or transfected with siRNA against NEDD9, and cell lysates were used to isolate mRNA for RNA-Seq. Separately, SULF1 expression was measured by immunoblot in HPAECs transfected with si-NEDD9 or scrambled control siRNA. Paraffin-embedded lung specimens from donor controls (N=5), patients with PAH (N=5), or the monocrotaline (MCT) (inflammatory) and SU-5416/hypoxia (angioproliferative) experimental rat models of PAH were analyzed by immunofluorescence to quantify SULF1, NEDD9, and endothelial PECAM-1. Results: In ALDO-treated HPAECs, si-NEDD9 decreased SULF1 mRNA quantity vs. Un-Tx (logFC -6.39, N=3, P<0.001, FDR<0.001). Transfection with si-NEDD9 also decreased SULF1 protein compared to si-Scr (14 ± 2.0 vs. 2.7 ± 1.0 arb. units, N=3, P<0.01) in vitro . In remodeled pulmonary arterioles from MCT-PAH and SU-5416/hypoxia-PAH, SULF1 was increased by 5.6-fold (P<0.01) and 6.6-fold (P<0.01), respectively, and correlated strongly with NEDD9 protein (r=0.91, N=9, P<0.001). The translational relevance of these data was assessed next: compared to donor controls, pulmonary endothelial SULF1 was increased in remodeled pulmonary arterioles from PAH patients in situ (0.03 ± 0.01 vs. 0.14 ± 0.03 arb. units, P<0.05). Conclusions: SULF1 is regulated by the scaffolding protein NEDD9 in HPAECs in vitro , and remodeled pulmonary arterioles from two experimental PAH models and PAH patients express increased NEDD9-SULF1. Identifying the molecular mechanism(s) regulating NEDD9-dependent regulation of SULF1 may have important implications for therapeutic target discovery to prevent vascular remodeling in PAH.

Radix Scutellariae Ameliorates Stress-Induced Depressive-Like Behaviors via Protecting Neurons through the TGFβ3-Smad2/3-Nedd9 Signaling Pathway.

Chronic stress can impair hippocampal neurogenesis, increase neuronal apoptosis, and cause depressive-like behaviors. Our previous studies found that Radix Scutellariae (RS) can rescue the stress-induced neuronal injury, but the mechanism is not clear. Here, we continued to investigate the underlying antidepressant mechanisms of the RS extract. A 7-week chronic unpredictable mild stress (CUMS) procedure was used to establish a murine depression model. 0.75 g/kg or 1.5 g/kg RS was administered daily to the mice during the last 4 weeks. Depressive-like behaviors were evaluated by the sucrose preference test (SPT), forced swimming test (FST), open field test (OFT), and tail suspension test (TST). The neuroprotective effect of RS was evaluated with the expression of hippocampal neuron-related markers and apoptosis-associated proteins by Nissl staining, immunohistochemistry, and western blot. Transforming growth factor-β3 (TGFβ3) pathway-related proteins were detected by western blot. Results showed that RS could ameliorate depressive-like behaviors, increase the expression of the antiapoptotic protein B-cell lymphoma 2 (BCL-2), reduce the expression of the proapoptotic protein BCL-2-associated X (BAX), and increase the number of doublecortin- (DCX-), microtubule-associated protein 2- (MAP2-), and neuronal nucleus- (NeuN-) positive cells in the hippocampus. Moreover, RS could reverse the CUMS-induced decrease of TGFβ3 protein, promote the phosphorylation of SMAD2/3, and increase the expression of downstream NEDD9 protein. These results suggest that RS could exert antidepressant effects via protecting neurons. And the molecular mechanism might be related to the regulation of the TGFβ3-SMAD2/3-NEDD9 pathway.

