NEDD9 mRNA Research Articles

Role of NEDD9 expression in non-metastatic HER2-positive breast cancer.

e12645 Background: Neural Precursor Cell Expressed Developmentally Downregulated Protein 9 (NEDD9) is a member of the associated substrate family. Our previous studies demonstrated the role of NEDD9 overexpression in early tumorigenesis in HER2-positive (HER2+) breast cancer in vivo. Tissue microarray-based analysis showed a strong correlation between NEDD9 mRNA and relapse-free survival (RFS) and pathologic complete response (pCR) rate in HER2+ breast cancers. In this study, NEDD9 expression in non-metastatic HER2+ breast cancer was evaluated by tissue microarray. Methods: A total of 64 patients with non-metastatic HER2+ cancer of the breast who had received neoadjuvant chemotherapy and had sufficient pathology specimens were included in the study. NEDD9 was stained by tissue microarray, and the expression was subclassified as low, intermediate, and high based on the NEDD9 expression score. The correlations of NEDD9 expression with the clinical/biological characteristics, treatment, and treatment response were analyzed by Fisher Exact test. Results: A total of 64 patients were included in the study, with a medium follow-up duration of 4.6 years. The median age was 54, 1 patient was male, 79.3% of patients had stage 2 disease, 1 patient had inflammatory breast cancer, and 37.5% of patients had ER/PR negative disease. The most common neoadjuvant chemotherapy regimen was TCHP (82.5%), 44.4% of patients underwent lumpectomy, and 67.7% of patients underwent axillary lymph node dissection (ALND). 54% of patients achieved complete pathologic response (pCR), all patients received adjuvant anti-HER2 treatment, 85.7% received adjuvant radiotherapy, and 74.6% received adjuvant endocrine therapy. The NEDD9 expression distribution for low, intermediate, and high were 37.5%, 50.0%, and 12.5%, respectively. NEDD9-high patients had higher tumor stage and grade than NEDD9-low/ intermediate patients (p = 0.004, p= 0.041). More NEDD9-high patients underwent ALND compared to NEDD9- low/intermediate patients (p = 0.09). There is no statistically significant difference in pCR rate between NEDD9 expression groups, although numerically more NEDD9-high patients achieved pCR (75%, 56.2%, and 43.5% in NEDD9-high, intermediate, and low patients). Conclusions: In non-metastatic HER2+ breast cancer, NEDD9 expression is associated with more advanced tumor grade and stage. There is no statistically significant difference in pCR rate between groups, although more patients achieved pCR with a higher level of NEDD9 expression. The sample size limits our study. Additional study is needed to evaluate the correlation between NEDD9 expression and pCR rate following neoadjuvant chemotherapy in HER2+ breast cancer.

PKM2 Expression as Biomarker for Resistance to Oxaliplatin-Based Chemotherapy in Colorectal Cancer.

The purpose of the current study is to investigate the prognostic significance of M2 isoform of pyruvate kinase (PKM2) mRNA expression loss in patients with operable colon cancer (CC). Two hundred sixty-two specimens from patients with stage-III or high-risk stage-II CC (group-A) treated with adjuvant fluoropyrimidine and oxaliplatin chemotherapy (FOLFOX), 118 specimens from metastatic CC patients (group-B) treated with FOLFOX, and 104 metastatic CC patients (group-C) treated with irinotecan-based chemotherapy were analyzed for PKM2, TS, ERCC1, MYC, and NEDD9 mRNA expression, as well as KRAS exon2 and BRAFV600E mutations. High PKM2 mRNA expression was correlated with left-sided located primaries (p = 0.001, group-A; p = 0.003, group-B; p = 0.001, group-C), high-grade tumors (p = 0.001, group-A; p = 0.017, group-B; p = 0.021, group-C), microsatellite-stable tumors (p < 0.001, group-A), pericolic lymph nodes involvement (p = 0.018, group-A), and cMYC mRNA expression (p = 0.002, group-A; p = 0.008, group-B; p = 0.006, group-C). High PKM2 mRNA expression was correlated with significantly lower disease free survival (DFS) (p = 0.002) and overall survival (OS) (p = 0.001) in the group-A. Similarly, PKM2 mRNA expression was associated with significantly decreased progression free survival (PFS) (p = 0.001) and OS (p = 0.001) in group-B. On the contrary, no significant association for the PKM2 mRNA expression has been observed with either PFS (p = 0.612) or OS (p = 0.517) in group-C. To conclude, the current study provides evidence for the prediction of PKM2 mRNA expression oxaliplatin-based treatment resistance.

