Expression Of Migration Inhibitory Factor Research Articles

Network pharmacology combined with experimental validation to investigate the effect of Rongjin Niantong Fang on chondrocyte apoptosis in knee osteoarthritis.

Knee osteoarthritis (KOA) is a chronic degenerative disease that affects the quality of life of middle‑aged and elderly individuals, and is one of the major factors leading to disability. Rongjin Niantong Fang (RJNTF) can alleviate the clinical symptoms of patients with KOA, but the molecular mechanism underlying its beneficial effects on KOA remains unknown. Using pharmacological analysis and in vitro experiments, the active components of RJNTF were analyzed to explore their potential therapeutic targets and mechanisms in KOA. The potential targets and core signaling pathways by which RJNTF exerts its effects on KOA were obtained from databases such as Gene Expression Omnibus, Traditional Chinese Medicine Systems Pharmacology and Analysis Platform. Subsequently, chondrocyte apoptosis was modeled using hydrogen peroxide (H2O2). Cell Counting Kit‑8 assay involving a poly [ADP‑ribose] polymerase‑1 (PARP1) inhibitor, DAPI staining, reverse transcription‑quantitative PCR, Annexin V‑FITC/PI staining and flow cytometry, western blotting and co‑immunoprecipitation analysis were used to determine the therapeutic efficacy of RJNTF on KOA and to uncover the molecular mechanism. It was found that PARP1‑knockdown lentivirus, incubation with PARP1 inhibitor PJ34, medium and high doses of RJNTF significantly reduced H2O2‑induced chondrocyte apoptosis. Medium and high doses of RJNTF downregulated the expression of cleaved caspase‑3, cleaved PARP1 and PAR total proteins, as well as nucleus proteins of apoptosis‑inducing factor (AIF) and migration inhibitory factor (MIF), and upregulated the expression of caspase‑3, PARP1 total protein, as well as the cytoplasmic expression of AIF and MIF, suggesting that RJNTF may inhibit chondrocyte apoptosis through the PARP1/AIF signaling pathway.

MIF in the cerebrospinal fluid is decreased during relapsing-remitting while increased in secondary progressive multiple sclerosis

BackgroundMacrophage migration inhibitory factor (MIF) is involved in the function of both the innate and adaptive immune systems and in neuroprotection and has recently been implicated in multiple sclerosis (MS). ObjectivesDetermination of MIF levels in the cerebrospinal fluid (CSF) of patients with distinct subtypes of MS and the cellular localization of MIF in human brain tissue. MethodsThe levels of MIF were investigated in CSF from patients with clinically isolated syndrome (CIS) (n = 26), relapsing-remitting MS (RRMS) (n = 22), secondary progressive MS (SPMS) (n = 19), and healthy controls (HCs) (n = 24), using ELISA. The effect of disease-modifying therapies in the RRMS and SPMS cohorts were examined. Cellular distribution of MIF in the human brain was studied using immunochemistry and the newly available OligoInternode database. ResultsMIF was significantly decreased in treatment-naïve CIS and RRMS patients compared to HCs but was elevated in SPMS. Interestingly, MIF levels were sex-dependent and significantly lower in women with CIS and RRMS. MIF expression in the human brain was localized to neurons, astrocytes, pericytes, and oligo5 oligodendrocytes but not in microglia. ConclusionThe finding that MIF was decreased in newly diagnosed CIS and RRMS patients but was high in patients with SPMS may suggest that MIF levels in CSF are regulated by local MIF receptor expression that affects the overall MIF signaling in the brain and may represent a protective mechanism that eventually fails.

Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

BackgroundThe danger-associated molecular patterns (DAMPs) are critical contributors to the progressive neuropathology and thereafter affect the functional outcomes following spinal cord injury (SCI). Up to now, the regulatory mechanisms on their inducible production from the living cells remain elusive, aside from their passive release from the necrotic cells. Thrombin is immediately activated by the damaged or stressed central nervous system (CNS), which potently mediates inflammatory astrocytic responses through proteolytic cleavage of protease-activated receptors (PARs). Therefore, SCI-activated thrombin is conceived to induce the production of DAMPs from astrocytes at lesion site.MethodsRat SCI model was established by the cord contusion at T8–T10. The expression of thrombin and macrophage migration inhibitory factor (MIF) was determined by ELISA and Western blot. The PAR1, PAR3, and PAR4 receptors of thrombin were examined by PCR and immunohistochemistry. Primary astrocytes were isolated and purified from the spinal cord, followed by stimulation with different concentrations of thrombin either for transcriptome sequencing or for analysis of thrombin-mediated expression of MIF and related signal pathways in the presence or absence of various inhibitors. The post-injury locomotor functions were assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale.ResultsMIF protein levels were significantly elevated in parallel with those of thrombin induced by SCI. Immunostaining demonstrated that PAR1 receptor, together with MIF, was abundantly expressed in astrocytes. By transcriptome sequencing and bioinformatical analysis of thrombin-stimulated primary astrocytes, MIF was identified to be dynamically regulated by the serine protease. Investigation of the underlying mechanism using various inhibitors revealed that thrombin-activated PAR1 was responsible for the MIF production of astrocytes through modulation of JNK/NFκB pathway. Administration of PAR1 inhibitor at lesion sites following SCI significantly reduced the protein levels of MIF and ameliorated functional deficits of rat locomotion.ConclusionSCI-activated thrombin is a robust inducer of MIF production from astrocytes. Exploring the roles of thrombin in promoting the production of DAMPs from astrocytes at lesion site will provide an alternative strategy for the clinical therapy of CNS inflammation.

