Role Of NEDD9 Research Articles

NEDD9 sustains hexokinase expression to promote glycolysis

Elevated rates of glycolysis in cancer cells support tumor growth, in a process that typically depends on oncogene-induced increases in the expression and/or activity of enzymes in the glycolytic pathway. The NEDD9 scaffolding protein is upregulated in many advanced tumors, with increased NEDD9 promoting the activity of SRC and other effectors that promote invasion and metastasis. We here define a new role for NEDD9 in support of glycolysis. NEDD9 knockdown significantly impaired glycolysis in multiple lung cancer cell lines This was accompanied by post-transcriptional downregulation of steady-state levels of hexokinases (HK1 and HK2), which catalyze early steps in the glycolytic cascade, key rate limiting enzyme phosphofructokinase (PFK1), and downstream glyceraldehyde phosphate dehydrogenase (GAPDH). In mice, protein levels of HK1, HK2, PFK1, and GAPDH were depressed in Krastm4Tyj/J/Trp53tm1Brn/J (KP) non-small cell lung tumors with null versus wild type Nedd9. Reciprocally, depletion of HK1 or HK2 elevated NEDD9 expression, as did the treatment of cells with 2-deoxyglucose (2DG), an inhibitor of glycolysis; whereas overexpression of hexokinases promoted NEDD9 dephosphorylation, associated with reduced NEDD9 activity. Together, these data for the first time suggest a negative feedback circuit involving NEDD9 and glycolytic enzymes that may contribute to NEDD9 action in promoting the aggressive growth of advanced tumors.

Melatonin Inhibits the Progression of Oral Squamous Cell Carcinoma via Inducing miR-25-5p Expression by Directly Targeting NEDD9.

Melatonin exerts anti-cancer roles in various types of cancers. However, to the best of our knowledge, its role in oral squamous cell carcinoma (OSCC) is unknown. The present study aimed to investigate the role of melatonin and its underlying mechanism in OSCC. MTT, colony formation, wound healing, and transwell invasion assays proved that melatonin played anti-tumor effects in OSCC cells by inhibiting cell viability, proliferation, migration, and invasion in a concentration-dependent manner. The RT-qPCR analysis showed that miR-25-5p was significantly upregulated after melatonin treatment. Further, miR-25-5p might be involved in melatonin-induced inhibitory effects on the biological behavior of OSCC. The expression of miR-25-5p was decreased in tumor tissues and OSCC cells detected by RT-qPCR. MTT assay, colony formation assay, and TUNEL staining indicated miR-25-5p overexpression inhibited OSCC cell viability, proliferation, and induced OSCC cell apoptosis. Furthermore, wound healing, transwell invasion assay, and animal experiments suggested that miR-25-5p might exert suppressive effects on the migration, invasion, and tumor formation of OSCC cells, while miR-25-5p knockdown exhibited the opposite effects in OSCC cells. Bioinformatics analysis, western blot analysis, and luciferase reporter assay suggested that neural precursor cell expressed developmentally downregulated protein 9 (NEDD9) was proved to be a putative target for miR-25-5p. The role of NEDD9 in inhibiting OSCC cell proliferation, invasion, and migration was verified with NEDD9 siRNA transfection. Thus, melatonin exerted anti-proliferative, anti-invasive, and anti-migrative effects on OSCC via miR-25-5p/NEDD9 pathway. Melatonin could be applied as a potential novel drug on treating OSCC.

NEDD9 promotes oncogenic signaling, a stem/mesenchymal gene signature, and aggressive ovarian cancer growth in mice.

