Martin Schlesinger

Vascular cell adhesion molecule-1 (VCAM-1)--an increasing insight into its role in tumorigenicity and metastasis.

Vascular cell adhesion molecule‐1 (VCAM‐1) first attracted attention more than two decades ago as endothelial adhesion receptor with key function for leukocyte recruitment in term of cellular immune response. The early finding of VCAM‐1 binding to melanoma cells, and thus a suggested mechanistic contribution to metastatic spread, was the first and for a long time the only link of VCAM‐1 to cancer sciences. In the last few years, hallmarked by a growing insight into the molecular understanding of tumorigenicity and metastasis, an impressive variety of VCAM‐1 functionalities in cancer have been elucidated. The present review aims to provide a current overview of VCAM‐1 relevance for tumor growth, metastasis, angiogenesis, and related processes. By illustrating the intriguing role of VCAM‐1 in cancer disease, VCAM‐1 is suggested as a new and up to now underestimated target in cancer treatment and in clinical diagnosis of malignancies.

Lysophosphatidylcholine Pretreatment Reduces VLA-4 and P-Selectin–Mediated B16.F10 Melanoma Cell Adhesion In vitro and Inhibits Metastasis-Like Lung Invasion In vivo

Lysophosphatidylcholine (LysoPC) is an important intermediate in degradation and biosynthesis of phosphatidylcholine (PC). Reduced plasma LysoPC levels observed in patients with advanced cancer indicate a deregulation of LysoPC metabolism in metastasis. Recent data showed strong antimetastatic effects of liposomes consisting of saturated PC in a murine pancreatic metastasis model. LysoPC, generated from saturated PC after accumulation of the liposomes in tumor tissue, might be contributing to these effects. Examining effects of high local concentrations of saturated LysoPC and investigating potential molecular mechanisms, fast removal of saturated LysoPC from medium by murine B16.F10 melanoma cells and radical shifts in tumor cell membrane fatty acid (FA) composition toward saturated FAs were observed in vitro. Scanning electron microscopy revealed remarkable morphologic surface changes of LysoPC-treated tumor cells, probably causing their impaired migratory ability on fibronectin. A LysoPC concentration exceeding a threshold of about 400 μmol/L, slightly above physiologic levels, strongly reduced VLA-4–mediated binding of B16.F10 cells to VCAM-1 as well as P-selectin–dependent interaction with activated platelets, although expression levels were not altered. These findings were reflected in a syngenic intravenous lung invasion model using repeatedly ex vivo LysoPC-treated (450 μmol/L) B16.F10 cells, resulting in significantly reduced lung metastasis-like lesions (−48.3%, P = 0.006). Prior application of 50 IU unfractionated heparin further reduced lung invasion (−81.6%, P = 0.043). Our work shows for the first time that saturated LysoPC in high concentrations reduces melanoma cell adhesion in vitro and hematogeneous dissemination in vivo by direct ex vivo tumor cell targeting. Mol Cancer Ther; 10(1); 186–97. ©2011 AACR.

Binding between heparin and the integrin VLA-4.

