Macrophage migration inhibitory factor engages PI3K/Akt signalling and is a prognostic factor in metastatic melanoma.
ABSTRACT: BACKGROUND: Macrophage migration inhibitory factor (MIF) is a widely expressed cytokine involved in a variety of cellular processes including cell cycle regulation and the control of proliferation. Overexpression of MIF has been reported in a number of cancer types and it has previously been shown that MIF is upregulated in melanocytic tumours with the highest expression levels occurring in malignant melanoma. However, the clinical significance of high MIF expression in melanoma has not been reported. METHODS: MIF expression was depleted in human melanoma cell lines using siRNA-mediated gene knockdown and effects monitored using in vitro assays of proliferation, cell cycle, apoptosis, clonogenicity and Akt signalling. In silico analyses of expression microarray data were used to correlate MIF expression levels in melanoma tumours with overall patient survival using a univariate Cox regression model. RESULTS: Knockdown of MIF significantly decreased proliferation, increased apoptosis and decreased anchorage-independent growth. Effects were associated with reduced numbers of cells entering S phase concomitant with decreased cyclin D1 and CDK4 expression, increased p27 expression and decreased Akt phosphorylation. Analysis of clinical outcome data showed that MIF expression levels in primary melanoma were not associated with outcome (HR = 1.091, p = 0.892) whereas higher levels of MIF in metastatic lesions were significantly associated with faster disease progression (HR = 2.946, p = 0.003 and HR = 4.600, p = 0.004, respectively in two independent studies). CONCLUSIONS: Our in vitro analyses show that MIF functions upstream of the PI3K/Akt pathway in human melanoma cell lines. Moreover, depletion of MIF inhibited melanoma proliferation, viability and clonogenic capacity. Clinically, high MIF levels in metastatic melanoma were found to be associated with faster disease recurrence. These findings support the clinical significance of MIF signalling in melanoma and provide a strong rationale for both targeting and monitoring MIF expression in clinical melanoma.
Project description:<h4>Background</h4>Macrophage migration inhibitory factor (MIF) has been proposed as a link between inflammation and tumorigenesis. Despite its potentially broad influence in tumour biology and prevalent expression, the value of MIF as a therapeutic target in cancer remains unclear. We sought to validate MIF in tumour models by achieving a complete inhibition of its expression in tumour cells and in the tumour stroma.<h4>Methods</h4>We used MIF shRNA-transduced B16-F10 melanoma cells implanted in wild-type and MIF-/- C57Bl6 mice to investigate the effect of loss of MIF on tumour growth. Cytokine detection and immunohistochemistry (IHC) were used to evaluate tumours ex vivo.<h4>Results</h4>Macrophage migration inhibitory factor shRNA inhibited expression of MIF protein by B16-F10 melanoma cells in vitro and in vivo. In vitro, the loss of MIF in this cell line resulted in a decreased response to hypoxia as indicated by reduced expression of VEGF. In vivo the growth of B16-F10 tumours was inhibited by an average of 47% in the MIF-/- mice compared with wild-type but was unaffected by loss of MIF expression by the tumour cells. Immunohistochemistry analysis revealed that microvessel density was decreased in tumours implanted in the MIF-/- mice. Profiling of serum cytokines showed a decrease in pro-angiogenic cytokines in MIF-/- mice.<h4>Conclusion</h4>We report that the absence of MIF in the host resulted in slower tumour growth, which was associated with reduced vascularity. While the major contribution of MIF appeared to be in the regulation of angiogenesis, tumour cell-derived MIF played a negligible role in this process.
