Block Tumor Metastasis Research Articles

Sema3E–Plexin D1 signaling drives human cancer cell invasiveness and metastatic spreading in mice

Semaphorin 3E (Sema3E) is a secreted molecule implicated in axonal path finding and inhibition of developmental and postischemic angiogenesis. Sema3E is also highly expressed in metastatic cancer cells, but its mechanistic role in tumor progression was not understood. Here we show that expression of Sema3E and its receptor Plexin D1 correlates with the metastatic progression of human tumors. Consistent with the clinical data, knocking down endogenous expression of either Sema3E or Plexin D1 in human metastatic carcinoma cells hampered their metastatic potential when injected into mice, while tumor growth was not markedly affected. Conversely, overexpression of exogenous Sema3E in cancer cells increased their invasiveness, transendothelial migration, and metastatic spreading, although it inhibited tumor vessel formation, resulting in reduced tumor growth in mice. The proinvasive and metastatic activity of Sema3E in tumor cells was dependent on transactivation of the Plexin D1-associated ErbB2/Neu oncogenic kinase. In sum, Sema3E-Plexin D1 signaling in cancer cells is crucially implicated in their metastatic behavior and may therefore be a promising target for strategies aimed at blocking tumor metastasis.

Abstract 3398: Double-faced Sema3E-PlexinD1 signaling inhibits tumor growth but promotes metastatic spreading

Abstract Semaphorins comprise a wide family of secreted and membrane-bound signals, originally found in neural development, and then implicated in a range of functions: from angiogenesis, to the immune response, to cancer progression. We have previously shown that high-affinity semaphorin receptors are found in the families of the plexins and of the neuropilins. Moreover, additional molecules interact with semaphorin receptors in cell-context dependent manner, featuring a complex scenario of multiple potential signaling pathways. The role of semaphorin signaling in tumor progression is currently under close scrutiny; it is known that cancer cells release semaphorins regulating their own behavior as well as that of cells in the tumor microenvironment, such as endothelial cells and recruited leukocytes. In this work, we focused on the function of Semaphorin 3E (Sema3E) and its high affinity receptor PlexinD1, and on their potential role to regulate cancer progression. Here we found that Sema3E and PlexinD1 are highly expressed in human metastatic tumors. Moreover, we provided experimental evidences that Sema3E released by tumor cells is a double-faced signal, acting via two distinctive signaling pathways. In paracrine manner, Sema3E-PlexinD1 signaling can elicit endothelial cell repulsion, thereby reducing vessel density and tumor growth. Conversely, Sema3E autocrine signaling in tumor cells thrusts their invasiveness, trans-endothelial migration, and metastatic spreading, through the trans-activation of PlexinD1-associated oncogenic kinases. Notably, by silencing the endogenous expression of either Sema3E or PlexinD1 the metastatic potential of cancer cells is significantly reduced, identifying this pathway as a major driver of the metastatic spreading. In sum, Sema3E autocrine signaling in cancer cells is crucially implicated to induce a metastatic behavior, and it appears as a promising target for strategies aimed at blocking tumor metastasis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3398.

MiR-218 Inhibits Invasion and Metastasis of Gastric Cancer by Targeting the Robo1 Receptor

MicroRNAs play key roles in tumor metastasis. Here, we describe the regulation and function of miR-218 in gastric cancer (GC) metastasis. miR-218 expression is decreased along with the expression of one of its host genes, Slit3 in metastatic GC. However, Robo1, one of several Slit receptors, is negatively regulated by miR-218, thus establishing a negative feedback loop. Decreased miR-218 levels eliminate Robo1 repression, which activates the Slit-Robo1 pathway through the interaction between Robo1 and Slit2, thus triggering tumor metastasis. The restoration of miR-218 suppresses Robo1 expression and inhibits tumor cell invasion and metastasis in vitro and in vivo. Taken together, our results describe a Slit-miR-218-Robo1 regulatory circuit whose disruption may contribute to GC metastasis. Targeting miR-218 may provide a strategy for blocking tumor metastasis.

Integrins in Bone Metastasis Formation and Potential Therapeutic Implications

Integrins constitute a family of cell surface receptors that are heterodimers composed of noncovalently associated alpha and beta subunits. Integrins bind to extracellular matrix proteins and immunogobulin superfamily molecules. They exert a stringent control on cell migration, survival and proliferation. However, their expression and functions are often deregulated in cancer, and many lines of evidence implicate them as key regulators during progression from primary tumor growth to metastasis. Here, we review the role of integrins in bone metastasis formation and present evidence that the use of integrin-targeted therapeutic agents may be an efficient strategy to block tumor metastasis.

