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* The Bay Pines Veterans Affairs Healthcare System, Research and Development, Bay Pines, FL 33744;
University of South Florida, Department of Surgery, Tampa, FL 33612;
Malcom Randall Veterans Affairs Medical Center, Department of Pathology, Gainesville, FL 32608;
University of Florida College of Medicine, Department of Pathology, Immunology, and Laboratory Medicine, Gainesville, FL 32610; and
¶ Yale University School of Medicine, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Recent data support the hypothesis that there is a link between chronic prostate inflammation and prostate cancer (3). Prostate cancer is the second most common malignancy in American men, with estimates projecting that >232,000 new cases were diagnosed in the United States in 2005. Epidemiological studies have determined an associated risk between prostate cancer in men with a prior history of certain sexually transmitted infections or prostatitis, suggesting that prostate inflammation may modulate prostate cancer growth (4, 5, 6).
Our research has focused on understanding the contribution of a unique proinflammatory cytokine, macrophage migration inhibitory factor (MIF)3 and its cell surface receptor (CD74) to prostate cancer (7, 8). MIF is constitutively expressed in luminal epithelial cells, which are continuously exposed to environmental insult. In the epithelial cell, intracellular MIF stores may maintain cell inflammatory response by acting in cooperation with glucocorticoids to control both the set point and the magnitude of the response (9). Although MIF is essential for innate immune response, any shift toward MIF up-regulation increases the likelihood of chronic inflammatory disease and cancer outgrowth (10). Various inflammatory cytokines are implicated in maintaining tumor cell growth and viability. MIF is unusual in its contribution to the growth and survival of a developing neoplasm, first and foremost, because it is constitutively expressed and stored, preformed, in cells (9). Secondly, MIF is unique because it functions in many processes associated with tumor survival including cell division, angiogenesis, and suppression of host-tumor cell immune surveillance (11). The precise mechanisms of MIF in tumorigenesis are still being investigated. However, MIF suppresses the action of the tumor-suppressor gene p53, resulting in promotion of cell growth and inhibition of apoptosis (12, 13).
CD74, the MIF receptor, is a type II transmembrane protein that is part of the MHC (14). CD74 functions to regulate the loading of exogenously derived peptides onto MHC class II heterodimers (15). However, recent studies demonstrated that CD74 is transiently expressed on the cell surface in diverse cells including monocytes, B cells, fibroblasts, and urothelial cells, as well as the prostate (8, 16, 17, 18). The exact function of cell surface CD74 has not been determined. However, MIF binds to the extracellular domain of CD74 with high affinity, and such binding is required for MIF activation of ERK 1/2 MAPK cascade (19). In addition, we recently demonstrated that inflammation induced increases in MIF-CD74 interaction at sites of inflammation, suggesting a physiological role for MIF-CD74 binding early in the inflammatory response (17). A recent study by Starlets et al. (20) documents MIF-CD74 induction of signaling cascade that activates NF-
B and induces DNA synthesis and cell entry into the S phase as well as Bcl-xL expression, suggesting that CD74 functions as a survival receptor.
The cytosolic domain of CD74 is very short and appears to lack intracellular signaling domains (20). Recent studies have identified recruitment of transmembrane CD44 as a potential accessory protein required for MIF-CD74 signal transduction (21, 22). In addition, we recently showed the formation of a molecular complex between MIF, CD74, and CD44 in HT-1376 bladder carcinoma cells (23). Prostate cancer loses expression of standard CD44 and overexpresses almost entirely variant forms, which arise from alternate splicing of exons 7–10 (24), thus suggesting that variant forms of CD44 (e.g., CD44 variant 9 (CD44v9)) may be involved in MIF-CD74-mediated signal transduction in prostate cancer.
Androgens are critical to the growth and differentiation of prostate epithelial cells (3). Removal of androgen normally results in apoptosis, but androgen-independent tumors have developed mechanisms that allow cells to survive the loss of androgen. Clinical prostate cancer progression may be the result of the regrowth of cells that adapted to the low-androgen environment and resumed proliferation. In vitro prostate cancer cell models showed that the regrowth of androgen-independent cells was associated with increased expression of cyclin D1, indicating that the emergence of androgen independence is associated with a release from cell cycle arrest (25).
