Beatriz E. Rendon

A novel, macrophage migration inhibitory factor suicide substrate inhibits motility and growth of lung cancer cells

Although chemokine and growth factor receptors are attractive and popular targets for cancer therapeutic intervention, structure-based targeting of the ligands themselves is generally not considered practical. New evidence indicates that a notable exception to this is macrophage migration inhibitory factor (MIF). MIF, an autocrine- and paracrine-acting cytokine/growth factor, plays a pivotal role in both the initiation and maintenance of neoplastic diseases. MIF possesses a nonphysiologic enzymatic activity that is evolutionarily well-conserved. Although small molecule antagonists of MIFs enzymatic active site have been reported to inhibit biological activities of MIF, universally high IC(50)s have limited their clinical appeal. Using a computational virtual screening strategy, we have identified a unique small molecule inhibitor that serves as a suicide substrate for MIF, resulting in the covalent modification of the catalytically active NH(2)-terminal proline. Our studies further reveal that this compound, 4-iodo-6-phenylpyrimidine (4-IPP), is approximately 5x to 10x times more potent in blocking MIF-dependent catalysis and lung adenocarcinoma cell migration and anchorage-independent growth than the prototypical MIF inhibitor, ISO-1. Finally, using an in silico combinatorial optimization strategy, we have identified four unique congeners of 4-IPP that exhibit MIF inhibitory activity at concentrations 10x to 20x lower than that of parental 4-IPP.

Regulation of Human Lung Adenocarcinoma Cell Migration and Invasion by Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is expressed and secreted in response to mitogens and integrin-dependent cell adhesion. Once released, autocrine MIF promotes the activation of RhoA GTPase leading to cell cycle progression in rodent fibroblasts. We now report that small interfering RNA-mediated knockdown of MIF and MIF small molecule antagonism results in a greater than 90% loss of both the migratory and invasive potential of human lung adenocarcinoma cells. Correlating with these phenotypes is a substantial reduction in steady state as well as serum-induced effector binding activity of the Rho GTPase family member, Rac1, in MIF-deficient cells. Conversely, MIF overexpression by adenovirus in human lung adenocarcinoma cells induces a dramatic enhancement of cell migration, and co-expression of a dominant interfering mutant of Rac1 (Rac1N17) completely abrogates this effect. Finally, our results indicate that MIF depletion results in defective partitioning of Rac1 to caveolin-containing membrane microdomains, raising the possibility that MIF promotes Rac1 activity and subsequent tumor cell motility through lipid raft stabilization.

Amplification of Tumor Hypoxic Responses by Macrophage Migration Inhibitory Factor–Dependent Hypoxia-Inducible Factor Stabilization

Low oxygen tension-mediated transcription by hypoxia-inducible factors (HIF) has been reported to facilitate tumor progression, therapeutic resistance, and metastatic adaptation. One previously described target of hypoxia-mediated transcription is the cytokine/growth factor macrophage migration inhibitory factor (MIF). In studies designed to better understand hypoxia-stimulated MIF function, we have discovered that not only is MIF induced by hypoxia in pancreatic adenocarcinoma but MIF is also necessary for maximal hypoxia-induced HIF-1alpha expression. Cells lacking MIF are defective in hypoxia- and prolyl hydroxylase inhibitor-induced HIF-1alpha stabilization and subsequent transcription of glycolytic and angiogenic gene products. Moreover, COP9 signalosome subunit 5 (CSN5), a component of the COP9 signalosome previously reported to functionally interact with MIF, has recently been shown to interact with and stabilize HIF-1alpha. Our results indicate that MIF interacts with CSN5 in pancreatic cancer cells and that MIF-depleted cells display marked defects in hypoxia-induced CSN5/HIF-1alpha interactions. This functional interdependence between HIF-1alpha and MIF may represent an important and previously unrecognized pro-tumorigenic axis.

Rho GTPase-dependent Signaling Is Required for Macrophage Migration Inhibitory Factor-mediated Expression of Cyclin D1

Our previous studies demonstrated that the proinflammatory peptide, macrophage migration inhibitory factor (MIF), functions as an autocrine mediator of both growth factor- and integrin-dependent sustained ERK MAPK activation, cyclin D1 expression, and cell cycle progression. We now report that MIF promotes the activation of the canonical ERK MAPK cascade and cyclin D1 expression by stimulating the activity of the Rho GTPase and downstream signaling to stress fiber formation. Rho-dependent stress fiber accumulation promotes the sustained activation of ERK and subsequent cyclin D1 expression during G1-S phase cell cycle progression. This pathway is reported to be dependent upon myosin light chain (MLC) kinase, integrin clustering, and subsequent activation of focal adhesion kinase, leading to sustained MAPK activity. Our studies reveal that recombinant MIF induces cyclin D1 expression in a Rho-, Rho kinase-, MLC kinase-, and ERK-dependent manner in asynchronous NIH 3T3 fibroblasts. Moreover, MIF–/– murine embryonic fibroblasts display aberrant cyclin D1 expression that is linked to defective Rho activity, stress fiber formation, and MLC phosphorylation. These results suggest that MIF is an integral autocrine mediator of Rho GTPase-dependent signaling events and provide mechanistic insight into how MIF regulates proliferative, migratory, and oncogenic processes.

