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[Cancer Research 61, 3314-3320, April 15, 2001]
© 2001 American Association for Cancer Research

Carcinogenesis

Prevention of Apoptosis by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in the MCF-10A Cell Line

Correlation with Increased Transforming GrowthFactor {alpha} Production1

John W. Davis, II2, Fredine T. Lauer, Andrew D. Burdick, Laurie G. Hudson and Scott W. Burchiel3

Toxicology Program, The University of New Mexico College of Pharmacy, Albuquerque, New Mexico 87131


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have recently reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits epidermal growth factor (EGF) withdrawal-induced apoptosis in the human mammary epithelial cell line MCF-10A. We hypothesized that TCDD-mediated inhibition of apoptosis was due to its ability to stimulate the EGF receptor (EGFR) pathway. Indeed, in the present studies, the EGFR inhibitor AG1478 was able to prevent TCDD-, EGF-, and transforming growth factor {alpha} (TGF-{alpha})-dependent cell recovery and inhibition of apoptosis. These effects were specific for an EGFR-mediated pathway because cotreatment with AG825, an erbB2 inhibitor, had little effect on apoptosis. In addition, TCDD was able to mimic the EGF and TGF-{alpha} signaling as demonstrated by increasing Akt and extracellular signal-regulated kinase 1,2 phosphorylation. These effects were dependent on EGFR activity because AG1478, but not AG825, was able to prevent EGF-, TGF-{alpha}, or TCDD-mediated Akt and extracellular signal-regulated kinase 1,2 phosphorylation. The ability of TCDD to stimulate the EGFR pathway and inhibit apoptosis may be due to the ability of TCDD to increase expression of TGF-{alpha}, a ligand for EGFR. Treatment with 10 nM TCDD increased TGF-{alpha} mRNA at 2 h and TGF-{alpha} protein at 6 h. These data suggest a mechanism whereby TCDD is able to inhibit apoptosis in human mammary epithelial cells by stimulating TGF-{alpha} production, resulting in an autocrine effect.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Breast cancer has become one of the most important diseases in the Western world. It has been estimated that one in eight women in the United States will develop breast cancer, and over 40,000 people per year are expected to die from the disease (1, 2, 3) . Breast cancer is a complex disease, and its etiology is likely due to numerous factors. Recently, significant progress has been made in understanding the genetic basis of this disease; however, genetics appear to account for only 10% of total breast cancer cases (4) . It has been postulated that environmental pollutants may be involved. The exposure of women to ubiquitous environmental pollutants such as TCDD4 and other halogenated aromatic hydrocarbons, as well as polycyclic aromatic hydrocarbons, has been well documented (5 , 6) . Moreover, many of these chemicals are resistant to metabolic breakdown, are extremely lipophilic in nature, and can be stored in human breast fat and milk (6 , 7) . Organochlorine pesticides are considered to be risk factors for breast cancer (8 , 9) . However, the link between exposure to environmental contaminants and breast cancer has yet to be established. Exposure of women to TCDD from an industrial accident in Seveso, Italy did not result in increased incidence of breast cancer (5 , 10) , whereas other reports demonstrate that exposure to polycyclic aromatic hydrocarbons is not statistically associated with breast cancer (11 , 12) .

Mammary tumorigenesis is a multistage, complex process in which overexpression of proto-oncogenes and their cognate signaling pathways have been implicated (13 , 14) . Previous studies have demonstrated that TCDD mimics growth factor stimulation and cell growth in the HMEC line MCF-10A. TCDD increased total tyrosine phosphorylation and increased PI3K activity (15) . In addition, withdrawal of EGF resulted in the induction of apoptosis, whereas treatment with TCDD prevented apoptosis and increased Akt phosphorylation (16) . These results argue that TCDD could act as a mammary tumor promoter by overstimulating the EGF signal transduction pathway, thus inhibiting apoptosis.

The inhibition of apoptosis is widely accepted as one possible mechanism of tumor promotion/progression (17) , and the PI3K/Akt pathway has been demonstrated to provide a powerful antiapoptotic stimuli (18) . Akt, also known as protein kinase B, is activated by many growth factors including EGF and IGF-I (19) and has been observed to be up-regulated in ovarian and breast carcinomas (18 , 20 , 21) . Activated Akt protects against apoptosis by numerous mechanisms including phosphorylating Bad, a member of the Bcl-2 family of proteins (22) , inhibiting the caspase pathway (23) , phosphorylating and inhibiting the proapoptotic FKHRL1 (24) and activating NF{kappa}B (25) . In addition to the PI3K/Akt pathway, growth factors are also able to suppress apoptosis through activation of the ras/raf/MAPK pathway (26 , 27) .