Based on our previous studies, our model shows that MIF-CD74-CD44 interaction is required for normal prostate inflammatory responses. Our laboratory was first in describing the association between increased MIF expression in prostate tumor tissue, as well as the association between elevated serum MIF and prostate cancer (7). In prostate cancer, cell surface association of MIF and CD74 may play an essential role in cell survival. Thus, our working hypothesis is that the blockade of MIF or its recently identified receptor (CD74) will impede or reduce prostate cancer cell growth. In this study, we demonstrated cell surface localization of the MIF receptor (CD74) in aggressive, androgen-independent (DU-145) prostate cancer cells and that biologically active MIF and/or the MIF receptor (CD74) are necessary for the growth and invasion of these cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Normal human prostate epithelial cells (PrEC) were obtained from Cambrex and cultured exclusively in prostate epithelial cell growth medium. Human prostate cancer cell lines (LNCaP, DU-145) were obtained from the American Type Culture Collection (ATCC). Benign prostate epithelial cells (BPH-1) were obtained from Dr. Simon Hayward (Vanderbilt University, Nashville, TN). These cells were routinely cultured in RPMI 1640 with Glutamax supplemented with 10% FBS (standard growth medium). All cells were routinely grown without antibiotics in a 5% CO2, 37°C humidified incubator. Cell proliferation was evaluated using a WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) to formazan assay (Chemicon International). WST-1 reagent was added to the culture medium (1/10 dilution), and absorbance was measured at 450 nm following incubation at 37°C for 3 h.
Cell lysis, Western blot, coimmunoprecipitation, and biotinylation of cell surface proteins
Cell lysates from 
2 x 107 subconfluent cells were prepared using lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl with 1% Triton X-100, 0.1% SDS, and 0.25% sodium deoxycholate) with the addition of general protease inhibitors (Sigma-Aldrich), and protein concentrations were determined using the MicroBCA protocol (Pierce). Cell lysate protein (15 µg) was separated by 4–12% Bis-Tris SDS-PAGE (Invitrogen Life Technologies) and transferred as described previously (23). Blots were blocked in blocking buffer (1% BSA, 10 mM PBS (pH 7.5), and 0.05% Tween 20) for 1 h at 37°C and incubated overnight with primary Ab (1/1000 dilution) at 4°C, followed by incubation with appropriate biotinylated secondary Ab and avidin-HRP (Vectastain ABC; Vector Laboratories). Individual protein bands were visualized by ECL (Pierce), and band intensities quantified using Kodak Image Station (Kodak). The following Abs were used in Western blotting: affinity purified goat anti-human MIF polyclonal Ab was obtained from R&D Systems (AF-289-PB); goat polyclonal anti-human CD74 was obtained from Santa Cruz Biotechnology (sc-5438); and monoclonal anti-human CD44v9 was used undiluted from hybridoma cell culture medium (HB-258; ATCC). CD44v9 amounts were examined rather than standard CD44 because variant forms are overexpressed in prostate cancer cell lines, and this variant represents the longest exon product within this sequence (24). 
-Actin immunostaining of stripped blots was used as a loading control. MIF-interacting proteins were detected by coimmunoprecipitation using immobilized MIF polyclonal Ab (50 µg) conjugated to agarose beads (Seize X protein G-agarose; Pierce). Cell lysate (250 µl) was diluted 1/1 with PBS and incubated with Ab-coupled gel overnight at 4°C with end-over-end mixing. The resin was then washed with PBS until no protein was detected by A280 in the flow through and the bound proteins eluted with 200 µl of 100 mM glycine (pH 2.5). The pH of the eluted fractions was neutralized by the addition of 1 M Tris (pH 9.5), 1/20 dilution. The coimmunoprecipitated samples were separated by SDS-PAGE, and CD74 or CD44v9 immunostaining bands were detected as described for Western blotting.
To biotinylate cell surface proteins, BPH-1, LNCaP, or DU-145 cells (3 x 107) were plated into T75 culture flasks and grown for 48 h in standard growth medium. Adherent cells were washed three times with ice-cold PBS (pH 8.0) and 3 ml of 10 mM sulfosuccinimidyl-6-(biotin-amido)hexanoate (Pierce) PBS (pH 8.0) added to flask. The reaction mixture was incubated for 2 h at 4°C (to reduce active internalization of biotin reagent), and then cells were gently scraped from the flask surface. Labeled cells were collected by centrifugation, and the resulting pellet was washed three times with 15 ml of ice-cold 100 mM glycine in PBS (pH 8.0). Cell lysis buffer with general protease inhibitor mixture was added and biotinylated proteins were purified from lysates using avidin-agarose (Sigma-Aldrich). The resulting avidin-purified biotinylated proteins were concentrated 10-fold using a rotary vacuum evaporator (Brinkman Instruments) and 15 µl of the concentrated purified proteins processed for SDS-PAGE were transferred to a polyvinylidene difluoride (PVDF) membrane as described above. Biotinylated proteins were detected using HRP-conjugated streptavidin and chemiluminescent substrate. mAbs were used in the immunoprecipitation of biotinylated proteins as follows: LN2 specific for the carboxy terminus of human CD74 was obtained from Santa Cruz Biotechnology (sc-6262), mAb specific for human MIF was obtained from R&D Systems (MAB289), and CD44v9 mAb (HB-258; ATCC) was purified from cell culture supernatant by protein G-agarose. Avidin-purified biotinylated proteins were precleared with 10% weight-to-volume ratio protein G-agarose slurry (Kirkegaard & Perry Laboratories). Biotinylated MIF, CD74, or CD44v9 were immunoprecipitated with 5 µg of respective Ab overnight at 4°C with rocking, followed by incubation with 10% protein G-agarose slurry for 2 h with rocking. Immune complexes were dissociated with 40 µl of 2x sample buffer (Invitrogen Life Technologies) and incubated at 100°C for 5 min. The eluates were removed, dithiothreitol was added, and samples were incubated at 100°C for 5 min before loading on SDS-PAGE gels as described for Western blotting. Biotinylated proteins were detected using HRP-conjugated streptavidin and chemiluminescent substrate (Pierce).