ZMYND10, an epigenetically regulated tumor suppressor, exerts tumor-suppressive functions via miR145-5p/NEDD9 axis in breast cancer

BackgroundRecent studies suggested that ZMYND10 is a potential tumor suppressor gene in multiple tumor types. However, the mechanism by which ZMYND10 inhibits breast cancer remains unclear. Here, we investigated the role and mechanism of ZMYND10 in breast cancer inhibition.ResultsZMYND10 was dramatically reduced in multiple breast cancer cell lines and tissues, which was associated with promoter hypermethylation. Ectopic expression of ZMYND10 in silenced breast cancer cells induced cell apoptosis while suppressed cell growth, cell migration and invasion in vitro, and xenograft tumor growth in vivo. Furthermore, molecular mechanism studies indicated that ZMYND10 enhances expression of miR145-5p, which suppresses the expression of NEDD9 protein through directly targeting the 3'-untranslated region of NEDD9 mRNA.ConclusionsResults from this study show that ZMYND10 suppresses breast cancer tumorigenicity by inhibiting the miR145-5p/NEDD9 signaling pathway. This novel discovered signaling pathway may be a valid target for small molecules that might help to develop new therapies to better inhibit the breast cancer metastasis.

Silencing NEDD9 by lentivirus-delivered shRNA inhibits the growth of BxPC-3 cells in vitro and in vivo.

BackgroundNeural precursor cell-expressed, developmentally downregulated protein 9 (NEDD9) is an invasion and metastasis-related gene. It has been proven to be highly expressed and closely associated with tumor proliferation and invasion in several types of human cancers including pancreatic adenocarcinoma. The present study was aimed to investigate and characterize the efficacy of silencing NEDD9 by lentivirus-delivered shRNA in pancreatic cancer (PC) BxPC-3 cells in vivo and in vitro.MethodsFive kinds of PC cell lines were used to determine the cell line which expressed NEDD9 the most with qRT-PCR and western blotting. Then, we transduced the lentivirus-delivered NEDD9 shRNA into the human PC BxPC-3 cells to obtain a stable cell line expressing shRNA targeting NEDD9. NEDD9 mRNA and protein expression were measured by qRT-PCR and western blotting, respectively. Cell proliferation, migration, and invasion were assessed by cell colony formation, scratch wound healing, and Transwell assays, respectively. Mouse tumor xenografts were established by injecting tumor cells into the right flank of BALB/c nude mice. The effects of silencing NEDD9 on the growth of BxPC-3 cells in vivo were also examined.ResultsAmong 5 kinds of PC cell lines, BxPC-3 cells were selected as the most suitab to carry out the following experiment. Transduction of lentivirus-delivered NEDD9 shRNA efficiently reduced NEDD9 expression in pancreatic adenocarcinoma BxPC-3 cells. Silencing NEDD9 by RNAi inhibited proliferation, migration, and invasion of BxPC-3 cells in cell culture. Importantly, it significantly reduced the growth of BxPC-3 cells in mouse xenografts.ConclusionsSilencing NEDD9 by lentivirus-delivered shRNA efficiently inhibited the growth of PC BxPC-3 cells both in vitro and in vivo, and may prove to be a potential new therapeutic agent for human PC.

SOX2 promotes hypoxia-induced breast cancer cell migration by inducing NEDD9 expression and subsequent activation of Rac1/HIF-1\u03b1 signaling

BackgroundHypoxia, a major condition associated with the tumor microenvironment, stimulates the migration of cancer cells. SOX2 is a powerful transcription factor that shows higher expression in several cancers, however, its role in hypoxia-induced breast cancer cell migration remains largely elusive.MethodsThe human breast cancer cell lines MDA-MB-231 and MDA-MB-468 were cultured under hypoxic conditions. The cell migration rate was determined using the wound-healing and transwell assays. The protein levels of SOX2, NEDD9 and HIF-1α were evaluated via western blotting analysis. The NEDD9 mRNA levels were evaluated using qPCR. The activation of Rac1 was detected with the pulldown assay. The binding of SOX2 to the NEDD9 promoter was checked using the luciferase reporter assay. We also transfected breast cancer cells with specific siRNA for SOX2, NEDD9 or the Rac1 inactive mutant (T17 N) to investigate the role of SOX2, NEDD9 and Rac1 in the response to hypoxia.ResultsHypoxia markedly increased SOX2 protein levels in a time-dependent manner. SiRNA-mediated disruption of SOX2 inhibited cell migration under hypoxic conditions. Hypoxia also significantly augmented the NEDD9 mRNA and protein levels. Interestingly, SOX2 is a positive transcriptional regulator of NEDD9. Knockdown of SOX2 inhibited hypoxia-induced NEDD9 mRNA and protein expressions. Furthermore, hypoxia-induced upregulation of Rac1 activity and HIF-1α expression was attenuated by SOX2 or NEDD9 silencing, and Rac1-T17 N abolished HIF-1α expression as well as cell migration in cells subjected to hypoxia.ConclusionsOur results highlight the essential role of SOX2 in breast cancer cell motility. The upregulation of SOX2 under hypoxic conditions may facilitate NEDD9 transcription and expression, and subsequent activation of Rac1 and HIF-1α expression. This could accelerate breast cancer cell migration.

