Lubor Borsig

Heparin and cancer revisited: Mechanistic connections involving platelets, P-selectin, carcinoma mucins, and tumor metastasis

Independent studies indicate that expression of sialylated fucosylated mucins by human carcinomas portends a poor prognosis because of enhanced metastatic spread of tumor cells, that carcinoma metastasis in mice is facilitated by formation of tumor cell complexes with blood platelets, and that metastasis can be attenuated by a background of P-selectin deficiency or by treatment with heparin. The effects of heparin are not primarily due to its anticoagulant action. Other explanations have been suggested but not proven. Here, we bring together all these unexplained and seemingly disparate observations, showing that heparin treatment attenuates tumor metastasis in mice by inhibiting P-selectin-mediated interactions of platelets with carcinoma cell-surface mucin ligands. Selective removal of tumor mucin P-selectin ligands, a single heparin dose, or a background of P-selectin deficiency each reduces tumor cell-platelet interactions in vitro and in vivo. Although each of these maneuvers reduced the in vivo interactions for only a few hours, all markedly reduce long-term organ colonization by tumor cells. Three-dimensional reconstructions by using volume-rendering software show that each situation interferes with formation of the platelet “cloak” around tumor cells while permitting an increased interaction of monocytes (macrophage precursors) with the malignant cells. Finally, we show that human P-selectin is even more sensitive to heparin than mouse P-selectin, giving significant inhibition at concentrations that are in the clinically acceptable range. We suggest that heparin therapy for metastasis prevention in humans be revisited, with these mechanistic paradigms in mind.

Synergistic effects of L- and P-selectin in facilitating tumor metastasis can involve non-mucin ligands and implicate leukocytes as enhancers of metastasis

P-selectin facilitates human carcinoma metastasis in immunodeficient mice by mediating early interactions of platelets with bloodborne tumor cells via their cell surface mucins, and this process can be blocked by heparin [Borsig, L., Wong, R., Feramisco, J., Nadeau, D. R., Varki, N. M. & Varki, A. (2001) Proc. Natl. Acad. Sci. USA 98, 3352–3357]. Here we show similar findings with a murine adenocarcinoma in syngeneic immunocompetent mice but involving a different P-selectin ligand, possibly a sulfated glycolipid. Thus, metastatic spread can be facilitated by tumor cell selectin ligands other than mucins. Surprisingly, L-selectin expressed on endogenous leukocytes also facilitates metastasis in both the syngeneic and xenogeneic (T and B lymphocyte deficient) systems. PL-selectin double deficient mice show that the two selectins work synergistically. Although heparin can block both P- and L-selectin in vitro, the in vivo effect of a single heparin dose given before tumor cells seems to be completely accounted for by blockade of P-selectin function. Thus, L-selectin on neutrophils, monocytes, and/or NK cells has a role in facilitating metastasis, acting beyond the early time points wherein P-selectin mediates interactions of platelet with tumor cells.

Cancer Cell Adhesion and Metastasis: Selectins, Integrins, and the Inhibitory Potential of Heparins

Cell adhesion molecules play a significant role in cancer progression and metastasis. Cell-cell interactions of cancer cells with endothelium determine the metastatic spread. In addition, direct tumor cell interactions with platelets, leukocytes, and soluble components significantly contribute to cancer cell adhesion, extravasation, and the establishment of metastatic lesions. Clinical evidence indicates that heparin, commonly used for treatment of thromboembolic events in cancer patients, is beneficial for their survival. Preclinical studies confirm that heparin possesses antimetastatic activities that lead to attenuation of metastasis in various animal models. Heparin contains several biological activities that may affect several steps in metastatic cascade. Here we focus on the role of cellular adhesion receptors in the metastatic cascade and discuss evidence for heparin as an inhibitor of cell adhesion. While P- and L-selectin facilitation of cellular contacts during hematogenous metastasis is being accepted as a potential target of heparin, here we propose that heparin may also interfere with integrin activity and thereby affect cancer progression. This review summarizes recent findings about potential mechanisms of tumor cell interactions in the vasculature and antimetastatic activities of heparin.

