Fatty acid modulation of tumor cell adhesion to microvessel endothelium and experimental metastasis

Tumor cell interaction with the endothelium of the vessel wall is a rate limiting step in metastasis. The fatty acid modulation of this interaction was investigated in low (LM) and high (HM) metastatic B16 amelanotic melanoma (B16a) cells. 12(S)-HETE increased the adhesion of LM cells to endothelium derived from pulmonary microvessels. All other monohydroxy and dihydroxy fatty acids were ineffective. LTB₄ induced a modest stimulation but LTC₄, LTD₄, LTE₄ as well as LXA₄ and LXB₄ were ineffective. The 12(S)-HETE enhanced adhesion of B16a cells was inhibited by pretreatment with 13(S)-HODE but not by 13(R)-, 9(S)-HODE or 13-OXO-ODE. 13(S)-HODE decreased adhesion of HM B16a cells to endothelium. 12(S)-HETE enhanced surface expression of integrin αIIbβ₃ and monoclonal antibodies against this integrin but not against α₅β₁, blocked enhanced but not basal adhesion to endothelium. Intravenous injection of 12(S)-HETE treated LM cells resulted in increased lung colonization (experimental metastasis). This effect was specific for 12(S)-HETE and was inhibited by 13(S)-HODE but not by other HODE's. 12(S)-HETE also enhanced lung colonization by HM cells and 13(S)-HODE decreased lung colonization by HM cells. Our results suggest a highly specific bidirectional modulation of metastatic phenotype and lung colonization by 12(S)-HETE and 13(S)-HODE.     

Galectin-9 suppresses tumor metastasis by blocking adhesion to endothelium and extracellular matrices

We previously described an inverse correlation between galectin-9 (Gal-9) expression and metastasis in patients with malignant melanoma and breast cancer. This study verified the ability of Gal-9 to inhibit lung metastasis in experimental mouse models using highly metastatic B16F10 melanoma and Colon26 colon cancer cells. B16F10 cells transfected with a secreted form of Gal-9 lost their metastatic potential. Intravenous Gal-9 administration reduced the number of metastases of both B16F10 and Colon26 cells in the lung, indicating that secreted Gal-9 suppresses metastasis. Analysis of adhesive molecule expression revealed that B16F10 cells highly express CD44, integrin alpha1, alpha 4, alpha V, and beta1, and that Colon26 cells express CD44, integrin alpha2, alpha 5, alpha V, and beta1, suggesting that Gal-9 may inhibit the adhesion of tumor cells to vascular endothelium and the extracellular matrix (ECM) by binding to such adhesive molecules. Indeed, Gal-9 suppressed the binding of hyaluronic acid to CD44 on both B16F10 and Colon26 cells, and also suppressed the binding of vascular cell adhesion molecule-1 to very late antigen-4 on B16F10 cells. Furthermore, Gal-9 inhibited the binding of tumor cells to ECM components, resulting in the suppression of tumor cell migration. The present results suggest that Gal-9 suppresses both attachment and invasion of tumor cells by inhibiting the binding of adhesive molecules on tumor cells to ligands on vascular endothelium and ECM.     

Prostaglandin E2 receptor modulation affects tumor cell adhesion to laminin.

We have shown previously that murine mammary adenocarcinoma cells both synthesize prostaglandin E2 (PGE2) and have a high affinity receptor for this ligand. Modulation of either PGE synthesis or PGE receptor function changes the metastatic potential of these cells. Because of the importance of laminin and laminin receptors to the metastatic process, we asked whether or not the PGE receptor participates in tumor cell-laminin interactions. As has been reported for many other tumor cells, laminin and the laminin-derived peptide PA22-2, containing the sequence IKVAV, mediate attachment of line 410.4 mammary tumor cells in vitro. We now demonstrate that the attachment of 410.4 cells to laminin or peptide PA22-2 was significantly inhibited by three PGE receptor antagonists, LE0101, SC19220, and sodium meclofenamate. LE0101 was most active, inhibiting tumor cell adhesion in a dose-dependent manner in the absence of nonspecific toxicity. These receptor antagonists had no effect on the PA22-2-mediated attachment of a PGE receptor negative tumor cell line, except at the highest concentration of LE0101 tested. No inhibition of adhesion to Type I collagen was seen. These results indicate that the PGE2 receptor modulates tumor cell adhesion to laminin which may subsequently affect the in vivo process of metastasis.     

Prostate tumor CXC-chemokine profile correlates with cell adhesion to endothelium and extracellular matrix

Though chemokines of the CXC family are thought to play key roles in neoplastic transformation and tumor invasion, information about CXC chemokines in prostate cancer is sparse. To evaluate the involvement of CXC chemokines in prostate cancer, we analyzed the CXC coding mRNA of both chemokine ligands (CXCL) and chemokine receptors (CXCR), using the prostate carcinoma cell lines PC-3, DU-145 and LNCaP. CXCR proteins were further evaluated by Western blot, CXCR surface expression by flow cytometry and confocal microscopy. The expression pattern was correlated to adherence of the tumor cells to an endothelial cell monolayer or to extracellular matrix components. Based on growth and adhesion capacity, PC-3 and DU-145 were identified to be highly aggressive tumor cells (PC-3>DU-145), whereas LNCaP belonged to the low aggressive phenotype. CXCL1, CXCL3, CXCL5 and CXCL6 mRNA, chemokines with pro-angiogenic activity, were strongly expressed in DU-145 and PC-3, but not in LNCaP. CXCR3 and CXCR4 surface level differed in the following order: LNCaP>DU-145>PC-3. The differentiation factor, fatty acid valproic acid, induced intracellular CXCR accumulation. Therefore, prostate tumor malignancy might be accompanied by enhanced synthesis of angiogenesis stimulating CXC chemokines. Further, shifting CXCR3 and CXCR4 from the cell surface to the cytoplasm might activate pro-tumoral signalling events and indicate progression from a low to a highly aggressive phenotype.     