Abstract 5197: Histone deacetylase inhibitors promote breast cancer metastasis by elevating NEDD9 expression

Abstract The approved pan-histone deacetylase (HDAC) inhibitors have brought impressive therapeutic benefits for patients with a few subtypes of hematologic malignancies. Favorable outcomes in breast cancer have not been observed, although multiple HDAC inhibitors have been therapeutically validated in preclinical breast cancer models. Here, we report that pan-HDAC inhibitors provoke breast cancer metastasis in vitro and in vivo, which will severely impede their clinical benefits. Given the role of HDAC inhibitors in the epigenetic control of transcription, we utilized RNA sequencing (RNA-seq) analysis to analyze global gene-expression changes following LBH589 treatment of MCF-7 cells. Pathway enrichment analysis of the 1591 identified genes based on GO analysis showed that 146 GO were significantly altered. Interestingly, most of the changes were related to cytoskeletal, cell junction, and biological adhesion pathways. Genes in the above mentioned GO were intersected to obtain seven genes by Venn analysis. NEDD9 was among the most significantly upregulated transcripts by LBH589, for which was suggested as a metastasis marker in the last decade. We substantiated this result by detecting the mRNA and protein levels of NEDD9 in HDAC inhibitors treated cells. As with the RNA-seq results, a notable upregulation of NEDD9 mRNA and protein were induced by HDAC inhibitors in breast cancer in vitro and in vivo. Mechanistically, pan-HDAC inhibitors enhance acetylation of H3K9 at the NEDD9 gene promoter to upregulate NEDD9 expression via inhibition of HDAC4, and activate FAK phosphorylation. Notably, we demonstrated that FAK inhibitor can reverse pan-HDAC inhibitor-induced breast cancer metastasis, which may improve HDAC inhibitors efficacy for breast cancer. In summary, we demonstrated an NEDD9-FAK signaling pathway, which was induced by pan-HDAC inhibitors, leading to breast cancer metastasis, and thereby caused limitation of the clinical benefits of HDAC inhibitors on breast cancer. We propose that HDAC4 has an upstream regulatory role in NEDD9 expression, leading to FAK phosphorylation and subsequent stimulation of breast cancer metastasis through epigenetic regulation. Our results suggest a need for increased isoform selective HDAC inhibitor research and development. Citation Format: Zong-long Hu, Ya-fang Wang, Yi Su, Yan-yan Shen, Yan-fen Fang, Jian Ding, Yi Chen. Histone deacetylase inhibitors promote breast cancer metastasis by elevating NEDD9 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5197.

NEDD9 Facilitates Hypoxia-Induced Gastric Cancer Cell Migration via MICAL1 Related Rac1 Activation.

