[Show abstract][Hide abstract] ABSTRACT: Gelatinase B/ matrix metalloproteinase-9 (MMP-9) (EC 18.104.22.168) cleaves many substrates and is produced by most cell types as a zymogen, proMMP-9, in complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1). Natural proMMP-9 occurs as monomers, homomultimers, and heterocomplexes, but our knowledge about the overall structure of proMMP-9 monomers and multimers is limited. We investigated biochemical, biophysical, and functional characteristics of zymogen and activated forms of MMP-9 monomers and multimers. In contrast to a conventional notion of a dimeric nature of MMP-9 homomultimers, we demonstrate that these are reduction-sensitive trimers. Based on the information from electrophoresis, atomic force microscopy (AFM) and transmission electron microscopy (TEM), we generated a 3D structure model of the proMMP-9 trimer. Remarkably, the proMMP-9 trimers possessed a 50-fold higher affinity for TIMP-1 than the monomers. In vivo, this finding was reflected in a higher extent of TIMP-1 inhibition of angiogenesis induced by trimers versus monomers. Our results show that proMMP-9 trimers constitute a novel structural and functional entity that is differentially regulated by TIMP-1.
[Show abstract][Hide abstract] ABSTRACT: According to established notion, one of the major angiogenesis-inducing factors, pro-matrix metalloproteinase-9 (proMMP-9), is supplied to the tumor microenvironment by tumor-associated macrophages (TAMs). Accumulated evidence, however, indicates that tumor-associated neutrophils (TANs) are also critically important for proMMP-9 delivery, especially at early stages of tumor development. To clarify how much angiogenic proMMP-9 is actually contributed by TAMs and TANs, we quantitatively evaluated TAMs and TANs from different tumor types, including human xenografts and syngeneic murine tumors grown in wild-type and Mmp9-knockout mice. Whereas host MMP-9 competence was required for full angiogenic potential of both normal and tumor-associated leukocytes, direct comparisons of neutrophils versus macrophages and TANs versus TAMs demonstrated that macrophages and TAMs secrete 40- to 50-fold less proMMP-9 than the same numbers of neutrophils or TANs. Correspondingly, the levels of MMP-9–mediated in vivo angiogenesis induced by neutrophils and TANs substantially exceeded those induced by macrophages and TAMs. MMP-9–delivering TANs were also required for development of metastasis-supporting intratumoral vasculature, characterized by ≥ 11-μm size lumens and partial coverage with stabilizing pericytes. Importantly, MMP-9–producing TAMs exhibit M2-skewed phenotype but do not express tissue inhibitor of metalloproteinases-1 (TIMP-1), a novel characteristic allowing them to secrete TIMP-1–free, neutrophil-like MMP-9 zymogen unencumbered by its natural inhibitor. Together, our findings support the notion whereby TANs, capable of immediate release of their pre-stored cargo, are the major contributors of highly angiogenic MMP-9, whereas tumor-influxing precursors of macrophages require time to differentiate, polarize into M2-skewed TAMs, shut down their TIMP-1 expression, and only then, initiate relatively low-level production of TIMP-free MMP-9 zymogen.