Inhibition of p62/SQSTM1 sensitizes small-cell lung cancer cells to cisplatin-induced cytotoxicity by targeting NEDD9 expression.

Drug resistance is the leading cause for rapid progression and relapse in small-cell lung cancer (SCLC) patients. Thus overcoming drug resistance still remains to be urgently resolved during SCLC treatment. Here, we found p62/SQSTM1 was enriched in SCLC spheroids, a subpopulation possessing cancer stem-like properties, which is responsible for cancer relapse and metastasis. Subsequent functional assays in vitro showed that short hairpin RNA (shRNA)-mediated p62 knockdown increased sensitivity of SCLC cell lines to cisplatin (DDP), whereas lentivirus-mediated p62 ectopic overexpression diminished DDP-induced cytotoxicity in both NCI-H446 and NCI-H1688 cell lines. Moreover, ectopic p62 overexpression promoted DDP resistance of NCI-H446 cells-derived tumor xenografts in immunodeficient mice in vivo, as indicated by accelerated tumor growth rate and reduced fluorescent activity of cleaved caspase-3. Gene expression profiling analysis revealed that p62 was positively correlated with neuronal precursor cell-expressed, developmentally downregulated gene 9 (NEDD9) expression level. Consistently, NEDD9 messenger RNA (mRNA) level was decreased upon p62 suppression by small interfering RNA (siRNA) and increased with p62 transient overexpression in SCLC cell lines, suggesting that p62 positively regulated NEDD9 mRNA. Depletion of NEDD9 by siRNA, to a large extent, reversed p62-overexpressed SCLC cells to DDP-induced cytotoxicity, implying NEDD9 might act as a downstream target which was in charge of p62-mediated DDP resistance. Taken together, our findings uncovered a previously unknown role of p62 in the regulation of SCLC drug resistance, assigning p62 as an attractive target for SCLC treatment.

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.

Loss of LKB1 Protein Expression Correlates with Increased Risk of Recurrence and Death in Patients with Resected, Stage II or III Colon Cancer.

PurposeThe purpose of this study was to investigate the prognostic significance of liver kinase b1 (LKB1) loss in patients with operable colon cancer (CC).Materials and MethodsTwo hundred sixty-two specimens from consecutive patients with stage III or high-risk stage II CC, who underwent surgical resection with curative intent and received adjuvant chemotherapy with fluoropyrimidine and oxaliplatin, were analyzed for LKB1 protein expression loss, by immunohistochemistry as well as for KRAS exon 2 and BRAFV600E mutations by Sanger sequencing and TS, ERCC1, MYC, and NEDD9 mRNA expression by real-time quantitative reverse transcription polymerase chain reaction.ResultsLKB1 expression loss was observed in 117 patients (44.7%) patients and correlated with right-sided located primaries (p=0.032), and pericolic lymph nodes involvement (p=0.003), BRAFV600E mutations (p=0.024), and TS mRNA expression (p=0.041). Patients with LKB1 expression loss experienced significantly lower disease-free survival (DFS) (hazard ratio [HR], 1.287; 95% confidence interval [CI], 1.093 to 1.654; p=0.021) and overall survival (OS) (HR, 1.541; 95% CI, 1.197 to 1.932; p=0.002), compared to patients with LKB1 expressing expressing tumors. Multivariate analysis revealed LKB1 expression loss as independent prognostic factor for both decreased DFS (HR, 1.217; 95% CI, 1.074 to 1.812; p=0.034) and decreased OS (HR, 1.467; 95% CI, 1.226 to 2.122; p=0.019).ConclusionLoss of tumoral LKB1 protein expression, constitutes an adverse prognostic factor in patients with operable CC.

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.

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.

Effects of lentivirus-mediated RNAi knockdown of NEDD9 on human lung adenocarcinoma cells in vitro and in vivo.

