An antibody for macrophage migration inhibitory factor suppresses tumour growth and inhibits tumour-associated angiogenesis

To verify the role of macrophage migration inhibitory factor (MIF) in tumourigenesis, we examined the effect of an anti-MIF antibody on tumour growth and angiogenesis. We inoculated murine colon adenocarcinoma cell line colon 26 cells subcutaneously into the flank in BALB/c mice. After nine days, we treated tumour-bearing mice with an anti-rat MIF antibody by intraperitoneal injection on days 9, 11, 13, 15, 17, 19 and 21. We found significant inhibition of tumour growth by this treatment from day 15 to day 22. Next, we implanted a chamber filled with colon 26 cells, which only passes soluble factors, in the subcutaneous fascia of the flank, and treated mice with the anti-rat MIF antibody at days 1, 3 and 5. By histological examination at day 6, angiogenesis within the subcutaneous fascia in contact with the chamber was markedly suppressed. In vitro, we added an anti-human MIF antibody to human umbilical vein endothelial cells (HUVEC) to evaluate its effect on cell growth by measurement of [3H]thymidine incorporation. We observed that the anti-MIF antibody significantly suppressed [3H]thymidine uptake by HUVEC. These results suggest the possibility that MIF is involved in tumourigenesis via promotion of angiogenesis.     

Receptor agonists of macrophage migration inhibitory factor

The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also promotes AMPK activation with potential benefits for response to myocardial infarction and ischemia-reperfusion. Structure-based molecular design has led to the discovery of not only antagonists, but also the first agonists of MIF-CD74 binding. The compounds contain a triazole core that is readily assembled via Cu-catalyzed click chemistry. The agonist and antagonist behaviors were confirmed via study of MIF-dependent ERK1/2 phosphorylation in human fibroblasts.     

Regulated secretion of macrophage migration inhibitory factor is mediated by a non-classical pathway involving an ABC transporter

The cytokine macrophage migration inhibitory factor (MIF) is inducibly secreted by immune cells and certain other cell types to critically participate in the regulation of the host immune response. However, MIF does not contain a N-terminal signal sequence and the mechanism of MIF secretion is unknown. Here we show in a model of endotoxin-stimulated THP-1 monocytes that MIF does not enter the endoplasmatic reticulum and that MIF secretion is not inhibited by monensin or brefeldin A, demonstrating that MIF secretion occurs via a non-classical export route. Glyburide and probenicide but not other typical inhibitors of non-classical protein export strongly block MIF secretion, indicating that the export pathway of MIF involves an ABCA1 transporter.     

Interplay between inflammation and cancer

Tumor-promoting inflammation is one of the hallmarks of cancer. It has been shown that cancer development is strongly influenced by both chronic and acute inflammation process. Progress in research on inflammation revealed a connection between inflammatory processes and neoplastic transformation, the progression of tumour, and the development of metastases and recurrences. Moreover, the tumour invasive procedures (both surgery and biopsy) affect the remaining tumour cells by increasing their survival, proliferation and migration. One of the concepts explaining this phenomena is an induction of a wound healing response. While in normal tissue it is necessary for tissue repair, in tumour tissue, induction of adaptive and innate immune response related to wound healing, stimulates tumour cell survival, angiogenesis and extravasation of circulating tumour cells. It has become evident that certain types of immune response and immune cells can promote tumour progression more than others. In this review, we focus on current knowledge on carcinogenesis and promotion of cancer growth induced by inflammatory processes.     

Macrophage migration inhibitory factor increases MMP-2 activity in DU-145 prostate cells

Macrophage migration inhibitory factor (MIF) is a cytokine expressed by a number of different cell types and has been detected in prostatic glandular epithelial cells by immunohistochemistry. The goal of this study was to determine if in vitro cultured prostate cells produce this protein and some of the effects of MIF on these cells. Proliferation of normal prostate cells, the BPH-1 and DU-145 established cell lines in the presence of MIF were assessed. ELISA was used to screen conditioned medium for the production of MIF, matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2). Zymogram electrophoresis gels determined the activities of secreted MMP-2. The amount of MIF in the conditioned medium detected after 72 h of growth in normal, BPH-1 and DU-145 cells was 2.9, 5.2 and 10.2 ng/ml/10(6)cells respectively. Exogenous addition of MIF (25 ng/ml) to cells cultured in vitro stimulated proliferation of all the cell types tested. MIF addition to proliferating DU-145 cells resulted in a two-fold increase in the relative amount of active MMP-2 as determined by zymogram gel analysis of conditioned medium.     