Neoplasia (New York, N.Y.) 10/2014; 16(10):771–788. · 5.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A proangiogenic function of macrophages has long been attributed to their matrix metalloproteinase-9 zymogen (proMMP-9). Although tissue-infiltrating macrophages of M2-phenotype are regarded as proangiogenic, it has not been proved that angiogenesis-inducing proMMP-9 is actually supplied by M2-macrophages. We evaluated angiogenic capacities of human monocytes, mature M0-macrophages, and polarized M1- and M2-macrophages. Only M2-macrophages induced angiogenesis in vivo at levels comparable with highly-angiogenic neutrophils previously shown to release a unique, TIMP-1-free proMMP-9. Macrophage differentiation was accompanied by induction of low-angiogenic, TIMP-1-encumbered proMMP-9. However, polarization towards M2-, but not M1-phenotype, caused a substantial downregulation of TIMP-1 expression, resulting in production of angiogenic, TIMP-deficient proMMP-9. Correspondingly, the angiogenic potency of M2-proMMP-9 was lost after its complexing with TIMP-1, whereas siRNA-downregulation of TIMP-1 in M0- and M1-macrophages rendered them angiogenic. Similar to human cells, murine bone marrow-derived M2-macrophages also shutdown TIMP-1 secretion and produced proMMP-9 unencumbered by TIMP-1. Providing proof-of-principle, the angiogenic capacity of murine M2-macrophages depended on their TIMP-free proMMP-9 since M2-macrophages generated from Mmp9-null mice were non-angiogenic although their TIMP-1 was severely downregulated. Our study provides a unifying molecular mechanism for high angiogenic capacity of TIMP-unencumbered proMMP-9 that would be uniquely produced in a pathophysiological microenvironment by influxing neutrophils and/or M2-polarized macrophages.
[Show abstract][Hide abstract] ABSTRACT: Intravasation, the active entry of primary tumor cells into the vasculature, remains the least studied step in the metastatic cascade. Protease-mediated escape and stromal invasion of tumor cells represent widely-accepted processes leading up to the intravasation step. However, molecular factors that contribute directly to tumor cell vascular penetration have not been identified. In this study, the in vivo role of the collagenolytic protease, MMP-1, in cancer cell intravasation and metastasis was analyzed by employing a highly-disseminating variant of human HEp3 epidermoid carcinoma, HEp3-hi/diss. Whereas naturally-acquired or experimentally-induced MMP-1 deficiency substantially suppressed HEp3-hi/diss intravasation, supplementation of recombinant MMP-1 to MMP-1-silenced primary tumors, restored their impaired vascular dissemination. Surprisingly, abrogation of MMP-1 production and activity did not affect significantly HEp3-hi/diss migration or matrix invasion, suggesting non-collagenolytic mechanisms underlying MMP-1-dependent cell intravasation. In support of such non-collagenolytic mechanisms, MMP-1 silencing in HEp3-hi/diss cells modulated the microarchitecture and integrity of the angiogenic vasculature in a novel microtumor model. Concomitantly, MMP-1 deficiency led to decreased levels of intratumoral vascular permeability, tumor cell intravasation and metastatic dissemination. Taking advantage of PAR1 deficiency of HEp3-hi/diss cells, we further demonstrate that endothelial PAR1 is a putative non-tumor-cell/non-matrix target, activation of which by carcinoma-produced MMP-1 regulates endothelial permeability and transendothelial migration. The inhibitory effects of specific PAR1 antagonists in live animals have also indicated that the mechanisms of MMP-1-dependent vascular permeability in tumors involve endothelial PAR1 activation. Together, our findings mechanistically underscore the contribution of a tumor MMP-1/endothelial PAR1 axis to actual intravasation events manifested by aggressive carcinoma cells.
