Distinct mechanisms of tumor invasion and metastasis

Most cancer deaths are caused by metastasis rather than the primary tumor. Cancer cells invade normal tissue as epithelial sheets or single cells by inducing expression of programs characteristic of developmental processes. Depending on their tissue of origin, cancer cells subsequently spread to distinct target organs where they seed secondary tumors (metastasis). Recent experimental evidence suggests that metastasis requires changes not only in cancer cells but also in the tumor microenvironment and in the metastatic target site. For example, a premetastatic niche is formed in target organs that attract cancer cells. Understanding the distinct mechanisms used by cancer cells to form metastasis will enable better patient evaluation and the design of innovative therapeutic approaches.     

Molecular mechanisms of tumor invasion and metastasis: an integrated view

As tumors progress to increased malignancy, cells within them develop the ability to invade into surrounding normal tissues and through tissue boundaries to form new growths (metastases) at sites distinct from the primary tumor. The molecular mechanisms involved in this process are incompletely understood but those associated with cell-cell and cell-matrix adhesion, with the degradation of extracellular matrix, and with the initiation and maintenance of early growth at the new site are generally accepted to be critical. This article discusses current knowledge of molecular events involved in these various processes. The potential role of adhesion molecules (eg. integrins and cadherins) has undergone a major transition over the last ten years, as it has become apparent that such molecules play a major role in signaling from outside to inside a cell, thereby controlling how a cell is able (or not) to sense and interact with its local environment. Similarly the roles of proteolytic enzymes and their inhibitors (eg. matrix metalloproteinases and TIMPs) have also expanded as it has become apparent that they not only have the abilities to break down the components of the extracellular matrix but also are involved in the release of factors which can affect the growth of the tumor cells positively or negatively. Recent work has highlighted the importance of the later, post-extravasational stages of metastasis, where adhesion and proteolysis are now known to play a role along with other processes such as apoptosis, dormancy, growth factor-receptor interactions and signal transduction. Recent work has also demonstrated that not only the immediate cellular microenvironment, in terms of specific cell-cell and cell-matrix interactions, but also the extended cellular microenvironment, in terms of vascular insufficiency and hypoxia in the primary tumor, can modify cellular gene expression and enhance metastasis. Mechanisms of metastasis appear to involve a complex array of genetic and epigenetic changes many of which appear to be specific both for different types of tumors and for different sites of metastasis. Our improved understanding of the expanded roles of the individual molecules involved has resulted in a mechanistic blurring of the previously described discrete stages of the metastatic process.     

Pak1 and Pak2 mediate tumor cell invasion through distinct signaling mechanisms

Pak kinases are thought to play critical roles in cell migration and invasion. Here, we analyze the roles of Pak1 and Pak2 in breast carcinoma cell invasion using the transient transfection of small interfering RNA. We find that although both Pak1 and Pak2 contribute to breast carcinoma invasion stimulated by heregulin, these roles are mediated by distinct signaling mechanisms. Thus, whereas the depletion of Pak1 interferes with the heregulin-mediated dephosphorylation of cofilin, the depletion of Pak2 does not. The depletion of Pak1 also has a stronger inhibitory effect on lamellipodial protrusion than does the depletion of Pak2. Interestingly, Pak1 and Pak2 play opposite roles in regulating the phosphorylation of the myosin light chain (MLC). Whereas the depletion of Pak1 decreases phospho-MLC levels in heregulin-stimulated cells, the depletion of Pak2 enhances MLC phosphorylation. Consistent with their opposite effects on MLC phosphorylation, Pak1 and Pak2 differentially modulate focal adhesions. Pak2-depleted cells display an increase in focal adhesion size, whereas in Pak1-depleted cells, focal adhesions fail to mature. We also found that the depletion of Pak2, but not Pak1, enhances RhoA activity and that the inhibition of RhoA signaling in Pak2-depleted cells decreases MLC phosphorylation and restores cell invasion. In summary, this work presents the first comprehensive analysis of functional differences between the Pak1 and Pak2 isoforms.     