Macrophage migration inhibitory factor activates hypoxia-inducible factor in a p53-dependent manner

Background

Macrophage migration inhibitory factor (MIF) is not only a cytokine which has a critical role in several inflammatory conditions but also has endocrine and enzymatic functions. MIF is identified as an intracellular signaling molecule and is implicated in the process of tumor progression, and also strongly enhances neovascularization. Overexpression of MIF has been observed in tumors from various organs. MIF is one of the genes induced by hypoxia in an hypoxia-inducible factor 1 (HIF-1)-dependent manner.

Methods/Principal Findings

The effect of MIF on HIF-1 activity was investigated in human breast cancer MCF-7 and MDA-MB-231 cells, and osteosarcoma Saos-2 cells. We demonstrate that intracellular overexpression or extracellular administration of MIF enhances activation of HIF-1 under hypoxic conditions in MCF-7 cells. Mutagenesis analysis of MIF and knockdown of 53 demonstrates that the activation is not dependent on redox activity of MIF but on wild-type p53. We also indicate that the MIF receptor CD74 is involved in HIF-1 activation by MIF at least when MIF is administrated extracellularly.

Conclusion/Significance

MIF regulates HIF-1 activity in a p53-dependent manner. In addition to MIF''s potent effects on the immune system, MIF is linked to fundamental processes conferring cell proliferation, cell survival, angiogenesis, and tumor invasiveness. This functional interdependence between MIF and HIF-1α protein stabilization and transactivation activity provide a molecular mechanism for promotion of tumorigenesis by MIF.     

Cells behaving badly: a theoretical model for the Fas/FasL system in tumour immunology

One proposed mechanism of tumour escape from immune surveillance is tumour up-regulation of the cell surface ligand FasL, which can lead to apoptosis of Fas receptor (Fas) positive lymphocytes. Based upon this 'counterattack', we have developed a mathematical model involving tumour cell-lymphocyte interaction, cell surface expression of Fas/FasL, and their secreted soluble forms. The model predicts that (a) the production of soluble forms of Fas and FasL will lead to the down-regulation of the immune response; (b) matrix metalloproteinase (MMP) inactivation should lead to increased membrane FasL and result in a higher rate of Fas-mediated apoptosis for lymphocytes than for tumour cells. Recent studies on cancer patients lend support for these predictions. The clinical implications are two-fold. Firstly, the use of broad spectrum MMP inhibitors as anti-angiogenic agents may be compromised by their adverse effect on tumour FasL up-regulation. Also, Fas/FasL interactions may have an impact on the outcome of numerous ongoing immunotherapeutic trials since the final common pathway of all these approaches is the transduction of death signals within the tumour cell.     

Optimization of N-benzyl-benzoxazol-2-ones as receptor antagonists of macrophage migration inhibitory factor (MIF)

The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also exhibits keto–enol tautomerase activity, believed to be vestigial in mammals. Starting from a 1 μM hit from virtual screening, substituted benzoxazol-2-ones have been discovered as antagonists with IC₅₀ values as low as 7.5 nM in a tautomerase assay and 80 nM in a MIF–CD74 binding assay. Additional studies for one of the potent inhibitors demonstrated that it is not a covalent inhibitor of MIF and that it attenuates MIF-dependent ERK1/2 phosphorylation in human synovial fibroblasts.     

Lectin induction of lymphokines in cultured murine leukocytes

Antigens induce sensitized lymphocytes to undergo mitosis and to secrete soluble products, termed lymphokines, which modulate the immune response. Plant lectins are known to act as polyclonal lymphocyte mitogens and, in some cases, stimulate lymphocytes to produce lymphokines. In an effort to explore the relationship of specific cell surface glycoconjugates to the induction of mitosis and the production of lymphokine activities we have examined the ability of the mitogenic lectins, concanavalin A and Wistaria floribunda mitogen, and the nonmitogenic hemagglutinin from Wistaria floribunda seeds to stimulate the production of macrophage migration inhibition factor (MIF), macrophage chemotactic factor (CF), and lymphotoxin (LT). Concanavalin A causes lymphocytes to produce MIF and LT but no detectable CF activities. W. floribunda mitogen induces lymphocytes to produce soluble substances which exhibit all three lymphokine activities. The nonmitogenic W. floribunda agglutinin causes lymphocytes to produce MIF and CF but no observable LT activity. Within the sensitivity of the assays employed, the results indicate that mitogenesis is not a corequisite of the expression of either macrophage migration inhibition factor or lymphocyte-derived chemotactic factor but it may be associated with the induction of lymphotoxin. It is also apparent that the expression of each lymphokine activity is independent of the expression of the other lymphokines studied.     