MIF genotype
Two MIF-promoter polymorphisms (–173 G to C transition and seven copies of the –794 CATT repeat) are associated with increased MIF expression and are strongly associated with protein production. These genotypes were determined in BPH-1, LNCaP, and DU-145 cells by isolating genomic DNA from 1 x 106 subconfluent using DNAzol (Invitrogen Life Technologies) and genotyping as described previously (26).
Tautomerase activity
For the purposes of detecting MIF activity in biological samples, we developed a modified tautomerase assay using human recombinant MIF produced as described previously as a standard native sequence (27). DU-145, LNCaP, and BPH-1 cells were cultured with standard growth medium in T75 flasks until 80% confluent. Cells were lysed using lysis buffer, and spent culture medium was collected; 4 mM L-3,4-dihydroxyphenylalanine methyl ester (Sigma-Aldrich) and 20 mM sodium m-periodate (Sigma-Aldrich) were freshly prepared in water. Samples to be tested (60 µl, diluted if necessary in 10 mM sodium phosphate/1 mM EDTA (pH 6.2)) were pipetted into a standard polystyrene ELISA plate. Two hundred microliters of substrate (L-dopachrome methyl ester), prepared by mixing 72 µl of periodate stock with 108 µl of dihydroxyphenylalanine in 1620 µl of 10 mM sodium phosphate/1 mM EDTA (pH 6.2), was added. The samples were mixed and the change of A475 due to dopachrome tautomerization was monitored for 2 min. Inhibition of MIF tautomerase activity was determined by addition of MIF antagonist (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1, final concentrations from 0.1 to 100 µM) to cell lysates just before measurement of tautomerase activity (28). Data are expressed as change of A475 nm per second per milligram of total protein. To assess the effects of blocking MIF activity on cell proliferation, DU-145, LNCaP, and BPH-1 cells (1 x 105 per well; 96-well plate) were plated in RPMI 1640 with 10% FBS for 6 h, and then treated with ISO-1 (final concentrations from 0.1 to 20 µM) for 18 h. Control cultures were treated with 0.1% DMSO. Cell proliferation was assessed by WST-1 assay, and data were expressed as percentage of control.
Antibody treatment
DU-145, LNCaP, and BPH-1 cells (1 x 104 per well of 96-well plate) were plated in RPMI 1640 with 10% FBS for 6 h, and then treated with either MIF (AF-289-PB) or CD74 Ab (sc-5438) goat polyclonal Ab at a final concentration of 2 µg/ml for 72 h total with cell proliferation and apoptosis assessed at 24 h intervals. Control cell cultures were treated with nonspecific goat IgG (Vector Laboratories). Cell proliferation was evaluated using the WST-1 to formazan assay (Chemicon International) as described above.
RNA interference (RNAi)
BPH-1, LNCaP, or DU-145 cells were seeded in 6-well tissue culture plates (2 x 105 cells per well) and grown in standard growth medium at 37°C in a CO2 incubator for 48 h, at which time they were 80% confluent. Cells were transfected with MIF RNAi (sc-37137; Santa Cruz Biotechnology) or CD74 RNAi (sc-35023; Santa Cruz Biotechnology) using LipofectAMINE 2000 transfection reagent (Invitrogen Life Technologies) following the manufacturer’s protocol. Following incubation at 37°C for 7 h in a CO2 incubator, 1 ml of RPMI 1640 with Glutamax and 20% FBS without antibiotics was added and growth continued for 18 h. The transfection medium was then removed and replaced with normal growth medium. Every 24 h for the next 72 h, cell proliferation was assayed using WST-1 dye metabolism as described above. Six wells were collected every 24 h; half the wells were processed for total protein, and the other half were processed for total RNA. Control wells were treated with scramble RNAi (sc-37007).
