Rosemary Bass

Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues

Extracellular proteases of the matrix metalloproteinase (MMP) and serine protease families participate in many aspects of tumour growth and metastasis. Using quantitative real-time RT–PCR analysis, we have undertaken a comprehensive survey of the expression of these enzymes and of their natural inhibitors in 44 cases of human prostate cancer and 23 benign prostate specimens. We found increased expression of MMP10, 15, 24, 25 and 26, urokinase plasminogen activator-receptor (uPAR) and plasminogen activator inhibitor-1 (PAI1), and the newly characterised serine proteases hepsin and matriptase-1 (MTSP1) in malignant tissue compared to benign prostate tissue. In contrast, there was significantly decreased expression of MMP2 and MMP23, maspin, and the protease inhibitors tissue inhibitor of metalloproteinase 3 (TIMP3), TIMP4 and RECK (reversion-inducing cysteine-rich protein with Kazal motifs) in the cancer specimens. The expression of MMP15 and MMP26 correlated positively with Gleason score, whereas TIMP3, TIMP4 and RECK expression correlated negatively with Gleason score. The cellular localisation of the expression of the deregulated genes was evaluated using primary malignant epithelial and stromal cell cultures derived from radical prostatectomy specimens. MMP10 and 25, hepsin, MTSP1 and maspin showed predominantly epithelial expression, whereas TIMP 3 and 4, RECK, MMP2 and 23, uPAR and PAI1 were produced primarily by stromal cells. These data provide the first comprehensive and quantitative analysis of the expression and localisation of MMPs and their inhibitors in human prostate cancer, leading to the identification of several genes involved in proteolysis as potential prognostic indicators, in particular hepsin, MTSP1, MMP26, PAI1, uPAR, MMP15, TIMP3, TIMP4, maspin and RECK.

A major fraction of endoplasmic reticulum-located glutathione is present as mixed disulfides with protein.

The tripeptide glutathione is the most abundant thiol/disulfide component of the eukaryotic cell and is known to be present in the endoplasmic reticulum lumen. Accordingly, the thiol/disulfide redox status of the endoplasmic reticulum lumen is defined by the status of glutathione, and it has been assumed that reduced and oxidized glutathione form the principal redox buffer. We have determined the distribution of glutathione between different chemical states in rat liver microsomes by labeling with the thiol-specific label monobromobimane and subsequent separation by reversed phase high performance liquid chromatography. More than half of the microsomal glutathione was found to be present in mixed disulfides with protein, the remainder being distributed between the reduced and oxidized forms of glutathione in the ratio of 3:1. The high proportion of the total population of glutathione that was found to be in mixed disulfides with protein has significant implications for the redox state and buffering capacity of the endoplasmic reticulum and, hence, for the formation of disulfide bonds in vivo.

Regulation of Urokinase Receptor Proteolytic Function by the Tetraspanin CD82

The high affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored cellular receptor (uPAR) promotes plasminogen activation and the efficient generation of pericellular proteolytic activity. We demonstrate here that expression of the tetraspanin CD82/KAI1 (a tumor metastasis suppressor) leads to a profound effect on uPAR function. Pericellular plasminogen activation was reduced by ∼50-fold in the presence of CD82, although levels of components of the plasminogen activation system were unchanged. uPAR was present on the cell surface and molecularly intact, but radioligand binding analysis with uPA and anti-uPAR antibodies revealed that it was in a previously undetected cryptic form unable to bind uPA. This was not due to direct interactions between uPAR and CD82, as they neither co-localized on the cell surface nor could be co-immunoprecipitated. However, expression of CD82 led to a redistribution of uPAR to focal adhesions, where it was shown by double immunofluorescence labeling to co-localize with the integrin α5β1, which was also redistributed in the presence of CD82. Co-immunoprecipitation experiments showed that, in the presence of CD82, uPAR preferentially formed stable associations with α5β1, but not with a variety of other integrins, including α3β1. These data suggest that CD82 inhibits the proteolytic function of uPAR indirectly, directing uPAR and α5β1 to focal adhesions and promoting their association with a resultant loss of uPA binding. This represents a novel mechanism whereby tetraspanins, integrins, and uPAR, systems involved in cell adhesion and migration, cooperate to regulate pericellular proteolytic activity and may suggest a mechanism for the tumor-suppressive effects of CD82/KAI1