The present studies were initiated to begin to delineate the mechanism(s) through which TCDD inhibits EGF withdrawal-induced apoptosis in the HMEC line, MCF-10A. Previous studies examining the effects of TCDD on mammary epithelial cell growth have used tumor-derived cell lines that over-express the estrogen receptor, a complicating factor given TCDD’s anti-estrogenic activity (28 , 29) . The MCF-10A cell line is an attractive model because these cells are estrogen receptor-negative cell line, and exhibit a near normal phenotype (30) . Similar to primary cultures of normal HMECs, the MCF-10A cell line does not grow tumors when injected in nude mice and has a strict requirement for EGF and IGF-I for growth in serum-free media (31) . We hypothesized that inhibition of apoptosis in MCF-10A cells occurs through an EGFR-dependent pathway. The data presented here demonstrate that TCDD is able to activate both Akt and Erk in the absence of EGF, effects that also occur after treatment with EGF and TGF-{alpha}, ligands for EGFR. The ability of TCDD to increase cell number, inhibit apoptosis, and activate downstream kinases is EGFR specific because these responses were attenuated when cells were cotreated with the EGFR inhibitor AG1478, but not when cells were cotreated with the erbB2 inhibitor AG825. TCDD also increased TGF-{alpha} expression, suggesting that inhibition of apoptosis by TCDD is due to increased autocrine signaling in the MCF-10A cell line and independent activation of two downstream kinases, Akt and Erk.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents.
All chemicals were purchased from Sigma (St. Louis, MO), unless otherwise indicated. TCDD was obtained from Cambridge Isotopes Laboratories (Andover, MD) at >99% purity and was maintained as a stock solution (300 µM) in anhydrous tissue culture grade DMSO. AG1478 and AG825 were purchased from Calbiochem (La Jolla, CA) and stored at -20°C in DMSO. The final concentration of DMSO in all experiments was 0.1%.

MCF-10A Cell Culture.
MCF-10A, a nontransforming, estrogen receptor-negative HMEC line, was grown on Vitrogen-coated (Collagen Corp., Palo Alto, CA) 100 x 20-mm dishes (Corning Glass, Corning, NY) in a 10% CO2 incubator as described previously (16) .

Cell Growth Determination.
MCF-10A cells were plated on Biocoat 96-well plates precoated with collagen I (Becton Dickinson, Bedford, MA) at 1 x 103 cells/well in SFIHE and refed with SFIHE 24 h later. The next day, medium was removed, and cells were treated as indicated in the figure legends. Cells were grown for the indicated time with media, and treatment changed every 48 h. Cell number was determined using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI) as per the supplied instructions.

Detection of Apoptosis.
MCF-10A cells were plated into Vitrogen-coated 6-well plates at 1 x 105 cells/well in SFIHE plus 2% FBS to allow for attachment. The next day, plating media were removed, and cells were maintained in SFIHE without FBS. Cultures were treated in SFH (no growth factors) as indicated in the figure legends. Apoptosis was determined by flow cytometry using a kit that employs FITC-conjugated annexin V (PharMingen, San Diego, CA) and has been used previously (16 , 32) . To distinguish between apoptosis and necrosis, cells that stained for PI or PI and annexin V were determined to be necrotic and were not counted as apoptotic.

Determination of Erk and Akt Activation by Western Blot.
MCF-10A cells were plated on Vitrogen-coated 100 x 20-mm dishes at 3.5 x 105 cells/dish in SFIHE plus 2% FBS. The cells were refed the following day and every 3 days thereafter with SFIHE without FBS. Subconfluent cells 5 days after plating were starved for 18 h in SFH and treated as indicated. Cells were washed twice with ice-cold PBS, and 200 µl of lysis buffer [62.5 mM Tris (pH 6.8), 2% SDS, and 10% glycerol] were added. Cells were then scraped into Eppendorf tubes, and soluble membranes and DNA were sheared by pulling the lysates through an 18-gauge needle. Ten µl of the lysate were set aside for protein concentration determination using the Pierce micro-BCA protein assay reagent (Rockford, IL), and to the remaining sample DTT and bromphenol blue were added to a final concentration of 50 mM and 0.1%, respectively, to the remaining sample. Protein (50 µg) was boiled and separated on 12% Tris-glycine gels using standard conditions. Proteins were transferred overnight to PolyScreen polyvinylidene difluoride membrane (New England Nuclear Life Sciences, Boston, MA). Total and phosphorylated Akt (Ser473) and Erk (Thr202/Tyr204) were detected using New England BioLabs (Beverly, MA) PhosphoPlus antibody kits as per supplied instructions and visualized with Renaissance Western Blot Chemiluminescence Reagent (New England Nuclear) directed toward a horseradish peroxidase-conjugated secondary antibody (Promega). To quantitate the extent of phosphorylation, chemiluminescent films were scanned, and band densities were determined using Kodak Digital Science Image System 440 scanner and software (Kodak, Rochester, NY).

