The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation-or rapamycininduced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.

BackgroundGastric cardia adenocarcinoma (GCA) is the most aggressive subtype of gastric carcinoma. New molecular markers and therapeutic targets are needed for diagnosis, prognosis and treatment of GCA. This study is to establish the E3 ubiquitin ligase Nedd4-1 as a prognostic biomarker to predict the survival and guide the treatment of GCA patients.MethodsExpression of Nedd4-1 in 214 GCA tumor samples was detected by immunohistochemistry staining (IHC) using tissue microarray assay (TMA). Association of Nedd4-1 with cumulative survival of the TNM stages I-III patients and clinicopathological characteristics was statistically analyzed. The role of Nedd4-1 in gastric cancer cell migration and invasion were determined by transwell and wound healing assays.ResultsNedd4-1 is overexpressed in 83% of the GCA tumors. The 5-year survival rate in Nedd4-1 negative GCA patients is as high as 96%. Log-rank analysis indicated that overexpression of Nedd4-1 is inversely correlated with cumulative survival (χ2 = 21.885, p <0.001). Multivariate logistic regression analysis showed that overexpression of Nedd4-1 is associated with an extremely low GCA survival rate with a hazard ratio (HR) = 0.068 (p = 0.008) in TNM stages I-III patients. Statistical analysis of association of Nedd4-1 overexpression with clinicopathological characteristics revealed that overexpression of Nedd4-1 is tightly associated with TNM stage (p < 0.001). Knockdown of Nedd4-1 in gastric cancer cell lines AGS and N87 dramatically inhibited the gastric cancer cell migration and invasion.ConclusionsOur results indicate that Nedd4-1 is an exceptional prognostic biomarker for GCA and suggest that Nedd4-1 may play an essential role in GCA metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-248) contains supplementary material, which is available to authorized users.

BackgroundEGFR-dependent cell migration plays an important role in lung cancer progression. Our previous study observed that the HECT E3 ubiquitin ligase NEDD4 is significantly correlated with tumor metastasis and required for migration and invasion signaling of EGFR in gastric cancer cells. However, how NEDD4 promotes the EGFR-dependent lung cancer cell migration is unknown. This study is to elucidate the mechanism by which NEDD4 mediates the EGFR lung cancer migration signaling.MethodsLentiviral vector-loaded NEDD4 shRNA was used to deplete endogenous NEDD4 in lung cancer cell lines. Effects of the NEDD4 knockdown on the EGFR-dependent or independent lung cancer cell migration were determined using the wound-healing and transwell assays. Association of NEDD4 with activated EGFR was assayed by co-immunoprecipitation. Co-expression of NEDD4 with EGFR or PTEN was determined by immunohistochemical (IHC) staining in 63 lung adenocarcinoma tissue samples. Effects of NEDD4 ectopic expression or knockdown on PTEN ubiquitination and down-regulation, AKT activation and lysosomal secretion were examined using the GST-Uba pulldown assay, immunoblotting, immunofluorescent staining and a human cathepsin B ELISA assay respectively. The specific cathepsin B inhibitor CA-074Me was used for assessing the role of cathepsin B in lung cancer cell migration.ResultsKnockdown of NEDD4 significantly reduced EGF-stimulated cell migration in non-small cell lung carcinoma (NSCLC) cells. Co-immunoprecipitation assay found that NEDD4 is associated with EGFR complex upon EGF stimulation, and IHC staining indicates that NEDD4 is co-expressed with EGFR in lung adenocarcinoma tumor tissues, suggesting that NEDD4 might mediate lung cancer cell migration by interaction with the EGFR signaling complex. Interestingly, NEDD4 promotes the EGF-induced cathepsin B secretion, possibly through lysosomal exocytosis, as overexpression of the ligase-dead mutant of NEDD4 impedes lysosomal secretion, and knockdown of NEDD4 significantly reduced extracellular amount of cathepsin B induced by EGF. Consistent with the role of NEDD4, cathepsin B is pivotal for both basal and the EGF-stimulated lung cancer cell migration. Our studies propose a novel mechanism underlying the EGFR-promoted lung cancer cell migration that is mediated by NEDD4 through regulation of cathepsin B secretion.ConclusionNEDD4 mediates the EGFR lung cancer cell migration signaling through promoting lysosomal secretion of cathepsin B.

Our previous studies have shown that the HECT E3 ubiquitin ligase NEDD4 interacts with LC3 and is required for starvation and rapamycin-induced activation of autophagy. Here, we report that NEDD4 directly binds to SQSTM1 via its HECT domain and polyubiquitylates SQSTM1. This ubiquitylation is through K63 conjugation and is not involved in proteasomal degradation. Mutational analysis indicates that NEDD4 interacts with and ubiquitylates the PB1 domain of SQSTM1. Depletion of NEDD4 or overexpression of the ligase-defective mutant of NEDD4 induced accumulation of aberrant enlarged SQSTM1-positive inclusion bodies that are co-localized with the endoplasmic reticulum (ER) marker CANX, suggesting that the ubiquitylation functions in the SQSTM1-mediated biogenic process in inclusion body autophagosomes. Taken together, our studies show that NEDD4 is an autophagic E3 ubiquitin ligase that ubiquitylates SQSTM1, facilitating SQSTM1-mediated inclusion body autophagy.

