Regulation of cell adhesion: a collaborative effort of integrins, their ligands, cytoplasmic actors, and phosphorylation.

Integrins are large heterodimeric type 1 membrane proteins expressed in all nucleated mammalian cells. Eighteen α-chains and eight ß-chains can combine to form 24 different integrins. They are cell adhesion proteins, which bind to a large variety of cellular and extracellular ligands. Integrins are required for cell migration, hemostasis, translocation of cells out from the blood stream and further movement into tissues, but also for the immune response and tissue morphogenesis. Importantly, integrins are not usually active as such, but need activation to become adhesive. Integrins are activated by outside-in activation through integrin ligand binding, or by inside-out activation through intracellular signaling. An important question is how integrin activity is regulated, and this topic has recently drawn much attention. Changes in integrin affinity for ligand binding are due to allosteric structural alterations, but equally important are avidity changes due to integrin clustering in the plane of the plasma membrane. Recent studies have partially solved how integrin cell surface structures change during activation. The integrin cytoplasmic domains are relatively short, but by interacting with a variety of cytoplasmic proteins in a regulated manner, the integrins acquire a number of properties important not only for cell adhesion and movement, but also for cellular signaling. Recent work has shown that specific integrin phosphorylations play pivotal roles in the regulation of integrin activity. Our purpose in this review is to integrate the present knowledge to enable an understanding of how cell adhesion is dynamically regulated.

Targeting IL11 Receptor in Leukemia and Lymphoma: A Functional Ligand-Directed Study and Hematopathology Analysis of Patient-Derived Specimens.

PURPOSE: The IL11 receptor (IL11R) is an established molecular target in primary tumors of bone, such as osteosarcoma, and in secondary bone metastases from solid tumors, such as prostate cancer. However, its potential role in management of hematopoietic malignancies has not yet been determined. Here, we evaluated the IL11R as a candidate therapeutic target in human leukemia and lymphoma. EXPERIMENTAL DESIGN AND RESULTS: First, we show that the IL11R protein is expressed in a variety of human leukemia- and lymphoma-derived cell lines and in a large panel of bone marrow samples from leukemia and lymphoma patients, whereas expression is absent from nonmalignant control bone marrow. Moreover, a targeted peptidomimetic prototype (termed BMTP-11), specifically bound to leukemia and lymphoma cell membranes, induced ligand-receptor internalization mediated by the IL11R, and resulted in a specific dose-dependent cell death induction in these cells. Finally, a pilot drug lead-optimization program yielded a new myristoylated BMTP-11 analogue with an apparent improved antileukemia cell profile. CONCLUSIONS: These results indicate (i) that the IL11R is a suitable cell surface target for ligand-directed applications in human leukemia and lymphoma and (ii) that BMTP-11 and its derivatives have translational potential against this group of malignant diseases.

Design, development, and validation of a high-throughput drug-screening assay for targeting of human leukemia.

BACKGROUND: The authors developed an ex vivo methodology to perform drug library screening against human leukemia. METHODS: The strategy for this screening relied on human blood or bone marrow cultures under hypoxia; under these conditions, leukemia cells deplete oxygen faster than normal cells, causing a hemoglobin oxygenation shift. Several advantages were observed: 1) partial recapitulation of the leukemia microenvironment, 2) use of native hemoglobin oxygenation as a real-time sensor/reporter, 3) cost-effectiveness, 4) species specificity, and 5) a format that enables high-throughput screening. RESULTS: For a proof of concept, a chemical library (size, approximately 20,000 compounds) was screened against human leukemia cells. Seventy compounds were identified ("hit" rate, 0.35%; Z-factor = 0.71) that had activity, and 20 compounds were examined to identify 18 true-positive compounds (90%). Finally, the results demonstrated that carbonohydraxonic diamide group-containing compounds are potent antileukemia agents that induce cell death in leukemia cells and in patient-derived samples. CONCLUSIONS: The current results indicated that this unique functional assay can identify novel drug candidates and can help with the development of future applications in personalized drug selection for patients with leukemia.

In vivo targeting of activated leukocytes by a ß2-integrin binding peptide.