Heparin possesses antimetastatic effects that were related to various molecular mechanisms beyond anticoagulant activities. The ability of heparin to interfere with the function of adhesion receptors in the metastatic course appears as a promising therapeutic approach. This refers to numerous findings that heparin attenuates metastasis in a selectin-dependent manner. We recently demonstrated that heparin interferes with the integrin VLA-4 on murine melanoma cells binding to VCAM-1. To confirm this activity and to obtain further insight into molecular recognition of heparin by VLA-4, we investigated the inhibition of VLA-4 mediated binding of human melanoma MV3 cells to immobilised VCAM-1 by different heparins. The size of heparin has an important impact on inhibition. Unfractionated heparin (UFH) and tinzaparin, a low-molecular-weight heparin (LMWH) representing a mean of about 18-20 monomers, displayed high inhibitory activity. Fractionating tinzaparin to 14-18 monomers reduced inhibition slightly, while the pentasaccharide fondaparinux was without effects. To confirm molecular recognition of tinzaparin by VLA-4, a surface acoustic wave-biosensor was applied. A VLA-4 containing membrane preparation of MV3 cells was immobilised at the sensors to allow for detection of kinetic binding constants of tinzaparin compared to VCAM-1. Tinzaparin binds to VLA-4 with affinity in the low micromolar range (4.61 x 10(-6) M), which clearly indicates specific molecular recognition. Furthermore, tinzaparin displays a nearly identical k(off) compared to VCAM-1 (5.13 x 10(-3) s(-1) versus 3.44 x 10(-3) s(-1)) which is evident for interference with the ligand binding. The data provide evidence for a direct confirmation of heparin binding to VLA-4 and thus, contribute to understand the antimetastatic activity of heparin.

Saturated and mono-unsaturated lysophosphatidylcholine metabolism in tumour cells: a potential therapeutic target for preventing metastases

BackgroundMetastasis is the leading cause of mortality in malignant diseases. Patients with metastasis often show reduced Lysophosphatidylcholine (LysoPC) plasma levels and treatment of metastatic tumour cells with saturated LysoPC species reduced their metastatic potential in vivo in mouse experiments. To provide a first insight into the interplay of tumour cells and LysoPC, the interactions of ten solid epithelial tumour cell lines and six leukaemic cell lines with saturated and mono-unsaturated LysoPC species were explored.MethodsLysoPC metabolism by the different tumour cells was investigated by a combination of cell culture assays, GC and MS techniques. Functional consequences of changed membrane properties were followed microscopically by detecting lateral lipid diffusion or cellular migration. Experimental metastasis studies in mice were performed after pretreatment of B16.F10 melanoma cells with LysoPC and FFA, respectively.ResultsIn contrast to the leukaemic cells, all solid tumour cells show a very fast extracellular degradation of the LysoPC species to free fatty acids (FFA) and glycerophosphocholine. We provide evidence that the formerly LysoPC bound FFA were rapidly incorporated into the cellular phospholipids, thereby changing the FA-compositions accordingly. A massive increase of the neutral lipid amount was observed, inducing the formation of lipid droplets. Saturated LysoPC and to a lesser extent also mono-unsaturated LysoPC increased the cell membrane rigidity, which is assumed to alter cellular functions involved in metastasis. According to that, saturated and mono-unsaturated LysoPC as well as the respective FFA reduced the metastatic potential of B16.F10 cells in mice. Application of high doses of liposomes mainly consisting of saturated PC was shown to be a suitable way to strongly increase the plasma level of saturated LysoPC in mice.ConclusionThese data show that solid tumours display a high activity to hydrolyse LysoPC followed by a very rapid uptake of the resulting FFA; a mechanistic model is provided. In contrast to the physiological mix of LysoPC species, saturated and mono-unsaturated LysoPC alone apparently attenuate the metastatic activity of tumours and the artificial increase of saturated and mono-unsaturated LysoPC in plasma appears as novel therapeutic approach to interfere with metastasis.

Low molecular weight heparin tinzaparin antagonizes cisplatin resistance of ovarian cancer cells.

Low molecular weight heparin (LMWH) is routinely used for antithrombotic treatment of cancer patients. Preclinical- and clinical data suggest that LMWH has beneficial effects for cancer patients beyond the prevention of thrombosis, i.e. by inhibiting metastasis. It is, however, unclear whether heparin has an impact on the efficiency of chemotherapy in cancer patients. Here we show that a therapeutic dosage of LMWH tinzaparin reverses cisplatin resistance of A2780cis human ovarian cancer cells to the level of sensitive cells. This novel activity of tinzaparin is associated with intense transcriptional reprogramming. Our gene expression profiling experiments revealed that 3776 genes responded to tinzaparin treatment. For this reason tinzaparin has a complex impact on diverse biological processes. We discovered that tinzaparin inhibits the expression of genes that mediate cisplatin resistance of A2780cis cells. In contrast tinzaparin induced the expression of genes that antagonize drug resistance. This activity of tinzaparin is mediated by cell surface proteoglycans, since enzymatic cleavage of heparan sulfates prevented the reversal of cisplatin resistance. These data indicate that cell surface heparan sulfate proteoglycans play an important role for chemotherapy resistance. The results of this study shed a new light on LMWH application in cancer therapy and suggest tinzaparin as promising treatment option of ovarian cancer patients in combination with anticancer drugs. Future clinical trials are needed to validate these findings.

