Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L-like proteases in tumor microenvironment

Human cathepsin L along with cathepsin S, K, and V are collectively known as cathepsin L-like proteases due to their high homology. The overexpression and aberrant activity of each of these proteases has been implicated in tumorigenesis. These proteases contain propeptide domains that can potently inhibit both their cognate protease and other proteases within the cathepsin L-like subfamily. In this investigation, we have produced the cathepsin S propeptide recombinantly and have shown that it is a potent inhibitor of the peptidolytic, elastinolytic, and gelatinolytic activities of the cathepsin L-like proteases. In addition, we show that this peptide is capable of significantly attenuating tumor cell invasion in a panel of human cancer cell lines. Furthermore, fusion of an IgG Fc-domain to the COOH terminus of the propeptide resulted in a chimeric protein with significantly enhanced ability to block tumor cell invasion. This Fc fusion protein exhibited enhanced stability in cell-based assays in comparison with the unmodified propeptide species. This approach for the combined inhibition of the cathepsin L-like proteases may prove useful for the further study in cancer and other conditions where their aberrant activity has been implicated. Furthermore, this strategy for simultaneous inhibition of multiple cysteine cathepsins may represent the basis for novel therapeutics to attenuate tumorigenesis.

Invasion by the amphipod Gammarus pulex alters community composition of native freshwater macroinvertebrates

Assessing the effects of invading species on native community structure is often confounded by environmental factors and weakened by lack of replicated, long-term pre- and post-invasion monitoring. Here, we uncouple the community effects of a freshwater amphipod invader from environmental differences. In Irish rivers, the introduced Gammarus pulex replaces the native Gammarus duebeni celticus. However, the River Lissan in Northern Ireland is dissected by a weir that has slowed the upstream invasion by G. pulex. This allowed us in 2000 to sample three contiguous 150-m reaches that were (1) G. pulex dominated; (2) mixed Gammarus spp.; and (3) G. duebeni celticus only. In 2003, we resampled these reaches and one additional of mixed Gammarus species and one with only G. duebeni celticus further upstream. In temperature, conductivity, and pH, there were statistically significant but no biologically relevant differences among the five reaches of 2003, and between the three reaches surveyed in both years. Although there was evidence of recovery in macroinvertebrate diversity and richness in invaded reaches between years, continued upstream invasion was associated with sustained reductions in these community metrics as compared to un-invaded sites. Community ordination indicated (1) different associations of community composition attributed to the distribution, abundance, and biomass of the invader; and (2) increasing similarity of invaded communities over time. The impact mechanisms of G. pulex on macroinvertebrate community composition may include predation and competition. The consequences of the observed community changes for ecosystem functioning require further investigation.

CD44 enhances invasion of basal-like breast cancer cells by upregulating serine protease and collagen-degrading enzymatic expression and activity

Introduction: Basal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells.

Methods: CD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion.

Results: Expression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen.

Conclusion: HA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion.

Forecasting invasions: resource use by mussels informs invasion patterns along the South African coast

Invasive species are often more able to rapidly and efficiently utilise resources than natives, and comparing per capita resource use at different resource densities among invaders and trophically analogous natives could allow for reliable predictions of invasiveness. In South Africa, invasion by the Mediterranean mussel Mytilus galloprovincialis has transformed wave-exposed shores, negatively affecting native mussel species. Currently, South Africa is experiencing a second mussel invasion with the recent detection of the South American Semimytilus algosus. We tested per capita uptake of an algal resource by invading M. galloprovincialis, S. algosus, and the native Aulacomya atra at different algal concentrations and temperatures, representing the west and south coasts of South Africa, to examine whether their per capita resource use could be a predictor of their spread and subsequent invasiveness. Regardless of temperature, M. galloprovincialis was the most efficient consumer, significantly reducing algal cells compared to the other species when the resource was presented in both low and high starting densities. Furthermore, these findings aligned with a greater biomass of M. galloprovincialis on the shore in comparison with the other species. Resource use by the new invader S. algosus was dependent on the density of resource and, although this species was efficient at low algal concentrations at cooler temperatures, this pattern broke down at higher algal densities. This was once more reflected in lower biomass in surveys of this species along the cool west coast. We therefore forecast that S. algosus will be become established along the south coast; however, we also predict that M. galloprovincialis will maintain dominance on these shores.

A species invasion mediated through habitat structure, intraguild predation and parasitism.