Effect of cyclic RGD peptide on cell adhesion and tumor metastasis.

Several kinds of cyclic peptides containing an L-arginine-glycine-L-aspartic acid RGD sequence were synthesized by the liquid phase method, and we investigated their effects on the attachment of mouse B16 melanoma cells onto fibronectin-coated well. Cyclo (GRGDSPA) inhibited the cell attachment at a 20-fold lower concentration than the linear form. The cell adhesion was inhibited by the synthetic peptides with the following relative order of activity: cyclo (GRGDSPA) much greater than cyclo (GRGD) greater than cyclo (RGDS), cyclo (GRGDSP) greater than cyclo (GRGDS) greater than cyclo (RGDSP), cyclo (RGDSPA). Cyclo (GRGDSPA) was more effective at inhibiting cell attachment to vitronectin than it was at competing with fibronectin attachment, as reported in the case of GRGDSP. Moreover, cyclo (GRGDSPA) significantly reduced the formation of colonies in mice injected with B16-FE7 melanoma cells.     

Factors involved in cell adhesion to vascular endothelium

The adhesion of blood cells to endothelium can be studied in vitro using human endothelial cells in culture. This experimental model and radiometric techniques provide us with a simple system to quantify the adhesion of blood cells to endothelium. Normal human granulocytes isolated by density gradient adhere to normal endothelial cells in a proportion of 25%. Human promyelocytic cells (HL 60) induced by retinoic acid into mature cells adhere as well as normal granulocytes while the noninduced adhere poorly to endothelium. A small percentage of normal red cells attach to endothelial cells while red cells from patients with sickle cell anemia or diabetes mellitus have a significantly increased adhesion to endothelial cells (P greater than 0.001). This adhesion is statistically correlated with the extent and severity of vascular complications in diabetes mellitus (P less than 0.05). The addition of fibrinogen significantly increased (P less than 0.01) the adhesion of normal red cells, red cells from patients with sickle cell anemia or diabetes mellitus while gamma-globulins did not modify adhesion. Fibronectin potentiated the adhesion of normal red cells.     

Fatty acid and phospholipid chlorohydrins cause cell stress and endothelial adhesion

The oxidation of low-density lipoprotein (LDL) is thought to contribute to atherogenesis, which is an inflammatory disease involving activation of phagocytic cells. Myeloperoxidase, an enzyme which is able to produce hypochlorous acid (HOCl), is released from these phagocytic cells, and has been found in an active form in atherosclerotic plaques. HOCl can oxidize both the lipid and protein moiety of LDL, and HOCl-modified LDL has been found to be pro-inflammatory, although it is not known which component is responsible for this effect. As HOCl can oxidize lipids to give chlorohydrins, we hypothesized that phospholipid chlorohydrins might have toxic and pro-inflammatory effects. We have formed chlorohydrins from fatty acids (oleic, linoleic and arachidonic acids) and from phospholipids (stearoyl-oleoyl phosphatidylcholine, stearoyl-linoleoyl phosphatidylcholine and stearoyl-arachidonoyl phosphatidylcholine), and investigated various biological effects of these oxidation products. Fatty acid and phospholipid chlorohydrins were found to deplete ATP levels in U937 cells in a concentration-dependent manner, with significant effects observed at concentrations of 25 microM and above. Low concentrations (25 microM) of stearoyl-oleoyl phosphatidylcholine and stearoyl-arachidonoyl phosphatidylcholine chlorohydrins were also found to increase caspase-3 activity. Finally, stearoyl-oleoyl phosphatidylcholine chlorohydrin increased leukocyte adhesion to artery segments isolated from C57Bl/6 mice. These results demonstrate potentially harmful effects of lipid chlorohydrins, and suggest that they may contribute to some of the pro-inflammatory effects that HOCl-modified low density lipoprotein has been found to induce.     

Numerical study on the adhesion of a circulating tumor cell in a curved microvessel

Biomechanics and Modeling in Mechanobiology - The adhesion of a circulating tumor cell (CTC) in a three-dimensional curved microvessel was numerically investigated. Simulations were first performed...     

Control of tumor angiogenesis and metastasis through modulation of cell redox state

Redox reactions pervade all biology. The control of cellular redox state is essential for bioenergetics and for the proper functioning of many biological functions. This review traces a timeline of findings regarding the connections between redox and cancer. There is ample evidence of the involvement of cellular redox state on the different hallmarks of cancer. Evidence of the control of tumor angiogenesis and metastasis through modulation of cell redox state is reviewed and highlighted.     

