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Tumor Biology |
Departments of Obstetrics and Gynecology and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60612
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and C/EBPß, but not C/EBP
, significantly induced promoter II activity. Moreover, we demonstrated the presence of both C/EBPß and C/EBP but not C/EBP in a DNA-protein complex formed by the nuclear extract from TCM-treated HAFs and a probe containing this critical C/EBP binding element (-317/-304 bp). Finally, treatment of HAFs with TCM strikingly induced C/EBPß expression, whereas this did not affect the levels of C/EBP or C/EBP transcripts. In conclusion, malignant breast epithelial cells secrete factors, which induce aromatase expression in adipose fibroblasts via promoter II. This is, at least in part, mediated by a TCM-induced up-regulation and enhanced binding of C/EBPß to a promoter II regulatory element. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of the human P450arom (CYP19) gene is under the control of several distinct and partly tissue-specific promoters (12 , 13) . Three of these promoters (I.4, I.3, and II) are used in adipose tissue. Interestingly, in disease-free breast adipose tissue, P450arom is usually expressed at low levels via a distal promoter (I.4), whereas in the adipose tissue of the breast bearing a tumor, P450arom expression is increased through the activation of two proximal promoters, II and I.3 (14, 15, 16) . In addition to these in vivo observations, treatments of HAFs in culture with various hormones switch promoter use. For example, glucocorticoids plus cytokines induce P450arom expression via promoter I.4 in cultured primary HAFs, whereas treatment with a cAMP analogue switches the promoter use to II and I.3 (12 , 13) . We hypothesized that malignant breast epithelial cells interact with the surrounding adipose tissue fibroblasts to activate promoters II and I.3. The data presented in this report will serve to reconcile the in vivo and in vitro observations summarized above (12, 13, 14) . We report a novel epithelial-stromal interaction, which favors the induction of P450arom expression in HAFs by malignant epithelial cells via promoter II.
We and others have shown previously that breast cancer cells could stimulate aromatase expression in HAFs, which was suggestive of cross-talk between malignant epithelial cells and surrounding HAFs to favor estrogen production in breast tumors (17, 18, 19)
. We demonstrated recently that medium conditioned with malignant epithelial cells inhibited the differentiation of HAFs to mature adipocytes via the suppression of the essential adipogenic transcription factors C/EBP and PPAR
. C/EBPß and C/EBP/, on the other hand, were up-regulated in these undifferentiated murine fibroblasts treated with TCM (20)
. TCM-induced decreases in C/EBP or PPAR were sufficient to completely inhibit adipogenic differentiation of 3T3-L1 cells in our hands (20)
. This was in agreement with reports published previously (21
, 22)
. On the other hand, we were intrigued by the TCM-induced increases in C/EBPß and C/EBP/ mRNA levels in 3T3-L1 murine cells (20)
. In contrast to C/EBP or PPAR, ectopic expressions of C/EBPß or C/EBP were not sufficient to induce adipocyte differentiation in the absence of C/EBP or PPAR (23)
. Thus, we hypothesize that breast cancer-induced increases in C/EBPß or C/EBP/ levels do not affect adipocyte differentiation but may serve to increase aromatase expression in adipose fibroblasts surrounding the cancer. We used herein a model whereby human TCM is added to primary HAFs to understand the roles of C/EBP isoforms in the up-regulation of aromatase expression in undifferentiated fibroblasts. We chose to study the activation of promoter II, because work from three different laboratories demonstrated that the activity of this promoter was up-regulated in vivo in breast stroma bearing a carcinoma (14, 15, 16)
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T47D cells purchased from American Type Culture Collection (Rockville, MD) were initially grown in RPMI 1640 with 10% FBS containing 0.02 mM HEPES, whereas MCF-7 cells, prostate cancer cell line PC-3, and hepatocellular carcinoma cell line HepG2 (American Type Culture Collection) were grown in MEM with 10% FBS. Human normal mammary epithelial cells purchased from Clonetics, Inc. (Walkersville, MD) were grown in fully supplemented MEGM medium (Clonetics). Before shipment, these cells were passed twice and demonstrated to contain immunoreactive cytokeratins 14 and 18. In our hands, these cells were alive and dividing every 48–72 h. Cell-conditioned media from T47D, MCF-7, PC-3, HepG2, or normal mammary epithelial cells were collected to be used subsequently as treatments on HAFs. To collect conditioned media, cells were initially grown to confluence and switched to DMEM/F12 for a 12-h washout period; then, cells were incubated in DMEM/F12 for 24 h to allow accumulation of secreted factors in the medium.
RT-PCR Amplification.