Neural precursor cell expressed, developmentally downregulated 9 (NEDD9) supports oncogenic signaling in a number of solid and hematologic tumors. Little is known about the role of NEDD9 in ovarian carcinoma (OC), but available data suggest elevated mRNA and protein expression in advanced stage high grade cancers. We used a transgenic MISIIR-TAg mouse OC model combined with genetic ablation of Nedd9 to investigate its action in the development and progression of OC. A Nedd9−/− genotype delayed tumor growth rate, reduced incidence of ascites, and reduced expression and activation of signaling proteins including SRC, STAT3, E-cadherin and AURKA. Cell lines established from MISIIR-TAg;Nedd9−/− and MISIIR-TAg;Nedd9+/+ mice exhibited altered migration and invasion. Growth of these cells in a syngeneic allograft model indicated that systemic Nedd9 loss in the microenvironment had little impact on tumor allograft growth, but in a Nedd9 wild type background Nedd9−/− allografts exhibited significantly reduced growth, dissemination and oncogenic signaling compared to Nedd9+/+ allografts. Gene expression analysis revealed that Nedd9+/+ tumors exhibited more mesenchymal ‘stem-like’ transcriptional program, including increased expression of Aldh1a1 and Aldh1a2. Conversely, loss of Nedd9 resulted in increased expression of differentiation genes, including fallopian tube markers Foxj1, Ovgp1 and Pax8. Collectively, these data suggest that tumor cell-intrinsic Nedd9 expression promotes OC development and progression by broad induction of oncogenic protein signaling and stem/mesenchymal gene expression.

Involvement of NEDD9 in the invasion and migration of gastric cancer.

Recent studies have demonstrated that neural precursor cell expressed, developmentally downregulated 9 (NEDD9) is highly expressed in various tumor tissues and cell lines. However, research on the role of NEDD9 in gastric cancer (GC) is rare, and the potential mechanism in tumor progression has not yet been explored. In this study, we investigated the role and mechanism of NEDD9 in GC. The expression of NEDD9 in GC tissues and cell lines was measured by immunohistochemistry, qRT-PCR, and Western blot, respectively. Inhibiting NEDD9 expression was carried out by siRNA transfection, and upregulating of NEDD9 was via NEDD9 overexpression plasmid. The ability of proliferation, migration, and invasion was detected by MTT assay, scratch wound assay, and transwell assay, respectively. The expression of vimentin, E-cadherin, Zeb1, and Zeb2 was measured by Western blot and qRT-PCR. We found that NEDD9 expression was dramatically increased both in GC tissues and cell lines, and the expression was significantly related to GC development. Knockdown of NEDD9 in SGC-7901 strongly inhibited its malignant capacity in vitro. Meanwhile, upregulation of NEDD9 in GES-1 increased the malignant capacity. In addition, the expression of vimentin, Zeb1, and Zeb2 was positively correlated with NEDD9, while E-cadherin was opposite. Collectively, our findings suggest that NEDD9 acts as an oncogene and promotes GC metastasis via EMT.

Association NEDD9 with TGF-β-triggered epithelial-mesenchymal transition and cell invasion in prostate cancer cells: implications for cancer aggressiveness

NEDD9 is one of the Crk-associated substrate (Cas) family proteins that mediate downstream signaling processes including cytoskeletal organization, cell-cycle and tumorigenesis. While NEDD9 plays a crucial role in epithelial-mesenchymal transition (EMT), the functional mechanism underlying NEDD9-mediated EMT in prostate cancer (PCa) remains uncertain. Recently we confirmed that NEDD9 is predominantly involved in TGF-β-mediated EMT and cell motility in PCa cells. We herein discuss the functional role of NEDD9 in terms of pathogenesis of PCa.

Abstract 989: NEDD9 expression promotes epithelial ovarian cancer growth and dissemination