Molecular fractionation and characterization of a Candida albicans fraction that increases tumor cell adhesion to hepatic endothelium

Systemic candidiasis remains a major complication among patients suffering from hematological malignancies and favors the development of hepatic metastasis. To contribute to the understanding of the underlying mechanisms, the aim of this study was to identify molecules that may increase tumor cell adhesion to hepatic endothelial cells. To this end, a well-established in vitro model was used to determine the enhancement of tumor cell adhesion induced by Candida albicans and its fractions. Different fractions were obtained according to their molecular weight (M _r) (five) or to their isoelectric point (pI) (four), using preparative electrophoresis and preparative isoelectric focusing, respectively, followed by affinity chromatography. The fraction that most enhanced melanoma cell adhesion to endothelium had an M _r range from 45 to 66 kDa. It was characterized using two-dimensional electrophoresis, and 14 proteins were identified by peptide mass fingerprinting: Dor14p, Fba1p, Pdi1p, Pgk1p, Idh2p, Mpg1p, Sfa1p, Ape3p, Ilv5p, Tuf1p, Act1p, Eno1p, Qcr2p, and Adh1p. Of these, several are related to the immunogenic response, and the latter seven belonged to the most reactive fraction according to their pI range, from 5 to 5.6. These findings could represent a step forward in the search for new targets, to suppress the pro-metastatic effect of C. albicans.     

Shear stress modulates tumour cell adhesion to the endothelium

The adhesion of breast adenocarcinoma cells (MDA-MB-231) to human umbilical vein endothelial cells (HUVEC) was studied in whole blood and under varying flow conditions. This study was done on HUVEC either kept under static conditions or pre-conditioned in flow for 2 hours at a shear stress of 5 or 13 dyn/cm(2). Coverslips coated by HUVEC were placed in a parallel plate perfusion chamber and perfused at a shear rate of 300 or 1500 sec(-1) with heparin-anticoagulated blood containing 111In labelled MDA-MB-231 cells. We report here the optimal conditions for studying the adhesion of MDA-MB-231 to endothelial cells under shear constraints corresponding to those observed into small and medium sized arteries.     

Mesenchymal stem cell adhesion to cardiac microvascular endothelium: activators and mechanisms

Circulating stem cells home within the myocardium, probably as the first step of a tissue regeneration process. This step requires adhesion to cardiac microvascular endothelium (CMVE). In this study, we studied mechanisms of adhesion between CMVE and mesenchymal stem cells (MSCs). Adhesion was studied in vitro and in vivo. Isolated 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled rat MSCs were allowed to adhere to cultured CMVE in static and dynamic conditions. Either CMVE or MSCs were pretreated with cytokines [IL-1beta, IL-3, IL-6, stem cell factor, stromal cell-derived factor-1, or TNF-alpha, 10 ng/ml]. Control or TNF-alpha-treated MSCs were injected intracavitarily in rat hearts in vivo. In baseline in vitro conditions, the number of MSCs that adhered to CMVE was highly dependent on the flow rate of the superfusing medium but remained significant at venous and capillary shear stress amplitudes. Activation of both CMVE and MSCs with TNF-alpha or IL-1beta before adhesion concentration dependently increased adhesion of MSCs at each studied level of shear stress. Consistently, in vivo, activation of MSCs with TNF-alpha before injection significantly enhanced cardiac homing of MSCs. TNF-alpha-induced adhesion could be completely blocked by pretreating either CMVE or MSCs with anti-VCAM-1 monoclonal antibodies but not by anti-ICAM-1 antibodies. Adhesion of circulating MSCs in the heart appears to be an endothelium-dependent process and is sensitive to modulation by activators of both MSCs and endothelium. Inflammation and the expression of VCAM-1 but not ICAM-1 on both cell types have a regulatory effect on MSC homing in the heart.     

Vascular adhesion protein 1 mediates binding of immunotherapeutic effector cells to tumor endothelium

Tumor-infiltrating lymphocytes (TIL) can be used as an immunotherapeutic tool to treat cancer. Success of this therapy depends on the homing and killing capacity of in vitro-activated and -expanded TIL. Vascular adhesion protein 1 (VAP-1) is an endothelial molecule that mediates binding of lymphocytes to vessels of inflamed tissue. Here, we studied whether VAP-1 is involved in binding of TIL, lymphokine-activated killer (LAK) cells, and NK cells to vasculature of the cancer tissue. We demonstrated that VAP-1 is expressed on the endothelium of cancer vasculature. The intensity and number of positive vessels varied greatly between the individual specimens, but it did not correlate with the histological grade of the cancer. Using an in vitro adhesion assay we showed that VAP-1 mediates adhesion of TIL, LAK, and NK cells to cancer vasculature. Treatment of the tumor sections with anti-VAP-1 Abs diminished the number of adhesive cells by 60%. When binding of different effector cell types was compared, it was evident that different cancer tissues supported the adhesion of TIL to a variable extent and LAK cells were more adhesive than TIL and NK cells to tumor vasculature. These data suggest that VAP-1 is an important interplayer in the antitumor response. Thus, by up-regulating the expression of VAP-1 in tumor vasculature, it can be possible to improve the effectiveness of TIL therapy.     