Apoptosis assays
Apoptosis was assessed by caspase-3 activity and DNA laddering. For assessment of caspase-3 activity, 2 x 106 cells were plated into 12-well culture dishes 6 h before RNAi treatment. After 48 h, cells were dislodged mechanically and washed twice in PBS. Caspase-3 activity was assessed using the BD ApoAlert caspase-3 colorimetric assay kit (BD Biosciences) as described by the manufacturer. p-Nitroaniline (pNA) was measured using a plate reader (Bio-Tek Instruments) at 405 nm. z-DEVD-fmk, a specific inhibitor of caspase-3, was used to confirm assay specificity. For DNA-laddering analysis, 48 h Ab-treated cells were harvested from cell wells and cell culture medium. The resulting cell pellet was washed in PBS, and genomic DNA was isolated by the direct addition of DNAzol (Invitrogen Life Technologies). DNA (2 µg) was resolved on 1.5% agarose gels with a 10- to 0.5-kb DNA marker for fragment size reference (Kilobase DNA marker; GE Healthcare).
Matrigel invasion
Matrigel invasion assay was conducted in 24-well Transwell culture plates containing microporous (3.0 µm) membranes coated with Matrigel (Costar). In brief, DU-145, LNCaP, or BPH-1 cells (3 x 104 cells/well) were added to the upper chamber containing basal medium (RPMI 1640 with 0.1% BSA) with appropriate Ab (2 µg/ml), and transmigration toward a serum gradient (70% standard growth medium, 20% conditioned medium, and 10% FBS with appropriate Ab at 2 µg/ml) in the lower chamber continued for 18 h. Following the incubation period, the nonmigrating cells were removed from the upper chamber using a cotton swab, and the adherent cells on the lower surface of the insert were stained with toluidine blue, and total invading cells were counted in nine representative fields at x400 magnification. The data were expressed as average number of cells per treatment per x400 field ± SE and were analyzed by ANOVA; p < 0.05 was considered statistically significant.
Real-time PCR
Total RNA was isolated from RNAi-treated DU-145 cells and reverse transcribed as described previously (23). Real-time PCR determination of MIF, CD74, and CD44v9 cDNA amounts were determined using a SYBR green reagent and the appropriate PCR primers (MIF-PPH00548A, CD74-PPH05587A, and CD44v9-PPH00114A; SuperArray) normalized to an 18S rRNA internal standard (PPH05666A; SuperArray). Temperature cycling and real-time fluorescence measurements were performed using an Opticon Monitor system (MJ Research). The PCR conditions were as follows: initial incubation at 95°C for 15 min, followed by 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s with incubation at 85°C for 15 s before the plate was read. The relative quantification of gene expression was performed using the comparative CT (
CT) method following initial validation experiments that verified that the amplification efficiencies of MIF, CD74, CD44v9, and 18S rRNA were approximately equal as determined by CT with serial dilution of the control template. Plots of CT vs cDNA concentration were fit using least-squares regression analysis with the resulting slopes close to zero (MIF = –0.072, CD74 = 0.070, and CD44v9 = 0.069) indicating that relative changes in gene expression could be determined by CT (29). The CT was calculated as the difference between the normalized CT values (CT) of the treatment and the control samples: CT = CT treatment – CT control. CT was converted to fold change by the following formula: fold change = 2–CT (29).
ERK 1/2 activation
The effect of MIF or CD74 RNAi treatment on ERK 1/2 activation was examined by Western blotting with Abs specific for phosphorylated (activated) ERK 1/2 (AF-1018; R&D Systems). BPH-1, LNCaP, and DU-145 cells were treated with RNAi for 72 h, and Western blotting was performed as described above. The membrane was then stripped and reprobed with ERK 1/2 Ab specific for total ERK 1/2 to normalize for differences in protein loading (AF-1576; R&D Systems).