TGF-{alpha} Quantitative RT-PCR.
MCF-10A cells were plated on Vitrogen-coated 6-well plates as described for the annexin V assay. Fresh SFIHE were added 24 h after plating, and cells were cultured to approximately 75% confluence. Cells were then starved in SFH for 18 h, followed by treatment with 10 nM TCDD (or 0.1% DMSO) for the indicated times. After treatment, media were removed, and total RNA was harvested using 0.5 ml of Tri-Reagent (Sigma) as per the supplied instructions. TGF-{alpha} mRNA was quantitated using a competitive RT-PCR method described by Van den Heuvel et al. (33) , as modified from Gilliland et al. (34) . Internal standards were prepared specifically for TGF-{alpha} quantitation using a method described previously (35) . Primers used to generate the internal standard have the following sequences: (a) forward primer, 5'-TAA-TAC-GAC-TCA-CTA-TAG-GTC-AGT-TCT-GCT-TCC-ATG-CAA-CCC-ATT-GCT-ACA-GGC-ATC-GTG-GTG, and (b) reverse primer, 5'-TTT-TTT-TTT-TTT-TTT-TTT-TTT-CTG-AGT-GGC-AGC-AAG-CGG-CAT-CTT-ACG-GAT-GGC-ATG-ACA-G. TGF-{alpha} primers described previously by Adam et al. (36) are as follows: (a) forward primer, 5'-TCA-GTT-CTG-CTT-CCA-TGA-AAC-C; and (b) reverse primer, 5'-TTT-CTG-AGT-GGC-AGC-AAG-CG. All primers were purchased form Integrated DNA Technologies, Inc. (Coralville, IA), and all PCR reagents were obtained from Promega. Total RNA (300 ng) and a known amount of internal standard were added to a reaction that contained the following: 1 x reaction buffer [67 mM Tris (pH 8.8), 6.7 mM EDTA, 16.7 mM ammonium sulfate, 0.008% BSA, and 0.0035% ß-mercaptoethanol]; 5 mM MgCl2; 1 mM each deoxynucleotide triphosphate; 0.125 µg of oligo(dT)15; 7.5 units of RNasin; and 50 units of mouse mammary tumor virus reverse transcriptase in a final volume of 20 µl. cDNA was generated in a MJ Research (Waltham, MA) DNA Engine 96-well thermocycler using the following cycle: 42°C for 15 min and 95°C for 5 min. Primers specifically for TGF-{alpha} and its internal standard were added to the cDNA reaction at a final amount of 6 pmol for each primer along with 1 x reaction buffer, 4 mM MgCl2, and 1.5 units of Taq polyermase in a final volume of 50 µl, and the following thermocycler conditions were used: 94°C for 5 min, followed by 30 amplification rounds of denaturing (94°C for 45 s), annealing (60°C for 45 s), and elongation (72°C for 2 min). PCR products were diluted with 5 x sample buffer and electrophoresed on 2% NuSieve (3:1) agarose (FMC Bioproducts, Rockland, ME). Gels stained with ethidium bromide were visualized on a Kodak Digital Science Image Station 440 (Eastman Kodak, New Haven, CT), and bands were analyzed by densitometry using Kodak ID software. Molecules of TGF-{alpha} mRNA were calculated as described by Van den Heuvel et al. (33) . All samples were analyzed in duplicate.

TGF-{alpha} ELISA.
MCF-10A cells were plated on Vitrogen-coated 100 x 20-mm dishes at 3.5 x 105 cells/dish with SFIHE plus 2% FBS as described previously. Cells were treated as indicated, and after treatment, cells were rinsed once with PBS, scrape harvested into 300 µl of PBS, and centrifuged at 1000 rpm for 10 min. Cell pellets were frozen overnight at -80°C. Intracellular TGF-{alpha} protein levels were quantitated colormetrically using an ELISA kit (Oncogene Research Products, Cambridge, MA) as per the supplied instructions and normalized to total protein content. Briefly, cell pellets were resuspended in 800 µl of PBS, and 160 µl of Antigen Extraction Reagent (Oncogene Research Products, supplied in the kit) were added to lysed cells. After incubation on ice, lysates were cleared by centrifugation, and total protein concentration was determined with the micro-BCA protein reagent (Pierce).