Integrins and tumor invasion

Cell-extracellular matrix interactions are important in the process of tumor cell invasion and metastasis. In particular, the interactions of tumor cells with basement membranes of tissue epithelial, as well as vascular endothelial, cells are likely to represent key steps in the metastatic process. The interactions between cells and the connective tissue matrix are mediated by a large family of cell surface receptors, the integrins, which represent multiple receptors for extracellular matrix and basement membrane components. Here, I review recent progress in elucidating the roles of integrins in tumor cell invasion. Altered expression of this large family of receptors on invasive tumor cells, as compared with non-invasive cells, may represent a fundamental step in the progressive expression of the invasive phenotype.     

Biochemical mechanisms of cephaloridine nephrotoxicity

Large doses of the cephalosporin antibiotic, cephaloridine, produce acute proximal tubular necrosis in humans and in laboratory animals. Cephaloridine is actively transported into the proximal tubular cell by an organic anion transport system while transport across the lumenal membrane into tubular fluid appears restricted. High intracellular concentrations of cephaloridine are attained in the proximal tubular cell which are critical to the development of nephrotoxicity. There is substantial evidence indicating that oxidative stress plays a major role in cephaloridine nephrotoxicity. Cephaloridine depletes reduced glutathione, increases oxidized glutathione and induces lipid peroxidation in renal cortical tissue. The molecular mechanisms mediating cephaloridine-induced oxidative stress are not well understood. Inhibition in gluconeogenesis is a relatively early biochemical effect of cephaloridine and is independent of lipid peroxidation. Furthermore, cephaloridine inhibits gluconeogenesis in both target (kidney) and non-target (liver) organs of cephaloridine toxicity. Since glucose is not a major fuel of proximal tubular cells, it is unlikely that cephaloridine-induced tubular necrosis is mediated by the effects of this drug on glucose synthesis.     

Biochemical mechanisms of cyclosporine neurotoxicity

Proper management of chemotoxicity in transplant patients requires detailed knowledge of the biochemical mechanisms underlying immunosuppressant toxicity. Neurotoxicity is one of the most significant clinical side effects of the immunosuppressive undecapeptide cyclosporine, occurring at some degree in up to 60% of transplant patients. The clinical symptoms of cyclosporine-mediated neurotoxicity consist of decreased responsiveness, hallucinations, delusions, seizures, cortical blindness, and stroke-like episodes that mimic those clinical symptoms of mitochondrial encephalopathy. Clinical computed tomography (CT) and magnetic resonance imaging (MRI) studies have revealed a correlation between clinical symptoms of cyclosporine-mediated neurotoxicity and morphological changes in the brain, such as hypodensity of white matter, cerebral edema, metabolic encephalopathy, and hypoxic damages. Paradoxically, in animal models cyclosporine protects the brain from ischemia-reperfusion (I/R) injury. Interestingly, cyclosporine appears to mediate both neurotoxicity (under normoxic conditions) and I/R protection across the same range of drug concentration. Both toxicity and protection might arise from the intersection of cyclosporine with mitochondrial energy metabolism. This review addresses basic biochemical mechanisms of: 1) cyclosporine toxicity in normoxic brain, and 2) its protective effects in the same organ during I/R. The marked and unparallel potential of magnetic resonance spectroscopy (MRS) as a novel quantitative approach to evaluate metabolic drug toxicity is described.     

Biochemical mechanisms of fluorine action

Biochemical mechanisms of fluorine ion action accounting for the biological role and significance of fluorine for vital activity of the organism are investigated. Results of these investigations are generalized. This trace element is shown to participate at least in two vitally important systems of the organism: the adenylate cyclase system which accounts for the cell response to neuroendocrinological information and the immune protection system providing antimicrobic resistance. Available data permit considering that cytotoxic fluorine action is based on the ability to hinder protein synthesis in eukaryotes and to stimulate peroxidation processes of biomembrane lipids. Inorganic fluorine compounds are recommended to be used with the treatment-and-prophylactic purpose for certain pathologic states, associated with its insufficient or excessive arrival into the organism.     