Gene expression array analysis
A GEArray Q series human cancer (HS-044) and G-protein (HS-025) array kits were obtained from SuperArray. Included in these arrays are TNF-
and IL-1, which are genes associated with downstream activation by MIF (30). DU-145 cells were cultured and treated as described above for RNAi experiments. The cells were pooled from three separate experiments (2 x 105 cells per experiment) to decrease bias. After 72 h of RNAi treatment, cells were harvested, total RNA was isolated with TRIzol reagent (Invitrogen Life Technologies), and 5 µg of RNA was used as a template to generate biotin-16-dUTP-labeled cDNA probes (AmpoLabeling-LPR kit; SuperArray). The cDNA probes were denatured and hybridized to membrane at 60°C overnight, and the resulting image was analyzed using the GEArray analyzer program (SuperArray). In brief, spot intensity was normalized by subtraction of the background of the average intensity value of three blank spots. The averages of two ribosomal protein RPL13A spots were used as positive controls and set as baseline values with which the signal intensity of other spots was compared. The array data represents mean fold variation of pooled samples from two separate arrays. RNAi-induced differences in gene expression were considered as present when the mean fold change was 
2.0. Data are expressed as increased or decreased compared with controls (i.e., DU-145 cells treated with scramble RNAi, control vs DU-145 cells treated with message-specific RNAi).
ISO-1 treatment of DU-145 xenografts
All animal experiments were done according to regulations approved by the Institutional Animal Care and Use Committee. In brief, 250 µl of DU-145 human prostate carcinoma cells (5 x 106 cells) in Matrigel (BD Biosciences) were inoculated s.c. into the flank of 5- to 6-wk-old nude male mice weighing 20 to 25 g (Harlan). Immediately following cell implantation, ISO-1 (at 20 mg/kg) or vehicle (5% DMSO) was injected i.p. Mice were injected twice a week (Tuesday and Friday) for a total of 10 doses per mouse (n = 4 mice per group). The tumor size was measured by calipers and recorded weekly for a period of 35 days. Tumor volume was estimated using the formula: (width)2 x length/2, measured in millimeters. At the end of the treatment, the mice were sacrificed and the tumors were removed, weighed, and processed for immunohistochemistry and protein. Serum was collected for human MIF ELISA determination using our previously described assay protocol (8). Tumor size and MIF amounts, represented as mean ± SE, were analyzed by Student’s t test. Values of p < 0.05 were considered as statistically significant.
Immunohistochemical staining xenograft tumors
Excised tumors were fixed in 4% buffered formaldehyde. Immunohistochemical staining was done as previously described (17). In brief, paraffin-embedded 4-µm tumor sections were overlaid with goat anti-proliferating cell nuclear Ag (PCNA) or rat anti-CD34 Ab (sc-9857 or sc-18917, respectively; Santa Cruz Biotechnology). Sections were incubated with appropriate biotinylated secondary Abs (Vector Laboratories) and visualized with streptavidin-biotin conjugated with HRP (Vector Laboratories). CD34 immunostained sections were counterstained with H&E, whereas PCNA sections were not. For quantitative analysis, stained nuclei (PCNA) or cells (CD34) were counted in 10 areas from all tumors at x40 total magnification. The results were averaged for each treatment and expressed as mean ± SE. Data were analyzed by ANOVA; p < 0.05 was considered as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Western blot analyses showed that MIF, CD74 (MIF receptor), and CD44v9 (signaling component of the MIF membrane receptor complex) amounts were increased in prostate cancer cell lines compared with benign prostate cells (Fig. 1, A and B). Invasive androgen-independent cells (DU-145) synthesize more MIF, CD74, and CD44v9 than noninvasive CaP cells (LNCaP) or benign BPH-1 cells (p < 0.001, unpaired t test; Fig. 1, A and B). Moreover, determination of the MIF genotype in these cells showed that the increased protein expression seen in DU-145 cells was associated with the high-expression –173 G/C MIF polymorphism and the high-expression –794 6/7 CATT repeat polymorphism (25). The BPH-1 and LNCaP cells both contain the lower expressing G/G, 6/6 MIF polymorphisms.
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To test the relationship between relative amounts of cell surface CD74, thus leading to MIF/CD44v9/CD74 interactions and prostate cancer aggressiveness, BPH-1, LNCaP, and DU-145 cell surface proteins were labeled with biotin, and avidin-purified biotinylated cell lysates were immunoprecipitated with MIF, CD44v9, or CD74 mAbs and detected using HRP-labeled streptavidin. Prostate cancer cells express greater cell surface protein amounts than BPH-1 cells (Fig. 1D). Examination of the particular proteins of interest (CD74, MIF CD44v9) showed that only DU-145 cells express cell surface CD74. In addition, DU-145 cells express a greater amount of cell surface MIF than LNCaP cells and cell surface MIF was not detected in BPH-1 cells (Fig. 1D). Because a small amount of cell surface MIF was detected in LNCaP cells, it is possible that these cells also expressed cell surface CD74 (thus binding MIF) albeit at a much lower amount and, thus, below the CD74 immunoprecipitation detection level. The possibility that MIF may bind to other cell surface proteins, however, cannot be ruled out at this point. Finally there was no difference in the cell surface amounts of CD44v9 expressed in BPH-1, LNCaP, or DU-145 cells (Fig. 1D). These results suggest that increased cell surface expression of CD74 allows greater binding of secreted MIF molecules by CD74 and, if this is the case, greater activation of CD74-mediated signaling pathways (e.g., ERK activation). Increased cell surface expression of CD74 in DU-145 cells compared with LNCaP and benign cells suggests that this mechanism may be associated with androgen-independent prostate cancer cell growth.