Cooperative Regulation of Non-Small Cell Lung Carcinoma Angiogenic Potential by Macrophage Migration Inhibitory Factor and Its Homolog, d-Dopachrome Tautomerase

Tumor-derived growth factors and cytokines stimulate neoangiogenesis from surrounding capillaries to support tumor growth. Recent studies have revealed that macrophage migration inhibitory factor (MIF) expression is increased in lung cancer, particularly non-small cell lung carcinomas (NSCLC). Because MIF has important autocrine effects on normal and transformed cells, we investigated whether autocrine MIF and its only known family member, d-dopachrome tautomerase (D-DT), promote the expression of proangiogenic factors CXCL8 and vascular endothelial growth factor in NSCLC cells. Our results demonstrate that the expression of CXCL8 and vascular endothelial growth factor are strongly reliant upon both the individual and cooperative activities of the two family members. CXCL8 transcriptional regulation by MIF and D-DT appears to involve a signaling pathway that includes the activation of JNK, c-jun phosphorylation, and subsequent AP-1 transcription factor activity. Importantly, HUVEC migration and tube formation induced by supernatants from lung adenocarcinoma cells lacking either or both MIF and D-DT are substantially reduced when compared with normal supernatants. Finally, we demonstrate that the cognate MIF receptor, CD74, is necessary for both MIF- and D-DT-induced JNK activation and CXCL8 expression, suggesting its potential involvement in angiogenic growth factor expression. This is the first demonstration of a biological role for D-DT, and its synergism with MIF suggests that the combined therapeutic targeting of both family members may enhance current anti-MIF-based therapies.

Control of Tumor-Associated Macrophage Alternative Activation by Macrophage Migration Inhibitory Factor

Tumor stromal alternatively activated macrophages are important determinants of antitumor T lymphocyte responses, intratumoral neovascularization, and metastatic dissemination. Our recent efforts to investigate the mechanism of macrophage migration inhibitory factor (MIF) in antagonizing antimelanoma immune responses reveal that macrophage-derived MIF participates in macrophage alternative activation in melanoma-bearing mice. Both peripheral and tumor-associated macrophages (TAMs) isolated from melanoma bearing MIF-deficient mice display elevated proinflammatory cytokine expression and reduced anti-inflammatory, immunosuppressive, and proangiogenic gene products compared with macrophages from tumor-bearing MIF wild-type mice. Moreover, TAMs and myeloid-derived suppressor cells from MIF-deficient mice exhibit reduced T lymphocyte immunosuppressive activities compared with those from their wild-type littermates. Corresponding with reduced tumor immunosuppression and neo-angiogenic potential by TAMs, MIF deficiency confers protection against transplantable s.c. melanoma outgrowth and melanoma lung metastatic colonization. Finally, we report for the first time, to our knowledge, that our previously discovered MIF small molecule antagonist, 4-iodo-6-phenylpyrimidine, recapitulates MIF deficiency in vitro and in vivo, and attenuates tumor-polarized macrophage alternative activation, immunosuppression, neoangiogenesis, and melanoma tumor outgrowth. These studies describe an important functional contribution by MIF to TAM alternative activation and provide justification for immunotherapeutic targeting of MIF in melanoma patients.

Mechanisms of macrophage migration inhibitory factor (MIF)-dependent tumor microenvironmental adaptation

Since its activity was first reported in the mid-1960s, macrophage migration inhibitory factor (MIF) has gone from a cytokine activity modulating monocyte motility to a pleiotropic regulator of a vast array of cellular and biological processes. Studies in recent years suggest that MIF contributes to malignant disease progression on several different levels. Both circulating and intracellular MIF protein levels are elevated in cancer patients and MIF expression reportedly correlates with stage, metastatic spread and disease-free survival. Additionally, MIF expression positively correlates with angiogenic growth factor expression, microvessel density and tumor-associated neovascularization. Not coincidentally, MIF has recently been shown to contribute to tumoral hypoxic adaptation by promoting hypoxia-induced HIF-1alpha stabilization. Intriguingly, hypoxia is a strong regulator of MIF expression and secretion, suggesting that hypoxia-induced MIF acts as an amplifying factor for both hypoxia and normoxia-associated angiogenic growth factor expression in human malignancies. Combined, these findings suggest that MIF overexpression contributes to tumoral hypoxic adaptation and, by extension, therapeutic responsiveness and disease prognosis. This review summarizes recent literature on the contributions of MIF to tumor-associated angiogenic growth factor expression, neovascularization and hypoxic adaptation. We also will review recent efforts aimed at identifying and employing small-molecule antagonists of MIF as a novel approach to cancer therapeutics.