The aim of the present study was to investigate the biological behavior of lung adenocarcinoma A549 cells following transfection with NEDD9-specific lentiviral particles invitro and invivo. NEDD9-specific lentiviral particles were chemically synthesized and transfected into the human lung adenocarcinoma A549 cell line. NEDD9 mRNA and protein levels were determined by fluorescence quantitative RT-PCR and western blotting. Cell proliferation was evaluated using soft agar colony formation assays and flow cytometric analysis. Migration and invasion were evaluated by wound-healing and transwell assays and xenograft animal models. Transfection was successful, and expression levels of NEDD9 mRNA and protein in the lentivirus-NEDD9-siRNA group were downregulated. As indicated by soft agar colony formation assays, the number of clones in the siRNA group were significantly lower than the number of colonies in the blank and negative control groups (P<0.01). In addition, the percentage of cells in the S phase in the siRNA group was significantly lower than the percentages in the blank and negative control groups (P<0.05). Furthermore, as detected by cell migration and invasion assays, values of wound healing were increased and the number of invading cells were decreased in the siRNA group (both P<0.05). We also showed that lentivirus-mediated NEDD9-siRNA decreased the growth potential of subcutaneous A549 xenografts invivo. These data imply that knockdown of the NEDD9 gene results in suppression of tumor cell proliferation, migration, invasion and cell growth invitro and invivo. Lentivirus-mediated NEDD9-siRNA may have potential therapeutic utility for human lung adenocarcinoma.

Maternal pre‐gravid body mass index and adiposity influence umbilical cord gene expression at term in AGA infants

While maternal obesity is associated with unfavorable maternal and fetal outcomes, the influence of maternal obesity on fetal gene expression is less clear. Umbilical cords (UC) from 12 lean (pre‐gravid BMI &lt; 25) and 10 overweight/obese (OB, pre‐gravid BMI ≥25) women without gestational diabetes were collected at term and utilized for gene expression analysis using Human Primeview microarrays (Affymetrix). Although offspring birth weight and infant adiposity (at 2‐wk) did not differ between groups, expression of 232 transcripts was affected in UC from OB compared to lean mothers. GSEA analysis revealed an up‐regulation of genes related to metabolism, stimulus and defense response and inhibitory to insulin signaling in the OB group. We confirmed that Egr‐1, periostin, FosB, and NEDD9 mRNA expression was induced in UCs from OB moms, while endothelin receptor B, Klf10, Peg3 and Egln3 expression was decreased. mRNA expression of Egr‐1, FosB, MEST and SOCS1 was positively associated (p&lt;0.05) with mother's 1st trimester body fat mass (%). Metabolic and hormonal assessments were also assayed in mother's serum at 30 weeks of gestation and in UC plasma. Our data indicate that maternal obesity per se causes changes in UC gene expression favoring inflammation and insulin resistance, possibly predisposing infants of overweight moms to become overweight later on in life. Funding USDA ARS‐CRIS 6251–51000‐007–04S.

Expression of NEDD9 in pancreatic ductal adenocarcinoma and its clinical significance

The aim of this study was to investigate the expression and prognostic significance of NEDD9 in pancreatic ductal adenocarcinoma (PDA). Expressional levels of NEDD9 mRNA and protein in paired pancreatic cancer lesions and adjacent noncancerous tissues were examined by quantitative real-time PCR and western blotting. NEDD9 expression was analyzed by immunohistochemistry in 106 patients with PDA. The correlations between NEDD9 immunostaining levels and clinicopathologic factors, as well as the follow-up data of patients, were analyzed statistically. NEDD9 protein and mRNA levels were elevated in pancreatic carcinoma lesions compared with the paired adjacent noncancerous tissues. A high level of expression of NEDD9 was significantly correlated with clinical staging (P < 0.001), lymph node metastasis (P < 0.001), and histological differentiation (P < 0.001). Patients with a higher NEDD9 expression had a significantly shorter survival time than those patients with lower NEDD9 expression. The multivariate analysis revealed that NEDD9 could serve as an independent factor of poor prognosis. Our finding indicates that NEDD9 could be used as prognostic molecular marker and therapeutic target for PDA.