MIF-specific inhibitor (ISO-1) reduces MIF tautomerase activity and cell proliferation in DU-145 cells only
MIF is readily detectable within prostate cells in vitro and is actively secreted into the culture medium. However, it is not known whether intracellular and/or secreted MIF is biologically active. ISO-1 is a nontoxic inhibitor of MIF that binds to bioactive MIF at a catalytically active, tautomerase site within this cytokine (28). This is a useful target of MIF function, because the tautomerase activity can be measured by in vitro assay to determine the presence of bioactive MIF. MIF tautomerase activity was detected in cell lysates (Fig. 2A) and spent culture medium (Fig. 2B) from subconfluent BPH-1, LNCaP, and DU-145 cultures, indicating that MIF has biological activity both inside the cell and following secretion into the culture medium. In addition, intracellular MIF tautomerase activity was 2.0-fold greater in prostate cancer cells when compared with BPH-1 cells (0.0587 ± 0.002 change in A475 nm per second per milligram of total protein, DU-145 and 0.0447 ± 0.004 change in A475 nm per second per milligram of total protein, LNCaP vs 0.0240 ± 0.003 change in A475 nm per second per milligram of total protein, BPH-1). MIF-associated tautomerase activity in cell lysates and spent culture medium was reduced in a dose-dependent manner by increasing concentrations of the MIF antagonist ISO-1 (Fig. 2, A and B). Although ISO-1 reduced MIF in vitro the tautomerase activity in the cell lysates and in the culture medium of all tested cell lines (Fig. 2, A and B), inactivation of bioactive MIF using ISO-1 preferentially decreased proliferation of DU-145 cells (Fig. 2C). Therefore, blockade of MIF’s biological activity, either inside the cell or outside the cell (in the culture medium) resulted in decreased cell proliferation only in cells showing cell surface CD74 localization (DU-145), without affecting BPH-1 or androgen-dependent LNCaP cells.
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Neutralizing Abs to MIF or CD74 inhibited the proliferation of DU-145 cells with anti-MIF showing a significant decrease within 24 h of treatment (Fig. 3A, p < 0.05) and anti-CD74 decreasing cell numbers at 48 h (Fig. 3B, p < 0.01). Anti-MIF or anti-CD74 treatments also induced apoptosis in DU-145 cells (Fig. 4A), indicating that cell viability in DU-145 cells is dependent upon functional MIF-CD74 interactions. These treatments had no effect on BPH-1 or LNCaP cells that lacked cell surface expression of CD74. Additionally, inclusion of anti-MIF or anti-CD74 with DU-145 cells in the upper Transwell chamber reduced Matrigel invasion (Fig. 5). Therefore, blocking MIF-CD74 interactions with neutralizing Abs specifically affected androgen-independent DU-145 cells.
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The effects of MIF or CD74 inactivation at the mRNA level were assessed by RNAi treatment. RNAi treatment decreased the associated mRNA and protein in all cell lines (Fig. 6 and data not shown). Unexpectedly, MIF RNAi treatment resulted in a dramatic increase in CD74 mRNA and protein by DU-145 cells (Fig. 6, B and D), suggesting a coordinated regulation of expression to result in receptor up-regulation when the agonist amounts are depressed. As was seen with ISO-1 and neutralizing Ab treatment, MIF or CD74 RNAi treatment only affected proliferation of DU-145 cells (Fig. 6E). Reduction in MIF or CD74 mRNA and the associated decrease in detectable protein did not affect BPH-1 or LNCaP cell proliferation (data not shown). MIF or CD74 RNAi induced apoptosis only in DU-145 cells (Fig. 4B).