With field, laboratory, and modeling approaches, we examined the interplay among habitat structure, intraguild predation (IGP), and parasitism in an ongoing species invasion. Native Gammarus duebeni celticus (Crustacea: Amphipoda) are often, but not always, replaced by the invader Gammarus pulex through differential IGP. The muscle-wasting microsporidian parasite Pleistophora mulleri infects the native but not the invader. We found a highly variable prevalence of P. mulleri in uninvaded rivers, with 0–91% of hosts parasitized per sample. In addition, unparasitized natives dominated fast-flowing riffle patches of river, whereas parasitized individuals dominated slower- flowing, pooled patches. We examined the survivorship of invader and native in single and mixed-species microcosms with high, intermediate, and zero parasite prevalence. G. pulex survivorship was high in all treatments, whereas G. duebeni subsp. celticus survivorship was significantly lower in the presence of the invader. Further, parasitized G. duebeni subsp. celticus experienced near-total elimination. Models of the species replacement process implied that parasite-enhanced IGP would make invasion by G. pulex more likely, regardless of habitat and parasite spatial structure. However, where heterogeneity in parasite prevalence creates a landscape of patches with different susceptibilities to invasion, G. pulex may succeed in cases where invasion would not be possible if patches were equivalent. The different responses of parasitized and unparasitized G. duebeni subsp. celticus to environmental heterogeneity potentially link landscape patterns to the success or failure of the invasion process.

Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells

Using a validated tetracycline (tet)-regulated MCF7-founder (MCF7F) expression system to modulate expression of CD44 standard form (CD44s), we report the functional importance of CD44s and that of a novel transcriptional target of hyaluronan (HA)/CD44s signaling, EMS1/cortactin, in underpinning breast cancer metastasis. In functional experiments, tet-regulated induction of CD44s potentiated the migration and invasion of MCF7F cells through HA-supplemented Matrigel. EMS1/cortactin was identified by expression profiling as a novel transcriptional target of HA/CD44 signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast cancer cell lines. The mechanistic basis underpinning CD44-promoted transcription of EMS1/cortactin was shown to be dependent upon a NFB mechanism, since pharmacological inhibition of IKinase-2 or suppression of p65 Rel A expression attenuated CD44-induced increases in cortactin mRNA transcript levels. Overexpression of a c-myc tagged murine cortactin construct in the weakly invasive, CD44-deficient MCF7F and T47D cells potentiated their invasion. Furthermore, the functional importance of cortactin to CD44s-promoted metastasis was demonstrated by selective suppression of cortactin in CD44-expressing MCF7F-B5 and MDA-MB-231 breast cancer cells using RNAi, which was shown to result in attenuated CD44-promoted invasion and CD44-promoted adhesion to bone marrow endothelial cells (BMECs).

Agelastatin A: a novel inhibitor of osteopontin-mediated adhesion, invasion, and colony formation

Effective inhibitors of osteopontin (OPN)-mediated neoplastic transformation and metastasis are still lacking. (-)-Agelastatin A is a naturally occurring oroidin alkaloid with powerful antitumor effects that, in many cases, are superior to cisplatin in vitro. In this regard, past comparative assaying of the two agents against a range of human tumor cell lines has revealed that typically (-)-agelastatin A is 1.5 to 16 times more potent than cisplatin at inhibiting cell growth, its effects being most pronounced against human bladder, skin, colon, and breast carcinomas. In this study, we have investigated the effects of (-)-agelastatin A on OPN-mediated malignant transformation using mammary epithelial cell lines. Treatment with (-)-agelastatin A inhibited OPN protein expression and enhanced expression of the cellular OPN inhibitor, Tcf-4. (-)-Agelastatin A treatment also reduced beta-catenin protein expression and reduced anchorage-independent growth, adhesion, and invasion in R37 OPN pBK-CMV and C9 cell lines. Similar effects were observed in MDA-MB-231 and MDA-MB-435s human breast cancer cell lines exposed to (-)-agelastatin A. Suppression of Tcf-4 by RNA interference (short interfering RNA) induced malignant/invasive transformation in parental benign Rama 37 cells; significantly, these events were reversed by treatment with (-)-agelastatin A. Our study reveals, for the very first time, that (-)-agelastatin A down-regulates beta-catenin expression while simultaneously up-regulating Tcf-4 and that these combined effects cause repression of OPN and inhibition of OPN-mediated malignant cell invasion, adhesion, and colony formation in vitro. We have also shown that (-)-agelastatin A inhibits cancer cell proliferation by causing cells to accumulate in the G(2) phase of cell cycle.

RAN GTPase is an effector of the invasive/metastatic phenotype induced by osteopontin.

Osteopontin (OPN) is a phosphorylated glycoprotein that binds to alpha v-containing integrins and is important in malignant transformation and cancer. Previously, we have utilized suppressive subtractive hybridization between mRNAs isolated from the Rama 37 (R37) rat mammary cell line and a subclone rendered invasive and metastatic by stable transfection with an expression vector for OPN to identify RAN GTPase (RAN) as the most overexpressed gene, in addition to that of OPN. Here we show that transfection of noninvasive R37 cells with an expression vector for RAN resulted in increased anchorage-independent growth, cell attachment and invasion through Matrigel in vitro, and metastasis in syngeneic rats. This induction of a malignant phenotype was induced independently of the expression of OPN, and was reversed by specifically reducing the expression of RAN using small-interfering RNAs. By using a combination of mutant protein and inhibitors, it was found that RAN signal transduction occurred through the c-Met receptor and PI3 kinase. This study therefore identifies RAN as a novel effector of OPN-mediated malignant transformation and some of its downstream signaling events in a mammary epithelial model of cancer invasion/metastasis.