PDZ binding-kinase (PBK) (also named T-lymphokine-activated killer cell-originated protein kinase (TOPK)), a serine/threonine kinase, is tightly controlled in normal tissues but elevated in many tumors, and functions in tumorigenesis and metastasis. However, the signaling that regulates expression of PBK in cancer cells remains elusive. Here we show that atorvastatin (Lipitor), an inhibitor of hydroxymethylglutaryl co-enzyme A (HMG-CoA) reductase that is a rate-limiting enzyme of mevalonate pathway, down-regulates expression of PBK by impairing protein geranylgeranylation. The shRNA knockdown demonstrated that Yes-associated protein (YAP) mediates geranylgeranylation-regulated expression of PBK. Importantly, atorvastatin or the geranylgeranyltransferase I inhibitor GGTI-298 inhibited breast cancer cell proliferation through inactivation of YAP signaling and down-regulation of PBK. These findings have defined a new signaling pathway that regulated expression of PBK and identified PBK as a downstream target of the Hippo-YAP signaling, uncoverd a mechanism underlying the anti-cancer effect by inhibition of mevalonate pathway and geranylgeranylation, and provided a potential target for breast cancer targeted therapy.

Gastric cardia adenocarcinoma (GCA) is the most aggressive subtype of gastric cancer with a high metastatic rate. In this report, we collected tumor tissue samples from 214 GCA cases and examined expression of CYR61, a target gene product of the Hippo-YAP/TAZ pathway, in the GCA tumors by immunohistochemical (IHC) staining using the tissue microarray assay (TMA). The results have shown that CYR61 is overexpressed in 44% of the GCA tumor samples. Expression of CYR61 is inversely correlated with cumulative survival of GCA patients (p<0.001) and significantly associated only with metastatic pathological categories (with N category, p=0.052; with TNM stage, p=0.001). Furthermore, knockdown of CYR61 in gastric cancer AGS cells impairs the cancer cell migration and invasion, suggesting a driver role of CYR61 in metastasis. Thus, our studies have established CYR61 as a metastatic biomarker for prediction of poor prognosis of GCA and provided a potential molecular target for anti-metastatic therapy of GCA.

Background Gastric cardia adenocarcinoma (GCA) is an aggressive subtype of gastric cancer with a high metastatic rate. However, the metastatic biomarker of GCA has not been established. Methods To search for the biomarker for GCA metastasis, we here examined expression of the Hippo signaling effector WWTR1 (WW domain containing transcription regulator 1, commonly listed as TAZ) in tumor tissue samples from 214 GCA cases using the tissue microarray assay (TMA), and statistically analyzed association of the WWTR1 expression with metastasis-related pathological outcomes and cumulative survival of the GCA patients. Furthermore, shRNA knockdown was used to determine the role of WWTR1 in promoting cell migration in gastric cancer cells. Results The results have shown that WWTR1 is overexpressed in 66.4% of the GCA tumor samples. Expression of WWTR1 has a significant inverse correlation with cumulative survival of GCA patients (p < 0.01). WWTR1 positive patients had a mean survival of 56.9 ± 4.4 months, comparing to WWTR1 negative mean survival of 77.3 ± 5.9 months. More importantly, expression of WWTR1 significantly associated with tumor invasion and metastasis (in T stage, p = 0.031; N stage, p < 0.01; and TNM stage, p < 0.001). Furthermore, knockdown of WWTR1 impaired migration of gastric cancer AGS cells. Conclusions Our studies have identified WWTR1 as a metastatic biomarker of GCA for poor prognosis, defined a role of WWTR1 in driving metastasis of gastric cancer, and suggested WWTR1 as a potential target for anti-metastatic therapy of GCA.

The objective of the present study was to investigate the protective effect of ulinastatin on severe pulmonary infection under immunosuppression, and its molecular mechanism. Mice were treated with methylprednisolone and lipopolysaccharide (LPS) to establish the model of severe pulmonary infection under immunosuppression. Mice were randomly divided into group A (model group; treated with equal volumes of saline), group B (treated with 1×105 U/kg ulinastatin), and group C (normal control group). Bronchoalveolar lavage fluid (BALF) was collected, and the concentrations of cytokines in BALF were measured by enzyme-linked immunosorbent assay (ELISA). Pathological changes in lung tissues were observed by hematoxylin and eosin (H&E) staining. The mRNA levels of M1 and M2 macrophage markers in lung tissues were detected by real-time polymerase chain reaction (PCR). Specific protein levels in lung tissues were measured by western blotting. Apoptosis in lung tissues was detected by the terminal-deoxynucleoitidyl transferase mediated nick end-labeling (TUNEL) method. The concentrations of TNF-α, IL-6, and IL-1β in BALF, the mRNA levels of the three M1 macrophage markers, and the protein levels of p-Janus Kinase 2 (p-JAK2), p-signal transducer and activator of transcription-3 (p-STAT-3), cleaved caspase-9, and cleaved poly-ADP-ribose polymerase (PARP), and the number of apoptotic cells in lung tissues in group A were significantly higher than those in groups B and C (P<0.05), whereas the concentrations of IL-4, IL-10, and IL-13 and the mRNA levels of the three M2 macrophage markers were significantly lower than those in groups B and C (P<0.05). Immunofluorescence showed that the nuclei of lung epithelial macrophages in group A became smaller and moved towards the side of nuclear membranes. In conclusion, ulinastatin can improve the inflammatory response caused by severe infection under immunosuppression, which balances the inflammatory microenvironment and inhibits apoptosis at least partially through inhibiting JAK2/STAT-3 and/or caspase pathway activity, ultimately playing a role in lung protection.