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Molecular Biology and Genetics |
Laboratory of Immunophysiology, Department of Animal Sciences [W-H. S., J-H. Z., S. R. B., K. W. K.] and Department of Pathology, College of Medicine [G. G. F.], University of Illinois, Urbana, Illinois 61801, and Institut National de la Recherche Agronomique-Institut National de la Santé et de la Recherche Médicale U394, Unité de Recherches de Neurobiologie Intégrative, Institute François Magendie, 33077 Bordeaux, France [R. D.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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at 0.1 ng/ml and is reduced by 80% at 100 ng/ml. Similarly, both IL (interleukin) -1ß and IL-6 significantly reduce the ability of IGF-I to promote DNA synthesis. Flow cytometry confirmed that all three of the cytokines inhibit IGF-I-induced DNA synthesis by preventing cells from entering the S phase of the cell cycle, leading to G0/G1 arrest. Although none of the cytokines alone are cytotoxic to transformed epithelial cells in the absence of serum, TNF- significantly inhibits the antiapoptotic property of IGF-I in protecting MCF-7 cells from DNA fragmentation. TNF- and IL-1ß act by inhibiting the IGF-I receptor from tyrosine phosphorylating insulin receptor substrate-1 without affecting tyrosine kinase activity of the IGF-IR itself. These data support the novel idea that the major inhibitory properties of proinflammatory cytokines on growth of breast cancer cells are manifested prominently in the presence of growth factors. These data also highlight growth factor receptor adaptor molecules, such as insulin receptor substrate-1, rather than the receptors themselves as targets for antitumor therapeutic strategies. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3
(1)
, IL-1ß (2)
, and IL-6 (3)
cause tumor regression and increase median survival time in a variety of cancer patients. In contrast, elevated circulating concentrations of growth factors such as IGF-I are a surrogate risk for cancers of the breast (4)
, prostate (5)
, and colon (Ref. 6
; reviewed in Refs. 7
, 8
), which are the three most prevalent cancers in the United States. Inhibition of growth factor activation and site-directed delivery of proinflammatory cytokines are two biological strategies that are now being tested in cancer therapy. Unfortunately, there are almost no reports that form a conceptual basis to link these two strategies. In this study, we tested the novel idea that the three major proinflammatory cytokines inhibit breast cancer proliferation by impairing mitotic and antiapoptotic activities that are mediated by growth factor receptors.
Elevation in plasma IGF-I caused by injections of growth hormone into primates induces mammary hyperplasia (9)
. These effects can be mimicked in vitro, where IGF-I promotes the growth and survival of both primary (10)
and transformed (11)
mammary epithelial cells. In the absence of growth factors provided by FBS, the activity of TNF- is significantly impaired, as assessed by the ability of TNF to inhibit the growth and induce the death of breast cancer cells (12)
. Moreover, TNF- causes growth inhibition in hormone-dependent but not hormone-independent breast cancer cells (13)
. These data could be interpreted to indicate that cytokines suppress survival and growth of transformed cells by inhibiting the biological activity of growth factors. We have demonstrated that FBS acts like IGF-I to increase expression of the antiapoptotic protein Bcl-2 (14)
. Similarly, an antibody (IR3) directed against the human IGF-IR profoundly inhibits the ability of FBS to promote both cell proliferation (15)
and differentiation (16)
. This same IGF-IR antibody has now been found to augment chemotherapy in both breast (17)
and colon cancers (18)
, indicating that the IGF-IR may be a crucial target for cancer therapies.
In this report, we have taken advantage of earlier findings showing that IGF-I is a potent mitogen for transformed human mammary epithelial cells (19)
. We demonstrate that TNF-, IL-1ß, and IL-6 all dose-dependently inhibit IGF-I-promoted DNA synthesis. Flow cytometry confirmed that these cytokines suppress IGF-I-induced MCF-7 cell proliferation by preventing cells from entering the S phase of the cell cycle, leading to the arrest of these cells in the G0/G1 phase. Although none of the cytokines alone are cytotoxic in the absence of FBS, TNF- inhibits the ability of IGF-I to protect MCF-7 cells from DNA fragmentation. Both TNF- and IL-1ß inhibit the IGF-IR from tyrosine phosphorylating IRS-1 without affecting intrinsic tyrosine kinase activity of the receptor. These data support the novel idea that proinflammatory cytokines suppress growth of breast cancer cells by inhibiting activation of early adaptor molecules used by growth factor receptors to promote cell division. More importantly, these data form a conceptual basis for the use of both antigrowth factor and proinflammatory cytokine gene targeting strategies in combination therapy for inhibiting the uncontrolled proliferation of breast cancer cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and IL-1ß were purchased from Intergen (Purchase, NY), and recombinant human IL-6 was from Calbiochem-Novabiochem Co. (San Diego, CA). All of the antibodies were purified as immunoglobulins. They were rabbit anti-IRS-1, rabbit anti-IRS-2, and rabbit antiPI3k p85 (all from Upstate Biotechnology Inc., Waltham, MA); rabbit antiIGF-IRß (C-20; sc-713), goat anti-actin (I-19, sc-1616), HRP-linked donkey antigoat IgG (all from Santa Cruz Biotechnology Inc., Santa Cruz, CA), mouse antiphosphotyrosine PY20, and HRP-conjugated mouse PY20 monoclonal antibody (both from Transduction Laboratories, San Diego, CA); and HRP-linked donkey antirabbit and sheep antimouse IgG (both from Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Rainbow molecular weight markers were from Amersham Pharmacia Biotech, Inc.