15-Hydroxyeicosatetraenoic acid stimulates migration of human retinal microvessel endothelium in vitro and neovascularization in vivo.

We evaluated 15-hydroxyeicosatetraenoic acid (15-HETE), a major arachidonic acid product of vascular endothelium and leukocytes, for its effect on neovascularization. In a modified Boyden chamber assay, 15-HETE (10-7 M) stimulated human retinal microvessel endothelial cell migration by 42 +/- 10% (mean +/- S.E.M., p less than 0.01). 12-HETE, a major arachidonic acid metabolite of platelets, had no such effect. Further studies in the rabbit corneal pocket assay revealed that 15-HETE stimulated neovascularization in vivo. Concentrations at which the in vivo effects were observed are within the range generated by several cell types and are achievable in human serum. 15-HETE stimulation of human endothelial cell migration in vitro and neovascularization in vivo suggests that it may play a role in vasoproliferative disorders.     

Role of cell surface carbohydrate moieties in monocytic cell adhesion to endothelium in vitro

Monocyte adhesion to endothelium represents the first step in the emigration of this leukocyte from blood to tissue during such pathologic and physiologic processes as atherosclerotic plaque development, wound healing, and inflammation. We have examined the role of carbohydrate moieties in the binding of mononuclear cells to endothelium in vitro. Wheat germ agglutinin (WGA) completely inhibited binding of the human monocytic cell line U937 to pig or human endothelial cells (EC). The inhibition was abolished by the presence of N-acetyl glucosamine, a preferred ligand for WGA. This sugar itself, however, had no effect on monocytic cell binding to EC, suggesting that WGA is inhibiting the cell-cell interaction by binding to a distinct sugar moiety. We tested a series of simple and phosphorylated sugars for the ability to inhibit U937 cell binding to EC. Two phosphorylated disaccharides, lactose-1-phosphate and maltose-1-phosphate, but not 14 other sugars, caused complete suppression of monocyte adhesion to EC. Among the inactive sugars were mannose-6-phosphate and fructose-1-phosphate, which have been shown by others to markedly suppress lymphocyte adhesion to EC. A nonionic detergent, n-octyl-beta-D-glucopyranoside (octyl glucoside), which contains a sugar group as a hydrophilic moiety, also inhibited U937 cell or human monocyte binding to human or porcine EC. The inhibition was observed at a nontoxic concentration of octyl glucoside and appeared to be due to an effect on the monocytic cell rather than the EC. When suboptimal doses of WGA and octyl glucoside were added in combination to the U937 cell-EC adhesion assay, the level of inhibition was greatly reduced when compared with either of the inhibitors alone, suggesting an interaction between these two blocking agents. Lactose-1-phosphate, but not octyl glucoside or WGA, blocked neutrophil adhesion to EC. In summary, our results indicate that specific cell surface carbohydrate groups are required for the adhesion of monocytes to the endothelium.     

Cell adhesion and metastasis.

There is no single phenotypic trait whose exclusive expression correlates universally with metastatic behaviour. Nevertheless, several lines of evidence point towards adhesive phenomena as mediating some crucial steps in the malignant spread of particular tumour types. These steps include detachment from the primary, invasion of the ECM including include detachment from the primary, invasion of the ECM including structures such as the basement membrane and organ capsules, and lodgement in the vessels of remote organs. Furthermore, there is evidence that adhesive phenomena can contribute at least in part to the selective patterns of spread shown by a variety of tumour cell types. The molecular dissection of the adhesive events involved in these processes has only just begun. It is clear already, however, that several different determinants are likely to be involved and the clinical application of this knowledge is unlikely to be immediate.     

Site-specific metastasis formation: Chemokines as regulators of tumor cell adhesion,motility and invasion