Selectin-mucin interactions as a probable molecular explanation for the association of Trousseau syndrome with mucinous adenocarcinomas

Trousseau described spontaneous, recurrent superficial migratory thrombophlebitis associated with occult cancers, and this was later correlated with disseminated microangiopathy (platelet-rich clots in small blood vessels). Trousseau syndrome often occurs with mucinous adenocarcinomas, which secrete abnormally glycosylated mucins and mucin fragments into the bloodstream. Since carcinoma mucins can have binding sites for selectins, we hypothesized that selectin-mucin interactions might trigger this syndrome. When highly purified, tissue-factor free carcinoma mucin preparations were intravenously injected into mice, platelet-rich microthrombi were rapidly generated. This pathology was markedly diminished in P- or L-selectin-deficient mice. Heparin (an antithrombin-potentiating agent that can also block P- and L-selectin recognition of ligands) ameliorated this platelet aggregation, but had no additional effect in P- or L-selectin-deficient mice. Inhibition of endogenous thrombin by recombinant hirudin also did not block platelet aggregation. Mucins generated platelet aggregation in vitro in hirudinized whole blood, but not in platelet-rich leukocyte-free plasma nor in whole blood from L-selectin-deficient mice. Thus, Trousseau syndrome is likely triggered by interactions of circulating carcinoma mucins with leukocyte L-selectin and platelet P-selectin without requiring accompanying thrombin generation. These data may also explain why heparin ameliorates Trousseau syndrome, while vitamin K antagonists that merely depress thrombin production do not.

Altered tumor-cell glycosylation promotes metastasis

Malignant transformation of cells is associated with aberrant glycosylation presented on the cell-surface. Commonly observed changes in glycan structures during malignancy encompass aberrant expression and glycosylation of mucins; abnormal branching of N-glycans; and increased presence of sialic acid on proteins and glycolipids. Accumulating evidence supports the notion that the presence of certain glycan structures correlates with cancer progression by affecting tumor-cell invasiveness, ability to disseminate through the blood circulation and to metastasize in distant organs. During metastasis tumor-cell-derived glycans enable binding to cells in their microenvironment including endothelium and blood constituents through glycan-binding receptors – lectins. In this review, we will discuss current concepts how tumor-cell-derived glycans contribute to metastasis with the focus on three types of lectins: siglecs, galectins, and selectins. Siglecs are present on virtually all hematopoietic cells and usually negatively regulate immune responses. Galectins are mostly expressed by tumor cells and support tumor-cell survival. Selectins are vascular adhesion receptors that promote tumor-cell dissemination. All lectins facilitate interactions within the tumor microenvironment and thereby promote cancer progression. The identification of mechanisms how tumor glycans contribute to metastasis may help to improve diagnosis, prognosis, and aid to develop clinical strategies to prevent metastasis.

The role of platelet activation in tumor metastasis.

Platelets are highly reactive components of the circulatory system, which exert not only haemostatic activity but also contribute to the modulation of various pathological conditions including inflammation, atherosclerosis and cancer metastasis through the release of cytokines, chemokines and the presentation of several adhesion molecules. During cancer metastasis, the formation of platelet–tumor cell aggregates in the circulation facilitates immune evasion and the microvascular arrest of tumor cells at distant sites. Several adhesion molecules, such as integrins and glycoproteins, were shown to be involved in this process. Recent findings indicate that P-selectin is another main mediator of platelet–tumor cell interactions. Other effects of activated platelets on cancer progression are associated with a release of platelet-derived factors stimulating tumor growth and angiogenesis. Any interference in platelet–tumor cell interactions resulted in attenuation of cancer metastasis. The well recognized, albeit not fully characterized function of platelets during cancer progression defines platelets as potential targets for cancer therapy. Specifically, the rapid expression of P-selectin on the cell surface of activated platelets and its strong association with metastasis provide a rationale for P-selectin inhibition as an antimetastatic treatment.

Selectin Blocking Activity of a Fucosylated Chondroitin Sulfate Glycosaminoglycan from Sea Cucumber EFFECT ON TUMOR METASTASIS AND NEUTROPHIL RECRUITMENT

Heparin is an excellent inhibitor of P- and L-selectin binding to the carbohydrate determinant, sialyl Lewisx. As a consequence of its anti-selectin activity, heparin attenuates metastasis and inflammation. Here we show that fucosylated chondroitin sulfate (FucCS), a polysaccharide isolated from sea cucumber composed of a chondroitin sulfate backbone substituted at the 3-position of the β-d-glucuronic acid residues with 2,4-disulfated α-l-fucopyranosyl branches, is a potent inhibitor of P- and L-selectin binding to immobilized sialyl Lewisx and LS180 carcinoma cell attachment to immobilized P- and L-selectins. Inhibition occurs in a concentration-dependent manner. Furthermore, FucCS was 4–8-fold more potent than heparin in the inhibition of the P- and L-selectin-sialyl Lewisx interactions. No inhibition of E-selectin was observed. FucCS also inhibited lung colonization by adenocarcinoma MC-38 cells in an experimental metastasis model in mice, as well as neutrophil recruitment in two models of inflammation (thioglycollate-induced peritonitis and lipopolysaccharide-induced lung inflammation). Inhibition occurred at a dose that produces no significant change in plasma activated partial thromboplastin time. Removal of the sulfated fucose branches on the FucCS abolished the inhibitory effect in vitro and in vivo. Overall, the results suggest that invertebrate FucCS may be a potential alternative to heparin for blocking metastasis and inflammatory reactions without the undesirable side effects of anticoagulant heparin.