Measurement of DNA Synthesis.
Human MCF-7 and T-47D cells were trypsinized, washed three times (400 x g, at room temperature), adjusted to 5 x 104 cells/ml in culture medium, and plated into 96-well plates (Costar 3596; Corning Inc., Corning, NY) in a volume 200 µl/well. After incubation for 24 h, cells were treated in triplicate with different concentrations of recombinant human TNF-, IL-1ß, or IL-6 with or without 100 ng/ml of IGF-I. After 48 h, [3H]dThd (1µCi/well; 1 Ci = 37 GBq; ICN Pharmaceuticals, Inc., Costa Mesa, CA) was added, and the cells were incubated at 37°C for an additional 5 h. Cells were harvested immediately onto fiberglass filters with a PHD cell harvester (Cambridge Technology, Inc., Cambridge, MA). The filters were dried and then submerged in 3 ml of scintillation fluid, and [3H] radioactivity was determined on a Beckman LS 6000IC scintillation counter (Fullerton, CA).
Cell Cycle Analysis.
Human MCF-7 adenocarcinoma cells were seeded at 5 x 104 cells/ml in a volume of 2 ml in six-well Costar plates (Corning, Inc.) in maintenance medium for 24 h. After washing and incubating in culture medium for another 24 h, cells were treated in duplicate with either 1 or 5 ng/ml of TNF-, 50 ng/ml of IL-1ß, or IL-6 in the absence or presence of IGF-I (100 ng/ml). After 48 h, cells were trypsinized, washed once with PBS, and fixed with 80% ethanol at 4°C for 24 h. Fixed cells were washed three times and incubated for 1 h with a PI (Sigma Chemical Co.) solution (20 µg/ml) containing 0.1 mg/ml of RNase A (Sigma Chemical Co.). Cells were then subjected to cell cycle analysis on an EPICS XL flow cytometer (Coulter, Miami, FL). Cell debris was excluded on the basis of forward versus side scatter. Doublets and clumps were excluded by gating on a bivariate distribution of AUX (PI peak pulse) versus the PI integrated signal. Data from 10,000 events were collected in the final gated histograms.
Analysis of Cell Viability.
Cell survival was analyzed by measuring the proportion of unfixed MCF-7 cells that were positive for PI as assessed by flow cytometry. In these experiments, MCF-7 cells were plated in six-well culture plates at 5 x 104 cells/ml in a volume of 2 ml of maintenance medium for 24 h. Cells were washed and incubated in culture medium for another 24 h and exposed to different doses of TNF-, IL-1ß, or IL-6. After 48 h, the cells were trypsinized, washed with PBS, and subsequently incubated with 0.5 ml of PI at 2 µg/ml for 10 min. The PI-positive cell population was measured using an EPICS XL flow cytometer (Coulter).
Viable cells were measured after a 48-h treatment with different doses of TNF-, IL-1ß, or IL-6. MCF-7 cells were incubated for 4 h in 0.5 mg/ml of MTT (Sigma Chemical Co.) The precipitated blue formazan was solubilized by the addition of isopropyl alcohol/0.08 N HCl. Absorbance of the mixture was measured at 570 and 630 nm using an EL-310 microplate reader (Bio-Tek Instruments, Winooski, VT), as described previously by us (20)
. The absorbance value at 630 nm was subtracted from the value at 570 nm, and MTT conversion was calculated as relative units of MTT activity.
DNA fragmentation was used to measure the proportion of apoptotic cells, as described previously (21)
. MCF-7 cells were seeded into 24-well plates (Costar, Corning, Inc.) at 2 x 105 cells/ml in a volume of 1 ml and cultured for 24 h in maintenance medium. Cells were then washed three times and labeled with 2 µCi/0.5 ml/well of [3H]dThd in 37°C for another 24 h. After the cells were labeled with [3H]dThd, they were washed once and treated in duplicate for 48 h with different concentrations of TNF-, IL-1ß, or IL-6 with or without IGF-I (100 ng/ml) in culture medium. Trypsinized cells were collected by centrifugation at 1000 x g, and the resulting cell pellet was resuspended and lysed by addition of 0.5 ml Tris-Triton-EDTA solution [10 mM Tris HCl (pH 7.4), 1 mM EDTA, and 0.2% Triton X-100]. Lysates were vortexed vigorously and centrifuged at 16,000 x g for 10 min (4°C) to separate intact chromatin (pellet) from soluble low molecular weight DNA (supernatant). Supernatants (containing fragmented DNA) were transferred to a fresh tube, and the pellets were resuspended in 0.5 ml TTE. Intact chromatin DNA (pellet) and fragmented DNA (supernatant) were collected onto fiberglass filters, and samples were counted on Beckman LS 6000IC scintillation counter. The percentage of fragmented DNA was calculated as follows: % DNA fragmentation = [supernatant cpm/(pellet cpm + supernatant cpm)] x 100.