[Show abstract][Hide abstract] ABSTRACT: Specific cleavage of the transmembrane molecule, CUB domain-containing protein-1 (CDCP1), by plasmin-like serine proteases induces outside-in signal transduction that facilitates early stages of spontaneous metastasis leading to tumor cell intravasation, namely cell escape from the primary tumor, stromal invasion and transendothelial migration. We identified active β1 integrin as a biochemical and functional partner of the membrane-retained 70-kDa CDCP1 fragment, newly generated from its full-length 135-kDa precursor though proteolytic cleavage by serine proteases. Both in cell cultures and in live animals, active β1 integrin complexed preferentially with functionally activated, phosphorylated 70-kDa CDCP1. Complexing of β1 integrin the 70-kDa with CDCP1 fragment induced intracellular phosphorylation signaling, involving focal adhesion kinase-1 (FAK) and PI3 kinase (PI3K)-dependent Akt activation. Thus, inhibition of FAK/PI3K activities by specific inhibitors as well as short-hairpin RNA downregulation of β1 integrin significantly reduced FAK/Akt phosphorylation under conditions where CDCP1 was processed by serine proteases, indicating that FAK/PI3K/Akt pathway operates downstream of cleaved CDCP1 complexed with β1 integrin. Furthermore, this complex-dependent signaling correlated positively with high levels of tumor cell intravasation and dissemination. Correspondingly, abrogation in vivo of CDCP1 cleavage either by unique cleavage-blocking monoclonal antibody 10-D7 or by inhibition of proteolytic activity of plasmin-like serine proteases with aprotinin prevented β1 integrin/CDCP1 complexing and downstream FAK/Akt signaling concomitant with significant reduction of stromal invasion and spontaneous metastasis. Therefore, β1 integrin appears to serve as a motility-regulating partner mediating cross-talk between proteolytically cleaved, membrane-retained CDCP1 and members of FAK/PI3K/Akt pathway. This CDCP1 cleavage-induced signaling cascade constitutes a unique mechanism, independent of extracellular matrix remodeling, whereby a proteolytically cleaved CDCP1 regulates in vivo locomotion and metastasis of tumor cells through β1 integrin partnering. Our findings indicate that CDCP1 cleavage, occurring at the apex of a β1 integrin/FAK/PI3K/Akt signaling cascade, may represent a therapeutic target for CDCP1-positive cancers.Oncogene advance online publication, 3 December 2012; doi:10.1038/onc.2012.547.
[Show abstract][Hide abstract] ABSTRACT: Plasmin, one of the most potent and reactive serine proteases, is involved in various physiological processes, including embryo development, thrombolysis, wound healing and cancer progression. The proteolytic activity of plasmin is tightly regulated through activation of its precursor, plasminogen, only at specific times and in defined locales as well as through inhibition of active plasmin by its abundant natural inhibitors. By exploiting the plasminogen activating system and overexpressing distinct components of the plasminogen activation cascade, such as pro-uPA, uPAR and plasminogen receptors, malignant cells can enhance the generation of plasmin which in turn, modifies the tumor microenvironment to sustain cancer progression. While plasmin-mediated degradation and modification of extracellular matrix proteins, release of growth factors and cytokines from the stroma as well as activation of several matrix metalloproteinase zymogens, all have been a focus of cancer research studies for decades, the ability of plasmin to cleave transmembrane molecules and thereby to generate functionally important cleaved products which induce outside-in signal transduction, has just begun to receive sufficient attention. Herein, we highlight this relatively understudied, but important function of the plasmin enzyme as it is generated de novo at the interface between cross-talking cancer and host cells.
BioMed Research International 10/2012; 2012:564259. · 2.71 Impact Factor
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[Show abstract][Hide abstract] ABSTRACT: Data accumulated over the latest two decades have established that the serine protease urokinase-type plasminogen activator (uPA) is a potential therapeutic target in cancer. When designing inhibitors of the proteolytic activity of serine proteases, obtaining sufficient specificity is problematic since the topology of the proteases' active sites are highly similar. In an effort to generate highly specific uPA inhibitors with new inhibitory modalities, we isolated uPA-binding RNA aptamers by screening a library of 35 nucleotides long 2'-fluoro-pyrimidine RNA molecules using as bait a version of human pro-uPA lacking the epidermal growth factor-like and kringle domains. One pro-uPA binding aptamer sequence, referred to as upanap-126, proved to be highly specific for human uPA. Upanap-126 delayed the proteolytic conversion of human pro-uPA to active uPA, but did not inhibit plasminogen activation catalysed by two-chain uPA. The aptamer also inhibited the binding of pro-uPA to uPAR and the binding of vitronectin to the preformed pro-uPA/uPAR complexes both in cell-free systems and on cell surfaces. Furthermore, upanap-126 inhibited human tumour cell invasion in vitro, in the Matrigel assay, and in vivo, in the chick embryo assay of cell escape from microtumours. Finally, upanap-126 significantly reduced the levels of tumour cell intravasation and dissemination in the chick embryo model of spontaneous metastasis. Together, our findings demonstrate that utilisation of upanap-126 represents a novel multi-functional mechanistic modality for inhibition of uPA-dependent processes involved in tumour cell spread.