RhoC is essential for angiogenesis induced by hepatocellular carcinoma cells via regulation of endothelial cell organization

The angiogenesis induced by tumor cells is essential for metastasis of hepatocellular carcinoma. Available information suggests that RhoC participates in angiogenesis through regulation of vascular endothelial growth factor expression in tumor cells. For its broad functions in cell migration and cytoskeletal organization, we hypothesized that RhoC regulating angiogenesis does not exclusively depend on regulation of vascular endothelial growth factor expression. To address this question, in the present study, we used a retroviral small interfering RNA approach to selectively knockdown the expression of RhoC in a neovascularization model in vivo and in vitro. Our present results indicate that RhoC is the downstream regulator of vascular endothelial growth factor in endothelial cells and is essential for angiogenesis induced by vascular endothelial growth factor, notwithstanding that RhoC regulates the expression of vascular endothelial growth factor in tumor cells. Furthermore, we show that knockdown of RhoC is associated with the inhibition of invasion and migration but not apoptosis of endothelial cells. Knockdown of RhoC results in inhibition of endothelial cell organization through restraining the reorganization of F-actin filaments, which represses endothelial cell network and sprout formation. In conclusion, our results demonstrate that knockdown of RhoC inhibits angiogenesis induced by tumor cells not only through effecting the release of vascular endothelial growth factor, but also through inhibiting endothelial cell migration and organization, which implies that it blocks tumor metastasis by specifically inhibiting RhoC in endothelial cells.

Polyphosphate blocks tumour metastasis via anti-angiogenic activity

PolyP (inorganic polyphosphate) is a linear polymer of many tens or hundreds of orthophosphate residues found in a wide range of organisms, including bacteria, fungi, insects, plants and vertebrates. Despite its wide distribution in mammalian tissues and plasma, the biological functions of polyP on tumour metastasis and angiogenesis have not been previously examined. In the present study, we have shown that polyP effectively blocked in vivo pulmonary metastasis of B16BL6 cells by suppression of neovascularization, whereas it did not affect proliferation or adhesion to extracellular matrix proteins. PolyP not only inhibited bFGF (basic fibroblast growth factor)-induced proliferation and ERK (extracellular-signal-regulated kinase)/p38 MAPK (mitogen-activated protein kinase) activation of human endothelial cells, but also blocked the binding of bFGF to its cognate cell-surface receptor. Furthermore, polyP inhibited bFGF-induced in vitro and in vivo angiogenesis, suggesting that polyP possesses an anti-angiogenic activity. Since neovascularization is essential for tumour metastasis, our present findings clearly indicate that polyP has an in vivo anti-metastatic activity via its anti-angiogenic activity. Taken together with the fact that angiogenesis occurs under various normal and pathological conditions, our observations suggest that endogenous polyP may play a critical role during embryonic development, wound healing and inflammation, as well as in the progress of pathological diseases such as rheumatoid arthritis and cancer.

Selective Tumor Targeting by the Hypoxia-Activated Prodrug AQ4N Blocks Tumor Growth and Metastasis in Preclinical Models of Pancreatic Cancer

The antitumor activities and pharmacokinetics of the hypoxia-activated cytotoxin AQ4N and its metabolites were assessed in several preclinical models of pancreatic cancers. The cytotoxic effects of AQ4N prodrug and its bioreduced form, AQ4, were tested against multiple human tumor cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Nude mice bearing s.c. or orthotopically implanted human BxPC-3 or Panc-1 tumor cells were treated with AQ4N. Tumor growth inhibition, time to progression/end point, and liver metastasis were evaluated in treatment versus control groups. Plasma and tumor levels of AQ4N and its metabolites were quantitated by liquid chromatography-tandem mass spectrometry. In contrast to AQ4N, the bioreduced AQ4 metabolite displayed potent cytotoxicity in many human tumor lines, including those derived from human pancreatic adenocarcinomas. Single-agent administration of AQ4N significantly delayed tumor growth, progression, and survival in a manner comparable with gemcitabine in multiple pancreatic tumor models in vivo. Survival increases were accompanied by a reduction in incidence and spread of liver metastasis. Quantitation of AQ4N and its metabolites in tumor-bearing mice showed that the prodrug is rapidly cleared from the circulation by 24 h and neither of the bioreduced metabolites was detected in plasma. In contrast, AQ4N readily penetrated BxPC-3 tumors and the cytotoxic AQ4 metabolite rapidly accumulated in tumor tissues at high levels in a dose-dependent fashion. AQ4N undergoes rapid and selective conversion into the potent antineoplastic metabolite AQ4 in tumors in vivo and provides proof of principle for the use of hypoxia-activated prodrugs in the treatment against pancreatic cancers.