Western Blotting to Detect IGF-IR, IRS-1, and IRS-2.
Whole cell lysates were prepared from 1 x 106 MCF-7 cells in RIPA buffer [50 mM Tris-HCl (pH 7.4), 1% NP40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, containing freshly added 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 48 trypsin inhibitory units of aprotinin, 40 nM leupeptin, and 2 µg/ml pepstatin; Sigma Chemical Co.]. After centrifugation at 16,000 x g at 4°C for 15 min, the amount of protein in the supernatant was determined using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equal amounts of proteins (50 µg) were separated on 7.5% polyacrylamide gels and transferred to Trans-Blot polyvinylidene difluoride membranes (Bio-Rad Laboratories). The membranes were blocked for 1 h at room temperature with 1% BSA for IRS-1, IRS-2, and p85 or 5% skim milk for the IGF-IR and actin. Both BSA and skim milk were dissolved in Tris-buffered saline [20 mM Tris-HCl (pH 7.4) and 150 mM NaCl] supplemented with 0.1% Tween 20. The membranes were then incubated in the same blocking buffer with the indicated antibodies for 1 h at room temperature. Blots were incubated with HRP-labeled donkey antirabbit IgG (1:2,000 dilution for IRS-1, IRS-2, IGF-IR, and p85) or donkey antigoat IgG (1:2,000 dilution for actin). Immunoreactive bands were visualized using enhanced chemiluminescence detection reagents (ECL; Amersham Pharmacia Biotech, Inc.). NIH3T3 cell lysates were used as a positive control.
Immunoprecipitation.
MCF-7 cells were lysed in RIPA buffer, and equal amount of protein in cell lysates (
200 µg protein for IRS-1 precipitation and 500 µg protein for IGF-IR coimmunoprecipitation with IRS-1) were immunoprecipitated overnight at 4°C. All of the antibodies used for immunoprecipitation were preincubated with protein G-Sepharose at 4°C for 3 h. Immunoprecipitates were washed three times with RIPA buffer and separated on 7.5% polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes and subjected to Western blotting as described above. Intensity of protein bands on autoradiograms was quantified by scanning with an Agfa Duosacan T1200 scanner (NucleoTech, San Mateo, CA) followed by analysis using GelExpert 3.5 software (NucleoTech).
Statistical Analysis.
All of the statistical analyses were performed using Statistical Analysis System for Windows (22)
. All of the data, including standardized densitometric intensities from replicate autoradiograms, were analyzed as a completely randomized design using standard ANOVA procedures. Treatment differences were detected using multiple ad hoc comparisons, as assessed with Fisher’s least-significant-difference test. All of the experiments were independently replicated at least three times, and data were summarized as means ± SE. Two-sided Ps of P < 0.05 (*) or P < 0.01 (**) were considered statistically significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-1ß, and IL-6 Inhibit IGF-I-induced DNA Synthesis in Human MCF-7 Breast Adenocarcinoma Cells. inhibits proliferation (24)
and induces apoptosis (25)
of MCF-7 cells cultured in the presence of undefined growth factors like FBS. However, it remains unknown whether TNF- causes a similar cytostatic effect in breast cancer cells in the absence or presence of a defined recombinant growth factor like IGF-I. Therefore, we estimated DNA synthesis in MCF-7 cells after exposure to IGF-I in combination with various concentrations of proinflammatory cytokines by measuring cellular incorporation of [3H]dThd. Compared with cells incubated in medium alone, IGF-I (100 ng/ml) consistently increased DNA synthesis by 15-fold (Figs. 1, A–C)
. Analysis of [3H]dThd incorporation revealed a strong dose-dependent inhibition by TNF- of IGF-I-promoted DNA synthesis (Fig. 1A)
. TNF-, at a concentration as low as 0.1 ng/ml, significantly inhibited the ability of IGF-I to increase DNA synthesis. At 2 ng/ml, TNF- caused a 50% inhibition of DNA synthesis. Although the magnitude of DNA synthesis was much lower in the absence of IGF-I, TNF- additionally reduced basal [3H]dThd incorporation with a minimum significant dose of 0.5 ng/ml.