Aims and Hypothesis: NEDD9 is highly expressed in gastric cancer and has a significant involvement in its pathogenesis. However, the mechanism behind hypoxia-promoted cancer cell migration and its regulation because of NEDD9 is still unknown. The aim of this study is to investigate the involvement of NEDD9 in gastric cancer cell migration under hypoxia and explore the underlying potential molecular mechanisms.MethodsCell motility was measured by wound healing and transwell assay. NEDD9 and MICAL1 expressions were examined by western blot analysis. Interaction between NEDD9 and MICAL1 was assessed by immunohistochemistry and co-immunoprecipitation assay, respectively. Cells were transfected with plasmids or siRNA to upregulate or downregulate the expression of NEDD9 and MICAL1. Rac1, Cdc42, and RhoA activation was assessed by pulldown assay.ResultsThe mRNA and protein level of NEDD9 increased as a result of hypoxia in gastric cancer cell lines BGC-823 and SGC-7901 while decreased levels of NEDD9 caused reduced cell migratory potential in response to hypoxia. Hypoxia also caused the enhancement of MICAL1 expression. Furthermore, it was revealed that there is a positive correlation between NEDD9 and MICAL1 protein while hypoxia played role in increasing their interaction. Under hypoxic conditions, silencing of NEDD9 caused reduction in the stability of MICAL1 protein, while depletion of MICAL1 also inhibited the migration of NEDD9-overexpressing gastric cancer cells. In addition, silencing of NEDD9 or MICAL1 expression reversed the increased GTP forms of Rac1 and Cdc42 in hypoxic cells. However, only the upregulation of Rac1-GTP level was observed in gastric cancer cells that were already overexpressed by MICAL1.ConclusionIn all, it is concluded that MICAL1 is regulated by NEDD9 that facilitates hypoxia-induced gastric cancer cell migration via Rac1-dependent manner.

POSITIVE EXPRESSION OF NEDD9 IN HEAD AND NECK CANCER IS RELATED TO BETTER SURVIVAL PERIOD.

SUMMARYThe aim was to determine immunohistochemical expression of NEDD9 protein in head and neck squamous cell carcinoma (HNSCC) and the possible relation of its expression with primary tumor size (T), regional lymph node status (N), stage of disease (TNM) and survival period. A total of 131 patients with primary tumor localization in the area of oropharynx, hypopharynx and larynx, monitored for at least 5 years after initial surgical treatment were analyzed. The study included 128 male and three female patients, median age 62.0 (range 53.0-68.0) years. Of these, 105 (95%) patients showed positive NEDD9 expressed by dyed cytoplasm. There were no significant differences in NEDD9 expression according to TNM tumor status. Patients with positive NEDD9 expression had a significantly higher median (IQR) survival time 51.0 (15.0-60.0) months as compared to 22.5 (9.0-55.0) months in patients with negative NEDD9 expression (p=0.048). NEDD9 negative expression, controlled for the influence of other variables included in the Cox’s proportional hazards model, had a significant hazard ratio (HR) of 2.10 (95% CI: 1.23-3.58; p=0.006). The results of our study showed that NEDD9 expression might be an independent prognostic marker in patients with HNSCC regarding data on overall survival and mortality.

Estrogen receptor β promotes the vasculogenic mimicry (VM) and cell invasion via altering the lncRNA-MALAT1/miR-145-5p/NEDD9 signals in lung cancer.

While estrogen receptor β (ERβ) may impact the progression of non-small cell lung cancer (NSCLC), its linkage to alteration of the vasculogenic mimicry (VM) formation to influence the NSCLC cell invasion remains unclear. Here, we analyzed immunohistochemistry data from NSCLC tissues and found that ERβ-positive NSCLC female patients had worse survival outcomes than those of ERβ-negative NSCLC female patients. In vitro studies using multiple NSCLC cell lines also revealed that ERβ could increase the VM formation and cell invasion. Molecular mechanism dissection suggested that ERβ could increase the lncRNA-MALAT1 (MALAT1) expression via directly binding to the estrogen response elements (EREs) located on the promoter of MALAT1, which could then lead to (i) suppressing the miR145-5p and (ii) increasing the NEDD9 protein expression as miR145-5p can directly target the 3'-UTR of NEDD9-mRNA. A preclinical study using the in vivo mouse model further confirmed the in vitro cell lines data. Together, results from the above studies demonstrated that ERβ can promote NSCLC VM formation and cell invasion via altering the ERβ/MALAT1/miR145-5p/NEDD9 signaling. Targeting this newly identified signaling pathway with small molecules may help the development of novel therapies to better suppress the NSCLC metastasis.

