Could inhibition of metalloproteinases be used to block the process of metastasis?

Metastasis is a multisequential process that allows tumor cells to migrate to tissues distant from the primary tumor. Only a small number of cells escape from the primary tumor; however, the metastases generated are responsible for more than 90% of cancer deaths. Many metastatic processes initially require the total or partial start-up of a program for the transformation of tumor epithelial cells into mesenchymal cells (EMT). The launching of the EMT program is stimulated by cytokines and other elements produced by the diverse types of cells composing the tumor stroma. In parallel, a process of destabilization of the extracellular matrix (ECM) takes place by means of the synthesis of proteases of the matrix metalloproteinases (MMPs) family. EMC degradation allows the exportation of some tumor cells as mesenchymal cells to the circulatory system and their subsequent implantation in a tissue distant from the primary tumor. The blocking of these both processes appears as a hypothetical stop point in the metastatic mechanism. The present review deals with the different options to achieve the inhibition of MMPs, focusing on MMP7 as a target given its involvement in the metastatic processes of a wide variety of tumors.

C3G transgenic mouse models with specific expression in platelets reveal a new role for C3G in platelet clotting through its GEF activity.

We have generated mouse transgenic lineages for C3G (tgC3G) and C3GΔCat (tgC3GΔCat, C3G mutant lacking the GEF domain), where the transgenes are expressed under the control of the megakaryocyte and platelet specific PF4 (platelet factor 4) gene promoter. Transgenic platelet activity has been analyzed through in vivo and in vitro approaches, including bleeding time, aggregation assays and flow cytometry. Both transgenes are expressed (RNA and protein) in purified platelets and megakaryocytes and do not modify the number of platelets in peripheral blood. Transgenic C3G animals showed bleeding times significantly shorter than control animals, while tgC3GΔCat mice presented a remarkable bleeding diathesis as compared to their control siblings. Accordingly, platelets from tgC3G mice showed stronger activation in response to platelet agonists such as thrombin, PMA, ADP or collagen than control platelets, while those from tgC3GΔCat animals had a lower response. In addition, we present data indicating that C3G is a mediator in the PKC pathway leading to Rap1 activation. Remarkably, a significant percentage of tgC3G mice presented a higher level of neutrophils than their control siblings. These results indicate that C3G plays an important role in platelet clotting through a mechanism involving its GEF activity and suggest that it might be also involved in neutrophil development.

C3G forms complexes with Bcr-Abl and p38α MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion.

BACKGROUND: Previous studies by our group and others have shown that C3G interacts with Bcr-Abl through its SH3-b domain. RESULTS: In this work we show that C3G and Bcr-Abl form complexes with the focal adhesion (FA) proteins CrkL, p130Cas, Cbl and Abi1 through SH3/SH3-b interactions. The association between C3G and Bcr-Abl decreased upon Abi1 or p130Cas knock-down in K562 cells, which suggests that Abi1 and p130Cas are essential partners in this interaction. On the other hand, C3G, Abi1 or Cbl knock-down impaired adhesion to fibronectin, while p130Cas silencing enhanced it. C3G, Cbl and p130Cas-SH3-b domains interact directly with common proteins involved in the regulation of cell adhesion and migration. Immunoprecipitation and immunofluorescence studies revealed that C3G form complexes with the FA proteins paxillin and FAK and their phosphorylated forms. Additionally, C3G, Abi1, Cbl and p130Cas regulate the expression and phosphorylation of paxillin and FAK. p38α MAPK also participates in the regulation of adhesion in chronic myeloid leukemia cells. It interacts with C3G, CrkL, FAK and paxillin and regulates the expression of paxillin, CrkL and α5 integrin, as well as paxillin phosphorylation. Moreover, double knock-down of C3G/p38α decreased adhesion to fibronectin, similarly to the single silencing of one of these genes, either C3G or p38α. These suggest that C3G and p38α MAPK are acting through a common pathway to regulate cell adhesion in K562 cells, as previously described for the regulation of apoptosis. CONCLUSIONS: Our results indicate that C3G-p38αMAPK pathway regulates K562 cell adhesion through the interaction with FA proteins and Bcr-Abl, modulating the formation of different protein complexes at FA.