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nor IL-6 (Fig. 1C)
impaired [3H]dThd incorporation. However, when IGF-I was added, these cytokines caused a dose-dependent reduction in the ability of IGF-I to promote DNA synthesis. These data establish that all three of the proinflammatory cytokines inhibit the ability of IGF-I to induce incorporation of [3H]dThd, suggesting that the proinflammatory cytokines reduce the proportion of cells in the S phase of the cell cycle.
IGF-I-induced Cell Cycle Progression Is Impaired by TNF-, IL-1ß, and IL-6.
IGF-I is well known to act as a late G1 progression factor (29)
, and this is achieved by regulating expression of proteins at the G1-S transition point (30, 31, 32)
. Therefore, we tested the idea that proinflammatory cytokines inhibit cell cycle progression even earlier than the S phase. We first synchronized MCF-7 cells in G0/G1 by withdrawing FBS for 24 h, which led to accumulation of 80% of the cells in G0/G1 (Fig. 2A)
. In the absence of IGF-I, none of the proinflammatory cytokines affected the percentage of cells in the G0/G1, S, or G2/M phases of the cell cycle (P > 0.10). When compared with medium alone (16.6 ± 3.4%), IGF-I reduced (P < 0.01) the proportion of cells in the sub-G0/G1 peak (4.7 ± 1.7%). However, none of the proinflammatory cytokines affected the proportion of cells in this hypodiploid peak (P > 0.10).
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65%. These cycling cells increased the proportion of cells in both the S (from 8% to 15%) and G2/M (from 12 to 20%) phases of the cell cycle. However, TNF- (5 ng/ml; Fig. 2B
), IL-1ß (50 ng/ml; Fig. 2C
), and IL-6 (50 ng/ml; Fig. 2D
) reduced the ability of IGF-I to promote exit from G0 by 89% (P < 0.01), 72% (P < 0.01), and 45% (P < 0.05), respectively. Consistent with the [3H]dThd data in Fig. 1
, TNF-, IL-1ß, and IL-6 reduced the increase in S phase cells caused by IGF-I by 32% (P < 0.01), 32% (P < 0.01), and 13% (P > 0.10), respectively. Similarly, the ability of IGF-I to increase the proportion of cells in the G2/M phase was reduced by 29% (TNF-; P < 0.01), 23% (IL-1ß; P < 0.01), and 21% (IL-6; P < 0.05). When total cycling cells (S plus G2/M phases) were analyzed, IGF-I increased (P < 0.01) this proportion of cells from 20.0 ± 2.4% to 31.9 ± 4.1%. In the absence of IGF-I, none of the proinflammatory cytokines affected the percentage of total cycling cells (P > 0.10). However, in the presence of IGF-I, TNF- (5 ng/ml), IL-1ß, and IL-6 reduced the proportion of cycling cells from 31.9 ± 4.1% to 22.0 ± 2.9% (P < 0.01), 22.4 ± 2.3% (P < 0.01), and 25.8 ± 1.0% (P < 0.05), respectively. These experiments show that all three of the proinflammatory cytokines act very early in the cell cycle to impair the ability of IGF-I to promote cell cycle progression. Reduced numbers of cells exiting G0 probably account for fewer cells synthesizing DNA and undergoing mitosis.
TNF-, but not IL-1ß or IL-6, Inhibits Antiapoptotic Activity of IGF-I.
IGF-I promotes both cell proliferation and survival (20
, 33)
, perhaps through different domains of the IGF-IR (34)
. In these experiments, we used three different techniques to determine whether proinflammatory cytokines affect cell survival in the absence of IGF-I. After culture in medium alone for 48 h, 18.4 ± 1.8% of MCF-7 cells were positive for PI (Fig. 3A)
, indicating that their cytoplasmic membranes were permeable to this dye. TNF-, at concentrations ranging from 0.1 to 100 ng/ml, did not affect the proportion of unfixed cells that were positive for PI (Fig. 3A)
. Identical conclusions with TNF- were reached when total live cells were measured using an MTT-based assay (Fig. 3A)
. Similarly, neither IL-1ß (1100 ng/ml) nor IL-6 (1100 ng/ml) affected (P > 0.10), the proportion of cells positive for either PIor MTT (data not shown). Finally, we used a sensitive, radioactive assay (21
, 25)
to measure the proportion of cells with fragmented DNA. For this purpose, DNA in MCF-7 cells was prelabeled with [3H]dThd during a 24 h-culture period. We then incubated the labeled cells with cytokines for 48 h and measured the amount of [3H]dThd released into the cytoplasm in the form of fragmented DNA. DNA fragmentation (Fig. 3, B–D)
confirmed the presence of 2025% apoptotic cells in control medium. However, results of all three of the assays established that none of the proinflammatory cytokines, in the absence of IGF-I, reduced the survival of MCF-7 epithelial cells. In the presence of IGF-I, the amount of fragmented DNA was reduced 4-fold, from 25% to 6% (P < 0.01; Figs. 3, B–D
). TNF- significantly inhibited, in a dose-dependent fashion, the ability of IGF-I to reduce DNA degradation (Fig. 3B)
. At 20 ng/ml, TNF- doubled DNA fragmentation. However, neither IL-1ß (Fig. 3C)
nor IL-6 (Fig. 3D)
impaired the ability of IGF-I to reduce DNA fragmentation. These data establish that none of the cytokines are directly cytotoxic to MCF-7 cells. The cytotoxic activity of TNF-, but not IL-1ß or IL-6, can only be detected when IGF-I or perhaps other growth factors are present.