Statistical Analysis.
Data were analyzed for statistical difference (P < 0.05) between control and treated groups using SigmaStat statistical software (Jandel Scientific, San Rafael, CA). ANOVA followed by Dunnett’s t tests was performed on sample means.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TCDD Increases MCF-10A Cell Number and Inhibits Apoptosis via an EGFR Pathway.
The present studies were undertaken to further characterize the signaling pathway(s) responsible for TCDD-dependent MCF-10A cell growth and inhibition of apoptosis. MCF-10A cells were plated on collagen-coated 96-well plates as described in "Material and Methods." Cells were treated with 10 nM TCDD (or 0.1% DMSO) for 3 days in SFH (no insulin or EGF), and cell growth was determined using a colorimetric assay. Treatment of MCF-10A cells with TCDD increased absorbance by almost 2-fold (Fig. 1A)Citation . Because removal of EGF from the growth media resulted in cell death, and TCDD has been shown to increase MCF-10A cell number in the absence of EGF (16) , we hypothesized that the EGFR pathway was at least in part required for these effects. To determine whether EGFR activity is required, cells were cotreated with 1 µM AG1478, a specific inhibitor of EGFR (37) . As expected, AG1478 completely abolished TCDD-dependent cell growth, whereas the structurally similar erbB2 inhibitor AG825 (38) had very little effect on cell growth (Fig. 1A)Citation . More importantly, TCDD-dependent MCF-10A cell growth mimics physiological ligands for the EGFR. As demonstrated in Fig. 1BCitation , EGF is able to increase cell growth, which is reversed by AG1478 but not by AG825. In addition, another EGFR ligand, TGF-{alpha}, had almost identical effects as TCDD and EGF (Fig. 1C)Citation . The results suggest that EGFR signaling is required for MCF-10A growth and/or suppression of cell death and that TCDD is able to mimic this growth factor receptor pathway. In addition, this appears to be EGFR specific because inhibition of erbB2 had little or no effect on cell growth.



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  		Fig. 1. TCDD increases MCF-10A cell number via an EGFR-dependent mechanism. MCF-10A cells were plated on Biocoat 96-well plates precoated with collagen I at 1 x 103 cells/well in SFIHE and refed with SFIHE 24 h later. The next day, media were removed, and cells were treated for 3 days in SFH (no growth factors) with (A) DMSO (0.1%), 10 nM TCDD, or TCDD in the presence of 1 µM AG1478 or 10 µM AG825. Cell growth was determined as described in "Materials and Methods." B, MCF-10A cells were treated as described in A with EGF (10 ng/ml) ± AG1478 or AG825. C, MCF-10A cells were treated as described in A with TGF-{alpha} (10 ng/ml) ± AG1478 or AG825. Results shown are the mean ± SE of the mean (SE) for absorbance readings (n = 6) from a representative experiment in which significant differences were observed in at least three separate experiments. *, significantly different from inhibitor treatment in each group (P < 0.05).

 
Given that TCDD is able to mimic EGF’s growth effects (Fig. 1)Citation and has previously been demonstrated to inhibit EGF withdrawal-induced apoptosis in this cell line (16) , it seemed likely that TCDD-dependent suppression of apoptosis would occur through an EGFR-specific pathway. To determine whether this indeed is the case, MCF-10A cells were cultured and grown in Vitrogen-coated 6-well plates and treated with 10 nM TCDD in the presence or absence of AG1478 or AG825 for 3 days. Cells were analyzed for apoptosis as determined by annexin V binding using flow cytometry as described in "Materials and Methods." When MCF-10A cells were cultured for 3 days in the absence of EGF (SFH), between 15% and 20% of the cells were annexin V positive/PI negative. However, treatment with 10 nM TCDD resulted in an almost 50% reduction of apoptotic cells (Fig. 2A)Citation . As expected, cotreatment with the specific EGFR inhibitor AG1478 attenuated the TCDD response, whereas AG825 plus TCDD was not different from TCDD alone. Similar to cell growth data, the EGFR ligands EGF and TGF-{alpha} protected cells from growth factor withdrawal-induced apoptosis (Fig. 2, B and C)Citation . Finally, cotreatment with AG1478 but not AG825 completely attenuated the response. The results presented in Figs. 1Citation and 2Citation argue that inhibition of apoptosis by TCDD in the HMEC line MCF-10A is mediated through an EGFR-mediated pathway. Furthermore, the actions of TCDD mirror those of other EGFR ligands, further evidence that TCDD is able to exert its actions by stimulating the EGFR signal transduction pathway.