The metastatic spread of tumors is a well-coordinated process in which different types of cancers tend to form metastases in defined organs. The formation of site-specific metastases requires full compatibility between the intrinsic properties of the tumor cells and the tumor microenvironment. It was recently found that chemokines which are expressed in specific loci promote the adhesion, migration and invasion of tumor cells that express the corresponding receptor(s). Of the different members of the family, the CXCL12 chemokine and its cognate CXCR4 receptor are the prototypes of this process, although other members of the family (e.g. CCR7 and CCR10) also play a role in determination of the metastatic spread. This commentary addresses the fundamental roles of chemokines and their receptors in site-specific metastasis, with emphasis on CXCL12-CXCR4. The article also describes some of the efforts that were performed thus far in order to identify the intracellular components involved in this process. The focus is put on the roles played by proteins that regulate adhesion and migration of tumor cells in response to CXCL12, including mainly focal adhesion kinase (FAK), Pyk2/RAFTK and members of the Rho family of GTPases (RhoA, Rac, Cdc42). This is followed by discussion of open questions that need to be addressed in future research, and of the potential therapeutic implications of the findings that are available to date in this field.Key words: adhesion, cancer, chemokines, CXCL12, CXCR4, invasion, metastasis, migrationThe metastatic spread of tumors is not random. While certain types of cancer preferentially metastasize to particular organs, others “favor” different remote sites for metastasis formation. To form metastasis, tumor cells have to successfully complete a defined set of events that requires adequate properties of the tumor cells and of the target organs.^1–4 Of the different steps involved in this complex process, major emphasis was recently given to the events occurring at the target site, in which tumor cell arrest is followed by extravasation through the vessel wall, migration and invasiveness in the host organ, and finally by tumor cell proliferation and angiogenesis. Together, these steps enable the “seeding” of the cancer cells in distinct target organs and formation of site-specific metastases.^1–4Therefore, to succeed in the metastatic process, appropriate tumor cell properties need to join forces with a tumor-supporting microenvironment at the target site. In line with the “Seed and Soil” theory postulated by Stephaen Paget more than a century ago, an ample number of studies now indicate that full compatibility between the tumor cells and their surrounding milieu at the target organ is essential for site-specific metastasis formation.^1–3The extensive research that was performed on the metastatic process has led to identification of pivotal microenvironmental factors that may dictate the failure or success of the metastatic cascade. In this context, the focus was put on members of the chemokine superfamily. Chemokines are low molecular weight proteins whose activities are exerted primarily in the immunological context, where they induce the migration of leukocytes in response to chemotactic gradients. Chemokines regulate leukocyte homing to lymphoid organs in the course of normal hematopoiesis (homeostatic chemokines) or promote the extravasation of leukocytes to damaged/infected sites in inflammation (inflammatory chemokines). As such, chemokines that are released at specific sites either constitutively (usually homeostatic chemokines) or inducibly (mainly inflammatory chemokines), promote the adhesive properties of leukocytes that express the corresponding receptors, and activate the motility apparatus of such cells. Due to their chemotactic properties, the chemokines are viewed as indispensable regulators of leukocyte homing to specific sites in the body, and of the immune integrity of the host.^5–8Concurrently with the identification of the roles played by chemokines in immunity and inflammation, it became evident that specific chemokines are expressed in organs that are preferential sites of metastasis formation. These observations raised the possibility that chemokines that are present at specific organs promote the adhesion and migration of tumor cells that express the corresponding receptor(s), by that supporting tumor cell invasion and the establishment of metastases at these specific loci. In such a case, the chemokines may promote the “seeding” process of cancer cells in specific target organs and thereafter may also increase their ability to propagate at these sites. Such activities of the chemokines may thus contribute to site-specific metastasis formation, and may constitute an important determinant of the metastatic cascade.Along this rationale, the study by Zlotnik and his colleagues was the first to establish a firm link between chemokines and site-specific metastasis formation.⁹ In their study, the researchers focused on the chemokine CXCL12 which is expressed in lymph nodes, liver, lungs and bone, organs which constitute preferential metastatic sites in breast cancer. The study has demonstrated that breast tumor cells express CXCR4, the corresponding receptor for CXCL12 and that the chemokine induced migration and invasion properties in the tumor cells. Furthermore, by interfering with the intact activity of the CXCL12-CXCR4 axis, the authors have shown that formation of metastases in preferred organs was significantly inhibited, indicating that this chemokine and its receptor significantly dictate the specificity of the metastatic spread of breast tumor cells.In the years that followed, the above study was pursued by an extraordinarily high number of investigations, together establishing a new concept in the field of site-specific metastasis formation. Our current understanding of the metastatic process suggests a very important role to the chemokine-chemokine receptor axis, whereby chemokines that are produced at specific organs increase the adhesive, migratory and invasive properties of tumor cells that have reached these sites and express the corresponding receptor(s), by that promoting site-specific metastasis formation.^10–16The initial observations that were made on the contribution of the CXCL12-CXCR4 pair in breast cancer were soon followed by the demonstration that CXCR4 is expressed by almost all cancer types, suggesting that the CXCL12-CXCR4 pair may be involved in site-specific metastasis formation in a large number of malignant diseases. Alongside with the CXCL12-CXCR4 axis, other chemokines and their receptors were implied in organ-specific metastasis: it was suggested that CCR7 expression by tumor cells facilitates lymph node infiltration by the cancer cells and that CCR10 is involved in skin-directed metastasis by melanoma cells.