Analysis of SM4 sulfatide as a P-selectin ligand using model membranes

Carcinoma tumor cells express highly glycosylated mucins acting as ligands for selectin adhesion receptors and thus facilitating the metastatic process. Recently, a sulfated galactocerebroside SM4 was detected as solely P-selectin ligand on MC-38 colon carcinoma cells. Here we characterize the functionality of SM4 as selectin ligand using model membrane approaches. SM4 was found concentrated in lipid rafts of MC-38 cells indicating a local clustering that may increase the avidity of P-selectin recognition. To confirm this, SM4 was incorporated at various concentrations into POPC model membranes and lateral clustering was analyzed by fluorescence microscopy and found to be comparable to glycolipids carrying the sLe(x) epitope. SM4 containing liposomes were used as cell models, binding to immobilized P-selectin. Quartz crystal microbalance data confirmed SM4/P-selectin liposome binding that was inhibited dose-dependently by heparin. Comparable binding characteristics of SM4 and sLe(x) liposomes underscore the similarity of these epitopes. Thus, clustering of SM4 on tumor cells is a principle for binding P-selectin.

The inhibition of the integrin VLA-4 in MV3 melanoma cell binding by non-anticoagulant heparin derivatives

INTRODUCTION The integrin VLA-4-mediated binding is important for the metastatic dissemination of melanoma cells. Recently we found that heparin possesses a binding capacity to VLA-4. This could contribute to the heparin function to attenuate metastasis in a selectin-dependent manner. Aiming to a purposive, anti-adhesive heparin application, structural requirements of heparin for VLA-4 recognition have to be elucidated. MATERIALS AND METHODS A series of non-anticoagulant heparin derivatives were investigated concerning their inhibitory capacities for VLA-4 mediated binding of human melanoma MV3 cells to VCAM-1 under physiological flow conditions in vitro. A surface acoustic wave biosensor was applied to detect kinetic constants of selected derivatives binding to both, VLA-4 or P- and L-selectin. RESULTS Experimental metastasis of MV3 cells in mice confirmed the relevance of VLA-4 for metastatic dissemination. LMWHs (enoxaparin, tinzaparin) efficiently blocked VLA-4 cell binding, dominantly via the integrin`s α-chain. Desulfation at 2-O-position, N-acetylation or a size smaller than tetradecasaccharide disfavoured VLA-4 inhibition. Glycol-splitting of heparin and thus higher chain flexibility is a tolerable parameter. A derivative with 50% 6-O-desulfation appeared promising and exceeded tinzaparin in VLA-4 inhibition, both compounds displayed binding affinities to VLA-4 in the low micromolar range. CONCLUSIONS These findings provide structure-activity relationships for heparin VLA-4 binding, which partly differ from P- and L-selectin requirements. The data confirm that anti-coagulative and anti-adhesive function of heparin can be distinguished favouring applications of non-anticoagulant heparins in antimetastatic approaches without the risk of bleeding complications. The 50% 6-O-desulfated heparin-derivative appears promising to further evaluate the interference with selectin and VLA-4 binding functions in vivo.

The role of VLA-4 binding for experimental melanoma metastasis and its inhibition by heparin.