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, IL-1ß, and IL-6 Inhibit IGF-I-driven DNA Synthesis in T-47D Cancer Cells.2.5 fold (Fig. 4)
. At a concentration as low as 1 pg/ml, both TNF- (Fig. 4A)
and IL-1ß (Fig. 4B)
modestly but significantly (P < 0.05) inhibited the ability of IGF-I to promote [3H]dThd incorporation. IL-6 caused a similar reduction (P < 0.05) at 10 pg/ml (Fig. 4C)
. In the absence of IGF-I, higher concentrations of IL-1ß and IL-6, but not TNF-, reduced [3H]dThd incorporation. These data confirm that IGF-I promotes DNA synthesis in T47D cells (35)
and extend the concept that the ability of IGF-I to promote DNA synthesis is inhibited by all three of the proinflammatory cytokines.
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and IL-1ß, but not IL-6, Reduce the Ability of IGF-I to Tyrosine-Phosphorylate IRS-1.
. The 170 kDa IRS-2 protein was detectable, but required much longer exposures (data not shown). These observations support earlier findings that although both IRS-1 and IRS-2 can be detected in MCF-7 cells (37)
, IRS-1 is the major docking molecule (36)
. More importantly, the latter workers found that IRS-1 is the predominant signaling molecule that is phosphorylated on tyrosine by IGF-I in both MCF-7 cells and in primary estrogen receptor-positive breast cancer cells. To determine whether proinflammatory cytokines prevent the IGF-IR from activating IRS-1, MCF-7 cells were cultured with TNF- (1 and 10 ng/ml), IL-1ß (50 ng/ml), or IL-6 (50 ng/ml) for 48 h, followed by IGF-I stimulation for 5 min. In the absence of IGF-I, with or without cytokine treatment, very little IRS-1 protein was phosphorylated on tyrosine residues (Fig. 5)
. Stimulation with IGF-I for 5 min induced a 15-fold increase in the ratio of tyrosine phosphorylation of IRS-1 versus the mass of IRS-1 in the immunoprecipitates. TNF- reduced in a dose-dependent fashion the ability of IGF-I to cause tyrosine phosphorylation of IRS-1 (Fig. 5B)
. Similarly, at a dose of 50 ng/ml, IL-1ß significantly inhibited the ability of IGF-I to tyrosine phosphorylate IRS-1 (Fig. 5C)
. In contrast, IL-6 (50 ng/ml) failed to inhibit IGF-I-induced tyrosine phosphorylation of IRS-1 (Fig. 5D)
.
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(10 ng/ml) or IL-1ß (50 ng/ml; data not shown). Collectively, these data establish that TNF- and IL-1ß impair the ability of IGF-I to tyrosine phosphorylate IRS-1 but not Shc. This is likely to be important, because the predominant signaling molecule activated by IGF-I in estrogen receptor-positive breast cancer cells is IRS-1 (36)
.
TNF- and IL-1ß Inhibit IGF-IR-associated IRS-1 Tyrosine Phosphorylation without Impairing Autophosphorylation of the IGF-IR.