Expression and Modulation of Carbohydrate-Binding Protein Galectin-3 in Multiple Myeloma Cells by Combined Treatment with GCS-100 and Dexamethasone.

Galectins belong to the family of mammalian lectins sharing an evolutionary conserved sequence in their carbohydrate recognition binding (CRD) domains and exhibit a characteristic affinity for beta-galactoside-sugars. Based on their structure and CRD, galectins have been categorized into three different categories: 1) Prototype (Gal-1, 2, 5, 7, 10, 11, 13, and 14), 2) Chimera type (Gal-3), and 3) Tandem repeat type (Gal-6, 8, 9, and 12). In particular, Galcetin-3 (Gal-3) is associated with growth, differentiation, adhesion, RNA processing, and apoptosis ( Apoptosis , 2005, 10: 267–75). Previous studies show that Gal-3 is expressed on tumor cell surface and mediate the transformation and metastasis of tumor cells in vivo ( Apoptosis , 2005, 10: 267–75). Here, we show that Gal-3 is differentially expressed in multiple myeloma (MM) cell lines and purified patient cells, as determined by both western blotting and polymerase chain reaction (PCR). Importantly, Gal-3 shares structural similarities with the anti-apoptotic protein Bcl-2: both proteins are rich in proline, glycine, and alanine amino acid residues in their NH-2-terminal domains and contain anti-death motif Asp-Trp-Gly-Arg (NWGR) in their C-terminal domains ( Proc Natl Acad Sci USA , 1996, 93: 6737–42). Gal-3, like Bcl-2, is therefore a potential therapeutic target. In this context, modified citrus pectins (MCP) have been reported to bind and inhibit Gal-3-induced human umbilical endothelial cell migration and micro vessel tube formation ( Am J Pathol. 2000, 156:899–909) as well as block tumor growth and metastasis (J Natl Cancer Inst. 2002, 94:1854–62). GCS-100 is a MCP currently under clinical development. Here we show that GCS-100 binds to human recombinant Gal-3. In MM cells, GCS-100 triggers apoptosis and enhances anti-tumor activity of the conventional agent Dexamethasone. We examined whether GCS-100-, Dex- or GCS-100 + Dex-induced MM cell death modulates Gal-3 or Bcl-2 expression. Treatment of MM.1S cells with either GCS-100 or Dex alone does not alter Gal-3 or Bcl-2 expression. However, combined treatment of MM cells with sub-toxic concentrations of GCS-100 and Dex markedly attenuates Gal-3 expression, without any changes in Bcl-2 levels, suggesting that alterations in Gal-3 are specific. Previous studies showed that Gal-3 inhibits mitochondrial pro-apoptotic protein cytochrome-c (cyto-c), and our results show that GCS-100 + Dex triggers the release of cyto-c from mitochondria to cytosol. Together, these data suggest that the combination of GCS-100 + Dex overcomes the inhibitory effect of Gal-3 on cyto-c, thereby inducing apoptosis. Since Gal-3 is widely expressed in MM cells, its inhibition may augment the anti-tumor activity of therapeutic regimens.