MIF/SCL3A2 depletion inhibits the proliferation and metastasis of colorectal cancer cells via the AKT/GSK-3β pathway and cell iron death.

This study investigated the mechanisms of migration inhibitory factor (MIF) and solute carrier family 3 member 2 (SLC3A2) in colorectal cancer progression. The levels of MIF and SLC3A2 expression in cells were measured by RT‐qPCR. SW480 and SW620 cells were transfected with sh‐MIF and sh‐SLC3A2, respectively. MIF, SLC3A2, GPX4, E‐cadherin and N‐cadherin expression were detected by immunofluorescence (IF). CCK8 and Transwell assays were performed to detect cell proliferation and migration. Co‐immunoprecipitation (CoIP) was used to measure the binding activity of MIF and SLC3A2. Finally, a nude mouse tumorigenicity assay was used to confirm the functions of MIF and SLC3A2 in colorectal cancer. Results showed that the levels of MIF and SLC3A2 expression were up‐regulated in colorectal cancer cells. Inhibition of MIF or SLC3A2 expression prevented cell proliferation, migration, epithelial‐mesenchymal transition (EMT) and invasion. In addition, knockdown of MIF and SLC3A2 promoted iron death in SW480 and SW620 cells. CoIP results showed that MIF and SLC3A2 directly interact with each other. Knockdown of both MIF and SLC3A2 inhibited tumour growth and metastasis via the AKT/GSK‐3β pathway in vivo. The Akt/GSK‐3β pathway was found to participate in regulating MIF and SLC3A2 both in vivo and in vitro. MIF and SLC3A2 might be potential biomarkers for monitoring the treatment of colorectal cancer.

Biomimetic Nanosystems for the Synergistic Delivery of miR-144/451a for Oral Squamous Cell Carcinoma

Background:The miR-144/451a cluster acts as a tumor suppressor in various tumors by synergistically inhibiting the proliferation, migration, and invasion of oral squamous cell carcinoma (OSCC).Aims:To achieve the synergistic delivery of the miR-144/451a cluster for OSCC treatment by constructing chitosan nanoparticles (CAs) camouflaged with macrophage membranes.Study Design:A cell-culture study.Methods:CAs were prepared using the ionic cross-linking method, and biomimetic nanoparticles coloaded with the miR-144/451a cluster (miR-144-source of macrophage-derived exosomes [MEXO]/CA-miR-451a) were prepared using the uptake–efflux method. The MEXO was detected by a bicinchoninic acid assay. The as-prepared biomimetic nanoparticles were then characterized to determine their protective effects on microRNAs (miRNAs). Moreover, the influence of the miR-144-MEXO/CA-miR-451a nanoparticles on the proliferation, migration, and invasion of OSCCs was evaluated. Finally, the effects of the biomimetic system on the expression of calcium-binding protein 39 (CAB39) and migration inhibitory factor (MIF) were detected using the real-time polymerase chain reaction and Western blot.Results:After coating the CAs with MEXO, their particle size increased from 113.1 ± 3.4 nm to 143.2 ± 14 nm, and their surface potential decreased from 26.34 ± 0.4 mV to −10.3 ± 1.6 mV. The expression of the MEXO marker protein was also observed on the biomimetic nanoparticles’ surface. The system can protect miRNAs from RNase A degradation. Compared with the CAs cotransfected with free miR-144/451a cluster, CAs that are coloaded with miR-144-MEXO/CA-miR-451a nanoparticles substantially reduced the viability (p < 0.001), migration (p = 0.023), and invasion (p = 0.004) of OSCC. These findings revealed the successful construction of biomimetic nanoparticles coloaded with the miR-144/451a cluster. CAB39 and MIF expression in OSCC treated with miR-144-MEXO/CA-miR-451a nanoparticles have significantly decreased compared with the miR-144/451a group (p < 0.05). Thus, the nanoparticles can effectively improve the inhibitory effects of the miR-144/451a cluster on OSCC.Conclusion:This study provided a new idea for the application of gene cotransfection to tumor treatment.

