The polycomb group protein B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) is dysregulated in various cancers, and its upregulation strongly correlates with an invasive phenotype and poor prognosis in patients with nasopharyngeal carcinomas. However, the underlying mechanism of Bmi-1-mediated invasiveness remains unknown. In the current study, we found that upregulation of Bmi-1 induced epithelialmesenchymal transition (EMT) and enhanced the motility and invasiveness of human nasopharyngeal epithelial cells, whereas silencing endogenous Bmi-1 expression reversed EMT and reduced motility.

The Bmi-1 oncoprotein regulates proliferation and oncogenesis in human cells. Its overexpression leads to senescence bypass in human fibroblasts and immortalization of human mammary epithelial cells. In this study, we report that compared with normal nasopharyngeal epithelial cells (NPEC), Bmi-1 is overexpressed in nasopharyngeal carcinoma cell lines. Importantly, Bmi-1 was also found to be overexpressed in 29 of 75 nasopharyngeal carcinoma tumors (38.7%) by immunohistochemical analysis. In contrast to nasopharyngeal carcinoma, there was no detectable expression of Bmi-1 in noncancerous nasopharyngeal epithelium. Moreover, high Bmi-1 expression positively correlated with poor prognosis of nasopharyngeal carcinoma patients. We also report that the overexpression of Bmi-1 leads to bypass of senescence and immortalization of NPECs, which normally express p16INK4a and exhibit finite replicative life span. Overexpression of Bmi-1 in NPECs led to the induction of human telomerase reverse transcriptase activity and reduction of p16INK4a expression. Mutational analysis of Bmi-1 showed that both RING finger and helix-turn-helix domains of it are required for immortalization of NPECs. Our findings suggest that Bmi-1 plays an important role in the development and progression of nasopharyngeal carcinoma, and that Bmi-1 is a valuable marker for assessing the prognosis of nasopharyngeal carcinoma patients. Furthermore, this study provides the first cellular proto-oncogene immortalized nasopharyngeal epithelial cell line, which may serve as a cell model system for studying the mechanisms involved in the tumorigenesis of nasopharyngeal carcinoma. (Cancer Res 2006; 66(12): 6225-32)

The microRNA miR-125b is dysregulated in various human cancers but its underlying mechanisms of action are poorly understood. Here, we report that miR-125b is downregulated in invasive breast cancers where it predicts poor patient survival. Hypermethylation of the miR-125b promoter partially accounted for reduction of miR-125b expression in human breast cancer. Ectopic restoration of miR-125b expression in breast cancer cells suppressed proliferation, induced G 1 cell-cycle arrest in vitro, and inhibited tumorigenesis in vivo. We identified the ETS1 gene as a novel direct target of miR-125b. siRNA-mediated ETS1 knockdown phenocopied the effect of miR-125b in breast cell lines and ETS1 overexpression in invasive breast cancer tissues also correlated with poor patient prognosis. Taken together, our findings point to an important role for miR-125b in the molecular etiology of invasive breast cancer, and they suggest miR-125b as a potential theranostic tool in this disease. Cancer Res; 71(10); 3552-62. Ó2011 AACR.

Epstein–Barr virus (EBV) is implicated as an aetiological factor in B lymphomas and nasopharyngeal carcinoma. The mechanisms of cell-free EBV infection of nasopharyngeal epithelial cells remain elusive. EBV glycoprotein B (gB) is the critical fusion protein for infection of both B and epithelial cells, and determines EBV susceptibility of non-B cells. Here we show that neuropilin 1 (NRP1) directly interacts with EBV gB23–431. Either knockdown of NRP1 or pretreatment of EBV with soluble NRP1 suppresses EBV infection. Upregulation of NRP1 by overexpression or EGF treatment enhances EBV infection. However, NRP2, the homologue of NRP1, impairs EBV infection. EBV enters nasopharyngeal epithelial cells through NRP1-facilitated internalization and fusion, and through macropinocytosis and lipid raft-dependent endocytosis. NRP1 partially mediates EBV-activated EGFR/RAS/ERK signalling, and NRP1-dependent receptor tyrosine kinase (RTK) signalling promotes EBV infection. Taken together, NRP1 is identified as an EBV entry factor that cooperatively activates RTK signalling, which subsequently promotes EBV infection in nasopharyngeal epithelial cells.