BACKGROUND: In immunopathological conditions, clinical diagnosis is commonly made on the basis of patient symptoms, measurement of blood leukocyte levels or proinflammatory biomarkers, non-specific radiological findings and, regarding infection, microbiological analysis. Nevertheless, frequently the exact spatial location of inflammation or even infection cannot be reliably identified, despite the use of up-to-date clinical, laboratory and imaging techniques. For this reason, new tools are warranted for use in advanced diagnosis and therapy targeting in patients. OBJECTIVE: The peptide CPCFLLGCC (LLG), known to bind activated ß2-integrins in vitro, was fused with green fluorescent protein (GFP) to test the ability of LLG fusions to target and bind activated leukocytes in vivo. METHODS: A murine skin scratch inflammation model was chosen for the convenient non-surgical detection of GFP. RESULTS: The murine skin lesion inflammation model demonstrated in vivo targeting of LLG-GFP to sites of inflammation. Targeting by LLG-GFP fusion construct depends on the ability of the LLG-moiety to bind activated leukocytes. Control construct unable to bind ß2-integrins appeared biologically inert. CONCLUSION: The data support the possibility of using this fluorescently labeled peptide as a tool for both the detection of and targeting to inflammatory sites characterized by robust leukocyte activation.

Combinatorial targeting and discovery of ligand-receptors in organelles of mammalian cells.

Phage display screening allows the study of functional protein-protein interactions at the cell surface, but investigating intracellular organelles remains a challenge. Here we introduce internalizing-phage libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and target-specific organelles as a tool to select ligand peptides and identify their intracellular receptors. We demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of internalizing phage into cells. We also show that an internalizing-phage construct displaying an established mitochondria-specific localization signal targets mitochondria, and that an internalizing-phage random peptide library selects for peptide motifs that localize to different intracellular compartments. As a proof-of-concept, we demonstrate that one such peptide, if chemically fused to penetratin, is internalized receptor-independently, localizes to mitochondria, and promotes cell death. This combinatorial platform technology has potential applications in cell biology and drug development.

Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment.

Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.

Vascular ligand-receptor mapping by direct combinatorial selection in cancer patients.

Molecules differentially expressed in blood vessels among organs or between damaged and normal tissues, are attractive therapy targets; however, their identification within the human vasculature is challenging. Here we screened a peptide library in cancer patients to uncover ligand-receptors common or specific to certain vascular beds. Surveying ~2.35 x 10(6) motifs recovered from biopsies yielded a nonrandom distribution, indicating that systemic tissue targeting is feasible. High-throughput analysis by similarity search, protein arrays, and affinity chromatography revealed four native ligand-receptors, three of which were previously unrecognized. Two are shared among multiple tissues (integrin α4/annexin A4 and cathepsin B/apolipoprotein E3) and the other two have a restricted and specific distribution in normal tissue (prohibitin/annexin A2 in white adipose tissue) or cancer (RAGE/leukocyte proteinase-3 in bone metastases). These findings provide vascular molecular markers for biotechnology and medical applications.

Targeting neuropilin-1 in human leukemia and lymphoma.

Targeted drug delivery offers an opportunity for the development of safer and more effective therapies for the treatment of cancer. In this study, we sought to identify short, cell-internalizing peptide ligands that could serve as directive agents for specific drug delivery in hematologic malignancies. By screening of human leukemia cells with a combinatorial phage display peptide library, we isolated a peptide motif, sequence Phe-Phe/Tyr-Any-Leu-Arg-Ser (F(F)/(Y)XLRS), which bound to different leukemia cell lines and to patient-derived bone marrow samples. The motif was internalized through a receptor-mediated pathway, and we next identified the corresponding receptor as the transmembrane glycoprotein neuropilin-1 (NRP-1). Moreover, we observed a potent anti-leukemia cell effect when the targeting motif was synthesized in tandem to the pro-apoptotic sequence (D)(KLAKLAK)2. Finally, our results confirmed increased expression of NRP-1 in representative human leukemia and lymphoma cell lines and in a panel of bone marrow specimens obtained from patients with acute lymphoblastic leukemia or acute myelogenous leukemia compared with normal bone marrow. These results indicate that NRP-1 could potentially be used as a target for ligand-directed therapy in human leukemias and lymphomas and that the prototype CGFYWLRSC-GG-(D)(KLAKLAK)2 is a promising drug candidate in this setting.

A chimera of green fluorescent protein with gelatinase binding and tumor targeting peptide.