Project description:Melanoma is believed to be a highly immunogenic tumor and recent developments in immunotherapies are promising. IFN-? produced by immune cells has a crucial role in tumor immune surveillance; however, it has also been reported to be pro-tumorigenic. In the current study, we found that IFN-? enhances the expression of CD74, which interacts with its ligand, macrophage migration inhibitory factor (MIF), and thereby activates the PI3K/AKT pathway in melanoma, promoting tumor survival. IFN-? increased phosphorylation of AKT Ser473 and upregulated total cell surface expression of CD74 in human melanoma cell lines tested. CD74 was highly expressed in melanoma tissues. Moreover, the expression of CD74 on tumor cells correlated with plasma IFN-? levels in melanoma patient samples. In our analysis of melanoma cell lines, all produced MIF constitutively. Blockade of CD74-MIF interaction reduced AKT phosphorylation and expression of pro-tumorigenic molecules, including IL-6, IL-8, and BCL-2. Inhibition of CD74-MIF interaction significantly suppressed tumor growth in the presence of IFN-? in our xenograft mouse model. Thus, we conclude that IFN-? promotes melanoma cell survival by regulating CD74-MIF signaling, suggesting that targeting the CD74-MIF interaction under IFN-?-stimulatory conditions would be an effective therapeutic approach for melanoma.
Project description:Macrophage migration inhibitory factor (MIF), a proinflammatory and immunoregulatory chemokine, plays important roles in cancer-related biological processes. However, few studies have focused on the clinical relevance of MIF and cyclin D1 expression in hepatocellular carcinoma cells (HCCs). In this study, MIF and cyclin D1 expression levels in HCC tissues and cell lines were significantly upregulated compared with adjacent normal tissues or a normal liver cell line. In HCC specimens, MIF expression positively correlated with cyclin D1 expression. Additionally, MIF and cyclin D1 expression positively correlated with tumor size. MIF knockdown inhibited the proliferation of PLC and HepG2 cells and promoted apoptosis. However, small interfering RNA (siRNA) against MIF did not influence the cell cycle in these cells. In an in vivo xenograft model, MIF knockdown reduced the tumor growth rate. The expression levels of Bcl-2, p-caspase-3, BIM and Bax were upregulated, while the expression levels of cyclin D1, p-Akt and p-ERK were downregulated in MIF-knockdown cells. These findings indicate that MIF siRNA reduces proliferation and increases apoptosis in HCC cells. MIF knockdown inhibits the expression of growth-related proteins and induces the expression of apoptosis-related proteins, supporting a role for MIF as a novel therapeutic target for HCC.
Project description:Deficiency of the tumour suppressor merlin leads to the development of schwannomas, meningiomas and ependymomas occurring spontaneously or as a part of the hereditary disease Neurofibromatosis type 2 (NF2). Merlin loss is also found in a proportion of other cancers like mesothelioma, melanoma, breast cancer and glioblastoma. The tumour suppressor/transcription factor p53 regulates proliferation, survival and differentiation and its deficiency plays a role in the development of many tumours. 53 can be negatively regulated by FAK, PI3K/AKT and MDM2 and possibly positively regulated by merlin in different cell lines. In this study we investigated the role of p53 in merlin-deficient tumours. Using our in vitro model of primary human schwannoma cells we have previously demonstrated that FAK is overexpressed/activated and localises into the nucleus of schwannoma cells increasing proliferation. AKT is strongly activated via platelet-derived growth factor (PDGF) - and insulin-like growth factor 1 (IGF1) - receptors increasing survival. Here we investigated p53 regulation and its role in proliferation and survival of human primary schwannoma cells using western blotting, immunocytochemistry, immunohistochemistry and proliferation, survival and transcription factor assays. In human primary schwannoma cells p53 was found to be downregulated while MDM2 was upregulated leading to increased cell proliferation and survival. p53 is regulated by merlin involving FAK, AKT and MDM2. Merlin reintroduction into schwannoma cells increased p53 levels and activity, and treatment with Nutlin-3, a drug which increases p53 stability by disrupting the p53/MDM2 complex, decreased tumour growth and reduced cell survival. These findings are important to dissect the mechanisms responsible for the development of merlin-deficient tumours and to identify new therapeutic targets. We suggest that Nutlin-3, possibly in combination with FAK or PI3K inhibitors, can be employed as a novel treatment for schwannoma and other merlin-deficient tumours.