Abstract NEDD9 (Neural precursor cell Expressed, Developmentally Downregulated 9) is a scaffolding protein with roles in focal adhesion signaling, cell motility and regulation of centrosome function. NEDD9 has been reported as an important player in development and progression of several different solid tumors; however, its role in gynecologic cancers is poorly understood. In this study we investigated the role of NEDD9 in development and progression of epithelial ovarian cancer (EOC) using murine models. Three mouse strains were used: MISIIR-TAg mice that develop spontaneous ovarian carcinomas; MISIIR-TAg-Low mice that express the TAg transgene, but do not develop tumors; and Nedd9 null mice. To determine the effects of loss of Nedd9 on ovarian carcinoma development, MISIIR-TAg and Nedd9-/- mice were crossed and spontaneous ovarian tumor growth in MISIIR-TAg;Nedd9-/- and MISIIR-TAg;Nedd9+/+ mice was monitored and quantified by longitudinal magnetic resonance imaging (MRI). MISIIR-TAg-Nedd9-/- mice, compared to MISIIR-TAg-Nedd9+/+ mice, exhibited delayed tumor development and decreased tumor burden. Correspondingly, microarray analysis of tumors showed that several key oncogenic signaling pathways were upregulated in MISIIR-TAg-Nedd9+/+ mice compared to MISIIR-TAg-Nedd9-/- mice. Murine ovarian carcinoma (MOVCAR) cell lines were established from MISIIR-TAg-Nedd9-/- and MISIIR-TAg-Nedd9+/+ mice and essential tumorigenic features compared using in vitro assays. Interestingly, although these cells displayed no differences in adhesion or proliferation, Nedd9-/- MOVCAR cells were more aggressive than Nedd9+/+ cells in assays of migration and invasion. However, when grown as orthotopic allografts in permissive MISIIR-TAg-Low;Nedd9+/+ hosts, in vivo tumor growth was delayed and the number of metastatic tumor nodules was decreased in mice engrafted with Nedd9-/- cells compared to those injected with Nedd9+/+ cells. To study the potential role of Nedd9 in the tumor microenvironment, immune cell populations were compared in: 1) orthotopic allografts of Nedd9+/+ MOVCAR cells grown in MISIIR-TAg-Low;Nedd9-/- and MISIIR-TAg-Low;Nedd9+/+ hosts; or 2) spontaneous ovarian tumors in MISIIR-TAg-Nedd9-/- and MISIIR-TAg-Nedd9+/+ mice. In the allograft model, the number and total volume of metastatic tumor nodules was not significantly different in Nedd9-/- or Nedd9+/+ hosts; however, genotype-associated differences in tumor infiltrating NK-cell numbers were observed. Comparison of the spontaneous tumor models showed that the number of tumor infiltrating macrophages and NK-cells were decreased in MISIIR-TAg-Nedd9-/- mice. Collectively, these data suggest that Nedd9 promotes development and progression of EOC, through induction of oncogenic signaling and by pro-tumorigenic alterations of the immune cell microenvironment. Citation Format: Rashid Gabbasov, Laura E. Bickel, Shane W. O'Brien, Samuel Litwin, Sachiko Seo, Erica A. Golemis, Denise C. Connolly. NEDD9 expression promotes epithelial ovarian cancer growth and dissemination. [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 989. doi:10.1158/1538-7445.AM2014-989

Abstract A66: NEDD9 promotes cell invasion through modulation of ARF6 activity and endocytic recycling

Abstract The adhesion scaffolding protein NEDD9 was identified as potential pro-metastatic gene in several cancers. The molecular mechanisms of NEDD9-driven metastasis are still unknown. In this study, we show that expression of NEDD9 positively correlates with the invasive stage of breast cancer. We show that NEDD9 localizes to invadopodia and endosomes. Notably, NEDD9 depleted cells have increased levels of inactive surface receptors due to increase in fast recycling of Rab4 and Rab5 positive vesicles. Mechanistically, we found that NEDD9 binds to and scaffolds the Arf6 specific GAP - ASAP3, decreasing Arf6 activity. Thus, depletion of NEDD9 leads to activation of Arf6. Inhibition of Arf6 or re-expression of NEDD9 in shNEDD9 cells was sufficient to restore recycling rates, decrease the number of Rab4 and 5 positive vesicles and the invasive properties of tumor cells. Thus, in this work, we uncover the mechanistic basis of NEDD9-driven invasion and identify a new role for NEDD9 in Arf6-dependant endocytosis. Citation Format: Elena N. Pugacheva, Yuriy Loskutov, Sarah McLaughlin, Polina Kozyulina, Varvara Kozyreva, Ryan Ice, Mark Culp, Robert Wysolmerski, Scott Weed, Alexey Ivanov. NEDD9 promotes cell invasion through modulation of ARF6 activity and endocytic recycling. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A66.