Feedback activation of STAT3 limits the response to PI3K/AKT/mTOR inhibitors in PTEN-deficient cancer cells

The PI3K/AKT/mTOR signaling pathway is constitutively active in PTEN-deficient cancer cells, and its targeted inhibition has significant anti-tumor effects. However, the efficacy of targeted therapies is often limited due to drug resistance. The relevant signaling pathways in PTEN-deficient cancer cells treated with the PI3K/mTOR inhibitor BEZ235 were screened using a phosphokinase array, and further validated following treatment with multiple PI3K/AKT/mTOR inhibitors or AKT knockdown. The correlation between PTEN expression levels and STAT3 kinase phosphorylation in the tissue microarrays of gastric cancer patients was analyzed by immunohistochemistry. Cell proliferation and clonogenic assays were performed on the suitably treated PTEN-deficient cancer cells. Cytokine arrays, small molecule inhibition and knockdown assays were performed to identify related factors. PTEN-deficient tumor xenografts were established in nude mice that were treated with PI3K/AKT/mTOR and/or STAT3 inhibitors. PTEN deficiency was positively correlated with low STAT3 activity. PI3K/mTOR inhibitors increased the expression and secretion of macrophage migration inhibitory factor (MIF) and activated the JAK1/STAT3 signaling pathway. Both cancer cells and in vivo tumor xenografts showed that the combined inhibition of PI3K/AKT/mTOR and STAT3 activity enhanced the inhibitory effect of BEZ235 on the proliferation of PTEN-deficient cancer cells. Our findings provide a scientific basis for a novel treatment strategy in cancer patients with PTEN deficiency.

Evaluating macrophage migration inhibitory factor 1 expression as a prognostic biomarker in colon cancer.

Several studies indicate that macrophage migration inhibitory factor 1 plays a role for tumor progression in colon cancer. We investigated whether determination of migration inhibitory factor 1 mRNA expression levels in lymph nodes of colon cancer patients could be used as a prognostic marker. Expression levels of migration inhibitory factor 1 and carcinoembryonic antigen mRNAs were assessed in primary tumors and regional lymph nodes of 123 colon cancer patients (stages I-IV), and in colon cancer- and immune cell lines using quantitative reverse transcriptase-polymerase chain reaction. Expression of migration inhibitory factor 1 protein was investigated by two-color immunohistochemistry and immunomorphometry. Migration inhibitory factor 1 mRNA was expressed at 60 times higher levels in primary colon cancer tumors compared to normal colonic tissue (medians 8.2 and 0.2 mRNA copies/18S rRNA unit; p < .0001). A highly significant difference in mRNA expression levels was found between hematoxylin-eosin positive lymph nodes and hematoxylin-eosin negative lymph nodes (p < .0001). Migration inhibitory factor 1 and carcinoembryonic antigen proteins were simultaneously expressed in many colon cancer-tumor cells. Kaplan-Meier survival model and hazard ratio analysis, using a cutoff level at 2.19 mRNA copies/18S rRNA unit, revealed that patients with lymph nodes expressing high levels of migration inhibitory factor 1 mRNA had a 3.5-fold (p = .04) higher risk for recurrence, associated with a small, but significant, difference in mean survival time (7 months, p = .03) at 12 years of follow-up. Although migration inhibitory factor 1 mRNA expression levels were related to severity of disease and lymph node analysis revealed that colon cancer patients with high levels had a shorter survival time after surgery than those with low levels, the difference was small and probably not useful in clinical practice.

Inhibition of microRNA-451 is associated with increased expression of Macrophage Migration Inhibitory Factor and mitigation of the cardio-pulmonary phenotype in a murine model of Bronchopulmonary Dysplasia

BackgroundMacrophage migration inhibitory factor (MIF) has been implicated as a protective factor in the development of bronchopulmonary dysplasia (BPD) and is known to be regulated by MicroRNA-451 (miR-451). The aim of this study was to evaluate the role of miR-451 and the MIF signaling pathway in in vitro and in vivo models of BPD.MethodsStudies were conducted in mouse lung endothelial cells (MLECs) exposed to hyperoxia and in a newborn mouse model of hyperoxia-induced BPD. Lung and cardiac morphometry as well as vascular markers were evaluated.ResultsIncreased expression of miR-451 was noted in MLECs exposed to hyperoxia and in lungs of BPD mice. Administration of a miR-451 inhibitor to MLECs exposed to hyperoxia was associated with increased expression of MIF and decreased expression of angiopoietin (Ang) 2. Treatment with the miR-451 inhibitor was associated with improved lung morphometry indices, significant reduction in right ventricular hypertrophy, decreased mean arterial wall thickness and improvement in vascular density in BPD mice. Western blot analysis demonstrated preservation of MIF expression in BPD animals treated with a miR-451 inhibitor and increased expression of vascular endothelial growth factor-A (VEGF-A), Ang1, Ang2 and the Ang receptor, Tie2.ConclusionWe demonstrated that inhibition of miR-451 is associated with mitigation of the cardio-pulmonary phenotype, preservation of MIF expression and increased expression of several vascular growth factors.

