FTY720, a potent immunomodulator, becomes phosphorylated in vivo (FTY-P) and interacts with sphingosine-1-phosphate (S1P) receptors. Recent studies showed that FTY-P affects vascular endothelial growth factor (VEGF)-induced vascular permeability, an important aspect of angiogenesis. We show here that FTY720 has antiangiogenic activity, potently abrogating VEGF-and S1P-induced angiogenesis in vivo in growth factor implant and corneal models. FTY720 administration tended to inhibit primary and significantly inhibited metastatic tumor growth in a mouse model of melanoma growth. In combination with a VEGFR tyrosine kinase inhibitor PTK787/ ZK222584, FTY720 showed some additional benefit. FTY720 markedly inhibited tumor-associated angiogenesis, and this was accompanied by decreased tumor cell proliferation and increased apoptosis. In transfected HEK293 cells, FTY-P internalized S1P 1 receptors, inhibited their recycling to the cell surface, and desensitized S1P receptor function. Both FTY720 and FTY-P apparently failed to impede VEGF-produced increases in mitogen-activated protein kinase activity in human umbilical vascular endothelial cells (HUVEC), and unlike its activity in causing S1PR internalization, FTY-P did not result in a decrease of surface VEGFR2 levels in HUVEC cells. Pretreatment with FTY720 or FTY-P prevented S1P-induced Ca 2+ mobilization and migration in vascular endothelial cells. These data show that functional antagonism of vascular S1P receptors by FTY720 potently inhibits angiogenesis; therefore, this may provide a novel therapeutic approach for pathologic conditions with dysregulated angiogenesis.

Collagen XVIII (c18) is a triple helical endothelial/epithelial basement membrane protein whose noncollagenous (NC)1 region trimerizes a COOH-terminal endostatin (ES) domain conserved in vertebrates, Caenorhabditis elegans and Drosophila. Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types. This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein. The motility-inducing and mitogen-activated protein kinase–stimulating activities of c18 NC1 were blocked by its physiologic cleavage product ES monomer, consistent with a proteolysis-dependent negative feedback mechanism. These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis.

The p210 bcr-abl protein tyrosine kinase (PTK) appears to be directly responsible for the initial manifestations of chronic myelogenous leukemia (CML). In contrast to the extensive characterization of the PTK and its effects on cell function, relatively little is known about the nature of the protein tyrosine phosphatases (PTPs) that may modulate p210 bcr-abl-induced signalling. In this study, we have demonstrated that expression of PTP1B is enhanced specifically in various cells expressing p210 bcr-abl, including a cell line derived from a patient with CML. This effect on expression of PTP1B required the kinase activity of p210 bcr-abl and occurred rapidly, concomitant with maximal activation of a temperature-sensitive mutant of the PTK. The effect is apparently specific for PTP1B since, among several PTPs tested, we detected no change in the levels of TCPTP, the closest relative of PTP1B. We have developed a strategy for identification of physiological substrates of individual PTPs which utilizes substrate-trapping mutant forms of the enzymes that retain the ability to bind to substrate but fail to catalyze efficient dephosphorylation. We have observed association between a substrate-trapping mutant of PTP1B (PTP1B-D181A) and p210 bcr-abl, but not v-Abl, in a cellular context. Consistent with the trapping data, we observed dephosphorylation of p210 bcr-abl, but not v-Abl, by PTP1B in vivo. We have demonstrated that PTP1B inhibited binding of the adapter protein Grb2 to p210 bcr-abl and suppressed p210 bcr-abl-induced transcriptional activation that is dependent on Ras. These results illustrate selectivity in the effects of PTPs in a cellular context and suggest that PTP1B may function as a specific, negative regulator of p210 bcr-abl signalling in vivo.

