In breast cancer bone metastasis, tumor cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone stimulate tumor growth. The A v B 3 integrin is an adhesion receptor expressed by breast cancer cells and osteoclasts. It is implicated in tumor cell invasion and osteoclast-mediated bone resorption. Here, we hypothesized that the therapeutic targeting of tumor A v B 3 integrin would prevent bone metastasis formation. We first showed that, compared with mock-transfected cells, the i.v. inoculation of A v B 3 -overexpressing MDA-MB-231 breast cancer cells in animals increased bone metastasis incidence and promoted both skeletal tumor burden and bone destruction. The direct inoculation of A v B 3 -overexpressing transfectants into the tibial bone marrow cavity did not however enhance skeletal tumor burden and bone destruction, suggesting that A v B 3 controls earlier events during bone metastasis formation. We next examined whether a nonpeptide antagonist of A v B 3 (PSK1404) exhibits meaningful antitumor effects in experimental breast and ovarian cancer bone metastasis. A continuous PSK1404 treatment, which inhibited osteoclast-mediated bone resorption in an animal model of bone loss, substantially reduced bone destruction and decreased skeletal tumor burden. Importantly, a short-term PSK1404 treatment that did not inhibit osteoclast activity also decreased skeletal tumor burden and bone destruction. This dosing regimen caused a profound and specific inhibition of bone marrow colonization by green fluorescent protein, A v B 3 -expressing tumor cells in vivo and blocked tumor cell invasion in vitro. Overall, our data show that tumor A v B 3 integrin stands as a therapeutic target for the prevention of skeletal metastases. [Cancer Res 2007;67(12):5821-30]
Ezrin, a membrane cytoskeleton linker, is involved in cellular functions, including epithelial cell morphogenesis and adhesion. A mutant form of ezrin, ezrin T567D, maintains the protein in an open conformation, which when expressed in Madin-Darby canine kidney cells causes extensive formation of lamellipodia and altered cell-cell contacts at low cell density. Furthermore, these cells do not form tubules when grown in a collagen type I matrix. While measuring the activity of Rho family GTPases, we found that Rac1, but not RhoA or Cdc 42, is activated in ezrin T567D-expressing cells, compared with cells expressing wild-type ezrin. Together with Rac1 activation, we observed an accumulation of E-cadherin in intracellular compartments and a concomitant decrease in the level of E-cadherin present at the plasma membrane. This effect could be reversed with a dominant negative form of Rac1, N17Rac1. We show that after a calcium switch, the delivery of E-cadherin from an internalized pool to the plasma membrane is greatly delayed in ezrin T567D-producing cells. In confluent cells, ezrin T567D production decreases the rate of E-cadherin internalization. Our results identify a new role for ezrin in cell adhesion through the activation of the GTPase Rac1 and the trafficking of E-cadherin to the plasma membrane. INTRODUCTIONThe ezrin, radixin, and moesin (ERM) proteins act as linkers between the plasma membrane and the actin cytoskeleton. They have been implicated in the organization of specific domains of the plasma membrane, such as the apical domain of epithelial cells, the immunological synapse, or the formation of uropods Crepaldi et al., 1997;Serrador et al., 1997;Allenspach et al., 2001;Delon et al., 2001;Roumier et al., 2001). Their functions are regulated by changes in their conformation. ERM proteins exist in the cytoplasm as dormant monomers in which the F-actin cytoskeleton and the plasma membrane binding sites are masked. This closed conformation is due to an intramolecular N-to C-ERM association domain (ERMAD) interaction Gary and Bretscher, 1995). Thus, abrogation of the N/C-ERMAD interaction is required to "open up" the molecules and to expose their cryptic binding sites . Two factors have been implicated in the activation of ERM proteins. The binding to phosphatidylinositol 4,5-biphosphate is required for their interaction with actin in vitro and with membrane proteins in vivo (Nakamura et al., 1999;Barret et al., 2000). In addition, phosphorylation of a conserved threonine residue in the C-ERMAD, T567, inhibits the N/C-ERMAD interaction in vitro (Matsui et al., 1998;Nakamura et al., 1999) and in vivo, converts inactive oligomers to active monomers . Expression of an ezrin mutant, ezrin T567D, which mimics its phosphorylation, induces lamellipodia formation in nonconfluent cells and tufts of microvilli in confluent cells. Moreover, production of ezrin T567D perturbs the organization of epithelial cell monolayers (Yonemura et al., 1999;Gautreau et al., 2000).A number of studies have implicated ERM proteins in...