Biochemical mechanisms of memory storage

A series of studies on Hermissenda classical conditioning has lead to a discovery that the biophysical events (accumulation of Ca2+ and depolarization in B cell) found during memory acquisition are clearly distinct from those (suppression of K-currents, IA and ICa2+K+) detected in the retention phase of memory. Biochemical analysis of eyes isolated shortly after (a few hours) training revealed increased phosphorylation of a 20,000 M.W. protein which is very likely one of the substrates for both Ca/CaM-dependent protein kinase and C-kinase and possibly a locus of convergence for conditioned stimulus and unconditioned stimulus pathways. Furthermore, conditioning-specific changes in the two K+ currents have been reproduced by simultaneous activation of the CaM-kinase pathway (via iontophoretic injection of CaM-kinase II plus Ca2+-load or IP3 injection) and the C-kinase pathway (via bath application of phorbol-ester or diacylglycerol analog plus Ca2+-load). Therefore, synergistic interaction between the two Ca2+-dependent phosphorylation systems in the identified B cell is considered to be critically important for acquisition of associative memory. Evidence also has been obtained for similar biophysical changes and molecular mechanisms during retention of classical conditioning in the mammalian brain. Further work will be needed to uncover the biochemical mechanism(s) responsible for transforming short-term into long-lasting memory.     

Biochemical and molecular mechanisms regulating apoptosis

In eukaryotes, the regulation of tissue cell numbers is a critical homeostatic objective that is achieved through tight control of apoptosis, mitosis and differentiation. While much is known about the genetic regulation of cell growth and differentiation, the molecular basis of apoptosis is less well understood. Genes involved in both cell proliferation and apoptosis reflect the role of some stimuli in both of these processes, the cell response depending on the overall cellular milieu. Recent research has given fascinating insights into the complex genetic and molecular mechanisms regulating apoptosis. A picture is emerging of the initiation in certain cells, after an apoptotic trigger, of sequential gene expression and specific signal transduction cascades that guide cells along the cell death pathway. Changes in gene expression precede the better known biochemical and morphological changes of apoptosis. It seems possible that, as a result of increased understanding of the cellular events preceding cell death, apoptosis may become more amenable to manipulation by appropriate drug- and gene-based therapies.     

Modes and mechanisms of a Daphnia invasion

Whether exotic species invade new habitats successfully depends on (i) a change in the invaded habitat that makes it suitable for the invader and (ii) a genetic change in the invading taxon that enhances its fitness in the new habitat, or both. We dissect the causes of invasions of Swiss lakes, north of the Alps, by Daphnia galeata (a zooplankter typical of eutrophic lakes, e.g. those south of the Alps, which are also warmer) by comparing the fitness performance of eight geographically distributed clones that were fed algal-food typical of oligotrophic versus eutrophic conditions at two temperatures. Daphnia longispina, native to oligotrophic Swiss lakes, served as a reference. Daphnia galeata requires eutrophic food to persist, whereas D. longispina survives and grows on oligotrophic food but does even better on eutrophic food. Invasion by D. galeata is further explained because invading clones from the north perform better on eutrophic food and at cooler temperatures than native clones from the south, suggesting a local response to countergradient selection. Our data support the hypothesis that populations of the invader in northern lakes are dominated by well-adapted genotypes. Our results illustrate how environmental change (i.e. eutrophication) and local adaptation can act together to drive a successful invasion.     