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, IL-6, jun (AP-1), TNF- were down-regulated at least 2.5-fold by both MIF RNAi and CD74 RNAi (Table I). While expression of two genes (GRM-8 and opioid receptor 1 (OPR- 1)) were up-regulated at least 2.5-fold by MIF RNAi and CD74 RNAi (Table I). MIF RNAi specifically down-regulated the expression of cyclin D1 (5.2-fold, CCN-D1) and cyclooxygenase-2 (6.9-fold, COX-2), but up-regulated the expression of p27 (3.5-fold, CDKN-1B). CD74 RNAi specifically down-regulated the expression of cyclin E2 (3.9-fold, CCN-E2), but specifically up-regulated the expression of type 1 collagen (2.8-fold, COL1-1), heat shock protein 90 kDa (3.7-fold, HSPCA), prolactin (2.6-fold, PRL) and prostaglandin E receptor (4.2-fold, PTGER-1). Although all of these genes are known to be expressed in the prostate, these results are the first to document a requirement for MIF and/or CD74 expression in the regulation of the aforementioned genes. The gene expression changes in Bcl-2, cyclin D1, and cyclin E2 detected by arrays were verified by Western blot analysis. MIF RNAi resulted in decreased protein amounts of both Bcl-2 and cyclin D1, whereas CD74 RNAi treatment resulted in decreased amounts of Bcl-2, cyclin D1, and cyclin E2 (Fig. 8).
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We examined whether blocking MIF using the antagonist ISO-1 could inhibit the in vivo growth and neovascularization of androgen-independent DU-145 prostate tumors. The dose of ISO-1 used in this study was based on a previous report (31). The mean tumor volume after 35 days of treatment was significantly smaller in the ISO-1 treated animals (300.3 ± 58.6 mm3) compared with the controls (776.8 ± 82.6 mm3; Fig. 9A; p < 0.01). In addition, total tumor weight after treatment in the ISO-1-treated animals was significantly smaller than in the control mice (p < 0.05; Fig. 9B). Serum MIF and total tumor MIF amounts were determined using a human MIF ELISA, which we previously determined does not detect mouse MIF (7, 8). ISO-1 treatment did not change the total tumor human MIF per protein content (491.9 ± 80.2 vs 421.2 ± 40.6 ng/mg in control and treated, respectively), but ISO-1 treatment did significantly reduce total human serum MIF (1.54 ng/ml controls, 0.89 ng/ml with ISO-1 treatment; p < 0.05), indicating that ISO-1 treatment may have decreased tumor-secreted MIF.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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and IL-1, and as such, it has been suggested that MIF is a key initiator of the inflammatory cascade (30, 32, 33). In addition, MIF is an autocrine regulator of cell proliferation and differentiation. Serum-starved NIH/3T3 cells secreted endogenous MIF as a consequence of serum stimulation resulting in proliferation of quiescent cells. This response was associated with phosphorylation and subsequent activation of ERK 1/2 (34, 35). Inability to modulate the MIF autocrine loop may result in localized chronic inflammation, and the resultant epithelial cell injury may play a role in carcinogenesis. Our current results suggest that cell surface expression of CD74 is a key step in MIF-mediated signal transduction and may play an important role in androgen-independent prostate cancer. It is difficult to account for the absence of a MIF response under "normal" physiological conditions, considering the ubiquitous expression of this protein. All prostate epithelial cell lines examined release MIF into the culture medium (data not shown), suggesting that bioactive MIF is readily available in the local environment. In addition, increased MIF-CD74 and MIF-CD44v9 complex formation was observed in prostate cancer cells. However, cell surface expression of CD74 was only observed in androgen-independent (DU-145) prostate cancer cells. This is an intriguing and novel observation, but whether this finding applies to other androgen-independent prostate cancer cells remains to be determined. CD44v9, although increased in prostate cancer cells (36), was not increased at the cell surface. Taken together, these data suggest that although CD44 may be necessary for CD74 signaling (21, 22), it is not a regulating mechanism because it is present in overabundance and in steady-state levels at the cell surface. Therefore, these results suggest that up-regulation of cell surface CD74 is the necessary step in mediating MIF-CD74-activated signal transduction. Thus, blocking MIF-CD74 interactions (presumably at the cell surface) either by pharmacologic inhibition of MIF (e.g., ISO-1) or decreasing MIF and/or CD74 would have little effect on normal cell function (indicating that these cells are not dependent on MIF-mediated signal transduction) but may provide a potent inhibition of cancer cell proliferation (indicating that these cells are dependent on MIF-mediated signal transduction). Therefore, it is possible that in normal prostate epithelial cells, MIF may be important for optimal cell growth under certain specific conditions (e.g., inflammation), whereas in cancer cells, there is a reliance upon MIF-dependent ERK activation to sustain cell proliferation resulting in increased MIF and CD74 expression. In fact, our present findings confirm this hypothesis because blocking MIF protein (anti-MIF), message (RNAi), and/or activity (ISO-1) had little or no effect on benign BPH-1 cells or androgen-dependent LNCaP cells but resulted in decreased DU-145 cell proliferation and viability.