Molecular Cancer Research 10/2012; · 4.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: After neoplastic cells leave the primary tumor and circulate, they may extravasate from the vasculature and colonize tissues to form metastases. β1 integrins play diverse roles in tumorigenesis and tumor progression, including extravasation. In blood cells, activation of β1 integrins can be regulated by "inside-out" signals leading to extravasation from the circulation into tissues. However, a role for inside-out β1 activation in tumor cell metastasis is uncertain. Here we show that β1 integrin activation promotes tumor metastasis and that activated β1 integrin may serve as a biomarker of metastatic human melanoma. To determine whether β1 integrin activation can influence tumor cell metastasis, the β1 integrin subunit in melanoma and breast cancer cell lines was stably knocked down with shRNA and replaced with wild-type or constitutively-active β1. When tumor cells expressing constitutively-active β1 integrins were injected intravenously into chick embryos or mice, they demonstrated increased colonization of the liver when compared to cells expressing wild-type β1 integrins. Rescue expression with mutant β1 integrins revealed that tumor cell extravasation and hepatic colonization required extracellular ligand binding to β1 as well as β1 interaction with talin, an intracellular mediator of integrin activation by the Rap1 GTPase. Furthermore, shRNA-mediated knock down of talin reduced hepatic colonization by tumor cells expressing wild-type β1, but not constitutively-active β1. Overexpression in tumor cells of the tumor suppressor, Rap1GAP, inhibited Rap1 and β1 integrin activation as well as hepatic colonization. Using an antibody that detects activated β1 integrin, we found higher levels of activated β1 integrins in human metastatic melanomas compared to primary melanomas, suggesting that activated β1 integrin may serve as a biomarker of invasive tumor cells. Altogether, these studies establish that inside-out activation of β1 integrins promotes tumor cell extravasation and colonization, suggesting diagnostic and therapeutic approaches for targeting of β1 integrin signaling in neoplasia.
PLoS ONE 10/2012; 7(10):e46576. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The CUB domain-containing protein-1 (CDCP1) is a transmembrane molecule that has recently been implicated in cancer progression. In this study we have established a novel mechanism for initiation of CDCP1-mediated signaling in vivo and demonstrated that specific 135→70-kDa processing of cell-surface CDCP1 by extracellular serine proteases is a prerequisite for CDCP1-dependent survival of cancer cells during metastasis. The in vivo cleavage of CDCP1 triggers a survival program involving recruitment of Src and PKCδ, Src-mediated phosphorylation of cell-surface-retained 70-kDa CDCP1, activation of Akt and suppression of PARP1-induced apoptosis. We demonstrate in vivo that phosphorylated Src, PKCδ and Akt all constitute activated elements of a CDCP1-signaling axis during tissue colonization of tumor cells. Preventing in vivo cleavage of CDCP1 with unique anti-CDCP1 antibodies, serine protease inhibitors or genetic modulation of the cleavage site in the CDCP1 molecule completely abrogated survival signaling associated with the 70-kDa CDCP1, and induced PARP1 cleavage and PARP1-mediated apoptosis, ultimately resulting in substantial inhibition of tissue colonization by tumor cells. The lack of CDCP1 cleavage in the lung tissue of plasminogen-knockout mice along with a coordinated reduction in tumor cell survival in a lung retention model, and importantly rescue of both by in vivo supplied plasmin, indicated that plasmin is the crucial serine protease executing in vivo cleavage of cell-surface CDCP1 during early stages of lung colonization. Together, our findings indicate that in vivo blocking of CDCP1 cleavage upstream from CDCP1-induced pro-survival signaling provides a potential mechanism for therapeutic intervention into metastatic disease.