Phase I Trial of a Monoclonal Antibody Specific for αvβ3 Integrin (MEDI-522) in Patients with Advanced Malignancies, Including an Assessment of Effect on Tumor Perfusion

At present, a variety of agents targeting tumor angiogenesis are under clinical investigation as new therapies for patients with cancer. Overexpression of the alpha(v)beta(3) integrin on tumor vasculature has been associated with an aggressive phenotype of several solid tumor types. Murine models have shown that antibodies targeting the alpha(v)beta(3) integrin can affect tumor vasculature and block tumor formation and metastasis. These findings suggest that antibodies directed at alpha(v)beta(3) could be investigated in the treatment of human malignancies. The current phase I dose escalation study evaluated the safety of MEDI-522, a monoclonal antibody specific for the alpha(v)beta(3) integrin, in patients with advanced malignancies. Twenty-five patients with a variety of metastatic solid tumors were treated with MEDI-522 on a weekly basis with doses ranging from 2 to 10 mg/kg/wk. Adverse events were assessed weekly; pharmacokinetic studies were done; and radiographic staging was done every 8 weeks. In addition, dynamic computed tomography imaging was done at baseline and at 8 weeks in patients with suitable target lesions amenable to analysis, to potentially identify the effect of MEDI-522 on tumor perfusion. Treatment was well tolerated, and a maximum tolerated dose was not identified by traditional dose-limiting toxicities. The major adverse events observed were grade 1 and 2 infusion-related reactions (fever, rigors, flushing, injection site reactions, and tachycardia), low-grade constitutional and gastrointestinal symptoms (fatigue, myalgias, and nausea), and asymptomatic hypophosphatemia. Dynamic computed tomography imaging suggested a possible effect on tumor perfusion with an increase in contrast mean transit time from baseline to the 8-week evaluation with increasing doses of MEDI-522. No complete or partial responses were observed. Three patients with metastatic renal cell cancer experienced prolonged stable disease (34 weeks, >1 and >2 years) on treatment. With this weekly schedule of administration, and in the doses studied, MEDI-522 seems to be without significant toxicity, may have effects on tumor perfusion, and may have clinical activity in renal cell cancer. These findings suggest the MEDI-522 could be further investigated as an antiangiogenic agent for the treatment of cancer.

Angiostatin directly inhibits human prostate tumor cell invasion by blocking plasminogen binding to its cellular receptor, CD26

Previous studies demonstrate that one of the six plasminogen type 2 glycoforms, plasminogen 2ɛ, enhances invasiveness of the 1-LN human prostate tumor cell line in an in vitro model. Binding of plasminogen 2ɛ to CD26 on the cell surface induces a Ca 2+ signaling cascade which stimulates the expression of matrix metalloproteinase-9, required by these cells to invade Matrigel®. We now report that angiostatin, a fragment derived from plasminogen which prevents endothelial cell proliferation, is also a potent, direct inhibitor of 1-LN tumor cell invasiveness. We studied the effect of individual plasminogen 2 glycoform-derived angiostatins and found that only angiostatin 2ɛ binds to CD26 on the surface of 1-LN cells at a site also recognized by plasminogen 2ɛ. As a result, the plasminogen 2ɛ-induced Ca 2+ signaling cascade is inhibited, the expression of matrix metalloproteinase-9 is suppressed, and invasion of Matrigel® by 1-LN cells is blocked. Angiostatin 2ɛ is also the only angiostatin glycoform which is able to inhibit in vitro endothelial cell proliferation and tubule formation. These studies suggest that, in addition to its ability to inhibit tumor vascularization, angiostatin 2ɛ may also directly block tumor metastasis.

Anti-idiotype x anti-CD44 bispecific antibodies inhibit invasion of lymphoid organs by B cell lymphoma.

The demonstration that Abs to adhesion molecules can block tumor metastasis suggested their use for therapy. However, such Abs affect nonmalignant cells as well. To circumvent this adverse effect, we proposed the use of bispecific Abs that bind simultaneously to an adhesion receptor and to a tumor-specific Ag. Such bifunctional Abs bind more avidly to tumor cells that coexpress both target Ags than to normal cells. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. Therefore, we produced bispecific Abs with specificity to the adhesion molecule, CD44, and to an idiotypic determinant of the murine B cell lymphoma, 38C-13. These anti-Id x anti-CD44 bispecific Abs blocked 38C-13 cell adhesion to hyaluronic acid, while not affecting adhesion of Id-negative cells. In vivo studies demonstrated that the bispecific Abs inhibited lymphoma cell dissemination to the lymph nodes, bone marrow, and spleen, and prolonged survival of tumor-bearing mice. Migration of 38C-13 cells to the lymphoid organs was inhibited by the bispecific Abs. Thus, the bispecific Ab-mediated reduction in metastasis resulted, at least in part, from reduced homing to these organs. In contrast to anti-CD44 monospecific Abs, the anti-Id x anti-CD44 bispecific Abs did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and tumor-specific Ags may selectively block tumor metastasis in a way which may leave at least part of the immune system intact.