SPOT-004 NEDD9 is crucial for tumour progression in renal cell carcinoma mouse models

IntroductionRenal cell carcinoma (RCC) is characterised by high lethality in advanced stages. Vastly resistant to radio- and chemotherapy, RCCs respond to targeted therapies such as tyrosine kinase inhibitors, mTOR antagonists and immune checkpoint inhibitors. However, limited response rates and emerging resistance mechanisms demand new treatment strategies.Scaffolding protein NEDD9 (neural precursor cell expressed, developmentally down regulated 9) is frequently overexpressed in various cancer types and associated with tumour aggressiveness. In vivo,high NEDD9 expression is associated with adverse clinical outcome and in vitro, NEDD9 promotes aggressiveness in RCC cells. Thus, we systematically characterised the role of NEDD9 in RCC both in vitro and in vivo with the goal to establish basis for new treatment strategies.Material and methodsUsing RNAi, we generated NEDD9-proficient and -deficient syngeneic (RENCA) and xenograft (786-O) tumour models via subcutaneous and orthotopic transplantations as well as tail vein injections. Tumour growth was monitored dynamically using calliper and MRI imaging. Extensive in vitro studies were performed to analyse NEDD9 and its associated oncogenic signalling cascades including Western blot, proliferation, migration and gene expression analyses. Tissue microarrays (TMAs) of 92 RCC patients were stained for NEDD9 and analysed using automated quantitative analysis (AQUA) technology to associate NEDD9 protein expression with clinical outcome.Results and discussionsNEDD9 is highly expressed in RCC cells and NEDD9 depletion significantly impairs migration and proliferation in both human and murine RCC cells. This is accompanied by reduced signalling of oncogenic pathways, particularly activation of ERK1/2. NEDD9 tumour promoting role was confirmed in vivo where NEDD9-deficient murine RCC cells exhibited significantly reduced tumour growth after subcutaneous and orthotopic syngeneic transplantation as well as inhibited lung metastatic seeding capacity after tail vein injection.In human RCC cells, only NEDD9 down-regulation was sufficient to completely abolish tumour growth in subcutaneous, orthotopic and lung seeding xenograft models. In line with our in vitro and in vivo results, we found high NEDD9 expression in human RCC tissue to be significantly associated with shorter overall survival.ConclusionWe show for the first time a crucial role for NEDD9 in RCC tumour progression in vivo, which potentially offers new therapeutic approaches.

Targeting epithelial to mesenchymal transition in prostate cancer by a novel compound, plectranthoic acid, isolated from Ficus microcarpa

Epithelial-to-mesenchymal transition (EMT) plays a crucial role in prostate cancer (PCa) metastasis. This has led to a surge in the efforts for identification of safer and more effective compounds which can modulate EMT and consequently inhibiting migration and invasion of PCa cells. We reported previously that Plectranthoic acid (PA), a natural compound isolated from the extracts of Ficus microcarpa, has the capability to induce cell cycle arrest and apoptosis in PCa cells. Here, we determined the effects of PA on EMT, migration, and invasion of PCa cells. Inhibition of EMT induced by different mitogens was effectively inhibited by PA treatment with subsequent decrease in migration of PCa cells. Employing a PCa cell culture model of TGF-β-induced EMT, we showed that PA has the ability to reverse EMT. PA treatment was associated with induction of epithelial markers and decrease in the expression of mesenchymal markers in PCa cells. Proteomic analysis identified Rac1 as the major cadherin signaling protein modulated with PA treatment. In silico studies indicated that PA docked to the CH domain of NEDD9 protein with an estimated free binding energy of -7.34 Kcal/moL. Our studies revealed significant inhibition of Rac1/NEDD9 pathway in PA treated cells thereby providing a molecular basis of the inhibitory effect of PA on PCa cell migration and invasion. In conclusion, our data suggest that PA should be investigated further as an adjuvant treatment in human PCa cells, given its potential as an anti-invasive agent.