Key role of MIF-related neuroinflammation in neurodegeneration and cognitive impairment in Alzheimer\u2019s disease

BackgroundMacrophage Migration Inhibitory Factor (MIF) is a potent proinflammatory cytokine that promotes the production of other immune mediators. MIF is produced by most cell types in the brain including microglia, astrocytes and neurons. Enhanced expression of MIF might contribute to the persistent activation of glial, chronic neuroinflammation and neurodegeneration. Here, we investigated the effect of MIF on inflammatory markers and spatial learning in a mouse model of sporadic AD and on tau pathology in AD patients.MethodsWe examined the effects of MIF deficiency and pharmacological MIF inhibition in vitro and in vivo. In vitro, quantitative PCR and ELISA were used to assess cytokine production of STZ-treated glial cells. In vivo, C57BL/6 mice were subjected to intracerebroventricular streptozotocin injection (3 mg/kg, ICV-STZ). Neuroinflammation and contextual learning performance were assessed using quantitative PCR and fear conditioning, respectively. Pharmacological MIF inhibition was achieved with intraperitoneal injections of ISO-1 (daily, IP, 20 mg/kg in 5% DMSO in 0.9% NaCl) for 4 weeks following ICV-STZ injection. The findings from ISO-1 treated mice were confirmed in MIF knockout C57BL/6. To assess the role of MIF in human AD, cerebrospinal fluid levels of MIF and hyperphosphorylated tau were measured using ELISA.ResultsAdministration ICV-STZ resulted in hippocampal dependent cognitive impairment. MIF inhibition with ISO-1 significantly improved the STZ-induced impairment in contextual memory performance, indicating MIF-related inflammation as a major contributor to ICV-STZ-induced memory deficits. Furthermore, inhibition of the MIF resulted in reduced cytokine production in vitro and in vivo.In human subjects with AD at early clinical stages, cerebrospinal fluid levels of MIF were increased in comparison with age-matched controls, and correlated with biomarkers of tau hyper-phosphorylation and neuronal injury hinting at MIF levels as a potential biomarker for early-stage AD.ConclusionsThe present study indicates the key role of MIF in controlling the chronic cytokine release in neuroinflammation related to tau hyperphosphorylation, neurodegeneration, and clinical manifestations of AD, suggesting the potential of MIF inhibition as therapeutic strategy to slow down neurodegeneration and clinical disease progression.

Biphasic MIF and SDF1 expression during podocyte injury promote CD44-mediated glomerular parietal cell migration in focal segmental glomerulosclerosis.

Glomerular parietal epithelial cell (PEC) activation, as revealed by de novo expression of CD44 and cell migration toward the injured filtration barrier, is a hallmark of podocyte injury-driven focal segmental glomerulosclerosis (FSGS). However, the signaling pathway that mediates activation of PECs in response to podocyte injury is unknown. The present study focused on CD44 signaling, particularly the roles of two CD44-related chemokines, migration inhibitory factor (MIF) and stromal cell-derived factor 1 (SDF1), and their common receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), in the NEP25/LMB2 mouse podocyte-toxin model of FSGS. In the early phase of the disease, CD44-positive PECs were locally evident on the opposite side of the intact glomerular tuft and subsequently increased in the vicinity of synechiae with podocyte loss. Expression of MIF and SDF1 was first increased in injured podocytes and subsequently transferred to activated PECs expressing CD44 and CXCR4. In an immortalized mouse PEC (mPEC) line, recombinant MIF and SDF1 (rMIF and rSDF1, respectively) individually increased CD44 and CXCR4 mRNA and protein levels. rMIF and rSDF1 stimulated endogenous MIF and SDF1 production. rMIF- and rSDF1-induced mPEC migration was suppressed by CD44 siRNA. However, MIF and SDF1 inhibitors failed to show any impact on proteinuria, podocyte number, and CD44 expression in NEP25/LMB2 mice. Our data suggest that injured podocytes upregulate MIF and SDF1 that stimulate CD44 expression and CD44-mediated migration, which is enhanced by endogenous MIF and SDF1 in PECs. This biphasic expression pattern of the chemokine-CD44 axis in podocytes and PECs may be a novel mechanism of "podocyte-PEC cross-talk" signaling underlying podocyte injury-driven FSGS.