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  		Fig. 2. TCDD-dependent inhibition of apoptosis requires EGFR activity. MCF-10A cells were plated into Vitrogen-coated 6-well plates at 1 x 105 cells/well in SFIHE plus 2% FBS to allow for attachment. The next day, plating media were removed, and cells were allowed to equilibrate for 1 day in SFIHE without FBS. Twenty-four h later, media were removed, and the cells were treated in SFH (no growth factors) for 3 days with (A) DMSO (0.1%) or 10 nM TCDD ± 1 µM AG1478 or 10 µM AG825. Apoptosis was determined as described in "Materials and Methods." B, MCF-10A cells were treated as described in A with EGF (10 ng/ml) ± AG1478 or AG825. C, MCF-10A cells were treated as described in A with TGF-{alpha} (10 ng/ml) ± AG1478 or AG825. Results shown are the mean ± SE for cell stained positively for annexin V and negatively for PI obtained in triplicate from a representative experiment in which significant differences were observed in at least three separate experiments. *, significantly different from inhibitor treatment in each group (P < 0.05).

 
TCDD-mediated Akt and Erk1,2 Phosphorylation Correlates with Inhibition of Apoptosis.
We have demonstrated by annexin V staining that TCDD protects against EGF withdrawal-induced apoptosis in a manner that is dependent on EGFR but not erbB2 activity (Fig. 2)Citation . In addition, previously published results have indicated that TCDD stimulates Akt phosphorylation and activation (16) . Growth factor receptors can signal through numerous kinase pathways, such as the PI3K/Akt and the ras/raf/MAPK pathways, which in turn may result in an inhibition of apoptosis (26 , 27 , 39 , 40) . The ability of TCDD to increase phosphorylation of Akt and Erk was examined under growth factor-deprived conditions using antibodies that recognize Akt phosphorylated at Ser473 and Erk1,2 phosphorylated at Thr202 and Tyr204. MCF-10A cells were grown and treated as described in "Materials and Methods." Treatment of cells with concentrations of up to 10 nM TCDD transiently increased Akt phosphorylation, with the greatest effect occurring at 6 h (Fig. 3A)Citation . This is similar to EGF and TGF-{alpha}, which increased Akt phosphorylation after 15 min. Likewise, TCDD also stimulated Erk1,2 phosphorylation at similar time points and concentrations, whereas EGFR ligands were also able to increase Erk1,2 activity (Fig. 3B)Citation . It should also be noted that neither TCDD, EGF, nor TGF-{alpha} had any effect on the other MAPKs, c-Jun NH2-terminal kinase or p38MAPK (data not shown). Finally, when these blots were probed with antibodies directed against the total population of each kinase (bottom panels in Fig. 3, A and BCitation ), the levels were unchanged. These results suggest that TCDD inhibits apoptosis in an EGF-like manner, possibly through the phosphorylation of Akt and/or Erk1,2.



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  		Fig. 3. Concentration-response analysis of TCDD-mediated phosphorylation of Akt and Erk1,2. MCF-10A cells were plated on Vitrogen-coated 100 x 20-mm dishes at 3.5 x 105 cells/dish in SFIHE plus 2% FBS. The cells were refed the following day and fed every 3 days thereafter with SFIHE without FBS. Subconfluent cells 5 days after plating were starved for 18 h in SFH and treated as indicated. Total cell lysate proteins (50 µg) were resolved on a 12% polyacrylamide gel, transferred to polyvinylidene difluoride membrane, and probed with (A) anti-phospho Ser473-Akt antibody or (B) anti-phospho Thr202/Tyr204-Erk1,2 antibody (top panels in A and B, respectively). Total Akt or Erk1,2 protein was determined by washing the blot extensively and reprobing with total Akt or Erk1,2 antibodies (bottom panels in A and B, respectively). Lanes 1–4, cells were treated for 6 h in the presence of 0.1% DMSO and 1, 3, or 10 nM TCDD; Lanes 5–8, cells were treated for 8 h in the presence of 0.1% DMSO and 1, 3, or 10 nM TCDD; Lane 9, cells were treated for 15 min with 10 ng/ml EGF (0.1% DMSO); Lane 10, cells were treated for 15 min with 10 ng/ml TGF-{alpha} (0.1% DMSO). Blots were quantitated as stated in "Materials and Methods," and the band intensities are as follows (shown as fold induction over DMSO control): A: Lane 1, 1.0; Lane 2, 1.4; Lane 3, 2.0; Lane 4, 2.5; Lane 5, 1.0; Lane 6, 1.6; Lane 7, 1.4; Lane 8, 1.6; Lane 9, 3.1; Lane 10, 3.1; B: Lane 1, 1.0; Lane 2, 3.1; Lane 3, 11.0; Lane 4, 16.2; Lane 5, 1.0; Lane 6, 1.3; Lane 7, 0.7; Lane 8, 1.6; Lane 9, 21.2; Lane 10, 20.7. The figure shown is from a representative experiment repeated at least three times with similar results.