^10–13The possibility that chemokines and their receptors contribute to site-specific metastasis has led to an extensive research in many cancer types. Not only that the expression of CXCR4 was detected in a large number of cancer types, in many of the cases CXCL12 induced adhesion, migration and invasion by the tumor cells. These in vitro findings suggested that CXCL12 promotes tumor cell activities that are required for the completion of steps that are essential for disease progression, leading to formation of metastases at specific organs. Indeed, such roles were confirmed for the CXCL12-CXCR4 pair in several of the tumor cell systems that were analyzed, by studies in animal model systems showing that impairment or induction of the activities of this axis significantly affected metastasis formation. Finally, the roles of CXCL12-CXCR4 in elevating site-specific establishment of metastases were substantiated in specific malignant diseases by clinical studies, correlating the degree of CXCR4 expression and its specific pattern of intracellular localization with formation of metastases at remote and specific organs in cancer patients. Together, these findings identified a key determinant that dictates the ability of specific tumor cells to establish metastases in a well-coordinated and site-directed process.^10–16However, as attractive as the role of the CXCL12-CXCR4 pair in organ-specific metastasis may be, several ambiguities still remain. The major difficulties emerge from the tumor-wide nature of CXCR4 expression, and from the implications of this phenomenon. The many different cancer types that express CXCR4 and adhere/migrate/invade in response to CXCL12 do not necessarily share the same metastatic pattern in cancer patients. Some form metastases in all or part of the organs that are enriched with CXCL12, while others establish metastases in other loci, in which CXCL12 is not a predominant constituent of the tissue. Obviously, it is possible that the CXCL12-CXCR4 pair acts alongside with other chemokine-chemokine receptor pairs, and that the end result is dictated by the equilibrium that exists in the target organs between different members of the family, acting on tumor cells that express the corresponding receptor/s. Therefore, the final consequence may reflect the activities of several chemokines and their receptors, including for example CCR7, CCR10 and/or CXCR7, the recently identified receptor for CXCL12.^16,17 However, based on the multifactorial nature of malignant diseases, it is quite obvious that chemokines and their receptors are not the only determinants of site-specific metastasis formation, and that intrinsic properties of the tumor cells other than chemokine receptors come into play, by responding to additional microenvironmental stimuli at specific body targets (Fig. 1).Open in a separate windowFigure 1Chemokines and additional microenvironmental factors in site-specific metastasis. Chemokines are important contributors to site-specific metastasis formation and they constitute a part of a very complex microenvironment that interacts with tumor cells that have reached the potential metastatic sites. In this respect, only a full compatibility between the chemokine/additional microenvironmental factor (one or more) and the corresponding properties of the tumor cells would support a successful and full-blown metastatic process. (A) The microenvironment counterpart of the process: successful establishment of metastasis is dictated by factors that are expressed at the microenvironment of the potential metastatic site(s), chemokines and others. (#1) Full compatibility between the factors that are found at the potential metastatic site (chemokines such as CXCL12 and others) and receptors that are expressed by the tumor cells supports the successful establishment of metastases. (#2, #3) The lack of essential chemokine/s, or of other tumor-supporting factor(s) reduces the efficacy of metastasis formation, or leads to failure of this process, despite the fact that receptors for both are expressed by the tumor cells. (B) The tumor counterpart of the process: successful establishment of metastasis is dictated by the array of receptors that are expressed by the tumor cells. Although the potential metastatic site is enriched with the essential tumor-promoting chemokines/additional microenvironmental factors, the metastatic process can not reach its maximal potential or fails, because the tumor cells lack the expression of the required receptor(s) for these factors (or express non-functional receptors). (C) The microenvironment at the potential metastatic site is diverse and complex, including a large array of pro-malignancy factors [chemokine(s) and other(s)] whose activities complement each other, therefore together amplifying the metastatic process. (#1) A microenvironment enriched with all the potential tumor-supporting factors can interact with tumor cells that express functional receptors for all these factors, together leading to the most intensified levels of metastasis. (#2, #3, #4) A microenvironment that lacks one or more of the essential factors would not enable the formation of full-blown metastatic process, although the tumor cells express the required receptors.The take home message is therefore that the roles played by chemokine receptors and their ligands in dictating the metastatic spread of tumors are important, but that they are one of several determinants that are involved in the site-specific metastatic process. The implications are mainly at the therapeutic arena, where the inhibition of the CXCL12-CXCR4 pair, for example, may prove inefficient in preventing the establishment of metastases. In addition, when the CXCL12-CXCR4 pair is considered as a test case, we need to clearly characterize the levels at which this pair acts, in order to identify potential targets for inhibition. One approach would be to inhibit the expression of CXCL12 and/or of CXCR4, by that taking the risk of impairing immune activities that are absolutely dependent on this pair. An alternative attitude would be to down-regulate the factors that induce CXCR4 expression by the tumor cells and/or the intracellular mechanisms leading to CXCL12-induced adhesive, migratory and invasive properties in tumor cells.The latter approach is challenging because it demands precise understanding of the mechanisms that regulate the expression of CXCR4 and of the signaling pathways it induces in tumor cells, together providing