Overexpression of NEDD9 in renal cell carcinoma is associated with tumor migration and invasion

Scaffold protein neural precursor cell expressed, developmentally downregulated 9 (NEDD9) is a member of the Crk-associated substrate protein family and is known to be a biomarker in multiple cancer types. It serves a critical function in regulating cell proliferation, migration, invasion and survival. The objective of this study was to evaluate the potential effects of NEDD9 in renal cell carcinoma (RCC). The expression of NEDD9 was analyzed by immunohistochemistry, western blotting and reverse transcription-quantitative polymerase chain reaction. NEDD9 protein and mRNA levels were significantly upregulated in RCC tissues compared with normal tissues (P<0.001). Furthermore, the NEDD9 immunostaining level was significantly associated with primary tumor stage and tumor, node, metastasis stage (P<0.05). High NEDD9 expression resulted in significantly lower survival rates for patients compared with normal NEDD9 expression (P<0.01). In addition, wound healing and transwell assays indicated that NEDD9 depletion by small interfering RNA significantly attenuated the migration and invasion of RCC cells (P<0.001). The present data suggested that NEDD9 may be a novel target for prevention and treatment of RCC metastasis and recurrence.

Abstract 1834: Constitutive Nedd9 null genotype promotes lung cancer aggressiveness

Abstract Non-small cell lung cancer (NSCLC) has a low survival rate, with metastasis contributing to the vast majority of deaths. The NEDD9 (HEF1/Cas-L) protein has been reported to be elevated in expression and to promote metastasis in a large subset of lung cancers and in other malignancies. NEDD9 functions as a scaffold for multiple critical effectors in integrin/FAK/SRC and receptor tyrosine kinase signaling cascades, as well as for the mitotic kinase Aurora-A, with overexpression thought to sustain signaling by these pro-oncogenic proteins. Previous studies demonstrated depletion of NEDD9 by RNAi reduced the growth and invasion of established lung cell lines and tumors, based on cell culture and xenograft assays. We have now investigated the consequences of a null genotype for Nedd9 from the earliest stages of tumor formation, crossing Nedd9 null mice to a 129S/Sv-Krastm3Tyj/Trp53tm1Brn (KP) model in which Kras mutation is induced specifically in lung tissue by inhalation of adenovirus bearing the Cre gene. Unexpectedly, based on in vivo imaging, the Nedd9 null genotype significantly accelerated tumor growth in KP mice. Pathological examination of tissues indicated Nedd9 null genotype also was associated with higher invasive capacity in vivo, including direct invasion to the heart, as well as elevated proliferation rate, decreased apoptotic activity, and changes in the expression of proteins such as vimentin, associated with mesenchymal status. Although Nedd9 function has been implicated in immune cell activity, we found minimal differences in tumor infiltration of myeloid cells or macrophages based on Nedd9 genotype. Rather, use of Reverse Phase Protein Array (RPPA) analysis to characterize signaling in isolated tumors suggested tumor intrinsic changes in glycolytic and proliferative signaling pathways, which were subsequently validated using direct functional assays. These results for the first time imply that NSCLC tumor progression beyond early stages in the absence of Nedd9 requires reprogramming of intrinsic tumor signaling to compensate for the absence of this protein, emphasizing activities distinct from those associated with elevated Nedd9 in late stage tumors. Citation Format: Alexander Deneka, Meghan Kopp, Anna S. Nikonova, Anna Gaponova, Alexandra Nagele, Harvey Hensley, Erica Golemis. Constitutive Nedd9 null genotype promotes lung cancer aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1834. doi:10.1158/1538-7445.AM2017-1834

Retinoic acid regulates Schwann cell migration via NEDD9 induction by transcriptional and post-translational mechanisms