To determine whether proinflammatory cytokines target the IGF-IR as well as IRS-1, we precipitated ß chains of the IGF-IR from whole cell lysates, separated the IGF-IR precipitable proteins on 7.5% polyacrylamide gels, transferred the proteins to PDVF membranes, and blotted with antibodies to phosphotyrosine. After stripping the phosphotyrosine blots, antibodies to IRS-1 or the IGF-IR were used to determine total mass of the IGF-IR-bound proteins. The upper portion of Figs. 6, A–C
, show representative autoradiograms of PY-IRS-1, PY-IGF-IR, mass of IRS-1 (IRS-1), and mass of ß chains of the IGF-IR (IGF-IRß). As expected, IGF-I induced a 10–13-fold increase in the amount tyrosine-phosphorylated IRS-1 relative to the IGF-IR (Fig. 6
; PY-IRS-1:IGF-IR). TNF- (Fig. 6A)
and IL-1ß (Fig. 6B)
reduced IGF-IR-precipitable IRS-1 tyrosine phosphorylation by 50% (P < 0.01). IL-6 produced a modest inhibition (18%), but this trend was not significant (P > 0.10). Reduction in the PY-IRS-1:IGF-IR ratio by TNF- (Fig. 6A)
and IL-1ß (Fig. 6B)
may be related to reduced mass of IRS-1 that associates with the IGF-IR (IRS-1:IGF-1R). However, neither TNF- (Fig. 6A)
nor IL-1ß (Fig. 6B)
caused a statistically significant reduction (P > 0.10) in the mass of IRS-1 that associated with the IGF-IR. Finally, although IGF-I consistently caused a 15-fold increase in autophosphorylation of the IGF-IR (Fig. 6
; PY-IGR-IR:IGF-IR), none of the proinflammatory cytokines impaired tyrosine phosphorylation of the ß chains of the receptor. These results establish that tyrosine phosphorylation of IRS-1 is significantly reduced by proinflammatory cytokines in a manner that is independent of changes in tyrosine autophosphorylation of the IGF-IR.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-1ß, and IL-6 impair cell cycle progression. In this scenario, proinflammatory cytokines act on breast cancer cells primarily by inhibiting IGF-I-induced growth signals (Figs. 1
and 4
) and impairing exit from G0/G1 (Fig. 2)
rather than directly suppressing cellular growth. Similarly, proinflammatory cytokines reduce cell survival only in the presence of IGF-I (Fig. 3)
. In this new model, TNF- and IL-1ß directly target and inhibit important growth factor receptor-mediated signaling events, such as tyrosine phosphorylation of IRS-1 (Fig. 5)
. We additionally show that both TNF- and IL-1ß impair tyrosine phosphorylation of the IRS-1 that specifically associates with the IGF-IR without affecting intrinsic tyrosine kinase activity of the receptor (Fig. 6)
. Therefore, proinflammatory cytokine receptors are unlikely to directly induce cell death or impair cellular growth. Instead, our data are more consistent with a model in which TNF- and IL-1ß act by inhibiting down-stream signaling events after activation of growth factor receptors.
The broad goal of this project is to explore molecular events that occur when cells are simultaneously exposed to both a hormone and a cytokine. We chose to study two of the earliest stages of IGF-IR activation: autophosphorylation of the receptor and tyrosine phosphorylation of its major docking molecule. The IGF-IR plays an important role in mutagenesis and tumor promotion (reviewed in Refs. 40, 41, 42
). Similar to earlier results (27)
, we found that IL-1ß inhibits the ability of IGF-I to promote growth of MCF-7 cells, and we extend this property to TNF- and IL-6. Unexpectedly, none of the three cytokines reduced IGF-IR tyrosine phosphorylation (Fig. 6)
, suggesting that tyrosine kinase activity of the receptor remains intact. This observation is different from that of Costantino et al. (27)
, who concluded that IL-1ß inhibits autophosphorylation of the IGF-IR. However, these workers used an enzyme-linked immunoabsorbent assay to measure IGF-IR autophosphorylation. In this assay, proteins that associate with the IGF-IR, such as IRS-1, would also be captured with the IGF-IR-specific antibody. In our experiments, we used specific antibodies to both the IGF-IR and IRS-1 in coimmunoprecipitation assays. These specific proteins were separated and Western blotted with antiphosphotyrosine antibodies to identify which component in the IGF-IR/IRS-1 complex is affected by cytokines. Our data confirm early work (27)
by showing that IL-1ß inhibits IGF-I-stimulated growth of breast cancer cells. However, these new data clearly show that IGF-IR autophosphorylation is not the target of proinflammatory cytokines in antagonizing the growth-promoting effects of IGF-I.
The IRS proteins are well known to serve as docking molecules that transmit downstream signals after activation of the IGF-IR. IRS-1 can also act in an IGF-IR-independent manner to regulate cancer cell adhesion and motility (43)
. Here we establish that IRS-1 is much easier to detect than IRS-2 in MCF-7 cells (Fig. 5A)
, and this finding is in agreement with the tyrosine phosphorylation results of others (36)
. IGF-I causes tyrosine phosphorylation of both the IGF-IR and IRS-1 to a similar extent (Fig. 5, B–D)
. In contrast to the IGF-IR, TNF- (Fig. 5B)
and IL-1ß (Fig. 5C)
significantly disrupt tyrosine phosphorylation of IRS-1. This observation implies that IRS-1, but not the IGF-IR, could be one of the inhibitory targets of these cytokines. However, pretreatment with either TNF- (10 ng/ml) or IL-1ß (50 ng/ml) caused only a 20–30% inhibition in tyrosyl phosphorylation of IRS-1 (Fig. 5, B and C)
. This modest inhibition contrasts with the ability of these cytokines to impair IGF-I-induced DNA synthesis (up to 80%). Considering that IRS proteins are common substrates for other receptors such as IL-4 (44)
and IL-10 (45)
, we then quantified tyrosine phosphorylation of the IRS-1 protein that specifically associates with the IGF-IR. The inhibition by cytokines of IGF-I-induced tyrosine phosphorylation on IGF-IR-precipitable IRS-1 is more prominent than the inhibition of unbound IRS-1 [48% versus 33% for TNF- (Fig. 6A)
; 48% versus 18% for IL-1ß (Fig. 6B)
; and 18% versus 6% for IL-6 (Fig. 6C)
]. IL-6 was the least effective inhibitor of IGF-I-induced cell cycle progression (Fig. 2D)
, and it also caused the most modest inhibition in the association of tyrosine phosphorylated IRS-1 with the IGF-IR (Fig. 6C)
. These observations support the idea that TNF- and IL-1ß, and to a more limited extent IL-6, suppress growth factor-stimulated cell cycle progression by suppressing tyrosine phosphorylation of that portion of IRS-1 that specifically associates with the growth factor receptor.