the correct conditions for successful “seeding” of the tumor cells at the target organ. Although a number of studies have identified factors that induce CXCR4 expression by tumor cells,^10–16 this avenue of research is just at its beginning. Furthermore, the molecular mechanisms that are induced by CXCL12 via CXCR4 are far from being elucidated, and only in a limited number of cases there is initial understanding of the intracellular mediators involved in tumor cell adhesion, migration and invasion in response to this chemokine.Thus far, many of the different studies that addressed the mechanisms involved in CXCL12-induced adhesion/migration/invasion show that actin filaments are polymerized following stimulation by the chemokine. Beyond this point, most of the investigations were sporadic and have analyzed several potential regulators of adhesion and migration. These included integrins, focal adhesion kinase (FAK), Pyk2/RAFTK, paxillin, Vav and members of the Rho family of GTPases, including RhoA, Rac1 and Cdc42. The number of studies that looked in depth into these issues, and directly associated the activation of these elements with functional regulation of chemokine-induced tumor cell invasion, through increased adhesion and migration, is surprisingly small. This is mainly so when one considers the importance of this field of research and its potential therapeutic implications.However, a more intensive and relatively informative research was performed on the mechanisms involved in CXCL12-CXCR4-induced adhesion/migration/invasion in two of the malignant diseases, breast cancer^9,18–23 and melanoma.^24–27 The studies in these two systems have provided initial insights into the complex net of interactions that exists between the different proteins that coordinate processes of cell adhesion and motility in response to CXCL12. In breast cancer, the different studies provided evidence to the direct involvement of FAK, Pyk2 and phosphatidylinositol 3 kinase (PI3K) in CXCL12-induced migration of the tumor cells.^18–20 In parallel, the studies by the group of Ganju have shown that CXCL12 upregulated the phosphorylation of a large number of proteins that are involved in formation of focal adhesions and in tumor cell motility: FAK, Pyk2, paxillin, Crk and Crk-L.^18,19 This group has also shown that PI3K was directly associated with the tyrosine phosphatase SHP2 and that SHP2 had an important role in the regulation of tumor cell chemotaxis. Combined with their observations regarding the roles of Cbl in control of motility, the authors suggested that stimulation of breast tumor cells by CXCL12 leads to migration processes that require FAK, Pyk2, PI3K, Cbl and SHP2. Furthermore, the authors suggested that Cbl, SHP2 and PI3K form a multimeric complex in response to CXCL12 stimulation, and that this complex is important for tumor cell motility.¹⁹ Since CXCL12 was also found to up-regulate matrix metalloproteinases (MMP) 2 and 9 in breast tumor cells,¹⁹ it is possible that the chemokine leads to increased tumor cell migration which is accompanied with matrix degradation, together supporting site-specific invasion and metastasis formation.In parallel to the studies that were done in breast cancer, major efforts were put by Texido and his colleagues to decipher the mechanisms involved in CXCL12-induced metastasis formation by melanoma cells. This group has provided a sequential and well-designed series of studies on the mechanisms contributing to CXCL12-induced adhesion and migration of the tumor cells. These investigations focused mainly on the activation of RhoA, Rac1 and Cdc42, GTPases that control the dynamics of the actin cytoskeleton and serve as major regulators of cell motility. The authors have shown that CXCL12 triggered in melanoma cells the activation of RhoA, Rac1 and Cdc42, however only RhoA and Rac1 were directly involved in melanoma cell invasion in response to CXCL12.²⁴ CXCL12-induced activation of RhoA and Rac1 has led to up-regulation of MT1-MMP expression, then giving rise to processing of pro-MMP-2 to mature MMP-2.^24,25 Therefore, similar to the findings in breast cancer, the activation of melanoma cells by CXCL12 can increase cell motility and in parallel promote degradation of the extracellular matrix. Together, these two processes can give rise to increased invasion by the tumor cells, which is potently induced by the chemokine.Further analyses by the same group have shed light on the mechanisms that activate RhoA and Rac in melanoma cells. Their findings demonstrated that stimulation of melanoma cells by CXCL12 triggered the activation of Jak, being an upstream event leading to Vav stimulation.²⁵ The activation by CXCL12 induced the phosphorylation of Vav1 and Vav2, and Vav1 phosphorylation correlated with increased quantities of Rac, and to a lesser extent of RhoA. Moreover, interference with Vav1 and Vav2 expression in the cells impaired substantially the activation of Rac and RhoA in response to CXCL12 in the melanoma cells and inhibited tumor cell invasion.²⁵Together, these findings indicate that activation of Jak leads directly or indirectly to Vav activation. Thereafter, Vav-induced activation of RhoA and Rac follows, promoting not only tumor cell motility but also degradation of basement membranes through the activation of MT1-MMP and than of MMP-2.^24,25 This complex chain of events is tightly controlled and careful investigation of the RhoA-mediated pathway indicated that it is oppositely regulated by specific Gα proteins: The stimulation of melanoma cells by CXCL12 has led to coupling of Gα_i to CXCR4, followed by Vav-RhoA activation and stimulation of tumor cell invasion; On the other hand, activation of Gα₁₃ by different measures gave rise to p190RhoGAP-mediated inactivation of RhoA, and to impairment of invasion,²⁶ in a process not involving Vav regulation. Similar indications were also found for Gα₁₂, where the expression of a constitutively active variant of this G protein induced defective RhoA activation.²⁶ Therefore, these findings indicate that activated Gα₁₃ and Gα₁₂ trigger similar RhoA-related functional responses in melanoma cells, and play important roles in regulating the metastatic spread in melanoma.The above findings suggest that it may be possible to inhibit the establishment of site-specific metastasis by targeting well-defined components that regulate tumor cell adhesion/migration/invasion in response to chemokines. Such selected intracellular molecules actually act as balancing factors, and their levels of expression and/or activities may dictate, at least to some extent the success or failure of metastasis formation. Accordingly, using again the CXCL12-CXCR4 axis as a test case, it was shown that breast tumor cell treatment by the tumor suppressor Slit has led to inhibition of breast cancer adhesion, chemotaxis and chemoinvasion.