Macrophage migration inhibitory factor plays a role in the regulation of microfold (M) cell-mediated transport in the gut

It has been shown previously that certain bacteria rapidly (3 h) up-regulated in vivo microfold cell (M cell)-mediated transport of Ag across the follicle-associated epithelium of intestinal Peyer's patch. Our aim was to determine whether soluble mediators secreted following host-bacteria interaction were involved in this event. A combination of proteomics and immunohistochemical analyses was used to identify molecules produced in the gut in response to bacterial challenge in vivo; their effects were then tested on human intestinal epithelial cells in vitro. Macrophage migration inhibitory factor (MIF) was the only cytokine produced rapidly after in vivo bacterial challenge by CD11c(+) cells located beneath the M cell-rich area of the follicle-associated epithelium of the Peyer's patch. Subsequently, in vitro experiments conducted using human Caco-2 cells showed that, within hours, MIF induced the appearance of cells that showed temperature-dependent transport of microparticles and M cell-specific bacterium Vibrio cholerae, and acquired biochemical features of M cells. Furthermore, using an established in vitro human M cell model, we showed that anti-MIF Ab blocked Raji B cell-mediated conversion of Caco-2 cells into Ag-sampling cells. Finally, we report that MIF(-/-) mice, in contrast to wild-type mice, failed to show increased M cell-mediated transport following in vivo bacterial challenge. These data show that MIF plays a role in M cell-mediated transport, and cross-talk between bacteria, gut epithelium, and immune system is instrumental in regulating key functions of the gut, including M cell-mediated Ag sampling.     

Role of the metastasis‐promoting protein osteopontin in the tumour microenvironment

Osteopontin (OPN) is a secreted protein present in bodily fluids and tissues. It is subject to multiple post‐translational modifications, including phosphorylation, glycosylation, proteolytic cleavage and crosslinking by transglutamination. Binding of OPN to integrin and CD44 receptors regulates signalling cascades that affect processes such as adhesion, migration, invasion, chemotaxis and cell survival. A variety of cells and tissues express OPN, including bone, vasculature, kidney, inflammatory cells and numerous secretory epithelia. Normal physiological roles include regulation of immune functions, vascular remodelling, wound repair and developmental processes. OPN also is expressed in many cancers, and elevated levels in patients’ tumour tissue and blood are associated with poor prognosis. Tumour growth is regulated by interactions between tumour cells and their tissue microenvironment. Within a tumour mass, OPN can be expressed by both tumour cells and cellular components of the tumour microenvironment, and both tumour and normal cells may have receptors able to bind to OPN. OPN can also be found as a component of the extracellular matrix. The functional roles of OPN in a tumour are thus complex, with OPN secreted by both tumour cells and cells in the tumour microenvironment, both of which can in turn respond to OPN. Much remains to be learned about the cross‐talk between normal and tumour cells within a tumour, and the role of multiple forms of OPN in these interactions. Understanding OPN‐mediated interactions within a tumour will be important for the development of therapeutic strategies to target OPN.     

Involvement of macrophage migration inhibitory factor (MIF) in the mechanism of tumor cell growth.

BACKGROUND: Macrophage migration inhibitory factor (MIF) was recently rediscovered as a cytokine, pituitary hormone, and glucocorticoid-induced immunomodulator. MIF is constitutively expressed in various cells and enhances production of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1, and interferon gamma. Recently, it was reported that MIF mRNA was overexpressed in prostatic tumors, which suggests that MIF is a protein involved in tumor cell growth beyond inflammatory and immune responses. MATERIALS AND METHODS: We examined the expression of MIF in the murine colon carcinoma cell line colon 26 by Western and Northern blot analyses and immunohistochemistry. Next, we investigated the effects of transforming growth factor (TGF) beta, basic fibroblast growth factor (b-FGF), and platelet-derived growth factor (PDGF) on the expression of MIF mRNA. Furthermore, we examined whether MIF is involved in tumor cell proliferation, using an MIF anti-sense plasmid transfection technique. RESULTS: We demonstrated that MIF protein and its mRNA were highly expressed in colon 26 cells, using Western and Northern blot analyses, respectively. By immunohistochemical analysis, we found that MIF was localized largely in the cytoplasm of the tumor cells. In response to TGF-beta, b-FGF, and PDGF, MIF mRNA expression was significantly up-regulated. Following this, we transfected the cells with an anti-sense MIF plasmid, which revealed that this treatment induced significant suppression of cell proliferation. CONCLUSION: Although MIF plays multifunctional roles in a broad spectrum of pathophysiological states, little has been done to investigate the role of this protein in association with tumor growth. The current results suggest the possibility that MIF induces tumor cell growth in concert with other growth factors, which encouraged us to investigate a novel approach for tumor therapy using an anti-MIF antibody and an MIF anti-sense plasmid transfection technique.     