[Show abstract][Hide abstract] ABSTRACT: Tumor-associated neutrophils contribute to neovascularization by supplying matrix metalloproteinase-9 (MMP-9), a protease that has been genetically and biochemically linked to induction of angiogenesis. Specific roles of inflammatory neutrophils and their distinct proMMP-9 in the coordinate regulation of tumor angiogenesis and tumor cell dissemination, however, have not been addressed. We demonstrate that the primary tumors formed by highly disseminating variants of human fibrosarcoma and prostate carcinoma recruit elevated levels of infiltrating MMP-9-positive neutrophils and concomitantly exhibit enhanced levels of angiogenesis and intravasation. Specific inhibition of neutrophil influx by interleukin 8 (IL-8) neutralization resulted in the coordinated diminishment of tumor angiogenesis and intravasation, both of which were rescued by purified neutrophil proMMP-9. However, if neutrophil proMMP-9, naturally devoid of tissue inhibitor of metalloproteinases (TIMP), was delivered in complex with TIMP-1 or in a mixture with TIMP-2, the protease failed to rescue the inhibitory effects of anti-IL8 therapy, indicating that the TIMP-free status of proMMP-9 is critical for facilitating tumor angiogenesis and intravasation. Our findings directly link tumor-associated neutrophils and their TIMP-free proMMP-9 with the ability of aggressive tumor cells to induce the formation of new blood vessels that serve as conduits for tumor cell dissemination. Thus, treatment of cancers associated with neutrophil infiltration may benefit from specific targeting of neutrophil MMP-9 at early stages to prevent ensuing tumor angiogenesis and tumor metastasis.
American Journal Of Pathology 09/2011; 179(3):1455-70. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Urokinase-type plasminogen activator (uPA) and plasmin have long been implicated in cancer progression. However, the precise contributions of the uPA/plasmin system to specific steps involved in cancer cell dissemination have not been fully established. Herein, we have used a highly disseminating variant of the human PC-3 prostate carcinoma cell line, PC-hi/diss, as a prototype of aggressive carcinomas to investigate the mechanisms whereby pro-uPA activation and uPA-generated plasmin functionally contribute to specific stages of metastasis. The PC-hi/diss cells secrete and activate significant amounts of pro-uPA, leading to efficient generation of plasmin in solution and at the cell surface. In a mouse orthotopic xenograft model, treatment with the specific pro-uPA activation-blocking antibody mAb-112 significantly inhibited local invasion and distant metastasis of the PC-hi/diss cells. To mechanistically examine the uPA/plasmin-mediated aspects of tumor cell dissemination, the anti-pro-uPA mAb-112 and the potent serine protease inhibitor, aprotinin, were used in parallel in a number of in vivo assays modeling various rate-limiting steps in early metastatic spread. Our findings demonstrate that, by generating plasmin, activated tumor-derived uPA facilitates early stages of PC-hi/diss dissemination, specifically the escape from the primary tumor and tumor cell intravasation. Moreover, through a series of in vitro and in vivo analyses, we suggest that PC-hi/diss-invasive escape and dissemination may be enhanced by cleavage of stromal fibronectin by uPA-generated plasmin. Together, our findings point to inhibition of pro-uPA activation at the apex of the uPA/plasmin cascade as a therapy-valid approach to control onset of tumor escape and ensuing metastatic spread.
Neoplasia (New York, N.Y.) 09/2011; 13(9):806-21. · 5.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Within the last two decades of research, the accumulated evidence unequivocally demonstrates that proteolytic degradation
and modification of ECM proteins and proteolytic remodeling of stromal tissue play critical roles in cell invasion. With regard
to cancer biology, a particular class of proteolytic enzymes, i.e., matrix metalloproteinases (MMPs), is considered highly
important in facilitating overall tumor progression and metastasis. As the metastatic cascade represents a continuum of distinct
steps, some of which appear to be rate-limiting, the contribution of select MMPs at different stages of malignant disease
could be appreciated. Thus, secreted MMPs such as MMP-9 coming from nontumor cells have been functionally linked to tumor
progression, while other MMPs have been shown to be potent in cell–cell contact dissolutions during the early epithelial-to-mesenchymal
transitions. The membrane-tethered MMPs such as MT1-MMP appear to govern directional invasion of tumor cells across basement
membranes into surrounding collagen-enriched stroma. However, the identification of MMPs which preferentially facilitate the
later steps of the metastatic cascade, especially the transition from micrometastases to macrometastases, remains an unanswered
issue. In addition, progress in cancer stem cell (CSC) biology, including the formation of the premetastatic and vascular
niches and mechanisms of CSC trafficking, has indicated a high potential of MMP involvement. This review is focused mostly
on recent advances in our understanding of MMP physiology in metastatic spread, but also provides an outlook on the experimental
findings which paved the ground for newly developed concepts and hypotheses about the roles of MMPs in cancer progression
especially in regard to stromal tissue and matrix remodeling.