A requirement for Nedd9 in luminal progenitor cells prior to mammary tumorigenesis in MMTV-HER2/ErbB2 mice

Overexpression of the NEDD9/HEF1/Cas-L scaffolding protein is frequent, and drives invasion and metastasis in breast, head and neck, colorectal, melanoma, lung and other types of cancer. We have examined the consequences of genetic ablation of Nedd9 in the MMTV-HER2/ERBB2/neu mouse mammary tumor model. Unexpectedly, we found that only a limited effect on metastasis in MMTV-neu;Nedd9(-/-) mice compared with MMTV-neu;Nedd9(+/+) mice, but instead a dramatic reduction in tumor incidence (18 versus 80%), and a significantly increased latency until tumor appearance. Orthotopic reinjection and tail-vein injection of cells arising from tumors, coupled with in vivo analysis, indicated tumors arising in MMTV-neu;Nedd9(-/-) mice had undergone mutational selection that overcame the initial requirement for Nedd9. To better understand the defects in early tumor growth, we compared mammary progenitor cell pools from MMTV-neu;Nedd9(-/-) versus MMTV-neu;Nedd9(+/+) mice. The MMTV-neu;Nedd9(-/-) genotype selectively reduced both the number and colony-forming potential of mammary luminal epithelial progenitor cells, while not affecting basal epithelial progenitors. MMTV-neu;Nedd9(-/-) mammospheres had striking defects in morphology and cell polarity. All of these defects were seen predominantly in the context of the HER2/neu oncogene, and were not associated with randomization of the plane of mitotic division, but rather with depressed expression the cell attachment protein FAK, accompanied by increased sensitivity to small molecule inhibitors of FAK and SRC. Surprisingly, in spite of these significant differences, only minimal changes were observed in the gene expression profile of Nedd9(-/-) mice, indicating critical Nedd9-dependent differences in cell growth properties were mediated via post-transcriptional regulation of cell signaling. Coupled with emerging data indicating a role for NEDD9 in progenitor cell populations during the morphogenesis of other tissues, these results indicate a functional requirement for NEDD9 in the growth of mammary cancer progenitor cells.

Abstract P1-05-03: A requirement for neural precursor cell-expressed developmentally downregulated gene 9 during the initiation of mammary tumorigenesis in MMTV-neu mice

Abstract The Neural precursor cell-Expressed Developmentally Downregulated gene 9 (NEDD9; also HEF1, CAS-L) scaffolding protein regulates many signaling pathways associated with mitosis, survival, migration, and ciliary integrity. Within the last few years, elevated NEDD9 expression has been implicated in progression and metastasis of several types of cancer, including those of the lung, skin, and brain. We have investigated the consequences of introducing a Nedd9 genotype into the MMTV-neu mouse mammary tumor model, which in many respects recapitulates features of human HER2+ breast cancer. 80% of wild-type MMTV-Neu; Nedd9+/+ animals developed tumors with an average latency of 339 days, but only 18% of MMTV-Neu;Nedd9−/− animals developed tumors with an average latency of 416 days. This highly significant difference indicates that the Nedd9−/− genotype significantly prevents neu-dependent mammary tumor formation. HER2-positive human breast tumors and MMTV-neu-induced tumors originate from mammary luminal epithelial progenitor cells. We evaluated mammary progenitor cell populations from non-tumor bearing MMTV-neu;Nedd9−/− versus MMTV-neu;Nedd9+/+ 4-month old mice (2 months prior to appearance of tumors). Flow cytometry analysis indicated a significantly reduced CD24high;CD49flow luminal progenitor subpopulation in MMTV-neu;Nedd9−/− and Nedd9−/− mammary glands, that could be contributed to the tumorigenesis resistance in the MMTV-neu;Nedd9−/− model. The MMTV-neu;Nedd9−/−genotype negatively affected the Matrigel mammosphere colony-forming potential of luminal progenitor cells compared to MMTV-neu; Nedd9+/+cells, causing formation of fewer colonies with aberrant size and morphology. Quantification of mitotic division planes of MMTV-neu;Nedd9−/− mammospheres revealed no contribution to the observed defects. However, MMTV-neu;Nedd9−/− mammospheres had defective expression of signaling proteins governing cell attachment, with reduced levels of FAK and more cytoplasmic localization of SRC. The results reported above, together with other data, support three main conclusions. First, they revealed a very substantial requirement for Nedd9 during early stages of HER2/neu-dependent tumor formation. Second, these results are the first to demonstrate a role for Nedd9 in supporting the abundance and colony-forming potential of mammary luminal progenitor cells. Third, they indicate that the defects in mammosphere growth likely involve perturbation of crucial cellular attachment signaling pathways involving FAK and SRC. In sum, these data provide a strong justification for future analysis of NEDD9 in the defective signaling of transformed mammary epithelial progenitor cell populations that initiate human breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-03.