The expression and clinical significance of migration inhibitory factor in human pulmonary tissues with chronic obstructive pulmonary disease

Objective To investigate the expression of macrophage migration inhibitory factor (MIF) in pulmonary tissues from patients with chronic obstructive pulmonary disease (COPD) and the relationship with its clinical features. Methods One hundred and eighty patients who underwent pulmonary bullectomy lobectomy due to pneumatocele from January 2015 to September 2018 in Longgang Central Hospital were enrolled and classified into patients without COPD (control group)and patients with COPD (COPD group), with 90 patients each group. According to the lung function parameters, 90 patients with COPD were divided into the mild COPD group, the moderate COPD group, and the severe COPD group. The levels of mRNA and protein of MIF were measured with RT-PCR, ELISA and Western blot. One-way ANOVA, Pearson correlation analysis and SNK-q test were used to analyze the results with SPSS 18.0, and P<0.05 was considered statistically significant. Results The level of MIF in pulmonary tissues from the control group was obviously lower than those in the COPD group (P<0.05). The level of MIF in pulmonary tissues in the severe COPD group was obviously higher than those in pulmonary tissues in the mild COPD, moderate COPD and control groups (P<0.05). MIF was positively correlated with the lung function parameters (P<0.05). Conclusion The high expression of MIF in pulmonary tissues is closely related to the severity of COPD. Key words: Migration inhibitory factor; Chronic obstructive pulmonary disease; Real time-PCR; ELISA; Western blot

Upregulation of MIF as a defense mechanism and a biomarker of Alzheimer\u2019s disease

BackgroundMacrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine. Chronic inflammation induced by amyloid β proteins (Aβ) is one prominent neuropathological feature in Alzheimer’s disease (AD) brain.MethodsElisa, Western blot, and immunohistochemical staining analysis were performed to examine the level of MIF protein in CSF and brain tissues. MTT and LDH assays were used to examine the neurotoxicity, and the Morris Water Maze test was performed to examine the cognitive function in the MIF+/−/APP23 transgenic mice.ResultsMIF expression was upregulated in the brain of AD patients and AD model mice. Elevated MIF concentration was detected in the cerebrospinal fluid of AD patients but not in that of the patients suffering from mild cognitive impairment and vascular dementia. Reduced MIF expression impaired learning and memory in the AD model mice. MIF expression largely associates with Aβ deposits and microglia. The binding assay revealed a direct association between MIF and Aβ oligomers. Neurons instead of glial cells were responsible for the secretion of MIF upon stimulation by Aβ oligomers. In addition, overexpression of MIF significantly protected neuronal cells from Aβ-induced cytotoxicity.ConclusionOur study suggests that neuronal secretion of MIF may serve as a defense mechanism to compensate for declined cognitive function in AD, and increased MIF level could be a potential AD biomarker.

Relationship between MIF expression and obese-induced abolition of sevoflurane preconditioning-induced cardioprotection in mice

Objective To evaluate the relationship between macrophage migration inhibitory factor (MIF) expression and obese-induced abolition of sevoflurane preconditioning-induced cardioprotection in mice. Methods Forty-eight male C57BL/6J mice, aged 4 weeks, were divided into 2 groups(n=24 each)using a random number table method: normal diet group (Lean group) and high-fat diet group (Obese group). Lean group were fed a normal diet (10% kcal) for 12 weeks, while Obese group were fed a high-fat diet (60% kcal) for 12 weeks.The weight of mice was measured.Blood samples were collected from the tail vein for determination of blood glucose concentrations, and plasma concentrations of total cholesterol, triglyceride, insulin and leptin.After measurement of the parameters mentioned above, Lean group and Obese group were divided into 3 subgroups (n=8 each) using a random number table method: sham operation groups (L-Sham group, O-Sham group), myocardial ischemia-reperfusion groups (L-IR group, O-IR group) and sevoflurane preconditioning groups (L-IR+ Sev group, O-IR+ Sev group). The mice were anesthetized and their hearts were immediately removed and retrogradely perfused in a Langendorff apparatus with an oxygenated K-H solution at 37 ℃.Hearts were continuously perfused with K-H solution for 115 min in L-Sham and O-Sham groups.Hearts were subjected to global ischemia for 25 min, followed by 60-min reperfusion after being retrogradely perfused with K-H solution in L-IR and O-IR groups.In L-IR+ Sev and O-IR+ Sev groups, hearts were subjected to 3 cycles of 5-min perfusion with sevoflurane-containing K-H solution(final concentration 0.6 mmol/L)and 5-min washout, and then hearts were subjected to global ischemia for 25 min, followed by 60-min reperfusion.Left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and the maximum rate of increase or decrease in left ventricular pressure (±dp/dtmax) were recorded at the end of reperfusion.Hearts were obtained at the end of reperfusion for determination of myocardial infarct size and expression of MIF (by Western blot). Results Compared with Lean group, the weight, blood glucose, levels of plasma total cholesterol, triglyceride, insulin and leptin were significantly increased in Obese group (P 0.05). Conclusion The mechanism by which obese abolishes sevoflurane preconditioning-induced cardioprotection may be related to inducing MIF over-expression in mice. Key words: Macrophage migration-inhibitory factors; Obesity; Anesthetics, inhalation; Ischemic preconditioning; Myocardial reperfusion injury

MiR-608 exerts tumor suppressive function in lung adenocarcinoma by directly targeting MIF.