Loss of macrophage migration inhibitory factor impairs the growth properties of human HeLa cervical cancer cells

Objectives: This study aims to determine the role of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with cell proliferation and tumour growth in vivo. Materials and methods: Our team used RNA interference technology to knock down MIF expression in human HeLa cervical cancer cells and to establish a stable cell line lacking MIF function. Results: Our results showed that long‐term loss of MIF had little effect on cell morphology, but significantly inhibited their population growth and proliferation. The HeLa MIF‐knockdown cells retained normal apoptotic signalling pathways in response to TNF‐alpha treatment; however, they exhibited unique DNA profiles following doxorubicin treatment, suggesting that MIF may regulate a cell cycle checkpoint upon DNA damage. Our data also showed that knockdown of MIF expression in HeLa cells led to increased cell adhesion and therefore impaired their migratory capacity. More importantly, cells lacking MIF failed to either proliferate in soft agar or form tumours in vivo, when administered to nude mice. Conclusion: MIF plays a pivotal role in proliferation and tumourigenesis of human HeLa cervical carcinoma cells, and may represent a promising therapeutic target for cancer intervention.     

Antisense macrophage migration inhibitory factor (MIF) prevents anti-IgM mediated growth arrest and apoptosis of a murine B cell line by regulating cell cycle progression

Macrophage migration inhibitory factor (MIF) is involved in the generation of cell-mediated immune responses. Recently it has been reported that MIF also plays a role in cell proliferation and differentiation. In the present study, using a B-cell line, WEHI-231, and its stable MIF-antisense transfectant, WaM2, as a representative transfectant, we investigated the mechanism underlying regulation of the cell growth by MIF. WaM2 cells produced less MIF than vector control or parental WEHI-231 cells. Reduced and increased proportions were seen in G1 and S-phase cells, respectively, in WaM2 as compared with WEHI-231. Growth arrest and apoptosis after stimulation via surface Ig (sIg) were less prominent in WaM2 cells than those in WEHI-231. However, the addition of recombinant rat MIF did not reverse the inhibition of the growth arrest and apoptosis induced in WaM2 by cross-linking sIg. Almost the same amount of p27kip1 expression was detected in WaM2 cells as those in WEHI-231 and vector control cells. Cross-linking of sIg elevated the p27kip1 level equally in these cells irrespective of the MIF-antisense expression. Taken together, it seems that MIF plays a role in inducing apoptosis in B cells upon IgM cross-linking by regulating the cell cycle via a novel intracellular pathway.     

Xenopus laevis macrophage migration inhibitory factor is essential for axis formation and neural development

Macrophage migration inhibitory factor (MIF) is an immunoregulatory cytokine involved in both acquired and innate immunity. MIF also has many functions outside the immune system, such as isomerase and oxidoreductase activities and control of cell proliferation. Considering the involvement of MIF in various intra- and extracellular events, we expected that MIF might also be important in vertebrate development. To elucidate the possible role of MIF in developmental processes, we knocked down MIF in embryos of the African clawed frog Xenopus laevis, using MIF-specific morpholino oligomers (MOs). For the synthesis of the MOs, we cloned a cDNA for a Xenopus homolog of MIF. Sequence analysis, determination of the isomerase activity, and x-ray crystallographic analysis revealed that the protein encoded by the cDNA was the ortholog of mammalian MIF. We carried out whole mount in situ hybridization of MIF mRNA and found that MIF was expressed at high levels in the neural tissues of normal embryos. Although early embryogenesis of MO-injected embryos proceeded normally until the gastrula stage, their neurulation was completely inhibited. At the tailbud stage, the MO-injected embryos lacked neural and mesodermal tissues, and also showed severe defects in their head and tail structures. Thus, MIF was found to be essential for axis formation and neural development of Xenopus embryos.