Schwann cell migration is essential during the regenerative response to nerve injury, however, the factors that regulate this phenomenon are not yet clear. Here we describe that retinoic acid (RA), whose production and signaling activity are greatly enhanced during nerve regeneration, increases Schwann cell migration. This is accompanied by the up-regulation of NEDD9, a member of the CAS family of scaffold proteins previously implicated in migratory and invasive behavior in gliomas, melanomas and the neural crest cells from which Schwann cells derive. This RA-induced NEDD9 accumulation is due to augmented mRNA levels, as well as an increase of NEDD9 protein stability. Although all NEDD9 phospho-isoforms present in Schwann cells are induced by the retinoid, the hormone also changes its phosphorylation status, thus altering the ratio between the different isoforms. Silencing NEDD9 in Schwann cells had no effect on basal migratory ability, but completely abrogated RA-induced enhanced migration. Collectively, our results indicate that RA could be a major regulator of Schwann cell migration after nerve injury, thus offering a new insight into peripheral nerve repair.

SiRNA Suppression of NEDD9 Inhibits Proliferation and Enhances Apoptosis in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is the most lethal of all genitourinary malignancies. NEDD9/HEF1/Cas-L is a member of the Cas protein family and is known as a biomarker in multiple cancer types. In this study, we demonstrate for the first time that NEDD9 was upregulated in RCC tissue and cell lines. Immunohistochemical analysis and quantitative RT-PCR analysis showed low expression of NEDD9 in normal renal tissues and high expression in RCC tissues. In addition, in vitro experiments show that expression of NEDD9 was upregulated in RCC cell lines. Through MTT assay, we observed that NEDD9 knockdown inhibited cell proliferation. Furthermore, flow cytometry analysis showed that NEDD9 downregulation induced apoptosis. Together, our data suggest that abnormal NEDD9 protein expression may be a marker for RCC, and NEDD9 knockdown suppresses cell growth.

Expression of NEDD9 in hepatocellular carcinoma and its clinical significance.

Neural precursor cell expressed, developmentally downregulated 9 (NEDD9) plays an integral role in natural and pathological cell biology. Overexpression of NEDD9 protein has been correlated with poor prognosis in various types of cancer. However, few available data address the precise function of the NEDD9 gene in hepatocellular carcinoma (HCC). In the present study, we investigated NEDD9 expression in 40 primary human HCC tissues compared with matched adjacent non-tumor hepatic tissues using RT-qPCR and western blot analysis. Immunohistochemistry was performed to analyze the correlations between NEDD9 expression and clinicopathological factors. Statistical analyses were applied to derive prognostic values of NEDD9 in HCC. The results showed that the NEDD9 mRNA and protein expression levels in HCC tissues were significantly higher than those in matched adjacent non-tumor hepatic tissues. High NEDD9 expression was correlated with larger tumor size, advanced tumor grade, metastasis, intrahepatic venous invasion and high UICC TNM stages in HCC patients. Patients with high NEDD9 expression levels exhibited poorer recurrence-free and overall survival than those with a low NEDD9 expression. Additionally, NEDD9 expression status was an independent prognostic factor for survival. This correlation remained significant in patients with early-stage HCC or with normal serum AFP levels. The results of this study suggest that NEDD9 may be a valuable prognostic biomarker for HCC, including early-stage and AFP-normal patients.

Elevated expression of NEDD9 is associated with metastatic activity in gastric cancer.

ObjectiveTo investigate the protein and mRNA expression of NEDD9 in gastric cancer (GC) tissues, adjacent atypical hyperplasia tissues, and normal gastric mucosa tissues, and analyze its relationship with the pathological features and prognosis of GC.MethodsForty cases of GC tissues, 20 cases of adjacent atypical hyperplasia tissues, and 40 cases of normal gastric mucous tissues were collected. Immunohistochemistry and Western blot were used to examine the expression of NEDD9 protein in various tissues. Situ hybridization and reverse transcription polymerase chain reaction were applied to detect the expression of NEDD9 mRNA in various tissues. The correlation of NEDD9 expression with invasion and metastasis of GC was analyzed.ResultsThe protein expression level of NEDD9 was significantly higher in GC tissues than in adjacent atypical hyperplasia tissues and normal gastric mucous tissues (P<0.05). The protein expression level of NEDD9 was positively related to the invasion depth of carcinoma and tumor lymph node metastasis (P<0.05), but unrelated to age, sex, tumor size, and clinical classification of cancer (P<0.05). The mRNA expression level of NEDD9 was also significantly higher in GC tissues than in adjacent atypical hyperplasia tissues and normal gastric mucous tissues (P<0.05), and positively related with the tumor lymph node metastasis and invasion depth of carcinoma (P<0.05).ConclusionNEDD9 is involved in the occurrence and development of GC, and it may be an important biological marker of GC metastasis and infiltration.