Most of the evidence showing that TNF- is cytotoxic to MCF-7 cells in vitro measured cell viability at time points >48 h. Those reports that measured TNF- killing at time points <48 h almost invariably used growth factors in the culture medium. For example, most experiments used at least 5% FBS (24
, 46, 47, 48, 49, 50, 51)
, supplemental insulin (48
, 52)
, or 0.5 µg/ml BSA (25
, 53)
, which is generally contaminated with numerous unidentified growth factor proteins (54)
. Our results are consistent with these findings, but they offer a different interpretation of the data. We show that TNF-, IL-1ß, and IL-6 do not directly cause DNA fragmentation of MCF-7 cells in the absence of growth factors (Fig. 3)
. Instead, TNF- acts on breast cancer cells to impair the survival-promoting activity of IGF-I (Fig. 3B)
. Similarly, all three of the proinflammatory cytokines impair IGF-I from acting as a late G1 progression factor (Fig. 2)
, leading to a reduction in the ability of IGF-I to promote DNA synthesis (Fig. 1)
. These new data are consistent with a model in which prototypical inflammatory cytokines suppress progression through the cell cycle of breast cancer cells by interfering with the mitotic and antiapoptotic roles of IGF-I.
Different mechanisms might be involved in negative regulation of IRS-1, including increased activity of tyrosine phosphatases (55)
, elevated release of IRS proteins from a multiprotein complex into the cytosol (56)
, or IRS-1 degradation (57)
. Furthermore, there are nearly 100 potential Ser/Thr-phosphorylation sites in IRS proteins, and increasing evidence has implicated Ser/Thr phosphorylation of IRS proteins as a major negative-feedback mechanism. For example, serine phosphorylation inhibits IRS-1 binding to the insulin receptor (58)
, triggers IRS-1 degradation (59)
, and inhibits virus-induced IRS-1 translocation to the nucleus (60)
. Particularly, inhibitors of Ser/Thr phosphatases mimic the ability of TNF- to reduce insulin-stimulated tyrosine phosphorylation of IRS-1 (61)
. However, serine phosphorylation of IRS proteins can have a dual function. For example, protein kinase B-induced serine phosphorylation enables IRS-1 to remain phosphorylated on tyrosine by protecting IRS-1 from protein tyrosine phosphatase-induced dephosphorylation (62)
. Indeed, recent data (63)
suggest that insulin, IGF-I, and TNF- can induce IRS-1 phosphorylation at Ser307, which is located on the COOH terminus of the PTB domain. Our preliminary data (not shown) confirm this observation and suggest that IRS-1 serine phosphorylation at Ser307 induced by IGF-I is even more remarkable than that caused by TNF-.
TNF- can inhibit tyrosine phosphorylation of IRS-1 without affecting tyrosine kinase activity of the insulin receptor (61)
. Similarly, early work demonstrated that substitution of Tyr960 of the insulin receptor with Phenylalanine led to a reduction in glycogen synthase activity, amino acid uptake, and [3H]dThd incorporation after stimulation with insulin (64)
. Although autophosphorylation remained intact, this mutated insulin receptor failed to tyrosine phosphorylate pp185 (IRS protein). Our data show a similar phenomenon after stimulation of the IGF-IR, and establish that TNF- and IL-1ß inhibit growth factor receptor signaling by targeting IRS-1 rather than the IGF-IR itself.
Experiments in this report show that TNF- and IL-1ß suppress the ability of IGF-I to promote association of tyrosine phosphorylated IRS-1 protein with the IGF-IR. There are numerous protein-protein interaction domains in IRS-1, including an NH2 terminus PH domain followed by a PTB domain (65)
. The PH domain interacts with both insulin and IGF-I receptors in the presence of low levels of the receptor (66)
. Similarly, the juxtamembrane region of the ß subunit of both insulin and IGF-I receptors contain an NPEY motif, which is important for specific binding of the PTB domain in IRS proteins and, thus, the recruitment of IRS proteins (67)
. A generally accepted idea regarding early IGF-I signaling events is that the interaction of the PTB domain of IRS-1 with the phosphorylated NPEY-motif in the juxtamembrane region of IGF-IR (67)
and the subsequent tyrosine phosphorylation of IRS-1 depend on a tyrosine kinase active IGF-IR (68)
. However, this direct evidence comes mostly from studies in a yeast hybrid system (68
, 69)
. Our data suggest that IRS-1 binds to the IGF-IR in the absence of exogenous IGF-I and that IGF-I treatment does not increase this association. These results imply that the association between the IGF-IR and IRS-1 may be regulated differently in MCF-7 cells.