Numerous studies have determined that reducing tumor MIF results in decreased cell proliferation (11), induction of apoptosis (13) and reduction in the synthesis and secretion of other growth factors (23, 37, 38). In accordance with these findings, the treatments used in the present study were found to suppress DU-145 cell proliferation and induce apoptosis. The molecular mechanism(s) associated with MIF function in the prostate are not fully understood. However, because DU-145 cells appear to have abundant (compared with other prostate cells examined in this study) cell surface localization of CD74, it would be expected that alteration of MIF and/or CD74 should also affect the expression and secretion of other proinflammatory molecules. Interestingly, our results show decreased expression of IL-1, TNF-, and IL-6, with either MIF or CD74 RNAi treatment lending evidence for MIF, through binding with cell surface CD74, as a modulator of cytokine production in prostate cancer cells.
MIF-CD74 interaction activates the ERK 1/2 signaling pathway, presumably through interaction with CD44 (21). Interestingly, there is no information as to the other pathways and/or signaling molecules (aside from ERK activation) that are affected by MIF-CD74 interaction, although a recent report suggests that MIF engagement of CD74 augments B cell survival by increasing the expression of Bcl-xL (20). We determined whether MIF and/or CD74 deficiency influenced downstream targets of MAPK using targeted array analysis. The results presented in this study document significant changes in the expression of known MIF downstream signaling molecules that affect prostate cancer cell viability and emergence of androgen-independent cell growth, such as cyclin D1 (39), p27 (40), and bcl-2 (41). Cyclin D1 is a critical regulator involved in cell cycle progression through the G1 phase into the S phase, thereby controlling cell proliferation, and its expression is attributed to ERK 1/2 activation (42). Our studies document that inhibition of the MIF and/or CD74 decreases cyclin D1 gene expression. A number of studies have shown that the induction of cyclin D1 expression requires activation of the MAPK pathway and that cyclin D1 expression is temporally preceded by the activation of a class of genes known as immediate-early genes, such as jun (AP-1) and Egr-1 (42, 43). The present study documents that inhibition of MIF and/or CD74 also results in decreased expression of jun and Egr-1, suggesting that MIF-CD74 interaction resulting in ERK 1/2 activation is a key initiator of DU-145 cyclin D1-regulated cell proliferation.
Based upon data obtained in the present study, we suggest that cell surface expression of CD74 is the rate-limiting member of the MIF-signal transduction complex. In DU-145 cells, large quantities of MIF are constitutively synthesized and secreted. The MIF-CD74 complex (in association with CD44v9) activates MAPK signal transduction pathways resulting in up-regulation of cyclin D1 and sustained cell proliferation.
In the DU-145 xenograft model, ISO-1 treatment suppressed angiogenesis and in vivo tumor growth. This is the first report of the inhibitory effects of ISO-1 treatment on in vivo tumor growth. Our results, therefore, support the hypothesis that the therapeutic effect of blocking MIF on tumor growth is attributable in part to the inhibition of angiogenesis.
In summary, the present study shows increased MIF and CD74 expression in the aggressive androgen-independent DU-145 prostate cancer cell line. In addition, this cell line had significant amounts of cell surface CD74, an event that was not observed for benign or androgen-dependent cell lines. Blocking MIF-CD74 interaction selectively decreased ERK 1/2 activation, decreased cell proliferation, and increased apoptosis in DU-145 cells only, while being ineffective in benign cells (BPH-1) or androgen-dependent cells (LNCaP). The emerging role of MIF and its receptor (CD74) in prostate tumorigenesis suggest that modulating this cytokine’s activity may result in new, selective, therapeutic modalities for men with androgen-independent prostate cancer.
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1 Supported by Veterans Affairs Merit Review Program (to K.L.M.-S. and P.L.V.), the Bay Pines Foundation (to K.L.M.-S. and P.L.V.), and National Institutes of Health (to R.B. and L.L.). 
2 Address correspondence and reprint requests to Dr. Katherine L. Meyer-Siegler, Bay Pines Veterans Affairs Health Care System, Research and Development (151), 10000 Bay Pines Boulevard, Bay Pines, FL 33744. E-mail address: Katherine.Siegler{at}med.va.gov
3 Abbreviations used in this paper: MIF, macrophage inhibitory factor; CD44v9, CD44 variant 9; PrEC, human prostate epithelial cells; PVDF, polyvinylidene difluoride; RNAi, RNA interference; PCNA, proliferating cell nuclear Ag; p-ERK, phosphorylated ERK; pNA, p-nitroaniline.
Received for publication June 7, 2006. Accepted for publication October 2, 2006.
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