We investigated the significance of erythropoietin receptor (EPOR) expression following treatment with recombinant human erythropoietin (rHuEPO; epoetin A) and the effect of recombinant epoetins (epoetin A, epoetin B, and darbepoetin A) alone or in combination with anticancer therapy on tumor growth in two well-established preclinical models of breast carcinoma (MDA-MB-231 and MCF-7 cell lines). Expression and localization of EPOR under hypoxic and normoxic conditions in MDA-MB-231 and MCF-7 cells were evaluated by immunoblotting, flow cytometry, and immunohistochemistry. EPOR binding was evaluated using [125 I]rHuEPO. Proliferation, migration, and signaling in MDA-MB-231 and MCF-7 cells following treatment with rHuEPO were evaluated. Tumor growth was assessed following administration of recombinant epoetins alone and in combination with paclitaxel (anticancer therapy) in orthotopically implanted MDA-MB-231 and MCF-7 breast carcinoma xenograft models in athymic mice. EPOR expression was detected in both tumor cell lines. EPOR localization was found to be exclusively cytosolic and no specific [ 125 I]rHuEPO binding was observed. There was no stimulated migration, proliferation, or activation of mitogen-activated protein kinase and AKT following rHuEPO treatment. In mice, treatment with recombinant epoetins alone and in combination with paclitaxel resulted in equivalent tumor burdens compared with vehicle-treated controls. Results from our study suggest that although EPOR expression was observed in two well-established breast carcinoma cell lines, it was localized to a cytosolic distribution and did not transduce a signaling cascade in tumors that leads to tumor growth. The addition of recombinant epoetins to paclitaxel did not affect the outcome of paclitaxel therapy in breast carcinoma xenograft models. These results show that recombinant epoetins do not evoke a physiologic response on EPOR-bearing tumor cells as assessed by numerous variables, including growth, migration, and cytotoxic challenge in preclinical in vivo tumor models. [Mol Cancer Ther 2006;5(2):347 -55]

No abstract

The bcr-abl chimeric oncoprotein exhibits deregulated protein tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive human leukemias, such as chronic myelogenous leukemia (CML). Recently we have shown that the levels of the protein tyrosine phosphatase PTP1B are enhanced in p210 bcr-ablexpressing cell lines. Furthermore, PTP1B recognizes p210 bcr-abl as a substrate, disrupts the formation of a p210 bcr-abl͞Grb2 complex, and inhibits signaling events initiated by this oncoprotein PTK. In this report, we have examined whether PTP1B effects transformation induced by p210 bcrabl. We demonstrate that expression of either wild-type PTP1B or the substrate-trapping mutant form of the enzyme (PTP1B-D181A) in p210 bcr-abl-transformed Rat-1 fibroblasts diminished the ability of these cells to form colonies in soft agar, to grow in reduced serum, and to form tumors in nude mice. In contrast, TCPTP, the closest relative of PTP1B, did not effect p210 bcr-abl-induced transformation. Furthermore, neither PTP1B nor TCPTP inhibited transformation induced by v-Abl. In addition, overexpression of PTP1B or treatment with CGP57148, a small molecule inhibitor of p210 bcr-abl, induced erythroid differentiation of K562 cells, a CML cell line derived from a patient in blast crisis. These data suggest that PTP1B is a selective, endogenous inhibitor of p210 bcr-abl and is likely to be important in the pathogenesis of CML.

Background: Implantation of foreign materials into mice and humans has been noted to result in the appearance of soft tissue sarcomas at the site of implantation. These materials include metal replacement joints and Dacron vascular grafts. In addition, occupational exposure to nickel has been shown to result in an increased risk of carcinogenesis. The molecular mechanisms of foreign bodyinduced carcinogenesis are not fully understood. Materials and Methods: In order to gain insight into these mechanisms, we implanted nickel sulfide into wild type C57BL/6 mice as well as a mouse heterozygous for the tumor suppressor gene, p53.

Inhibition of endothelial cell growth by fumagillin has been assumed to be mediated by inhibition of the molecular target methionine aminopeptidase 2 (MetAp2). New data show that depletion of MetAp2 by siRNA does not inhibit endothelial cell growth. Moreover, MetAp2-depleted endothelial cells remain responsive to inhibition by either fumagillin or a newly identified MetAp2 enzyme inhibitor. These data suggest that MetAp2 function is not required for endothelial cell proliferation.