¹⁸ The activity of Slit was mediated by repression of FAK and Pyk2 phosphorylation, inhibition of PI3K and MAPK activation and reduced activities of MMP-2 and MMP-9.¹⁸ Also in breast cancer, it was found that the bisphosphonate Zoledronic acid (ZOL) inhibited the chemotaxis of tumor cells to CXCL12, in a process mediated by inhibition of RhoA and decreased expression of CXCR4.²¹ Based on their results, that authors suggested that the ZOL-induced reduction in RhoA activation has led to disorganization of the actin cytoskeleton, that was accompanied by a loss of stress fibers. These experiments in breast cancer manifest the potential strength of maneuvers that inhibit determinants that are required for completion of tumor cell invasion in processes triggered by chemokines via their receptors.Another example for the potential use of approaches that are based on key molecules in chemokine-induced processes and of their potential use for limitation of metastasis formation was provided in melanoma cells. In this case, the researchers addressed the possibility that inhibition of stimuli that activate Gα₁₃ in melanoma cells may reduce CXCL12-induced RhoA activation, and thus may limit the invasive properties of the tumor cells. Indeed, in this system the expression of a constitutively active form of Gα₁₃ (Gα₁₃QL) in melanoma cells has led to inhibition of RhoA activation in the tumor cells, as well as to inefficient formation of stress fibers and reduced generation of focal contacts. Importantly, although the over-expression of Gα₁₃QL in the tumor cells did not affect the formation of primary tumors, it did lead to a substantial inhibition in lung metastasis formation and to prolonged survival of the mice.²⁶ Together, the findings on Slit and ZOL in breast cancer, and on Gα₁₃QL in melanoma illustrate the potential therapeutic applicability of approaches that target specific components that act as determinants of adhesion, migration and invasion by tumor cells.The therapeutic potential of such approaches requires a very precise identification of the mechanisms involved in the ability of chemokines to increase adhesive, migratory and invasive properties in tumor cells. Moreover, the current information available in this field suggests that the processes are complex and probably are tumor cell- and/or tumor type-specific. To give one example, RhoA was found to be important in activation of melanoma cell migration and invasion, and possibly also in breast cancer.^21,24,25 However, such roles for RhoA are not trivial, since the regulatory mechanisms mediated by RhoA and signaling by members of the G12 family of heterotrimeric G proteins may differ in a variety of tumor cell types. Specifically, it was found that G12 proteins play in breast cancer opposite roles to those described in melanoma cells: Gα₁₂ and Gα₁₃ promoted breast tumor cell invasion, and Gα₁₂ signaling was required for metastasis²³ (but not for formation of primary tumors). Taken together with recent studies in prostate cancer, glioblastoma and Jurkat T cells, and with studies suggesting a role for G12-mediated signaling in RhoA activation,^23,28–30 it is possible that the activities of this family of Rho GTPases and of heterotrimeric G proteins depend on the cellular milieu of the tumor cells, and therefore are tumor cell- and/or tumor type-specific. However, one should also consider the fact that the mechanisms may depend on the experimental system used (some of the above did not include stimulation by CXCL12 or any other chemokine) and whether stimulation by a chemokine was included and not, further emphasizing the need for more intensive research in this respect.Overall, the above information illustrates the importance of chemokines and their receptors in site-specific metastatic dissemination and calls for improved characterization of the mechanisms by which they induce adhesion, motility and invasion by tumor cells, leading to site-specific metastasis. Therapeutic approaches that aim at the inhibition of specific intracellular molecular elements require very definite identification of the events occurring downstream of receptor triggering by the chemokine. It is essential to identify the full cascade of events that takes place and to keep in mind that the pathways may be different, or even opposite, in various tumor cells and in different malignancies. It is also very important to take the research one step further, and to elucidate by direct experimental means the roles of specific targets, and of their inhibitors in in vivo model tumor systems. It is also essential to determine whether in cancer patients there are associations between such elements and disease course and progression.To conclude, the design of therapeutic approaches aimed at inhibition of chemokine-induced metastatic spread and localization depends ultimately on integrated and multidisciplinary research that is based on extensive and thorough experimentations. Only such combined efforts may lead to improved understanding of basic mechanisms taking place in chemokine-mediated processes of site-specific metastasis, and to the development of better therapeutic means in the future.     

Fatty acid modulation of MCF-7 human breast cancer cell proliferation,apoptosis and differentiation

Epidemiological studies suggest that dietary polyunsaturated fatty acids (PUFA) may influence breast cancer progression and prognosis. In order to study potential mechanisms of action of fatty acid modulation of tumor growth, we studied, in vitro, the influence of n-3 and n-6 fatty acids on proliferation, cell cycle, differentiation and apoptosis of MCF-7 human breast cancer cells. Both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) inhibited the MCF-7 cell growth by 30% and 54%, respectively, while linoleic acid (LA) had no effect and arachidonic acid (AA) inhibited the cell growth by 30% (p < 0.05). The addition of vitamin E (10uM) to cancer cells slightly restored cell growth. The incubation of MCF-7 cells with PUFAs did not alter the cell cycle parameters or induce cell apoptosis. However, the growth inhibitory effects of EPA, DHA and AA were associated with cell differentiation as indicated by positive Oil-Red-O staining of the cells. Lipid droplet accumulation was increased by 65%, 30% and 15% in the presence of DHA, EPA and AA, respectively; (p < 0.05). These observations suggest that fatty acids may influence cellular processes at a molecular level, capable of modulating breast cancer cell growth.