The MIF Homologue D-Dopachrome Tautomerase Promotes COX-2 Expression through β-Catenin–Dependent and –Independent Mechanisms

The cytokine/growth factor, macrophage migration inhibitory factor (MIF), contributes to pathologies associated with immune, inflammatory, and neoplastic disease processes. Several studies have shown an important contributing role for MIF-dependent COX-2 expression in the progression of these disorders. We now report that the MIF homologue, D-dopachrome tautomerase (D-DT), is both sufficient and necessary for maximal COX-2 expression in colorectal adenocarcinoma cell lines. D-DT–dependent COX-2 transcription is mediated in part by β-catenin protein stabilization and subsequent transcription. Also contributing to D-DTs regulation of COX-2 expression are the activities of both c-jun-N-terminal kinase and the MIF-interacting protein, Jab1/CSN5. Interestingly, D-DT–dependent β-catenin stabilization is regulated by COX-2 expression, suggesting the existence of an amplification loop between COX-2- and β-catenin–mediated transcription in these cells. Because both COX-2- and β-catenin–mediated transcription are important contributors to colorectal cancer (CRC) disease maintenance and progression, these findings suggest a unique and novel regulatory role for MIF family members in CRC pathogenesis. Mol Cancer Res; 8(12); 1601–9. ©2010 AACR.

Negative Regulation of AMP-activated Protein Kinase (AMPK) Activity by Macrophage Migration Inhibitory Factor (MIF) Family Members in Non-small Cell Lung Carcinomas

Background: Macrophage migration inhibitory factor (MIF) family members promote AMP-activated protein kinase (AMPK) activation in nontransformed cells. Results: MIF family members cooperatively inhibit AMPK in lung adenocarcinoma cell lines. Conclusion: Inhibition of AMPK activation in lung adenocarcinoma cells by MIF family members promotes efficient mTOR activation. Significance: Therapeutic targeting of MIF family members may benefit lung cancer patients. AMP-activated protein kinase (AMPK) is a nutrient- and metabolic stress-sensing enzyme activated by the tumor suppressor kinase, LKB1. Because macrophage migration inhibitory factor (MIF) and its functional homolog, d-dopachrome tautomerase (d-DT), have protumorigenic functions in non-small cell lung carcinomas (NSCLCs) but have AMPK-activating properties in nonmalignant cell types, we set out to investigate this apparent paradox. Our data now suggest that, in contrast to MIF and d-DTs AMPK-activating properties in nontransformed cells, MIF and d-DT act cooperatively to inhibit steady-state phosphorylation and activation of AMPK in LKB1 wild type and LKB1 mutant human NSCLC cell lines. Our data further indicate that MIF and d-DT, acting through their shared cell surface receptor, CD74, antagonize NSCLC AMPK activation by maintaining glucose uptake, ATP production, and redox balance, resulting in reduced Ca2+/calmodulin-dependent kinase kinase β-dependent AMPK activation. Combined, these studies indicate that MIF and d-DT cooperate to inhibit AMPK activation in an LKB1-independent manner.

MIF family members cooperatively inhibit p53 expression and activity.

The tumor suppressor p53 is induced by genotoxic stress in both normal and transformed cells and serves to transcriptionally coordinate cell cycle checkpoint control and programmed cell death responses. Macrophage migration inhibitory factor (MIF) is an autocrine and paracrine acting cytokine/growth factor that promotes lung adenocarcinoma cell motility, anchorage-independence and neo-angiogenic potential. Several recent studies indicate that the only known homolog of MIF, D-dopachrome tautomerase (D-DT - also referred to as MIF-2), has functionally redundant activities with MIF and cooperatively promotes MIF-dependent pro-tumorigenic phenotypes. We now report that MIF and D-DT synergistically inhibit steady state p53 phosphorylation, stabilization and transcriptional activity in human lung adenocarcinoma cell lines. The combined loss of MIF and D-DT by siRNA leads to dramatically reduced cell cycle progression, anchorage independence, focus formation and increased programmed cell death when compared to individual loss of MIF or D-DT. Importantly, p53 mutant and p53 null lung adenocarcinoma cell lines were only nominally rescued from the cell growth effects of MIF/D-DT combined deficiency suggesting only a minor role for p53 in these transformed cell growth phenotypes. Finally, increased p53 activation was found to be independent of aberrantly activated AMP-activated protein kinase (AMPK) that occurs in response to MIF/D-DT-deficiency but is dependent on reactive oxygen species (ROS) that mediate aberrant AMPK activation in these cells. Combined, these findings suggest that both p53 wildtype and mutant human lung adenocarcinoma tumors rely on MIF family members for maximal cell growth and survival.