 
AG1478, but not AG825, Attenuates TCDD-dependent Akt and Erk1,2 Activation.
TCDD-dependent suppression of apoptosis in the MCF-10A cell line appears to occur through an EGFR signaling pathway because the specific EGFR inhibitor AG1478 was able to repress not only the effects of TCDD, but also the effects of EGF and TGF{alpha}. In addition, TCDD and the EGFR ligands were able to activate two known downstream kinases of the EGFR, Akt and Erk. To determine whether EGFR signaling is required for Akt and Erk phosphorylation, MCF-10A cells were pretreated with 1 µM AG1478 or 10 µM AG825 (or 0.1% DMSO) for 30 min, followed by 10 nM TCDD for 6 h or EGF or TGF-{alpha} (10 ng/ml) for 15 min. The Western blot in Fig. 4ACitation demonstrates that the ability of TCDD to increase Akt phosphorylation is slightly diminished by the EGFR inhibitor AG1478, but not by the erbB2 inhibitor AG825 (compare Lanes 1–4, top panel). Although the effect is slight, it has been very consistent. Additionally, these effects mirror those seen when EGF or TGF-{alpha} was used to stimulate Akt (Lanes 5–7 and Lanes 8–10, respectively). As with Akt, TCDD-dependent Erk1,2 phosphorylation is also decreased by pretreatment with AG1478 (Fig. 4BCitation , Lanes 1–4). Also, inhibition of EGFR completely blocked EGF- and TGF-{alpha}-dependent Erk1,2 phosphorylation, whereas erbB2 blockade had no effect. These results suggest that growth factor-dependent inhibition of apoptosis occurs through an EGFR-specific pathway, probably by activation of one or more downstream kinases, namely, Akt and Erk. More importantly, inhibition of apoptosis by TCDD mimics the effects of the EGFR ligands, suggesting a mechanism through which this occurs.



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  		Fig. 4. TCDD-mediated phosphorylation of Akt and Erk1,2 is EGFR dependent. MCF-10A cells were plated and cultured as indicated in Fig. 3Citation . Total cell lysate proteins (50 µg) were resolved on a 12% polyacrylamide gel, and Western blotting was carried out as indicated in Fig. 3Citation for (A) phospho-Ser473 Akt or (B) phospho-Thr202/Tyr204 Erk1,2. The respective total kinases were detected as described in Fig. 3Citation . Lanes 1 and 2, 6-h treatment with DMSO (0.1%) or 10 nM TCDD; Lanes 3 and 4, DMSO or TCDD after 30-min pretreatment with 1 µM AG1478 or 10 µM AG825; Lanes 5–7, 15-min treatment with 10 ng/ml EGF or EGF after pretreatment with AG1478 or AG825; Lanes 8–10, 15-min treatment with 10 ng/ml TGF-{alpha} or TGF-{alpha} after pretreatment with AG1478 or AG825; Lanes 11 and 12, treatment with AG1478 or AG825 alone. Blots were quantitated as stated in "Materials and Methods," and the band intensities are as follows (shown as fold induction over DMSO control): A: Lane 1, 1.0; Lane 2, 2.9; Lane 3, 2.3; Lane 4, 3.2; Lane 5, 5.5; Lane 6, 1.9; Lane 7, 6.1; Lane 8, 5.2; Lane 9, 1.7; Lane 10, 7.0; Lane 11, 1.1; Lane 12, 1.9; B: Lane 1, 1.0; Lane 2, 2.7; Lane 3, 1.1; Lane 4, 4.4; Lane 5, 23.1; Lane 6, 5.2; Lane 7, 24.7; Lane 8, 23.0; Lane 9, 3.1; Lane 10, 26.4; Lane 11, 1.7; Lane 12, 1.0. The figure shown is from a representative experiment repeated at least three times with similar results.