A bacterial expression system for the inhibitory Nterminal domain of human tissue inhibitor of metalloproteinases 1 (N-TIMP-1) (Huang, W., Suzuki, K., Nagase, H., Arumugam, S., Van Doren, S. R., and Brew, K. (1996) FEBS Lett. 384, 155-161) has been used to produce 20 single-and double-site mutants that probe the roles of different residues in its inhibitory action on metalloproteinases. Mutations that produce the largest increases in the K i for a C-terminally truncated form of stromelysin 1, MMP-3(⌬C), but do not disturb the conformation involve substitutions of residues that are located in a ridge that is centered around the disulfide bond be- Most mutations that perturb the interaction with MMP-3 have parallel effects on the affinity of N-TIMP-1 for MMP-1 (interstitial collagenase) and MMP-2 (gelatinase A). However, the Thr 2 to Ala mutation produces an inhibitor that is 17-fold more effective against MMP-3 than MMP-1, suggesting that it is feasible to engineer TIMP-1 variants that are more specifically targeted to selected matrix metalloproteinases. The reactive site identified by these studies is a structurally constrained but elongated region of TIMP that can fit the matrix metalloproteinase substrate-binding site. Tissue inhibitors of metalloproteinases (TIMPs)1 are the only known protein inhibitors that are specific for the family of matrix metalloproteinases (MMPs), a group of endopeptidases that are responsible for the breakdown of components of connective tissue (1-3). Four paralogous TIMPs (TIMP-1, TIMP-2, TIMP-3, and TIMP-4), which differ in cellular expression, regulation, and localization, have been identified in mammals and birds (4 -9). All four TIMPs consist of two domains, each stabilized by three disulfide bonds (10). The N-terminal domain is responsible for their inhibitory action against active MMPs, and the C-terminal domain modulates the interaction of TIMPs with pro-MMPs (11). TIMPs also have growth factor activity for erythroid and other cells (12)(13)(14).MMPs play critical roles in biological processes associated with connective tissue turnover, and the balance between levels of TIMPs and active MMPs is important in normal processes such as tissue remodeling and wound healing. Imbalances in the activities of TIMPs and MMPs are associated with pathological conditions such as arthritis, tissue ulceration, and tumor cell invasion and metastasis. Because of their biomedical significance, the mechanisms by which TIMPs regulate the activity of MMPs in vivo and in vitro have been the focus of many previous studies. Aspects of the expression of TIMPs in different tissues or cells and its regulation have been investigated intensively (see Ref. 10 for a review) as well as the role of TIMPs in modulating the activation of MMPs (15). However, less progress has been made in determining the molecular basis of the inhibitory mechanism of TIMP.Previously, mutagenesis has been carried out in attempts to identify key residues for the inhibitory activity of TIMP-2 (16). Since there was no information av...

Methods are described for producing an active aminoterminal domain of tissue inhibitor of metalioproteinases-1 (N-TIMP-1) from inactive protein expressed as inclusion bodies in E. coli. Yields exceed 20 mg per litre of bacterial culture. Activity measurements, CD spectroscopy and NMR spectroscopy of the lSN-labeled protein show that it is fully active, homogeneous in conformation and suitable for high-resolution structural analysis. The affinity of N-TIMP-1 for matrix metalloproteinases 1, 2 and 3 is 6-8-fold less than that of the recombinant full-length protein, indicating that deletion of the Cterminal domain reduces the free energy of interaction by < 10%.

BackgroundGene Ontology (GO) annotation, which describes the function of genes and gene products across species, has recently been used to predict protein subcellular and subnuclear localization. Existing GO-based prediction methods for protein subcellular localization use the known accession numbers of query proteins to obtain their annotated GO terms. An accurate prediction method for predicting subcellular localization of novel proteins without known accession numbers, using only the input sequence, is worth developing.ResultsThis study proposes an efficient sequence-based method (named ProLoc-GO) by mining informative GO terms for predicting protein subcellular localization. For each protein, BLAST is used to obtain a homology with a known accession number to the protein for retrieving the GO annotation. A large number n of all annotated GO terms that have ever appeared are then obtained from a large set of training proteins. A novel genetic algorithm based method (named GOmining) combined with a classifier of support vector machine (SVM) is proposed to simultaneously identify a small number m out of the n GO terms as input features to SVM, where m <

The unregulated activities of matrix metalloproteinases (MMPs) are implicated in disease processes including arthritis and tumor cell invasion and metastasis. MMP activities are controlled by four homologous endogenous protein inhibitors, tissue inhibitors of metalloproteinases (TIMPs), yet different TIMPs show little specificity for individual MMPs. The large interaction interface in the TIMP-1⅐MMP-3 complex includes a contiguous region of TIMP-1 around the disulfide bond between Cys 1 and Cys 70 that inserts into the active site of MMP-3. The effects of fifteen different substitutions for threonine 2 of this region reveal that this residue makes a large contribution to the stability of complexes with MMPs and has a dominant influence on the specificity for different MMPs. The size, charge, and hydrophobicity of residue 2 are key factors in the specificity of TIMP. Threonine 2 of TIMP-1 interacts with the S1 specificity pocket of MMP-3, which is a key to substrate specificity, but the structural requirements in TIMP-1 residue 2 for MMP binding differ greatly from those for the corresponding residue of a peptide substrate. These results demonstrate that TIMP variants with substitutions for Thr 2 represent suitable starting points for generating more targeted TIMPs for investigation and for intervention in MMP-related diseases.The matrix metalloproteinases (MMPs) 1 are a family of about twenty Zn 2ϩ -dependent endopeptidases that have important roles in connective tissue turnover during physiological processes including development, morphogenesis, and wound healing (1, 2). Their activities in the extracellular matrix are stringently regulated through transcriptional control, zymogen activation, and the actions of four endogenous inhibitory proteins, tissue inhibitors of metalloproteinases (TIMPs) 1 to 4 (3-7). Normal matrix homeostasis is associated with an appropriate balance between the levels of TIMPs and active MMPs, whereas an imbalance involving excess MMP activity is linked with disease processes including arthritis, tumor cell metastasis, and tissue invasion and atherosclerosis (1, 2).Mammalian TIMPs have an N-terminal domain of about 125 amino acids and a smaller C-terminal domain of about 65 amino acids; each domain is stabilized by three disulfide bonds (8). The N-terminal domains of different TIMPs fold into a correct native structure which carries the inhibitory activity against MMPs (9 -11). Although correctly folded and functional C-terminal domains have not been described, truncation experiments indicate that this region is responsible for the interactions of TIMPs with pro-MMPs (12, 13). There is little specificity in the inhibitory actions of TIMPs on metalloproteinases, with the exception of the ability of TIMP-2 and TIMP-3 to inhibit membrane-type metalloproteinases-1 and -2, whereas TIMP-1 is a poor inhibitor of these enzymes (12-14). However, the interactions of TIMPs with pro-MMPs are more specific. For example, TIMP-2 and TIMP-4 form specific complexes with pro-MMP-2 (progelatinase A), whe...