Construction and characterization of a eukaryotic expression vector for small interfering RNA targeting the NEDD9 gene

The aim of this study was to construct a eukaryotic expression vector for a small interfering RNA (siRNA) targeting the neural precursor cell expressed, developmentally downregulated 9 (NEDD9) gene, and to investigate the effects of RNA interference (RNAi) on NEDD9 expression in human lung adenocarcinoma A549 cells. We used the siRNA design and analysis software to determine the target oligonucleotides according to the sequence of NEDD9 mRNA available in GenBank. Four siRNA sequences were obtained, and the corresponding cDNAs were synthesized and inserted into the pRNAT-CMV3.2 plasmid to construct the recombinant plasmids. These were transformed into the E. coli strain DH5α. The plasmids, after identification by PCR and DNA sequencing, were transfected into the A549 cell line via the liposome method. NEDD9 mRNA and protein in the cells were determined by fluorescence quantitative RT-PCR (FQ-PCR) and western blotting, respectively. The pRNAT-CMV3.2-transfected plasmid was used as a control. Four recombinant plasmids were identified by PCR and sequence analysis, which contained the correct insertion of the designed sequences in the plasmids. FQ-PCR and western blotting showed substantially decreased mRNA and protein expression of the NEDD9 gene in the transfected cells, compared with the control group. In conclusion, the recombinant plasmids expressing the siRNA targeting the NEDD9 gene were successfully constructed, and the siRNA expression vectors inhibited the expression of NEDD9 in A549 cells.

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.

Expression and clinical significance of NEDD9 in lung tissues

Neural precursor cell expressed, developmentally downregulated 9 (NEDD9) acts as a scaffold protein and belongs to a family of CAS (Crk-associated substrate) that regulates protein complexes controlling invasion and differentiation. Preclinical research for this gene was predominantly reported in melanomas, glioblastoma, and lymphoma. So we investigated the expression and significance of NEDD9 mRNA and protein in lung tissues. Specifically, we immunohistochemically compared NEDD9 expression and localization in 24 formalin-fixed and paraffin-embedded lung adenocarcinoma tissues with that of surrounding nonneoplastic tissue and five microscopically normal lungs. NEDD9 mRNA levels were quantitatively analyzed by fluorescent quantitative reverse transcription-polymerase chain reaction (FQ-PCR) in frozen tissue specimens of all tumors and 24 matched nonneoplastic lung parenchymas, and protein expression in 16 homogenates of matched neoplastic/nonneoplastic specimens was evaluated by Western blotting. The three techniques showed that NEDD9 is weakly expressed in nonneoplastic lung parenchyma and upregulated in lung adenocarcinoma. Moreover, FQ-PCR indicated a statistically significant correlation between NEDD9 upregulation and higher disease stages (I+II versus III+IV, p=0.001; high and middle versus low differentiation, p<0.001). Our results provide evidence that NEDD9 is upregulated in lung adenocarcinoma, and overexpression of NEDD9 protein has been strongly correlated with staging and differentiation grade and tumor size in lung adenocarcinoma, which demonstrated a poor prognosis.

Differential expression and dynamic changes of murine NEDD9 in progenitor cells of diverse tissues

NEDD9 is a scaffolding protein in the integrin signaling pathway that is involved in cell adhesion dynamics. Little is known of the cellular localization of NEDD9 expression during embryonic development. In the present study, we have analyzed NEDD9 mRNA expression in the mouse and identified new relevant expression sites. In addition, we have characterized NEDD9 protein expression pattern for the first time in mammals. At E9.5–E10.5, high levels of Nedd9 and the neurogenic transcription factor neurogenin-2 ( Ngn2) were found to largely overlap in two discrete domains of the trunk neural tube along its dorso-ventral axis, with Nedd9 extending to more ventral regions of the ventricular zone and Ngn2 differentially expressed in neuronally committed progenitors of the intermediate zone. At encephalic and trunk levels of the neural tube, NEDD9 was present in Sox2 + progenitor cell populations mostly generating Ngn2 + and/or Nurr1 + cells. A sharp down-regulation of NEDD9 expression was found in cells upon lineage commitment, as observed in Nurr1 + and Ngn2 + mesencephalic dopaminergic and brainstem neuronal progenitors. In other tissues/organs, i.e. prospective heart, retina, olfactory epithelium, gonads, cartilage, gut and pituitary gland, NEDD9 was found to be co-expressed with Sox2, RXR alpha and/or Nurr1-like proteins, suggesting that NEDD9 expression is confined to early progenitors involved in diverse organogenesis and that it may depend on the repertoire and levels of retinoic acid co-receptors expressed by those cells.