Anti-idiotype x anti-LFA-1 bispecific antibodies inhibit metastasis of B cell lymphoma.

Abs to adhesion molecules can block tumor metastasis. However, they may also block the function of normal cells. To circumvent this adverse effect, we proposed the use of bispecific Abs that bind simultaneously to an adhesion receptor and to a tumor-specific Ag. Such Abs bind more avidly to tumor cells that coexpress both target Ags than to normal cells. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. We therefore produced a bispecific Ab with specificity to the adhesion molecule LFA-1 and to the Id of the murine B cell lymphoma 38C-13. Here we demonstrate that this Ab blocked liver metastasis in mice carrying primary s.c. tumors and partially inhibited lymph node metastasis. Migration of 38C-13 cells to liver and lymph nodes was inhibited by the bispecific Ab, while migration to spleen was not affected. Hence, the bispecific Ab-mediated reduction in liver and lymph node metastasis resulted at least in part from reduced homing to these organs. In contrast to anti-LFA-1 monospecific Abs, the anti-Id x anti-LFA-1 bispecific Ab did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and against tumor-specific Ags may selectively block tumor metastasis in a way that may leave much of the immune system intact.

Calagualine inhibits nuclear transcription factors-κB activated by various inflammatory and tumor promoting agents

Calagualine derived from the fern of the genus Polypodium, commonly called calaguala, has had clinically documented medicinal uses in South America and Spain and been shown to block tumor metastasis, proliferation, and inflammation, all known to require the activation of nuclear transcription factor-κB (NF-κB). Therefore, we investigated the effect of calagualine on NF-κB activation induced by various inflammatory and tumor promoting agents. Calagualine blocked tumor necrosis factor (TNF)-induced activation of NF-κB through inhibition of phosphorylation and degradation of IκBα, an inhibitor of NF-κB. The effects of calagualine were not cell type-specific, as it blocked TNF-induced NF-κB activation in a variety of cells. NF-κB-dependent reporter gene transcription activated by TNF was also suppressed by calagualine. The TNF-induced NF-κB activation cascade involving TNFR1-TNF receptor-associated death domain-TNF receptor-associated factor 2 (TRAF2)-NF-κB-inducing kinase (NIK)-IκBα kinase was interrupted at the TRAF2 and NIK sites by calagualine, which would account for its suppression of NF-κB reporter gene expression. Calagualine blocked NF-κB activation induced by phorbol ester and lipopolysaccharide. Overall our results indicate that calagualine inhibits activation of NF-κB and this may provide a molecular basis for calagualine's ability to suppress inflammation and tumorigenesis.

Idiotype-specific inhibition of LFA-1-mediated cell adhesion by anti-idiotype×anti-LFA-1 bispecific antibodies

Adhesion molecules are involved in lymphoma dissemination. Antibodies to adhesion molecules may block tumor metastasis. However, such antibody treatment may block as well normal functions of the immune system. We tested the hypothesis that a bispecific antibody with specificity for an adhesion molecule and for a tumor specific antigen binds preferentially to tumor cells which coexpress both antigens and hence selectively blocks adhesion. A bispecific antibody was developed by somatic cell hybridization of two hybridomas, one producing a monoclonal antibody against the immunoglobulin idiotypic determinant of the murine B cell lymphoma 38C-13 and the other producing an antibody against the α subunit (CD11a) of the adhesion molecule LFA-1. The bispecific antibody, anti-idiotype×anti-LFA-1, was purified by affinity chromatography. The dual specificity of the hybrid hybridoma product was demonstrated by a radioimmunoassay devised for detection of bifunctional activity. The bispecific antibody was shown by flow cytometry to bind efficiently to 38C-13 cells that coexpress idiotype and LFA-1. It bound only weakly to idiotype-negative variants of 38C-13 that express only LFA-1. In binding assays to immobilized ICAM-1, the anti-idiotype×anti-LFA-1 was highly active in blocking 38C-13 cell adhesion. However, it did not effect adhesion of idiotype-negative tumor cells or of normal T lymphocytes. In summary, the bispecific antibody preferentially blocks adhesion of cells that coexpress the tumor specific antigen and the adhesion receptor. The present approach may provide a general way for the selective adhesion blockade of a specific cell population.