Project description:Isoliquiritigenin (ISL), a member of the flavonoids, is known to have anti-tumor activity in vitro and in vivo. The effect of ISL on reprogramming in cancer cells, however, remains elusive. In this study, we investigated the effect of ISL on reprogramming in human melanoma A375 cells. ISL (15 ?g/ml) significantly inhibited A375 cell proliferation, anchorage independent cell proliferation and G2/M cell cycle arrest after ISL exposure for 24 h. However, there were no significant changes in apoptosis rate. Terminal differentiation indicators (melanin content, melanogenesis mRNA expression, tyrosinase (TYR) activity) were all up-regulated by ISL treatment. In ISL-treated cells, glucose uptake, lactate levels and mRNA expression levels of GLUT1 and HK2 were significantly decreased, and accompanied by an increase in O2 consumption rate (OCR) and adenosine triphosphate (ATP) deficiency. Protein expression levels of mTORC2-AKT-GSK3? signaling pathway components (mTOR, p-mTOR, RICTOR, p-AKT, p-GSK3?) decreased significantly after ISL treatment. Co-treatment of ISL and the mTOR-specific inhibitor Ku-0063794 had a synergistic effect on the inhibition of proliferation, and increased melanin content and TYR activity. Glucose uptake and lactate levels decreased more significantly than treatment with ISL alone. These findings indicate that ISL induced reprogramming in A375 melanoma cells by activating mTORC2-AKT-GSK3? signaling.
Project description:NADPH oxidase 5 (NOX5) generated reactive oxygen species (ROS) have been implicated in signaling cascades that regulate cancer cell proliferation. To evaluate and validate NOX5 expression in human tumors, we screened a broad range of tissue microarrays (TMAs), and report substantial overexpression of NOX5 in malignant melanoma and cancers of the prostate, breast, and ovary. In human UACC-257 melanoma cells that possesses high levels of functional endogenous NOX5, overexpression of NOX5 resulted in enhanced cell growth, increased numbers of BrdU positive cells, and increased ?-H2AX levels. Additionally, NOX5-overexpressing (stable and inducible) UACC-257 cells demonstrated increased normoxic HIF-1? expression and decreased p27Kip1 expression. Similarly, increased normoxic HIF-1? expression and decreased p27Kip1 expression were observed in stable NOX5-overexpressing clones of KARPAS 299 human lymphoma cells and in the human prostate cancer cell line, PC-3. Conversely, knockdown of endogenous NOX5 in UACC-257 cells resulted in decreased cell growth, decreased HIF-1? expression, and increased p27Kip1 expression. Likewise, in an additional human melanoma cell line, WM852, and in PC-3 cells, transient knockdown of endogenous NOX5 resulted in increased p27Kip1 and decreased HIF-1? expression. Knockdown of endogenous NOX5 in UACC-257 cells resulted in decreased Akt and GSK3? phosphorylation, signaling pathways known to modulate p27Kip1 levels. In summary, our findings suggest that NOX5 expression in human UACC-257 melanoma cells could contribute to cell proliferation due, in part, to the generation of high local concentrations of extracellular ROS that modulate multiple pathways that regulate HIF-1? and networks that signal through Akt/GSK3?/p27Kip1 .
Project description:BACKGROUND AND PURPOSE: Macrophage migration inhibitory factor (MIF) is now known to be a pro-inflammatory cytokine associated with insulin resistance. Our aim was to investigate whether angiotensin converting enzyme 2 (ACE2) could modulate the expression of MIF and the insulin/Akt-endothelial nitric oxide (NO) synthase (eNOS) signalling in a human endothelial cell line (EAhy926). EXPERIMENTAL APPROACH: A recombinant plasmid encompassing human ACE2 gene was constructed and transfected into the EAhy926 cells. The mRNA, phosphorylation and protein levels of p22phox, MIF, Akt and eNOS in endothelial cells were determined by real-time PCR and Western blot analysis, respectively. KEY RESULTS: Gene transfer of ACE2 suppressed the expression of p22phox and MIF induced by angiotensin (Ang) II and Ang IV, accompanied by a decreased level of malondialdehyde in cells. In addition, Ang II diminished insulin-stimulated phosphorylation of Akt (at Ser(473)) and eNOS (at Ser(1177)) and NO generation, effects which were reversed by ACE2 gene transfer and anti-MIF treatment in endothelial cells. CONCLUSIONS AND IMPLICATIONS:The results reveal that gene transfer of ACE2 regulated Ang II-mediated impairment of insulin signalling and involved the Akt-eNOS phosphorylation pathway. These beneficial effects of ACE2 overexpression appear to result mainly from blocking MIF expression in endothelial cells, suggesting that the ACE2 gene may be a novel therapeutic target for diseases related to inflammation and insulin resistance.