 
TCDD Increases TGF-{alpha} mRNA and Protein Levels in the MCF-10A Cell Line.
TCDD-dependent inhibition of apoptosis appears to require signaling through the EGFR pathway. Furthermore, ligands for the EGFR also have similar effects, so it appears as though stimulation of this pathway occurs at the level of the receptor itself. Previous reports have demonstrated that TCDD is able to increase TGF-{alpha} production in human keratinocytes (41) . With this in mind, it was hypothesized that TCDD would have similar effects in HMECs. As demonstrated in Fig. 5ACitation , treatment of the MCF-10A cell line with 10 nM TCDD for 2 h resulted in more than a 3-fold increase in TGF-{alpha} mRNA. The ability of TCDD to increase TGF-{alpha} expression disappeared by 4 h. As expected, TCDD almost doubled intracellular TGF-{alpha} protein levels at 6 h as compared to DMSO-treated control cells (Fig. 5B)Citation . The ability of TCDD to enhance TGF-{alpha} protein was transient because shorter as well as longer treatment times had no effect on protein levels (data not shown). The timing of this is significant because activation of Akt and Erk1,2 occur at 6 h, and previously published work demonstrated that shorter treatment times did not result in Akt (16) and Erk activation (data not shown). Taken together, this suggests that TCDD is able to produce an autocrine signaling loop in MCF-10A cells via increased production of TGF-{alpha}, thereby inhibiting apoptosis.



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  		Fig. 5. TCDD increases TGF-{alpha} expression in MCF-10A cells. MCF-10A cells were plated and cultured as indicated in Fig. 3Citation . A, Cells were treated with DMSO (0.1%) or 10 nM TCDD for the indicated times, and TGF-{alpha} mRNA was quantitated using RT-PCR as described in "Materials and Methods." Results shown are the mean ± SE for molecules of TGF-{alpha} mRNA obtained in triplicate from a representative experiment in which significant differences were observed in at least three separate experiments. B, cells were treated with DMSO (0.1%) or 10 nM TCDD for 6 h, and intracellular TGF-{alpha} protein levels were quantitated colorimetrically as described in "Materials and Methods" and normalized to total protein content. Results shown are the mean ± SE for TGF-{alpha} production obtained in triplicate from a representative experiment in which significant differences were observed in at least three separate experiments. *, significantly different from DMSO (P < 0.05).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have recently shown that TCDD is able to inhibit apoptosis initiated by EGF withdrawal in a normal HMEC line (16) . Activation of growth factor receptors, particularly EGFR and IGF-receptor, and their cognate signaling pathways is a potential mechanism of mammary tumor promotion and progression (14) . In addition, the inhibition of apoptosis by growth factor signaling pathways is one possible mechanism of tumor promotion/progression (17 , 42) . TCDD is able to act as a rodent tumor promoter by inhibiting apoptosis in initiated livers (43) . It is with this in mind that we attempted to determine whether the ability of TCDD to inhibit apoptosis was in fact due to increased EGF signaling.

The specific EGFR inhibitor AG1478 blocked TCDD (10 nM)-dependent increased MCF-10A cell number in the absence of EGF. However, AG825, a structurally similar chemical that is specific for erbB2, did not (Fig. 1A)Citation . These results are similar to the growth effects seen by the EGFR ligands EGF and TGF-{alpha} (Fig. 1, B and C)Citation . In addition, TCDD-dependent, as well as EGF- and TGF-{alpha}-dependent, inhibition of apoptosis was also selectively blocked by AG1478 but not by AG825 (Fig. 2)Citation . Evidence for the involvement of the EGFR in mediating the effects of TCDD was observed when the ability of TCDD to stimulate Akt and Erk1,2 phosphorylation (Fig. 3)Citation was attenuated by AG1478 but not by AG825 (Fig. 4)Citation . Again, this is similar to the effects observed when MCF-10A cells are treated with EGF or TGF-{alpha}. Furthermore, EGFR involvement is likely, given that TCDD treatment resulted in a more than 3-fold increase in TGF-{alpha} mRNA and an almost 2-fold increase in TGF-{alpha} protein levels, potentially stimulating an autocrine signaling loop in the MCF-10A cell line (Fig. 5)Citation .

The ability of TCDD to positively regulate cell growth and inhibit apoptosis in HMECs differs from previously published reports. TCDD was found to inhibit IGF signaling, exerting an antiproliferative effect in MCF-7 cells (29) . However, MCF-7 is a malignant cell line that grows independently of added growth factors in culture and forms tumors when injected into nude mice. The MCF-10A cell line is an attractive model because it exhibits a normal phenotype, and, under serum-free conditions, its proliferation is dependent on addition of EGF and insulin (30) . Furthermore, preliminary data demonstrate that TCDD increases cell number in primary cultures of HMECs under similar growth factor-defined conditions.5 Finally, in utero and lactational exposure of female rats to TCDD resulted in an increase in mammary tumors, further demonstrating the possibility that TCDD could act as a mammary tumor promoter (44) .