Lung adenocarcinoma (LA) is considered as a highly aggressive disease with heterogeneous prognosis. The molecular mechanisms of LA progression remain elusive. Recent studies have shown that dysregulation of microRNAs (miRNAs) is prevalent in LA, playing a significant role in tumor progression. The present work aims to analyze the expression and function of miR-608 in LA. Quantitative Real-time polymerase chain reaction (qRT‑PCR) assay and Western blot were performed to detect expressions of miR-608 and migration inhibitory factor (MIF). Luciferase reporter assays were carried out to investigate the regulatory effect of miR-608 on MIF. The cell invasion and the migration capabilities were detected by transwell assay. QRT-PCR indicated that miR-608 expressions in LA tissues were markedly reduced than that of the normal tissues. Moreover, the expression of MIF, a potential target gene of miR-608, was inversely associated with miR-608 expression in LA. Furthermore, miR-608 overexpression could inhibit LA invasion and migration, which was reversed by MIF knockdown. Our study revealed the mechanisms that miR-608 suppressed LA invasion and migration by targeting MIF, suggesting that miR-608/MIF axis could be used as a potential prognostic biomarker and therapeutic target for LA.

Abstract 3560: Targeting the MIF receptor CD74 in bladder cancer tumorigenesis

Abstract Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that can stimulate cellular proliferation in multiple tumors. Our previous data indicates genetic deletion of MIF results in T1 stage arrest in the N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) induced bladder cancer model. Similarly, pharmacological inhibition of MIF results in lower stage tumors and reduced bladder weights when given therapeutically. CD74 is a human leukocyte antigen class II trafficking and processing protein that also functions as an extracellular receptor for MIF. Additionally, CD74 complexes with CD44 to act as the cognate MIF receptor complex, and upregulation of CD74 has been noted in high grade bladder cancer. We hypothesized CD74 would be upregulated in malignant bladder cancer cell lines, and knockout of CD74 would reduce bladder cancer tumorigenesis in the murine BBN model. The objectives of this study were to determine the effect of CD74 knockout on development and progression of urothelial cell carcinoma; as well as evaluate expression of the putative MIF signaling axis in BCa cell lines. BCa cell lines HTB-9, HTB-5, RT-4, T24 and HT-1376 and benign urothelial UROtsa cells were analyzed for MIF, CD74, and CD44 expression via quantitative PCR or western blot to assess expression of the MIF signaling axis. CD74 and CD44 expression by q-PCR were dramatically elevated in BCa cell lines vs. benign cell lines. Protein levels of CD74 and CD44 were elevated in multiple cell lines as well. MIF expression levels did not change between cell lines. To ascertain differences in BCa tumorigeneis, WT C57Bl/6J mice or CD74-/- mice were given 0.05% BBN in the drinking water for 22 weeks. Bladders were weighed and evaluated for pathologic staging by histology. After exposure to BBN, bladders from CD74-/- mice had a 33% reduction in weight vs. WT bladders (p=.062). Pathological analysis revealed lower stage tumors in bladders of CD74-/- mice (33% incidence; 6.7% pTa, 0% pTis, 6.7% pT1, 20% pT2, 6.7% pT3) as compared to bladders of WT mice (81.3% incidence; 0% pTa, 6.3% pTis, 25% pT1, 12.5% pT2, 37.5% pT3), which was a statistically significant reduction in tumor stage. Lower tumor stage correlated significantly with the CD74-/- genotype (p=0.02, Fisher's Exact Test). These data suggest CD74 likely mediates a portion of the effects of MIF in BCa, raising the possibility of both CD74 dependent, and independent MIF signaling pathways. Citation Format: Justin Penticuff, Sambantham Shanmugam, Joshua Warrick, Brian Chase, Erika Abbott, Benjamin L. Woolbright, John A. Taylor. Targeting the MIF receptor CD74 in bladder cancer tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3560.

Serum Levels of Migration Inhibitory Factor (MIF) and In Situ Expression of MIF and Its Receptor CD74 in Lepromatous Leprosy Patients: A Preliminary Report.