Acquisition of an invasive phenotype by cancer cells is a requirement for bone metastasis. Transformed epithelial cells can switch to a motile, mesenchymal phenotype by epithelial-mesenchymal transition (EMT). Recently, it has been shown that EMT is functionally linked to prostate cancer stem cells, which are not only critically involved in prostate cancer maintenance but also in bone metastasis. We showed that treatment with the non-peptide α(v)-integrin antagonist GLPG0187 dose-dependently increased the E-cadherin/vimentin ratio, rendering the cells a more epithelial, sessile phenotype. In addition, GLPG0187 dose-dependently diminished the size of the aldehyde dehydrogenase high subpopulation of prostate cancer cells, suggesting that α(v)-integrin plays an important role in maintaining the prostate cancer stem/progenitor pool. Our data show that GLPG0187 is a potent inhibitor of osteoclastic bone resorption and angiogenesis in vitro and in vivo. Real-time bioluminescent imaging in preclinical models of prostate cancer demonstrated that blocking α(v)-integrins by GLPG0187 markedly reduced their metastatic tumor growth according to preventive and curative protocols. Bone tumor burden was significantly lower in the preventive protocol. In addition, the number of bone metastases/mouse was significantly inhibited. In the curative protocol, the progression of bone metastases and the formation of new bone metastases during the treatment period was significantly inhibited. In conclusion, we demonstrate that targeting of integrins by GLPG0187 can inhibit the de novo formation and progression of bone metastases in prostate cancer by antitumor (including inhibition of EMT and the size of the prostate cancer stem cell population), antiresorptive, and antiangiogenic mechanisms.
We have previously described a 160-bp enhancer (BCE-1) in the bovine -casein gene that is activated in the presence of prolactin and extracellular matrix (ECM). Here we report the characterization of the enhancer by deletion and site-directed mutagenesis, electrophoretic mobility shift analysis, and in vivo footprinting. Two essential regions were identified by analysis of mutant constructions: one binds C/EBP- and the other binds MGF/STAT5 and an as-yet-unidentified binding protein. However, no qualitative or quantitative differences in the binding of these proteins were observed in electrophoretic mobility shift analysis using nuclear extracts derived from cells cultured in the presence or absence of ECM with or without prolactin, indicating that prolactin-and ECM-induced transcription was not dependent on the availability of these factors in the functional cell lines employed. An in vivo footprinting analysis of the factors bound to nuclear chromatin in the presence or absence of ECM and/or prolactin found no differences in the binding of C/EBP- but did not provide definitive results for the other factors. Neither ECM nor prolactin activated BCE-1 in transient transfections, suggesting that the chromosomal structure of the integrated template may be required for ECM-induced transcription. Further evidence is that treatment of cells with inhibitors of histone deacetylase was sufficient to induce transcription of integrated BCE-1 in the absence of ECM. Together, these results suggest that the ECM induces a complex interaction between the enhancer-bound transcription factors, the basal transcriptional machinery, and a chromosomally integrated template responsive to the acetylation state of the histones.It is now well established that the processes of development and differentiation depend on a cell's ability to correctly perceive its microenvironment (reviewed in references 1 and 43). A key component of this environment is the extracellular matrix (ECM). The ECM is an organized network of glycoproteins, proteoglycans, and glycosaminoglycans, components important for cell morphology as well as for signal transduction via cell surface integrins and ultimately for tissue-specific gene expression (reviewed in reference 43).The mammary gland appears to be particularly well suited for the study of ECM-induced differentiation and gene expression. In the adult animal, the gland develops after puberty and functionally differentiates in response to pregnancy. The mechanisms involved in these developmental processes are complex and guided by various hormones (54), growth factors (53), and the ECM (3). Milk protein expression is initiated at mid-pregnancy and correlates with the synthesis and deposition of a specialized laminin-rich ECM during alveolar development. Expression of these milk proteins can be used as markers for the differentiated state of the gland. In the last decade, a number of model systems using mammary epithelial cells to study ECM-dependent gene regulation have been developed. These range from primar...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.