The mechanism by which TCDD activates the EGFR pathway and leads to apoptosis is likely to be a complicated one. We have shown that overproduction of TGF-{alpha} by TCDD (Fig. 5)Citation occurs in MCF-10A cells and is one possible mechanism. The mechanism through which TCDD stimulates TGF-{alpha} production is complex. Choi et al. (41) demonstrated that 10 nM TCDD increased TGF-{alpha} mRNA and protein levels in kerotinocytes and, given the low concentration required for the effect, suggested a role for aryl hydrocarbon receptor, the receptor for TCDD (45 , 46) . In addition, others have shown that TCDD-stimulated TGF-{alpha} mRNA production occurs posttranscriptionally, possibly through message stabilization (47) .

TGF-{alpha} is very important in human mammary tumorigenesis. TGF-{alpha} can act as an autocrine factor in normal and immortalized HMECs, and its activity can be blocked using monoclonal antibodies to the EGFR (48) . In addition, TGF-{alpha} is overexpressed in over 50% of breast cancer cases (49) , and in some cases the EGFR is also coexpressed, implicating a functional role for a TGF-{alpha}/EGFR autocrine loop in tumors (48) . Furthermore, TGF-{alpha} is able to transform immortalized mammary cell lines (50) , and its expression correlates well with neoangiogenesis in mammary tumors (51) . Growth factor withdrawal provides a powerful apoptotic signal, and in cell models, growth factor-dependent inhibition of apoptosis has been observed to occur through two related pathways (27) . The PI3K/Akt pathway has been demonstrated to provide a powerful antiapoptotic stimulus (18) . Akt is activated by many growth factors including EGF and IGF-I (19) and is up-regulated in ovarian and breast carcinomas (18 , 20 , 21) . Activated Akt protects against apoptosis by numerous mechanisms, including phosphorylating Bad, a member of the Bcl-2 family of proteins (22) , inhibiting the caspase pathway (23) , phosphorylating and inhibiting the proapoptotic FKHRL1 (24) , and activating NF{kappa}B (25) . In addition to the PI3K/Akt pathway, growth factors are also able to suppress apoptosis through activation of the ras/raf/MAPK pathway (26 , 27) . MAPK activation can regulate apoptosis either directly by phosphorylating Bad (52) or through regulation of target gene expression such as p27kip1 and cyclin D1 (53) .

In summary, TCDD appears to stimulate an autocrine-signaling loop in MCF-10A cells, the model of which is depicted in Fig. 6Citation . The end result of this is an increase in cell number due to inhibition of apoptosis that is dependent and specific for the EGFR and is suppressed by AG1478. By overstimulating the EGFR pathway, it is possible that TCDD is able to regulate MCF-10A apoptosis by altering numerous kinases involved in the regulation of apoptosis. This regulation could occur at various levels including inhibition of proteases, proapoptotic proteins, and proapoptotic transcription factors such as FKHRL1 or the activation of antiapoptotic transcription factors such as NF{kappa}B. There are numerous examples of TCDD-dependent alteration of growth-regulatory genes, although the exact mechanism is still being elucidated (47 , 54) . We have presented evidence that TCDD can positively regulate the EGFR by increasing TGF-{alpha} levels, thus activating two distinct branches of that pathway. Additional studies are required to fully delineate the mechanism by which TCDD regulates human mammary cell growth and explore the signaling pathways by which it may act as a tumor promoter.



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  		Fig. 6. Proposed signaling pathway for TCDD-dependent EGF withdrawal-induced apoptosis in MCF-10A cells. See "Discussion" for details.

 


   ACKNOWLEDGMENTS
 
We thank Dr. Stephen P. Ethier (University of Michigan, Ann Arbor, MI) for supplying us with the MCF-10A cell line and Jennifer L. Lane for technical expertise in developing the TGF-{alpha} RT-PCR.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIEHS Grant 1-RO1-ES-07259. Back

2 Supported by NIEHS Grant 1-F32-ES-05895. Back

3 To whom requests for reprints should be addressed, at College of Pharmacy, 2502 Marble NE, Albuquerque, NM 87131. Phone: (505) 272-0920; Fax: (505) 272-6749; E-mail: burchiel{at}unm.edu Back

4 The abbreviations used are: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; EGF, epidermal growth factor; EGFR, EGF receptor; PI3K, phosphatidylinositol 3-kinase; HMEC, human mammary epithelial cell; TGF-{alpha}, transforming growth factor {alpha}; SFIHE, complete growth media; SFH, EGF- and insulin-deficient media; FBS, fetal bovine serum; PI, propidium iodide; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; IGF, insulin-like growth factor; FKHRL1, forkhead transcription factor; NF{kappa}B, nuclear factor {kappa}B; RT-PCR, reverse transcription-PCR. Back

5 S. L. Tannheimer and S. W. Burchiel, unpublished data. Back

Received 10/27/00. Accepted 2/13/01.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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