[Show abstract][Hide abstract] ABSTRACT: Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA's activity. In the present study we characterize the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active uPA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems, the antibodies inhibited tumour cell invasion and dissemination, providing evidence for the feasibility of pharmaceutical intervention with serine protease activity by targeting surface loops that undergo conformational changes during zymogen activation.
[Show abstract][Hide abstract] ABSTRACT: CUB-domain-containing protein 1 (CDCP1) is an integral membrane glycoprotein with potential as a marker and therapeutic target for a number of cancers. Here we examine mechanisms regulating cellular processing of CDCP1. By analyzing cell lines exclusively passaged non-enzymatically and through use of a panel of protease inhibitors, we demonstrate that full-length 135 kDa CDCP1 is post-translationally processed in a range of cell lines by a mechanism involving serine protease activity, generating a C-terminal 70-kDa fragment. Immunopurification and N-terminal sequencing of this cell-retained fragment and detailed mutagenesis, show that proteolytic processing of CDCP1 occurs at two sites, Arg-368 and Lys-369. We show that the serine protease matriptase is an efficient, but not essential, cellular processor of CDCP1 at Arg-368. Importantly, we also demonstrate that proteolysis induces tyrosine phosphorylation of 70-kDa CDCP1 and recruitment of Src and PKCdelta to this fragment. In addition, Western blot and mass spectroscopy analyses show that an N-terminal 65-kDa CDCP1 ectodomain is shed intact from the cell surface. These data provide new insights into mechanisms regulating CDCP1 and suggest that the biological role of this protein and, potentially, its function in cancer, may be mediated by both 70-kDa cell retained and 65-kDa shed fragments, as well as the full-length 135-kDa protein.
Journal of Biological Chemistry 08/2010; 285(34):26162-73. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A number of extensive reviews are available discussing the roles of MMPs in various aspects of cancer progression from benign tumor formation to overt cancer present with deadly metastases. This review will focus specifically on the evidence functionally linking the MMPs and tumor-induced angiogenesis in various in vivo models. Emphasis has been placed on the cellular origin of the MMPs in tumor tissue, the requirement of proMMP activation and the resulting proteolytic activity for the induction and progression of tumor angiogenesis, and the pleiotropic roles for some of the MMPs. The functional mechanisms of the angiogenic MMPs are discussed as well as their catalytic detection in complex biological systems. In addition, the contribution of active MMPs to metastatic spread and establishment of secondary metastasis will be discussed in view of the findings indicating that MMPs are involved in the preparation of pre-metastatic niches. Finally, the most recent evidence, indicating the pro-metastatic consequences of anti-angiogenic therapies employing MMP inhibitors will be presented as examples highlighting possible outcomes of interfering with the pleiotropic nature of the MMP functionality.
Biochimica et Biophysica Acta 01/2010; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the capacity of the adipokine leptin to promote angiogenesis by modulating the function of circulating angiogenic cells (CACs).