Role of NEDD9 in invasion and metastasis of lung adenocarcinoma

Treatment failure for lung adenocarcinoma is frequently due to lymph node metastasis and invasion to neighboring organs. The aim of the present study was to investigate the invasion- and metastasis-related gene, neural precursor cell expressed, developmentally downregulated 9 (NEDD9), in lung adenocarcinoma tissues and cell lines. The expression of NEDD9 was analyzed by the SP method of immunohistochemistry for 60 formalin-fixed and paraffin-embedded (FFPE) lung adenocarcinoma tissues in which 32 cases were metastastic and 28 were without metastases. NEDD9 mRNA expression and protein levels were quantified by fluorescence quantitative reverse transcription-polymerase chain reaction (FQ-PCR) and western blotting in the highly invasive lung adenocarcinoma cell lines A549 and 95D as well as in SPC-A-1 cells with low invasive potential. The immunostaining scores revealed a statistically significant difference between metastatic and non-metastatic lung adenocarcinomas (p<0.001). FQ-PCR and western blotting demonstrated that NEDD9 expression was higher in A549 and 95D compared to SPC-A-1 cells (P=0.003). Our results provide evidence that NEDD9 is upregulated in metastatic lung adenocarcinoma and in highly invasive lung adenocarcinoma cell lines, suggesting its potential involvement in regulating cell migration and invasion.

Enhanced Genetic Instability and Dasatinib Sensitivity in Mammary Tumor Cells Lacking NEDD9

Elevated expression of the NEDD9/HEF1/Cas-L scaffolding protein promotes tumor cell invasion and metastasis in multiple cancer cell types. Conversely, generation of mammary tumors in the mouse mammary tumor virus (MMTV)-polyoma virus middle T (PyVT) genetic model is delayed by a Nedd9(-/-) genotype. These activities arise from the role of NEDD9 in assembling complexes and supporting activity of cancer signaling proteins, including FAK, Src, Shc, and AKT, and would support evaluation of NEDD9 expression as an unambiguous biomarker for tumor aggressiveness. However, we here show that despite the initial delay in tumor growth, cells derived from MMTV-PyVT;Nedd9(-/-) tumors are characteristically hyperaggressive versus MMTV-PyVT;Nedd9(+/+) cells in anchorage-independent growth, in growth on three-dimensional matrix produced by tumor-associated fibroblasts, and in formation of tumors after mammary orthotopic reinjection and of lung metastases after tail vein injection. This reversal suggests the specific selection of MMTV-PyVT;Nedd9(-/-) cells for growth in an in vivo microenvironment. Indeed, MMTV-PyVT;Nedd9(-/-) cells have increased cell cycle, centrosomal, and mitotic defects, phenotypes compatible with the increased selection of these cells for aggressive growth. Intriguingly, in spite of their aggressive phenotype, MMTV-PyVT;Nedd9(-/-) cells persistently have low levels of Src activation and are hypersensitive to the Src kinase inhibitor dasatinib. These studies identify NEDD9 as a complex modulator of different aspects of mammary tumor growth.