Matrix metalloproteinases (MMPs) are enzymes that can hydrolyze almost all constituents of extracellular matrix. An MMP subgroup, the gelatinases, has been focused during last years, since over-expression of gelatinase A (MMP-2) and gelatinase B (MMP-9) has been linked with severe homeostasis disorders such as tumor growth, metastasis formation, and chronic inflammation. In this study, a phage display library-derived novel antigelatinolytic decapeptide, the CTT-peptide, was expressed as a carboxyl terminal, histidine-tagged fusion with the green fluorescent protein (CTT-GFP) in Escherichia coli. In addition, a biologically intact chimera, in which residues in the CTT-peptide critical for gelatinase binding were replaced with alanine (Ala-CTT-GFP), was constructed. The GFP-fusion proteins were purified to homogeneity with a simple one-step procedure utilizing nickel affinity chromatography. The purified chimeras were tested for their binding properties to 4beta-phorbol-12,13-butyrate (PdBu) activated, MMP-9 expressing THP-1 cells, and it was demonstrated that the CTT-GFP strongly bound to the cells, whereas Ala-CTT-GFP lacked the binding ability. Furthermore, the adherence of the CTT-GFP to MMP-9 expressing cells was demonstrated to be mediated by the CTT-moiety, since the binding could be dose-relatedly inhibited with increasing concentrations of synthetic soluble CTT-peptide. In conclusion, this novel tool, combining the gelatinase binding ability of the CTT-peptide with the fluorescing property of the GFP, should clearly improve both experimental and clinical studies of the role and function of gelatinases.

Identification of novel peptide inhibitors for human trypsins.

Human trypsin isoenzymes share extensive sequence similarity, but certain differences in their activity and susceptibility to inhibitors have been observed. Using phage display technology, we identified seven different peptides that bind to and inhibit the activity of trypsin-3, a minor trypsin isoform expressed in pancreas and brain. All of the peptides contain at least two of the amino acids tryptophan, alanine and arginine, whereas proline was found closer to the N-terminus in all but one peptide. All peptides contain two or more cysteines, suggesting a cyclic structure. However, we were able to make synthetic linear variants of these peptides without losing bioactivity. Alanine replacement experiments for one of the peptides suggest that the IPXXWFR motif is important for activity. By molecular modeling the same amino acids were found to interact with trypsin-3. The peptides also inhibit trypsin-1, but only weakly, if at all, trypsin-2 and -C. As trypsin is a highly active enzyme which can activate protease-activated receptors and enzymes that participate in proteolytic cascades involved in tumor invasion and metastasis, these peptides might be useful lead molecules for the development of drugs for diseases associated with increased trypsin activity.

Human Siglec-10 can bind to vascular adhesion protein-1 and serves as its substrate.

Leukocytes migrate from the blood into areas of inflammation by interacting with various adhesion molecules on endothelial cells. Vascular adhesion protein-1 (VAP-1) is a glycoprotein expressed on inflamed endothelium where it plays a dual role: it is both an enzyme that oxidizes primary amines and an adhesin that is involved in leukocyte trafficking to sites of inflammation. Although VAP-1 was identified more than 15 years ago, the counterreceptor(s) for VAP-1 on leukocytes has remained unknown. Here we have identified Siglec-10 as a leukocyte ligand for VAP-1 using phage display screenings. The binding between Siglec-10 and VAP-1 was verified by different adhesion assays, and this interaction was also consistent with molecular modeling. Moreover, the interaction between Siglec-10 and VAP-1 led to increased hydrogen peroxide production, indicating that Siglec-10 serves as a substrate for VAP-1. Thus, the Siglec-10-VAP-1 interaction seems to mediate lymphocyte adhesion to endothelium and has the potential to modify the inflammatory microenvironment via the enzymatic end products.

A novel and selective membrane type-1 matrix metalloproteinase (MT1-MMP) inhibitor reduces cancer cell motility and tumor growth.

Matrix metalloproteinases (MMPs), and especially membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14), play a role in cancer progression and can have a prognostic value. Various synthetic broad-spectrum MMP inhibitors have been developed but have had little success in cancer patient treatment owing to side effects. Until recently, selective targeting of specific MMPs has not been possible due to lack of specific inhibitors. Here we have developed a selective MT1-MMP peptide-inhibitor GACFSIAHECGA, which did not affect the activities of many other MMPs including MMP-1, -2, -3, -7, -8, -9, -10, -11, -12, -13, -15, -17 or -20. In a fluorescent peptide cleavage assay it displayed an IC(50) value of 150 microM. The peptide effectively inhibited the migration and invasion of cancer cell lines in vitro. Furthermore, in vivo the peptide reduced the growth of tongue carcinoma xenografts and prolonged the survival of mice. Overall these results suggest that selective MT1-MMP inhibitors may have utility as anticancer agents.

Role of leukemia cell invadosome in extramedullary infiltration.