Tumor attenuation by combined heparan sulfate and polyamine depletion

Cells depend on polyamines for growth and their depletion represents a strategy for the treatment of cancer. Polyamines assemble de novo through a pathway sensitive to the inhibitor, α-difluoromethylornithine (DFMO). However, the presence of cell-surface heparan sulfate proteoglycans may provide a salvage pathway for uptake of circulating polyamines, thereby sparing cells from the cytostatic effect of DFMO. Here we show that genetic or pharmacologic manipulation of proteoglycan synthesis in the presence of DFMO inhibits cell proliferation in vitro and in vivo. In cell culture, mutant cells lacking heparan sulfate were more sensitive to the growth inhibitory effects of DFMO than wild-type cells or mutant cells transfected with the cDNA for the missing biosynthetic enzyme. Moreover, extracellular polyamines did not restore growth of mutant cells, but completely reversed the inhibitory effect of DFMO in wild-type cells. In a mouse model of experimental metastasis, DFMO provided in the water supply also dramatically diminished seeding and growth of tumor foci in the lungs by heparan sulfate-deficient mutant cells compared with the controls. Wild-type cells also formed tumors less efficiently in mice fed both DFMO and a xylose-based inhibitor of heparan sulfate proteoglycan assembly. The effect seemed to be specific for heparan sulfate, because a different xyloside known to affect only chondroitin sulfate did not inhibit tumor growth. Hence, combined inhibition of heparan sulfate assembly and polyamine synthesis may represent an additional strategy for cancer therapy.

Heparin attenuates metastasis mainly due to inhibition of P- and L-selectin, but non-anticoagulant heparins can have additional effects

Heparin and low molecular weight heparin (LMWH) are widely used for treatment of cancer patients with thrombosis, a common complication of malignant disease. Several recent prospective clinical studies indicate that heparin might improve outcomes of human cancer. Meanwhile, experimental evidence from mouse models consistently demonstrates that heparin efficiently inhibits metastasis. We have previously shown that P- and L-selectin play independent roles in supporting the initial stages of hematogeneous metastasis. Heparin is a known potent inhibitor of such selectin-mediated interactions. Here we provide evidence that the absence of both P- and L-selectin (PL -/- mice) dramatically improved survival in an experimental metastasis model. The use of clinically acceptable amounts of heparin did not further aff5ct metastasis rates in such mice. However, a non-anticoagulant derivative of heparin with P- and L-selectin inhibitory properties reduced metastasis to similar levels as observed in PL -/- mice. The virtual elimination of metastasis by a single treatment with a modified heparin without anticoagulant activity strongly suggests that heparin primarily reduces metastatic disease by inhibiting P- and L-selectin interactions. However, such heparins could have further effects at higher doses.

Inflammatory chemokines and metastasis – tracing the accessory

The tumor microenvironment consists of stromal cells and leukocytes that contribute to cancer progression. Cross-talk between tumor cells and their microenvironment is facilitated by a variety of soluble factors, including growth factors and cytokines such as chemokines. Due to a wide expression of chemokine receptors on cells in the tumor microenvironment, including tumor cells, chemokines affect various processes such as leukocyte recruitment, angiogenesis, tumor cell survival, tumor cell adhesion, proliferation, vascular permeability, immune suppression, invasion and metastasis. Inflammatory chemokines are instrumental players in cancer-related inflammation and significantly contribute to numerous steps during metastasis. Recruitment of myeloid-derived cells to metastatic sites is mainly mediated by the inflammatory chemokines CCL2 and CCL5. Tumor cell homing and extravasation from the circulation to distant organs are also regulated by inflammatory chemokines. Recent experimental evidence demonstrated that besides leukocyte recruitment, tumor cell-derived CCL2 directly activated endothelial cells and together with monocytes facilitated tumor cell extravasation, in a CCL2- and CCL5-dependent manner. Furthermore, CX3CL1 expression in the bone facilitated metastasis of CX3CR1 expressing tumor cells to this site. Current findings in preclinical models strongly suggest that inflammatory chemokines have an important role during metastasis and targeting of the chemokine axis might have a therapeutic potential.