European hare (Lepus europaeus) invasion ecology: Implication for the conservation of the endemic Irish hare (Lepus timidus hibernicus)

European hare Lepus europaeus populations have undergone recent declines but the species has successfully naturalised in many countries outside its native range. It was introduced to Ireland during the mid-late nineteenth century for field sport and is now well established in Northern Ireland. The native Irish hare Lepus timidus hibernicus is an endemic subspecies of mountain hare L. timidus and has attracted major conservation concern following a long-term population decline during the twentieth century and is one of the highest priority species for conservation action in Ireland. Little is known about the European hare in Ireland or whether it poses a significant threat to the native mountain hare subspecies by compromising its ecological security or genetic integrity. We review the invasion ecology of the European hare and examine evidence for interspecific competition with the mountain hare for habitat space and food resources, interspecific hybridisation, disease and parasite transmission and possible impacts of climate change. We also examine the impact that introduced hares can have on native non-lagomorph species. We conclude that the European hare is an emerging and significant threat to the conservation status of the native Irish hare. Invasive mammal species have been successfully eradicated from Ireland before and immediate action is often the only opportunity for cost-effective eradication. An urgent call is issued for further research whilst the need for a European hare invasive Species Action Plan (iSAP) and Eradication strategy are discussed.

Fronts, jumps and secondary introductions suggested as different invasion patterns in marine species, with an increase in spread rates over time

Not all introduced (invasive) species in a region will spread from a single point of introduction. Long-distance dispersal or further introductions can obscure the pattern of spread, but the regional importance of such processes is difficult to gauge. These difficulties are further compounded when information on the multiple scale process of invasive species range expansion is reduced to one-dimensional estimates of spread (e. g. km yr(-1)). We therefore compared the results of two different metrics of range expansion: maximum linear rate of spread and accumulation of occupied grid squares (50 x 50 km) over time. An analysis of records for 54 species of introduced marine macrophytes in the Mediterranean and northeast Atlantic revealed cases where the invasion process was probably missed (e. g. Atlantic Bonnemaisonia hamifera) and suggested cases of secondary introductions or erratic jump dispersal (Dasysiphonia sp. and Womersleyella setacea). A majority of species analysed showed evidence for an accumulation of invaded sites without a clear invasion front. Estimates of spread rate are increasing for more recent introductions. The increase is greater than can be accounted for by temporally varying search effort and implies a historical increase in vector efficiency and/or a decreased resistance of native communities to invasion.

Cofilin immunolabelling correlates with depth of invasion in gastrointestinal endocrine cell tumors

Gastrointestinal endocrine cell tumors are a heterogeneous population of lesions believed to arise from neuroendocrine cells of the gastrointestinal mucosa. The current classification of these tumors is based on tumor size, microscopic features and clinical evidence of metastasis. Although diagnostic categories generally correlate with prognosis, molecular prognostic markers will be clinically useful adjuncts. Cofilin has been implicated in tumor invasion, and its immunolocalisation was studied in gastrointestinal endocrine cell tumors. The immunolocalisation of cofilin was studied by immunohistochemistry in 34 formalin-fixed, paraffin wax-embedded gastrointestinal endocrine cell tumors using a tissue microarray platform. A significant correlation was found between high cofilin immunolabelling and the depth of invasion (p

Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion

Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions. © European Molecular Biology Organization.

Type IVB pili are required for invasion but not for adhesion of Salmonella enterica serovar Typhi into BHK epithelial cells in a cystic fibrosis transmembrane conductance regulator-independent manner

The cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed as an epithelial cell receptor for the entry of Salmonella Typhi but not Salmonella Typhimurium. The bacterial ligand recognized by CM is thought to reside either in the S. Typhi lipopolysaccharide core region or in the type IV pili. Here, we assessed the ability of virulent strains of S. Typhi and S. Typhimurium to adhere to and invade BHK epithelial cells expressing either the wild-type CFTR protein or the Delta F508 CFTR mutant. Both S. Typhi and S. Typhimurium invaded the epithelial cells in a CFTR-independent fashion. Furthermore and also in a CFTR-independent manner, a S. Typhi pilS mutant adhered normally to BHK cells but displayed a 50% reduction in invasion as compared to wild-type bacteria. Immunofluorescence microscopy revealed that bacteria and CFTR do not colocalize at the epithelial cell surface. Together, our results strongly argue against the established dogma that CFTR is a receptor for entry of Salmonella to epithelial cells. (C) 2011 Elsevier Ltd. All rights reserved.