Project description: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.
Project description:Expression of programmed cell death ligand 1 (PD-L1) on tumor cells contributes to cancer immune evasion by interacting with programmed cell death 1 on immune cells. γ-Interferon (IFN-γ) has been reported as a key extrinsic stimulator of PD-L1 expression, yet its mechanism of expression is poorly understood. This study analyzed the role of CD74 and its ligand macrophage migration inhibitory factor (MIF) on PD-L1 expression, by immunohistochemical analysis of melanoma tissue samples and in vitro analyses of melanoma cell lines treated with IFN-γ and inhibitors of the MIF-CD74 interaction. Immunohistochemical analyses of 97 melanoma tissue samples showed significant correlations between CD74 and the expression status of PD-L1 (P < .01). In vitro analysis of 2 melanoma cell lines, which are known to secrete MIF constitutively and express cell surface CD74 following IFN-γ stimulation, showed upregulation of PD-L1 levels by IFN-γ stimulation. This was suppressed by further treatment with the MIF-CD74 interaction inhibitor, 4-iodo-6-phenylpyrimidine. In the analysis of melanoma cell line WM1361A, which constitutively expresses PD-L1, CD74, and MIF in its non-treated state, treatment with 4-iodo-6-phenylpyrimidine and transfection of siRNAs targeting MIF and CD74 significantly suppressed the expression of PD-L1. Together, the results indicated that MIF-CD74 interaction directly regulated the expression of PD-L1 and helps tumor cells escape from antitumorigenic immune responses. In conclusion, the MIF-CD74 interaction could be a therapeutic target in the treatment of melanoma patients.
Project description:BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine and mediator of acute and chronic inflammatory diseases. MIF is overexpressed in various tumours and has been suggested as a molecular link between chronic inflammation and cancer. MIF overexpression is observed in breast cancer but its causal role in the development of this tumour entity is unclear. METHODS: MIF levels in breast cancer cell lines were determined by ELISA and Western blot. CD74 was measured by Western blot, fluorescence microscopy and flow cytometry. Cell proliferation was studied by BrdU incorporation, cell adhesion by Matrigel adhesion assay, and cell invasion by migration assay through Matrigel-coated filters using the Transwell system. MIF expression in primary human breast cancers was measured by tissue microarray and a semi-quantitative immunoreactivity score (IRS) and comparison with histopathological parameters and patient outcome data. RESULTS: MIF was abundantly expressed in the non-invasive breast cancer cell lines MDA-MB-468 and ZR-75-1, but not in invasive MDA-MB-231 cells, which in turn expressed higher levels of the MIF-receptor CD74. Stimulation with exogenous MIF led to a dramatic upregulation of MIF secretion (50-fold) in MDA-MB-231 cells. Autocrine MIF promoted tumour cell proliferation, as indicated by blockade of MIF or CD74 in MDA-MB-231 and MDA-MB-468, and MDA-MB-231 invasiveness was enhanced by exogenous MIF. We correlated the expression of MIF with histopathological parameters and patient outcome data, using a tissue microarray of 175 primary invasive breast cancers and 35 normal control tissues. MIF was upregulated in breast cancer versus normal tissue (median IRS = 8 versus 6). MIF expression showed positive correlations with progesterone (p = 0.006) and estrogen (p = 0.028) receptor expression, markers of a favourable prognosis and a negative correlation to tumour size (p = 0.007). In line with these data, disease-specific overall (OS) as well as recurrence-free (RFS) survival was significantly improved in breast cancer patients with abundant cytosolic MIF expression compared to MIF low expressers (5-year OS = 67% versus 50%, p = 0.0019; 5-year RFS = 52% versus 36%, p = 0.0327). CONCLUSION: We conclude that intracellular expression of MIF in breast cancer cells is beneficial, whereas extracellular MIF may play a pro-oncogenic role in promoting breast cancer cell-stroma interactions.