INTRODUCTION Heparin is known to efficiently attenuate metastasis in various tumour models by different mechanisms including inhibition of tumour cell contacts with soluble and cellular components such as inhibition of heparanase or P- and L-selectin. We recently showed that heparin efficiently binds to VLA-4 integrin in melanoma cells in vitro. Here we describe VLA-4 integrin as a mediator of melanoma metastasis that is inhibited by the low molecular weight heparin (LMWH) Tinzaparin. MATERIALS AND METHODS sh-RNA-mediated knock-down of VLA-4 integrin in B16F10 murine melanoma cells (B16F10-VLA-4kd) was performed and cell binding characteristics were investigated in vitro. Experimental metastasis of B16F10-VLA-4kd and B16F10 cells and interference by Tinzaparin were analysed in mice. RESULTS VLA-4 knock-down of B16F10 cells resulted in loss of VCAM-1 binding, but preserved the capacity to bind platelets through P-selectin. The observed reduced metastasis of B16F10-VLA-4kd cells confirmed the role of VLA-4 in this process. However, loss of melanoma VLA-4 function hardly further affected reduction of metastasis in P-selectin deficient mice. Tinzaparin treatment of mice injected with B16F10 and B16F10-VLA-4kd cells significantly reduced metastasis suggesting its potential to block both P- and L-selectin and VLA-4 in vivo. The use of N-acetylated heparin, which has no VLA-4 binding activity but blocks P- and L-selectin was less efficient than Tinzaparin in mice injected with B16F10 cells and B16F10-VLA-4kd cells. CONCLUSION These findings provide evidence that heparin inhibits experimental melanoma metastasis primarily by blocking VLA-4 and P-selectin.

Contribution of very late antigen-4 (VLA-4) integrin to cancer progression and metastasis

The integrin “very late antigen-4” (VLA-4) is expressed by numerous cells of hematopoietic origin and possesses a key function in the cellular immune response, e.g., by mediating leukocyte tethering, rolling, binding, and finally transmigration of the vascular wall at inflammatory sites. Thus, VLA-4 is a valuable target in medical sciences to interfere with pathological inflammations. In addition, leukemic cells and different solid tumors, which express VLA-4, make use of these adhesive functions and confer VLA-4 a progressive role in the metastatic spread. With a growing insight into the molecular mechanisms for creating a tumor-friendly microenvironment at metastatic sites and various tumor host interactions, the multiple functions of VLA-4 became evident recently, e.g., in leukocyte recruitment to micrometastases, the protection of tumors from immune surveillance, or contribution to a chemoresistance. Nevertheless, despite accumulating evidence for several functions of VLA-4 in tumorigenicity, a therapeutic interference with VLA-4 in cancer sciences has not been developed yet to the clinical level, undoubtedly by a marked impact on the physiological immune response. This review gives an up to date insight into the multiple functional role of VLA-4 in cancer and introduces this integrin as a promising target worthwhile to attract attention in biomedical cancer research.

VCAM-1 directed target-sensitive liposomes carrying CCR2 antagonists bind to activated endothelium and reduce adhesion and transmigration of monocytes

Chemokines are critically involved in the development of chronic inflammatory-associated diseases such as atherosclerosis. We hypothesized that targeted delivery of compounds to the surface of activated endothelial cells (EC) interferes with chemokine/receptor interaction and thereby efficiently blocks inflammation. We developed PEGylated target-sensitive liposomes (TSL) encapsulating a CCR2 antagonist (Teijin compound 1) coupled with a specific peptide recognized by endothelial VCAM-1 (Vp-TSL-Tj). TSL were characterized for size (by dynamic light scattering), the amount of peptide coupled at the liposomal surface and Teijin release (by HPLC). We report that Vp-TSL-Tj binds specifically to activated EC in vitro and in situ, release the entrapped Teijin and prevent the transmigration of monocytes through activated EC. This is the first evidence that nanocarriers which transport and release chemokine inhibitors at specific pathological sites can reduce chemokine-dependent inflammatory processes.