Leprosy is a chronic disease caused by Mycobacterium leprae that affects the skin and peripheral nerves. It may present as one of two distinct poles: the self-limiting tuberculoid leprosy and the highly infectious lepromatous leprosy (LL) characterized by M. leprae-specific absence of cellular immune response. The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) enhance the bactericide activities of macrophages after interaction with its receptor, CD74. Importantly, MIF also possesses chemoattractant properties, and it is a key factor in situ for the activation of macrophages and in blood to promote leukocytes migration. MIF-mediated activation of macrophages is a key process for the elimination of pathogens such as Mycobacterium tuberculosis; however, its participation for the clearance of M. leprae is unclear. The aim of this study was to evaluate the serum levels of MIF as well as MIF and CD74 expression in skin lesions of LL and compare it with healthy skin (HSk) taken from subjects attending to dermatological consult. Samples of serum and skin biopsies were taken from 39 LL patients and compared with 36 serum samples of healthy subjects (HS) and 10 biopsies of HSk. Serum samples were analyzed by ELISA and skin biopsies by immunohistochemistry (IHC). IHC smears were observed in 12 100× microscopic fields, in which percentage of stained cells and staining intensity were evaluated. Both variables were used to calculate a semi-quantitative expression score that ranged from 0 to 3+. We found no differences in MIF levels between LL patients and HS in sera. In addition, MIF was observed in over 75% of cells with high intensity in the skin of patients and HSk. Although we found no differences in MIF expression between the groups, a CD74 score statistically higher was found in LL skin than HSk (p < 0.001); this was the result of a higher percentage of cells positive for CD74 (p < 0.001). As a conclusion, we found that CD74-positive cells are intensely recruited to the skin with LL lesions. In this manner, MIF signaling may be enhanced in the skin of LL patients due to increased expression of its receptor, but further studies are required.

Abstract 4789: A novel MIF signaling pathway drives the malignant character of pancreatic cancer by targeting NR3C2

Abstract Pancreatic cancers with aberrant expression of macrophage migration inhibitory factor (MIF) are particularly aggressive. To identify key signaling pathways that drive disease aggressiveness in tumors with high MIF expression, we analyzed the expression of coding and noncoding genes in high and low MIF-expressing tumors in multiple cohorts of pancreatic ductal adenocarcinoma (PDAC) patients. The key genes and pathways identified were linked to patient survival and were mechanistically, functionally, and clinically characterized using cell lines, a genetically engineered mouse model, and PDAC patient cohorts. Here, we report evidence of a novel MIF-driven signaling pathway that inhibits the orphan nuclear receptor NR3C2, a previously undescribed tumor suppressor that impacts aggressiveness and survival in PDAC. Mechanistically, MIF upregulated miR-301b that targeted NR3C2 and suppressed its expression. PDAC tumors expressing high levels of MIF displayed elevated levels of miR-301b and reduced levels of NR3C2. In addition, reduced levels of NR3C2 expression correlated with poorer survival in multiple independent cohorts of PDAC patients. Functional analysis showed that NR3C2 inhibited epithelial-to-mesenchymal transition and enhanced sensitivity to the gemcitabine, a chemotherapeutic drug used in PDAC standard of care. Furthermore, genetic deletion of MIF disrupted a MIF-mir-301b-NR3C2 signaling axis, reducing metastasis and prolonging survival in a genetically engineered mouse model of PDAC. Taken together, our results offer a preclinical proof of principle for candidate therapies to target a newly described MIF-miR-301b-NR3C2 signaling axis for PDAC management. Citation Format: Shouhui Yang, Peijun He, Liming Wang, Jian Wang, Aaron Schetter, Wei Tang, Naotake Funamizu, Katsuhiko Yanaga, Tadashi Uwagawa, Abhay R. Satoskar, Jochen Gaedcke, Markus Bernhardt, B. Michael Ghadimi, Matthias M Gaida, Frank Bergmann, Jens Werner, Thomas Ried, Nader Hanna, H. Richard Alexander, S. Perwez Hussain. A novel MIF signaling pathway drives the malignant character of pancreatic cancer by targeting NR3C2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4789. doi:10.1158/1538-7445.AM2017-4789

Molecular mechanisms of corticosteroid resistance in patients with chronic obstructive pulmonary disease