G-helix of Maspin Mediates Effects on Cell Migration and Adhesion

Maspin is a member of the serine protease inhibitor (serpin) superfamily that lacks protease inhibitory ability, although displaying tumor metastasis-suppressing activity resulting from its influence on cell migration, invasion, proliferation, apoptosis, and adhesion. The molecular mechanisms of these actions of maspin are as yet undefined. Here, we sought to identify critical functional motifs by the expression of maspin with point mutations at sites potentially involved in protein-protein interactions: the G α-helix (G-helix), an internal salt bridge or the P1 position of the reactive center loop. Our findings indicate that only mutations in the G-helix attenuated inhibition of cell migration by maspin and that this structural element is also involved in the effect of maspin on cell adhesion. The action of maspin on cell migration could be mimicked by a 15-mer G-helix peptide, indicating that the G-helix is both essential and sufficient for this effect. In addition, we provide evidence that the effects of the G-helix of maspin are dependent on β1 integrins. These data reveal that the major extracellular functions associated with the tumor suppressive action of maspin likely involve interactions in which the G-helix plays a key role.

Banking of fresh-frozen prostate tissue: Methods, validation and use

The question of banking of fresh frozen human prostatic tissue is addressed by a group of authors who have compared its value with what they call “pseudobanked” tissue. They then validate their methodology by a number of methods, including expression of hepsin, which they found to be significantly higher in malignant than in benign tissue. The theme of prostate cancer is continued by an English group looking at recent trends in the use of radical prostatectomy in England, and they make a number of interesting comments about this. Again in relation to prostate cancer, a group from Boston assess the use of steroids after prostate brachytherapy to reduce the risk of acute urinary retention.

Binding of extracellular maspin to β1 integrins inhibits vascular smooth muscle cell migration

Maspin is a serpin that has multiple effects on cell behavior, including inhibition of migration. How maspin mediates these diverse effects remains unclear, as it is devoid of protease inhibitory activity. We have previously shown that maspin rapidly inhibits the migration of vascular smooth muscle cells (VSMC), suggesting the involvement of direct interactions with cell surface proteins. Here, using immunofluorescence microscopy, we demonstrate that maspin binds specifically to the surface of VSMC in the dedifferentiated, but not the differentiated, phenotype. Ligand blotting of VSMC lysates revealed the presence of several maspin-binding proteins, with a protein of 150 kDa differentially expressed between the two VSMC phenotypes. Western blotting suggested that this protein was the β1 integrin subunit, and subsequently both α3β1 and α5β1, but not αvβ3, were shown to associate with maspin by coimmunoprecipitation. Specific binding of these integrins was also observed using maspin-affinity chromatography, using HT1080 cell lysates. Direct binding of maspin to α5β1 was confirmed using a recombinant α5β1-Fc fusion protein. Using conformation-dependent anti-β1 antibodies, maspin binding to VSMC was found to lead to a decrease in the activation status of the integrin. The functional involvement of α5β1 in mediating the effect of maspin was established by the inhibition of migration of CHO cells overexpressing human α5 integrin, but not those lacking α5 expression. Our observations suggest that maspin engages in specific interactions with a limited number of integrins on VSMC, leading to their inactivation, and that these interactions are responsible for the effects of maspin in the pericellular environment.

Avascular tumour growth dynamics and the constraints of protein binding for drug transportation.

The potential for the use of in-silico models of disease in progression monitoring is becoming increasingly recognised, as well as its contribution to the development of complete curative processes. In this paper we report the development of a hybrid cellular automaton model to mimic the growth of avascular tumours, including the infusion of a bioreductive drug to study the effects of protein binding on drug transportation. The growth model is operated within an extracellular tumour microenvironment. An artificial Neural Network based scheme was implemented that modelled the behaviours of each cell (proliferation, quiescence, apoptosis and/or movement) based on the complex heterogeneous microenvironment; consisting of oxygen, glucose, hydrogen ions, inhibitory factors and growth factors. To validate the growth model results, we conducted experiments with multicellular tumour spheroids. These results showed good agreement with the predicted growth dynamics. The outcome of the avascular tumour growth model suggested that tumour microenvironments have a strong impact on cell behaviour. To address the problem of cellular proteins acting as resistive factors preventing efficient drug penetration, a bioreactive drug (tirapazamine) was added to the system. This allowed us to study the drug penetration through multicellular layers of tissue after its binding to cellular proteins. The results of the in vitro model suggested that the proteins reduce the toxicity of the drug, reducing its efficacy for the most severely hypoxic fractions furthest from a functional blood vessel. Finally this research provides a unique comparison of in vitro tumour growth with an intelligent in silico model to measure bioreductive drug availability inside tumour tissue through a set of experiments.