Abstract 3257: NEDD9 is required for initiation of HER2/Neu-dependent mammary tumor development

Abstract Neural precursor cell-Expressed Developmentally Downregulated gene 9 (NEDD9; HEF1, CAS-L) is a scaffolding protein regulating mitosis, survival, and migration. Within the last 3 years, overexpression of NEDD9 has been implicated in the progression and metastasis of several cancers. Using a Nedd9 knockout strain, we have evaluated the in vivo requirement for NEDD9 in mammary tumorigenesis. Loss of Nedd9 increased the latency of tumor formation in the aggressive MMTV-Polyoma Virus middle T antigen (PyV(m)T) model of mammary tumorigenesis (Izumchenko et al. Cancer Res, 2009). We and others have shown that NEDD9 interacts with and positively regulates activity of key oncogenic signaling proteins such as Aurora-A kinase (AURKA) and Src in cell culture. Tumors harvested from MMTV-PyV(m)T;Nedd9−/− mice had greatly reduced activation of AURKA, SRC, and also FAK and AKT, with the lowest levels of activation correlating with the greatest delays in tumor onset. To explore the role of NEDD9 in a second highly physiological model of mammary tumorigenesis, Nedd9−/− mice were crossed to MMTV-neu (HER2; ErbB2) transgenic mice, and compared with MMTV-neu;Nedd9+/+ counterparts. Strikingly, after over 18 months, 89% of MMTV-neu;Nedd9+/+ mice developed tumors, in contrast to only 29% of MMTV-neu;Nedd9−/− mice. Between genotypes, there is no statistical difference between tumor growth rates or metastasis. Cell lines derived from tumors of both MMTV-neu;Nedd9+/+ and MMTV-neu;Nedd9−/− animals were then used for xenograft tumor growth and experimental metastasis assays. MMTV-neu;Nedd9−/− tumor-derived cell lines grew similarly to MMTV-neu;Nedd9+/+ counterparts as xenografts in the mammary fat pads of SCID mice, but metastasized to the lungs of SCID mice less readily following tail vein injection. These and other data indicate a novel role for NEDD9 in mammary tumor initiation distinct from the role controlling metastasis and progression of tumors in cooperation with the oncogenes FAK and SRC. Further, the work suggests that HER2-driven mammary tumorigenesis is dependent on NEDD9 at initial stages, but NEDD9 is not required for tumor maintenance, progression, or metastasis from primary organ sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3257.

Abstract B106: Nedd9 null status initially delays mammary tumor growth, then selects for hyper-aggressive mammary tumors

Abstract Altered expression of the HEF1/CAS-L/NEDD9 scaffolding protein has recently emerged as a candidate mechanism controlling tumor development, progression and metastasis. While some studies have identified elevated NEDD9 expression as pro-metastatic, other works have suggested a negative role in tumor progression. We have recently reported that the Nedd9 null genetic background significantly limits mammary tumor development in the MMTV-polyoma virus middle T (PyVmT) genetic model. Late-developing tumors are characterized by depressed activation of proteins including AKT, SRC, FAK, and ERK, emphasizing an important role of NEDD9 as a scaffolding protein for these pro-oncogenic proteins. However, NEDD9 is also regulates activation of the Aurora-A mitotic kinase (AurA). In vitro, knockdown of NEDD9 reduces AurA activation, suggesting in vivo loss of NEDD9 might be a potential source of genomic instability. We have evaluated a panel of cell lines derived from mammary tumors of MMTV-PyVmT;Nedd9−/− mice. Nedd9−/− cells injected orthotopically in mammary fat pads of SCID mice exhibited significantly faster growth of mammary tumors, and in a tail vein injection assay yielded a dramatically enhanced metastasis to the lungs. As hypothesized, Nedd9−/− cell lines had lower AurA kinase activity than Nedd9+/+ cells, and also had evidence of centrosomal and spindle abnormalities associated with deficient AurA function. Interestingly, the sensitivity of Nedd9−/− cells to small molecule inhibitors targeted at specific partner kinases and to chemotherapeutic agents used in breast cancer notably differed from that of Nedd9+/+ cells. Together, these data support the use of NEDD9 expression as a novel biomarker in establishing prognosis and drug responsiveness of tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B106.