Glucocorticoids are widely used for the treatment of chronic obstructive pulmonary disease (COPD) because of their antiinflammatory properties. However, their therapeutic effectiveness is significantly limited in COPD. Molecular mechanisms of steroid resistance include defective glucocorticoid receptor (GR) binding and translocation into the nucleus, increased expression of GRβ isoform, elevated expression of macrophage migration inhibitory factor (MIF), decreased expression of mitogenactivated protein kinase phosphatase 1 (MKP1) and histone deacetylase 2 (HDAC2). HDAC2 is involved in suppression of inflammatory genes by glucocorticoids, and its reduced activity and expression are the result of oxidative and nitrative stress induced by cigarette smoke. Oxidative stress causes activation of phosphoinositide3kinase δ (PI3Kδ) which leads to phosphorylation (activation) of Akt kinase, phosphorylation (inhibition) of glycogen synthase kinase 3β and phosphorylation (inactivation) of HDAC2. Understanding of the mechanisms leading to steroid resistance allowed identification drugs targeting this condition. Antidepressant nortriptyline and macrolide solithromycin reverse corticosteroid resistance through inhibition of Akt phosphorylation. Combination of glucocorticoid and longacting β2agonist increases GR nuclear translocation and inhibits Akt phosphorylation. The phosphodiesterase 4 inhibitor roflumilast in combination with dexamethasone improves steroid responsiveness through modulation of PI3Kδ, HDAC2, MKP1, MIF and GRβ expression. Investigation of the molecular mechanisms of steroid resistance can increase antiinflammatory properties of steroids and lead to more effective COPD treatment.

Alterations of Macrophage Migration Inhibitory Factor Expression in the Nervous System of the Rat Cystitis Model

Objective: We evaluated the changes in the migration inhibitory factor (MIF) expression with time in the bladder and spinal cord of a Sprague-Dawley rat cystitis model. Methods: The bladder created by injecting lipopolysaccharide (LPS) and the lumbosacral spinal cord were removed at 4, 24, and 48 h, and 7 days after the induction of chronic cystitis, and 24 h after the induction of acute cystitis. The MIF expression was estimated using immunohistochemical staining, and the MIF mRNA was quantitatively analyzed using real-time polymerase chain reaction. Results: The MIF expression in the urothelial cell was lowest at 24 h after LPS injection. As the degree of inflammation decreased, the MIF expression in the urothelial cell gradually increased 48 h and 7 days. The quantitative analysis of the MIF mRNA showed that the MIF expression was highest in both the bladder and the lumbosacral spinal cord 24 h after the cystitis became most severe. In chronic cystitis, the MIF mRNA expressions in the bladder and the lumbosacral spinal cord were 2.8 and 2.1 times significantly higher than in the normal control. Conclusion: MIF is involved in urinary symptoms and pain in cystitis, and in the mechanism that prolongs the symptoms of chronic cystitis by involving bladder tissue, and the nervous system.

Abstract B34: A Novel MIF-driven Signaling Drives Disease Aggressiveness by Targeting NR3C2 in Pancreatic Cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Pancreatic tumors with aberrant expression of macrophage migration inhibitory factor (MIF) are highly aggressive, and an increased MIF predicts poorer patient survival. We analyzed the expression of coding and non-coding genes in high and low MIF-expressing tumors in multiple cohorts of PDAC patients to identify key signaling pathways that drive disease aggressiveness in tumors with high MIF expression. The identified key genes and pathways were linked to patients’ survival and were mechanistically, functionally and clinically characterized using cell lines, genetically engineered mouse model and PDAC patient cohorts. Here, we report mechanistic, functional and clinical evidence of a novel MIF-driven signaling pathway that inhibits a previously undescribed tumor suppressor, nuclear-receptor-subfamily-3, group-C, member-2 (NR3C2), leading to enhanced disease aggressiveness and poorer survival in PDAC. MIF upregulated miR-301b, which then targeted and suppressed NR3C2 expression. Tumors with high MIF expression showed an elevated miR-301b and a reduced NR3C2 expression. Additionally, patients with a lower NR3C2 expression in tumors showed poorer survival in multiple independent cohorts of PDAC patients. NR3C2 inhibited epithelial-to-mesenchymal transition and enhanced sensitivity to chemotherapeutic drug gemcitabine. Furthermore, genetic deletion of MIF disrupted MIF-mir-301b-NR3C2 axis, which resulted in reduced metastasis and prolonged survival in genetically engineered mouse model of PDAC. These findings provide proof-of-principle that therapies targeting MIF-miR-301b-NR3C2 axis may improve disease outcome in PDAC Citation Format: Shouhui Yang, Peijun He, Jian Wang, Aaron Schetter, Wei Tang, Naotake Funamizu, Katsuhiko Yanaga, Tadashi Uwagawa, Abhay R. Satoskar, Jochen Gaedcke, Markus Bernhardt, B. Michael Ghadimi, Matthias M. Gaida, Frank Bergmann, Jens Werner, Thomas Ried, Nader Hanna, H. Richard Alexander, S. Perwez Hussain.{Authors}. A Novel MIF-driven Signaling Drives Disease Aggressiveness by Targeting NR3C2 in Pancreatic Cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B34.