Acute myelogenous leukemias (AMLs) are characterized by medullary and extramedullary invasion. We hypothesized that a supramolecular complex, the leukemia-cell invadosome, which contains certain integrins, matrix metalloproteinases (MMPs), and other as-yet unidentified proteins, is essential for tissue invasion and may be central to the phenotypic diversity observed in the clinic. Here we show that the specific binding of MMP-9 to leukocyte surface beta(2) integrin is required for pericellular proteolysis and migration of AML-derived cells. An efficient antileukemia effect was obtained by the hexapeptide HFDDDE, a motif of the MMP-9 catalytic domain that mediates integrin binding: HFDDDE prevented proMMP-9 binding, transmigration through a human endothelial cell layer, and extracellular matrix degradation. Notably, the functional protein anchorage between beta(2) integrin and proMMP-9 described in this study does not involve the enzymatic active sites targeted by known MMP inhibitors. Taken together, our results provide a biochemical working definition for the human leukemia invadosome. Disruption of specific protein complexes within this supramolecular target complex may yield a new class of anti-AML drugs with anti-invasion (rather than or in addition to cytotoxic) attributes.

An unrecognized extracellular function for an intracellular adapter protein released from the cytoplasm into the tumor microenvironment.

Mammalian cell membranes provide an interface between the intracellular and extracellular compartments. It is currently thought that cytoplasmic signaling adapter proteins play no functional role within the extracellular tumor environment. Here, by selecting combinatorial random peptide libraries in tumor-bearing mice, we uncovered a direct, specific, and functional interaction between CRKL, an adapter protein [with Src homology 2 (SH2)- and SH3-containing domains], and the plexin-semaphorin-integrin domain of beta(1) integrin in the extracellular milieu. Through assays in vitro, in cellulo, and in vivo, we show that this unconventional and as yet unrecognized protein-protein interaction between a regulatory integrin domain (rather than a ligand-binding one) and an intracellular adapter (acting outside of the cells) triggers an alternative integrin-mediated cascade for cell growth and survival. Based on these data, here we propose that a secreted form of the SH3/SH2 adaptor protein CRKL may act as a growth-promoting factor driving tumorigenesis and may lead to the development of cancer therapeutics targeting secreted CRKL.

Combinatorial targeting of the macropinocytotic pathway in leukemia and lymphoma cells.

Ligand-directed delivery of agents to leukemia and lymphoma cells has the potential to yield new mechanistic disease insights and targeted therapies. Here we set out to target the macropinocytotic pathway with a combinatorial approach. From the screening of acute T-lymphoblastic leukemia Molt-4 cells with a random phage-display peptide library, we isolated a phage displaying the sequence CAYHRLRRC. This peptide contains a lymph node-homing motif (Cys-Ala-Tyr) and a cell-penetrating motif (Arg-Leu-Arg-Arg). Binding of this ligand-directed phage to a large panel of leukemia/lymphoma cells and to patient-derived samples was much higher than to non-leukemia control cells. CAYHRLRRC phage internalization into Molt-4 cells is both energy- and temperature-dependent. Flow cytometry with fluorescein-labeled peptide and endocytosis blocking with specific inhibitors revealed that CAYHRLRRC is indeed taken up through macropinocytosis in Molt-4 and K562 human leukemia cells. Unexpectedly, the cell surface receptor for the CAYHRLRRC peptide is not a heparan sulfate proteoglycan as it would be predicted for other cell-penetrating peptides. Confirming this interpretation, a CAYHRLRRC-directed peptidomimetic-induced cell death in all the leukemia and lymphoma cells was evaluated, whereas a control transactivator of transcription protein (tat)-directed proapoptotic peptidomimetic was non-selective. In summary, the targeting peptide CAYHRLRRC is selectively internalized through macropinocytosis in leukemia and lymphoma cells and has potential as a drug lead for ligand-directed anti-leukemia therapies.

AlphaMbeta2 integrin modulator exerts antitumor activity in vivo.

BACKGROUND: Leukocyte immunomodulation has great clinical potential as a therapy of inflammatory conditions and cancer. We have recently developed leukocyte alphaMbeta2 integrin targeting small molecule (IMB-10) capable of inhibiting leukocyte migration and recruitment in vitro and in vivo. MATERIALS AND METHODS: The purpose of this study was to investigate the potential anticancer effects of IMB-10 using U937 histiocytic lymphoma, OCI-AML-3 acute myeloid leukemia and HSC-3 tongue squamous cell carcinoma xenografts in athymic nude mice lacking T-lymphocytes. RESULTS: IMB-10 therapy inhibited the growth of both leukemia and lymphoma xenografts and significantly prolonged the survival of the mice with lymphoma. Interestingly, IMB-10 also reduced host leukocyte infiltration in tumors and affected the invasion potential of squamous cell carcinomas. CONCLUSION: IMB-10 has potential as a therapy for leukocytic malignancies, particularly for lymphomas. Since it also inhibited HSC-3 carcinoma invasion, most likely by blockage of the tumor-infiltrating leukocytes, we suggest that the host inflammation process may affect tumor progression also in a T-cell independent manner.