MicroRNA-145 suppresses cell proliferation, invasion and migration in pancreatic cancer cells by targeting NEDD9.

MicroRNAs (miRNAs) represent a class of small non-coding RNAs regulating gene expression by inducing the degradation of RNA or interfering with translation. Aberrant miRNA expression has been described in several types of cancer in humans. In the present study, it was demonstrated that miR-145 is downregulated in pancreatic cancer tissues and the Panc-1 cell line. Restoration of miR-145 inhibited cell proliferation, invasion and migration in Panc-1 cells. Neural precursor cell expressed, developmentally down-regulated 9 (NEDD9) has been identified as a novel potential miR-145 target using bioinformatics. Using luciferase reporter constructs, it was observed that the NEDD9 3′-untranslated region is the location of the direct binding site for miR-145. Additionally, it was identified that miR-145 is inversely correlated with NEDD9 expression in pancreatic cancer tissues and that restoration of miR-145 in Panc-1 cells reduced NEDD9 mRNA and protein expression accompanied by inhibition of cell proliferation, invasion and migration. In conclusion, these findings indicate that miR-145 may be an effective target for pancreatic cancer therapy.

Abstract 4995: Role of MICAL1 in NEDD9 invasive signaling

Abstract Metastasis is the final step leading to patient death in most solid tumors, including head and neck squamous cell carcinoma (HNSCC). For metastasis to proceed, tumor cells must become mobile and invasive, losing their communal behavior. A goal of our work is to delineate the downstream effectors of growth factor signaling pathways that contribute to invasion. Using a phosphotyrosine proteomics screen we previously identified neural precursor cell expressed, developmentally down-regulated 9 (NEDD9; CasL) as a mediator of VEGF and IGF-1 signaling to invasion in HNSCC cells. NEDD9 tyrosine phosphorylation led to matrix metalloproteinase9 (MMP9) expression and secretion, invadopodia formation and enhanced invasion. To determine the molecular details of NEDD9 protein:protein interactions leading to invasion, we have generated a series of NEDD9 substrate domain tyrosine (Y to F, YXXP) and SH3 domain mutants. Mutation of all 13 YXXP motifs to FXXP blocked NEDD9 tyrosine phosphorylation, MMP9 expression and invadopodia formation. Of note, we found that NEDD9 co-immunoprecipitated with Molecule Interacting with CasL 1 (MICAL1). To further define this interaction and its downstream impact, stable NEDD9 or MICAL1 silenced HNSCC cell lines expressing select shRNAs were generated, with each exhibiting reduced migration and invasion. MICAL shRNA transfected cell lines also exhibited decreased MMP9 secretion, consistent with our findings that lack of MICAL1 decreases cell invasion and with findings of other labs that MICALs interact with Rab proteins altering intracellular trafficking. Further, we have verified that MICAL1 and NEDD9 form complexes in cells based on coimmunoprecipitation analyses. Better understanding of this pathway will undoubtedly lead to the identification of novel therapeutics for the prevention of invasion and metastasis. Supported by NIH R01CA134845 (SAR), P30 CA138313 and NIH/NCRR UL1 TR000062/TL1 TR000061 from the South Carolina Clinical &amp; Translational Research Institute (COH). Citation Format: Casey O. Holmes, Amanda M. Brock, Philip T. Sobash, Jessica A. Tiedeken, Steven A. Rosenzweig. Role of MICAL1 in NEDD9 invasive signaling. [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 4995. doi:10.1158/1538-7445.AM2014-4995