African swine fever virus protein p17 is essential for the progression of viral membrane precursors toward icosahedral intermediates.

The first morphological evidence of African swine fever virus (ASFV) assembly is the appearance of precursor viral membranes, thought to derive from the endoplasmic reticulum, within the assembly sites. We have shown previously that protein p54, a viral structural integral membrane protein, is essential for the generation of the viral precursor membranes. In this report, we study the role of protein p17, an abundant transmembrane protein localized at the viral internal envelope, in these processes. Using an inducible virus for this protein, we show that p17 is essential for virus viability and that its repression blocks the proteolytic processing of polyproteins pp220 and pp62. Electron microscopy analyses demonstrate that when the infection occurs under restrictive conditions, viral morphogenesis is blocked at an early stage, immediately posterior to the formation of the viral precursor membranes, indicating that protein p17 is required to allow their progression toward icosahedral particles. Thus, the absence of this protein leads to an accumulation of these precursors and to the delocalization of the major components of the capsid and core shell domains. The study of ultrathin serial sections from cells infected with BA71V or the inducible virus under permissive conditions revealed the presence of large helicoidal structures from which immature particles are produced, suggesting that these helicoidal structures represent a previously undetected viral intermediate.

C3G silencing enhances STI-571-induced apoptosis in CML cells through p38 MAPK activation, but it antagonizes STI-571 inhibitory effect on survival.

In this work we report evidences of a functional relationship between C3G and p38 MAPK in the apoptotic effect of STI-571 on the chronic myeloid leukemia (CML) cell line K562. This has been demonstrated by knocking down C3G and p38alpha using the interfering RNA approach, as well as through targeting p38 by its inhibitor SB203580. The results indicate that p38 is a mediator of the STI-571-induced apoptosis, while C3G plays a negative role on STI-571-mediated p38 activation through a Rap1-dependent mechanism. According to this, gene expression analysis in C3G silenced cells revealed an upregulation of a large number of genes involved in apoptosis. Some of these genes are also down-regulated (at the protein level) upon p38alpha knock-down, which further suggests a functional association between these two proteins. On the other hand, C3G knock-down reverts the STI-571-inhibitory effect on ERKs and Akt pathways in a Rap1-independent fashion. Moreover, C3G overexpression also increased both, basal and STI-571-induced apoptosis, in agreement with previous reports. Therefore, our results strongly suggest a dual regulatory role for C3G in CML cells, modulating both apoptosis and survival via Rap-dependent and independent mechanisms.

Characterization of p87C3G, a novel, truncated C3G isoform that is overexpressed in chronic myeloid leukemia and interacts with Bcr-Abl.

A novel C3G isoform, designated p87C3G, lacking the most amino terminal region of the cognate protein has been found to be overexpressed in two CML cell lines, K562 and Boff 210, both expressing Bcr-Abl p210. p87C3G expression is also highly augmented in patients diagnosed with chronic myeloid leukemia (CML) Ph+, in comparison with healthy individuals, and returns to basal levels after treatment with STI571. p87C3G co-immunoprecipitates with both CrkL and Bcr-Abl in CML cell lines and co-immunoprecipitation between p87C3G and Bcr-Abl was also detected in primary cells from CML patients. These interactions have been confirmed by in vitro pull down experiments. The interaction between p87C3G and Bcr-Abl involves the SH3-binding domain of p87C3G and the SH3 domain of Abl and depends mostly on the first polyproline region of p87C3G. Furthermore, we also demonstrated that p87C3G is phosphorylated in vitro by a Bcr-Abl-dependent mechanism. These results indicate that p87C3G overexpression is linked to CML phenotype and that p87C3G may exert productive functional interactions with Bcr-Abl signaling components suggesting the implication of this C3G isoform in the pathogenesis of chronic myeloid leukemia.