Cell-surface association between matrix metalloproteinases and integrins: role of the complexes in leukocyte migration and cancer progression.

Leukocyte motility is known to be dependent on both beta2-integrins and matrix metalloproteinases MMP-2/-9 or gelatinases, which mediate leukocyte adhesion and the proteolysis needed for invasion, respectively. Gelatinases not only play an important role in cell migration, tissue remodeling, and angiogenesis during development, but are also involved in the progression and invasiveness of many cancers, including leukemias. The concept that MMPs associate with integrins, as well as their importance in some physiologic and pathologic conditions, has been advanced previously but has not been examined on leukocytes. This review will examine mainly the function of the MMP-integrin complexes in normal leukocyte migration and the effect of integrin and broad-spectrum MMP inhibitors in tumor progression.

Novel peptide inhibitors of human kallikrein 2.

Human kallikrein 2 (hK2) is a serine protease produced by the secretory epithelial cells in the prostate. Because hK2 activates several factors participating in proteolytic cascades that may mediate metastasis of prostate cancer, modulation of the activity of hK2 is a potential way of preventing tumor growth and metastasis. Furthermore, specific ligands for hK2 are potentially useful for targeting and imaging of prostate cancer and for assay development. We have used enzymatically active recombinant hK2 captured by a monoclonal antibody exposing the active site of the enzyme to screen phage display peptide libraries. Using libraries expressing 10 or 11 amino acids long linear peptides, we identified six different peptides binding to hK2. Three of these were shown to be specific and efficient inhibitors of the enzymatic activity of hK2 toward a peptide substrate. Furthermore, the peptides inhibited the activation of the proform of prostate-specific antigen by hK2. Amino acid substitution analyses revealed that motifs of six amino acids were required for the inhibitory activity. These peptides are potentially useful for treatment and targeting of prostate cancer.

Stabilization of the activated alphaMbeta2 integrin by a small molecule inhibits leukocyte migration and recruitment.

Integrins are potential targets for the development of antiinflammatory agents. Here we develop a novel high-throughput assay by allowing a chemical library to compete with phage display peptide binding and identify a novel small-molecule ligand to the leukocyte-specific alpha(M)beta(2) integrin. The identified thioxothiazolidine-containing compound, IMB-10, had an unexpected activity in that it stabilized binding of alpha(M)beta(2) to its endogenous ligands proMMP-9 and fibrinogen. Single amino acid substitutions in the activity-regulating C-terminal helix and the underlying region in the ligand-binding I domain of the integrin suppressed the effect of IMB-10. A computational model indicated that IMB-10 occupies a distinct cavity present only in the activated form of the integrin I domain. IMB-10 inhibited alpha(M)beta(2)-dependent migration in vitro and inflammation-induced neutrophil emigration in vivo. Stabilization of integrin-mediated adhesion by a small molecule is a novel means to inhibit cell migration and may have a utility in treatment of inflammatory diseases involving leukocyte recruitment.

Human tongue carcinoma growth is inhibited by selective antigelatinolytic peptides.

Matrix metalloproteinases (MMP-2 and MMP-9, or gelatinases) are involved in tongue SCC invasion, metastasis and angiogenesis. We have recently shown that a novel and selective hydrophobic cyclic CTTHWGFTLC (CTT1) peptide is inhibitor for MMP-2 and MMP-9 (Koivunen et al., Nat Biotechnol 1999; 17:768-74). In this study, we demonstrate that both the new hydrophilic derivate GRENYHGCTTHWGFTLC (CTT2) peptide and the CTT1 peptide inhibited specifically the human tongue squamous cell carcinoma (HSC-3) cell-derived gelatinolytic activity and in vitro invasion and migration of these cells (p < or = 0.049). In situ zymography revealed that both peptides also inhibited clearly almost all of the gelatinolytic activity present in the human tongue SCC tissue sections, indicating that MMP-2 and MMP-9 are the major gelatinases detected in the tongue carcinomas. However, CTT2 did not inhibit the type I collagen degradation by human collagenases (MMP-1, MMP-8 and MMP-13). Furthermore, CTT2 reduced the blood vessel density (p < or = 0.043) and clearly improved the survival of the mice bearing human tongue carcinoma xenografts (p < or = 0.012). Overall, we suggest that CTT1 and CTT2 peptides being selective gelatinase inhibitors with significant anti-tumor properties could be useful to diminish the invasion and angiogenesis of human tongue carcinomas characterized by enhanced gelatinolytic activity in tumors.