Regulation of urokinase receptor function and pericellular proteolysis by the integrin α5β1

Interactions between the uPA receptor (uPAR) and various integrins, including alpha(5)beta(1), are known to modulate integrin-dependent cell adhesion, and we have shown that the integrin-associated tetraspanin protein CD82 down-regulates uPAR-dependent plasminogen activation by affecting alpha(5)beta(1) cellular localisation. Here we have investigated whether overexpression of alpha(5)beta(1) directly affects uPAR-dependent pericellular proteolysis. CHO cells overexpressing alpha(5)beta(1) were found to activate plasminogen at a rate up to 18-fold faster than B2CHO cells which are alpha(5)-deficient. This effect was dependent on the activation state of alpha(5)beta(1), as it was maximal in the presence of Mn(2+). To determine the role of uPAR-alpha(5)beta(1) interactions in this effect, we determined the adhesion of these cells to immobilised soluble uPAR (suPAR). Neither cell-type was found to adhere to suPAR, but both cell types were found to adhere to an anti-uPAR monoclonal antibody in a uPAR- and integrin-dependent manner. This adhesion was 10-fold greater in the absence of alpha(5)beta(1), possibly implicating the involvement of non-alpha(5)-integrins. Soluble forms of the various components were used to investigate the molecular basis of these effects, but no direct interactions could be demonstrated between alpha(5)beta(1) and either uPAR, uPA or uPA-uPAR complex. This suggests that assembly of these components on the plasma membrane is required to influence uPAR function, increasing uPAR-dependent pericellular proteolysis and decreasing uPAR-dependent cell adhesion. These interactions may be modified by other integrins, suggesting a complex interplay between uPAR and integrins on the cell surface with the potential to regulate invasive cell migration.

An in silico model to demonstrate the effects of Maspin on cancer cell dynamics

Most cancer treatments efficacy depends on tumor metastasis suppression, where tumor suppressor genes play an important role. Maspin (Mammary Serine Protease Inhibitor), an non-inhibitory serpin has been reported as a potential tumor suppressor to influence cell migration, adhesion, proliferation and apoptosis in in vitro and in vivo experiments in last two decades. Lack of computational investigations hinders its ability to go through clinical trials. Previously, we reported first computational model for maspin effects on tumor growth using artificial neural network and cellular automata paradigm with in vitro data support. This paper extends the previous in silico model by encompassing how maspin influences cell migration and the cell-extracellular matrix interaction in subcellular level. A feedforward neural network was used to define each cell behavior (proliferation, quiescence, apoptosis) which followed a cell-cycle algorithm to show the microenvironment impacts over tumor growth. Furthermore, the model concentrates how the in silico experiments results can further confirm the fact that maspin reduces cell migration using specific in vitro data verification method. The data collected from in vitro and in silico experiments formulates an unsupervised learning problem which can be solved by using different clustering algorithms. A density based clustering technique was developed to measure the similarity between two datasets based on the number of links between instances. Our proposed clustering algorithm first finds the nearest neighbors of each instance, and then redefines the similarity between pairs of instances in terms of how many nearest neighbors share the two instances. The number of links between two instances is defined as the number of common neighbors they have. The results showed significant resemblances with in vitro experimental data. The results also offer a new insight into the dynamics of maspin and establish as a metastasis suppressor gene for further molecular research.

A hybrid computational model for the effects of maspin on cancer cell dynamics

Cancer metastasis is a complex multistep process which allows cancer cells to establish new tumours in distant organs. The process of metastasis involves cell migration and invasion; it is what makes cancer a fatal disease. The efficiency of most cancer treatments depends on metastasis suppression. Maspin is a type II tumour metastasis suppressor which has multiple cellular effects. It has been described as a key regulatory protein in both the intracellular and extracellular environments. Maspin has been shown to reduce cell migration, invasion, proliferation and angiogenesis, and increase apoptosis and cell-cell adhesion in in vitro and in vivo experiments. The clinical data regarding the predictive effects of maspin expression are variable. To date, the whole cellular mechanisms that maspin uses to influence tumour cell behaviours have not been clearly defined. The diversity of the effects of maspin motivated us to develop an intelligent model to investigate its effects on cellular proliferation and migration. This paper reports a hybrid model of solid tumour growth in order to investigate the impact of maspin on the growth and evolutionary dynamics of the cancer cell. A feed-forward neural network was used to model the behaviours (proliferation, quiescence, apoptosis and/or movement) of each cell, which has been suggested as a suitable model of cell signalling pathways. Results show that maspin reduces migration by 10-40%, confirmed by published in vitro data. The model also shows a reduction in cell proliferation by 20-30% in the presence of maspin. So far, this is the first attempt to model the effect of maspin in a computational model to verify in vitro data. This will provide new insights into the tumour suppressive properties of maspin and inform the development of novel cancer therapies.