Two current biological antitumor strategies are being actively investigated for treatment of a variety of cancers: neutralization of endogenous growth factors (17
, 18)
and injection of exogenous bioactive cytokines (70
, 71)
. Our results offer a conceptual basis for combining cancer therapeutical strategies that use inhibitors of growth factors with site-directed delivery of proinflammatory cytokines. It as long been known that proinflammatory cytokines are expressed in the microenvironment of primary and metastatic tumors (72)
. Recently, Hambek et al. (73)
implanted mammary adenocarcinomas into nude mice. Combination therapy with TNF- and an antiepidermal growth factor receptor antibody caused significant inhibition of tumor growth, particularly in those adenocarcinomas that expressed low amounts of epidermal growth factor receptor. These findings are consistent with the idea that the cytostatic properties of TNF- in vivo may be mediated through inhibition of growth factor receptor activation.
Parenteral administration of high concentrations of these proinflammatory cytokines often leads to development of a condition known as the systemic inflammatory response syndrome. This clinical problem is now being circumvented by development of new technologies. For example, reduction in tumor growth and metastases has been achieved with novel tumor cell vaccines created by engineering tumor cells to synthesize and secrete cytokines within the tumor (74)
. Replacement of 29Arg of human TNF- with 29Val and introduction of a cell-adhesive 4Arg-5Gly-6Asp fragment leads to a reduction in the hypertensive properties of systemic TNF- (75)
. This genetically engineered TNF- has a longer half-life and exhibits greater antitumor activity compared with wild-type TNF- (76
, 77) . Similarly, application of a plasmid containing a noninflammatory IL-1ß fragment induces lymphoid reactive cells in the stroma surrounding hyperplastic mammary ductal alveolar structures (78)
and delays development of breast cancer in mice (79)
. Recently, isolated limb perfusion therapy with TNF- for locally advanced soft tissue sarcoma was approved by the European Medicine Evaluation Agency (70)
. All of these new approaches minimize inflammatory side effects of proinflammatory cytokines while retaining their antitumor activity.
Cytokines can inhibit tumor growth in the absence of significant side effects by local (80) or by intratumoral administration (81) through perfusion in a close circuit (1) , gene transfer (78) , or by engineering mutants of these proteins (82) . All of these new approaches necessitate re-evaluating the clinical application of cancer treatment with proinflammatory cytokines. Our experiments confirm that proinflammatory cytokines suppress growth factor-stimulated cell growth. We extend these findings to describe a novel mechanism by which proinflammatory cytokines directly target IRS-1 to reduce its tyrosine phosphorylation and impair its association with the IGF-IR. These data suggest that growth factor adaptor molecules, such as IRS-1, rather than the receptors themselves might serve as targets for antitumor therapies. Many types of cells express receptors for both growth factors and cytokines. Therefore, our model should contribute to a better understanding of the biological and intracellular events that occur in tumor cells after simultaneous exposure to both the growth factors and proinflammatory cytokines that are invariably expressed in the microenvironment of tumors.
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FOOTNOTES |
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1 Supported by grants from the NIH (MH-51569 and AI50442; to K. W. K.) and by the Pioneering Research Project in Biotechnology financed by the Japanese Ministry of Agriculture, Forestry, and Fisheries. 
2 To whom requests for reprints should be addressed, at University of Illinois, Laboratory of Immunophysiology, Department of Animal Sciences, 207 Edward R. Madigan Laboratory, 1201 West Gregory Drive, Urbana, IL 61801. Phone: (217) 333-5141; Fax: (217) 244-5617; Email: kwkelley{at}uiuc.edu
3 The abbreviations used are: TNF-, tumor necrosis factor ; IL, interleukin; IGF-I, insulin-like growth factor-I; IGF-IR, insulin-like growth factor-I receptor; IRS-1, insulin receptor substrate-1; FBS, fetal bovine serum; HRP, horseradish peroxidase; dThd, thymidine; PI, propidium iodide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RIPA, radioimmunoprecipitation; Shc, Src-homology collagen; PY-IRS-1, phosphorylated insulin receptor substrate-1; PY-IGF-IR, tyrosine phosphorylated insulin-like growth factor-I receptor; PTB, phospho-tyrosine-binding; PH, pleckstrin homology; PI3k, phosphatidylinositol 3'-kinase.
Received 2/21/02. Accepted 6/13/02.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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