Biochemical and cellular mechanisms of low-dose effects

Low-dose irradiation is usually considered to be rather ineffective in producing biologically relevant effects. Yet, individual radiation absorption events within cell nuclei or whole cells interact stochastically with subcellular structures due to the multiple ionizations along primary or secondary particle tracks, depending on ionization density. Whereas radiation effects are usually seen in the context of structure and function of DNA, other cellular effects, perhaps influencing DNA by secondary biochemical mechanisms, also warrant attention. Thus, previous work from this laboratory with bone marrow that was obtained from whole-body exposed mice, has shown that single or few instantaneous radiation absorption events per cell from gamma-rays produce an acute and temporary partial inhibition of the enzyme thymidine kinase; the effect appears within about 1 h after the event, reaches its maximum at approximately 4 h and disappears completely within another 6 h. This pattern of enzyme inhibition is fully concordant with the pattern of inhibition of uptake of tritiated thymidine or 125I-labelled deoxyuridine into the DNA; also concordant is a temporary increase in the concentration of free thymidine in the blood serum of the exposed mice. The particular response of thymidine kinase was considered to relate to some, thus far unknown, repair systems and/or to a defence mechanism of the hit cells. In order to further elucidate the role of the acute and temporary partial inhibition of thymidine kinase in cellular metabolism, experiments were carried out in which mice were acutely exposed to 0.01 or 0.1 Gy and again exposed to the same dose at different times up to 12 h after the first exposure. At regular time intervals after the second exposure bone marrow cells were obtained and thymidine kinase activity was studied by various assays. The results indicate that the first acute irradiation conditioned the cells in such a way that the second acute irradiation produced either an enhanced inhibition and recovery of thymidine kinase activity, or no effect at all was seen, when the second irradiation was given between about 3 and 8 h after the first irradiation. From 8 to 12 h after the first irradiation the cells apparently resumed their original state, so that the second irradiation produced effects quite similar to those seen after a single irradiation in unconditioned cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of different tumor escape mechanisms in several metastases from a melanoma patient undergoing immunotherapy

The cytotoxic activity of T cells selects the outgrowth of tumor cells that escape from immune surveillance by different strategies. The different mechanisms that interfere with immune recognition and limit vaccination efficiency are still poorly understood. We analysed six cell lines established from different metastases of melanoma patient UKRV-Mel-20 for specific characteristics known to have an impact on the tumor-T cell interaction: (1) alterations in the HLA class I phenotype, (2) expression of Fas/CD95, and (3) expression of specific cytokines and chemokines. One of the cell lines, UKRV-Mel-20f, exhibited an HLA class I haplotype loss and just this cell line was also characterised by the expression of Fas/CD95 and of relatively high levels of proinflammatory chemokines suggesting that the cytotoxic activity of tumor-infiltrating T cells might have selected the outgrowth of this tumor cell variant. All other cell lines analysed showed no alterations in HLA class I expression, but, in contrast to UKRV-Mel-20f, expressed much lower levels of Fas/CD95 and of proinflammatory chemokines and some of them produced high levels of immunosuppressive TGF-beta1. These results suggest that in patient UKRV-Mel-20, tumor cells interfere with T cell recognition by different strategies which might partially explain why this patient did not have a clinical response to an autologous tumor cell vaccine.     

Differential expressions of adhesive molecules and proteases define mechanisms of ovarian tumor cell matrix penetration/invasion

Epithelial ovarian cancer is an aggressive and deadly disease and understanding its invasion mechanisms is critical for its treatment. We sought to study the penetration/invasion of ovarian tumor cells into extracellular matrices (ECMs) using a fibroblast-derived three-dimensional (3D) culture model and time-lapse and confocal imaging. Twelve ovarian tumor cells were evaluated and classified into distinct groups based on their ECM remodeling phenotypes; those that degraded the ECM (represented by OVCAR5 cells) and those that did not (represented by OVCAR10 cells). Cells exhibiting a distinct ECM modifying behavior were also segregated by epithelial- or mesenchymal-like phenotypes and uPA or MMP-2/MMP-9 expression. The cells, which presented epithelial-like phenotypes, penetrated the ECM using proteases and maintained intact cell-cell interactions, while cells exhibiting mesenchymal phenotypes modified the matrices via Rho-associated serine/threonine kinase (ROCK) in the absence of apparent cell-cell interactions. Overall, this study demonstrates that different mechanisms of modifying matrices by ovarian tumor cells may reflect heterogeneity among tumors and emphasize the need to systematically assess these mechanisms to better design effective therapies.     

Sphingolipids in tumor metastases and angiogenesis

This review article summarizes data on the involvement of sphingolipids (sphingosine-1-phosphate, sphingosine-1-phosphocholine, neutral glycosphingolipids, and gangliosides) in tumor metastases and angiogenesis.