In vitro, leptin specifically promoted CAC adhesion to tubular endothelial structures and migration along outgrowing sprouts of endothelial cells. In vivo, stimulation of CACs with leptin increased their capacity to promote new vessel formation in the chorioallantoic membrane of chicken embryos and to improve neovascularization of ischemic murine hind limbs. These effects required the phosphorylation of alphavbeta5 integrins, which depended on the interaction of leptin with its receptor ObR, and on Janus kinase (JAK) 2- and phospholipase C (PLC) gamma-mediated activation of Src kinase. Protein tyrosine phosphatase 1B, a negative regulator of leptin signaling, was overexpressed in CACs from obese, hyperleptinemic individuals, and this was associated with insensitivity of CACs to the angiogenic effects of leptin. Weight loss (by 30+/-15 kg) normalized protein tyrosine phosphatase 1B expression in CACs and restored their responsiveness to leptin. A similar dose-dependent response was found after incubation of CACs from obese subjects with a protein tyrosine phosphatase 1B inhibitor ex vivo.
Our results point to the ObR-Src kinase-alphavbeta5 cross talk as a distinct novel component of the network of specific interactions between integrins and cytokine receptors in angiogenesis.
[Show abstract][Hide abstract] ABSTRACT: Despite significant progress and notable successes in tumor therapy, malignant disease remains an extremely difficult problem in today's health care setting. There is, however, an increasing application of new therapies targeting proteins specifically upregulated on tumor cells. These innovative therapeutic approaches are aimed at molecules that contribute to malignant development and progression but spare normal tissues. The CUB domain containing protein 1 (CDCP1) is such a tumor-associated protein and, thus, a potential candidate for targeted cancer immunotherapy. Herein, we describe the generation of function-blocking human antibodies against CDCP1 that were obtained from human scFv phage display libraries using subtractive panning protocols on CDCP1 expressing cancer cells and immunopurified CDCP1 protein. One of the isolated anti-CDCP1 antibodies, namely, C20Fc, efficiently blocked experimental metastasis of human carcinoma cells, including HeLa cells stably transfected with CDCP1 and prostate carcinoma cells PC-hi/diss naturally expressing CDCP1, in both chick embryo and mouse model systems. The C20Fc antibody also reduced colony formation of CDCP1 expressing cells in a soft agar assay for anchorage-independent cell growth. Specific targeting of CDCP1 by C20Fc mediated the delivery of a toxin-conjugated antibody complex, thus, providing evidence for antibody internalization and specific killing of CDCP1-positive tumor cells. Our findings indicate a functional role for CDCP1 in human cancer and underscore the therapeutic potential of function-blocking anti-CDCP1 antibodies targeting both primary and metastatic carcinoma cells.
[Show abstract][Hide abstract] ABSTRACT: To analyze the process of tumor cell intravasation, we used the human tumor-chick embryo spontaneous metastasis model to select in vivo high (PC-hi/diss) and low (PC-lo/diss) disseminating variants from the human PC-3 prostate carcinoma cell line. These variants dramatically differed in their intravasation and dissemination capacities in both chick embryo and mouse spontaneous metastasis models. Concomitant with enhanced intravasation, PC-hi/diss exhibited increased angiogenic potential in avian and murine models. PC-hi/diss angiogenesis and intravasation were dependent on increased secretion of vascular endothelial growth factor (VEGF), since treating developing tumors with a function-blocking anti-VEGF antibody simultaneously inhibited both processes without affecting primary tumor growth. PC-hi/diss cells were also more migratory and invasive, suggestive of heightened ability to escape from primary tumors due to matrix-degrading activity. Consistent with this suggestion, PC-hi/diss cells produced more of the serine protease urokinase-type plasminogen activator (uPA) as compared with PC-lo/diss. The functional role of uPA in PC-hi/diss dissemination was confirmed by inhibition of invasion, angiogenesis, and intravasation with specific function-blocking antibodies that prevented uPA activation and blocked uPA activity. These processes were similarly sensitive to aprotinin, a potent inhibitor of serine proteases, including uPA-generated plasmin. Thus, our comparison of the PC-3 intravasation variants points to key roles for the uPA-plasmin system in PC-hi/diss intravasation, possibly via (1) promoting tumor cell matrix invasion and (2) facilitating development of VEGF-dependent angiogenic blood vessels.
American Journal Of Pathology 10/2009; 175(4):1638-52. · 4.60 Impact Factor