Amplification of the untranslated 5' ends of P450arom transcripts from HAFs under various treatments was accomplished with exon-specific oligonucleotide pairs as described below. Five µg of DNase I-treated total RNA were used for reverse transcriptase reaction. Five µl of reverse transcriptase mixture were amplified using PCR. For the amplification of total P450arom transcripts, 5'-end sense primer from coding exon II (5'-TTG GAA ATG CTG AAC CCG AT-3') and 3'-end antisense primer complimentary to coding exon III (5'-CAG GAA TCT GCC CTG GGG AT-3') were used. To amplify promoter-specific 5'-untranslated sequences, primers for promoter II-specific sequence (5'-GCA ACA GGA GCT ATA GAT-3') and exon I.4 (5'-GTA GAA CGT GAC CAA CTG G-3') were used as 5'-end sense primers, together with an antisense primer complimentary to the coding exon III (5'-ATT CCC ATG CAG TAG CCA GG-3'). PCR conditions were as follows: denaturing at 95°C for 30 s, annealing at 55°C for amplification of promoter II-specific sequence or 58°C for amplification of exon I.4 and the coding region for 40 s, and extension at 72°C for 40 s for 30 cycles. GAPDH was chosen as an endogenous marker to check the integrity of cDNA. A 5'-end sense primer (5'-CGG AGT CAA CGG ATT TGG TCG TAT-3') and a 3'-end antisense primer (5'-AGC CTT CTC CAT GGT GGT GAA GAC-3') were used for amplifying a 306-bp-long sequence in GAPDH mRNA. PCR conditions were the same as those used for amplification of promoter II-specific fragments, except for the number of cycles (21)
and the quantity of reverse transcriptase mixture (0.5 µl). This RT-PCR method was described previously in greater detail (14)
.
Determination of Intracellular cAMP.
HAFs were plated in six-well, 35-mm culture dishes. After reaching confluence, HAFs were cultured either in serum-free DMEM/F12, DMEM/F12 containing 10% FBS, DMEM/F12 containing 10% FBS and forskolin (10 µM), DMEM/F12 containing 10% FBS and DEX (250 nM), or T47D cell conditioned DMEM/F12. Measurements were performed in triplicate replicates, and treatments were carried for 0, 12, 24, and 48 h. HAFs were lysed in a 0.1 M HCl solution after the removal of medium. Cell lysis mixture was centrifuged, and the supernatant was then used directly in the cAMP assay using Direct Cyclic AMP Enzyme Immunoassay kit (Assay Design, Inc., Ann Arbor, MI), following the protocol supplied by the vendor. Briefly, 50 µl of the pink-neutralizing reagent were added into each well, except for the total activity and blank wells. Samples (100 µl) were then added to appropriate wells. Fifty µl of the conjugate were added into each well, followed by the addition of 50 µl of the yellow antibody. After incubating at room temperature for 2 h on a shaker at 500 rpm, the plate was washed three times with 200 µl of washing buffer, followed by the addition of substrate solution 200 µl to each well. The stop solution (50 µl) was then added to each well, and the absorbance was read at 405 nm with correction to 570 nm. Results were obtained by plotting on the standard curve.
Transient Transfections and Luciferase Assays.
HAFs in primary culture were transfected using Lipofectamine Plus (Life Technologies, Inc., Grand Island, NY) with the following plasmids: (a) 1 µg of modified PGL3-Basic Luciferase reporter plasmid that contains serial deletion mutants of P450arom promoter II; (b) 0.2 µg of pcDNA3 expression plasmid (Invitrogen, Carlsbad, CA), which contains the cDNA of either C/EBP (human), C/EBPß (rat) or C/EBP (rat); and (c) 5 ng of pRL-CMV Renilla luciferase control reporter vectors that contain the cDNA encoding Renilla luciferase (Promega Corp., Madison, WI) as an internal control for transfection efficiency. The day before transfection, HAFs in primary culture were seeded into 35-mm dishes at 2 x 105 cell/dish. The transfection solution was made of 200 µl of OPTI-MEM I reduced-serum medium containing PLUS reagent (8 µl), precomplexed DNA (1.2 µg), and 5 µl of Lipofectamine reagent. After transfection for 6 h in transfection solution at 37°C in 5% CO2, medium was changed to antibiotic-free DMEM/F12 containing 10% FBS for overnight recovery. Cells were then switched to medium conditioned by normal breast epithelial cells or T47D cells for another 48 h. Luciferase and Renilla luciferase assays were performed using a dual-luciferase reporter assay system kit (Promega). Results are presented as the average of data from triplicate replicates and expressed as the ratio to the internal standard Renilla luciferase. The empty luciferase vector PGL3-Basic was arbitrarily assigned a unit of 1, and the rest of the results were expressed as multiples of the PGL3-Basic vector.
Northern Blotting.
Total RNA was isolated from HAFs in primary culture growing in: (a) DMEM/F12; (b) normal breast epithelial cell conditioned medium; or (c) TCM. Twenty µg of total RNA were used. cDNA probes for C/EBP, C/EBPß, and C/EBP were prepared from plasmids kindly provided by Drs. Steve McKnight (University of Texas Southwestern Medical Center, Dallas, TX), Gokhan Hotamisligil (Harvard Medical School, Boston, MA), and Gretchen Darlington (Baylor College of Medicine, Houston, TX).
Site-directed Mutagenesis.
To generate serial plasmids bearing mutated consensus-binding sequences for transcription factors of C/EBPs, SF-1 and CREB, site-directed mutagenesis was performed using the GeneEditor in vitro site-directed mutagenesis system (Promega), per the manufacturer’s instructions. A -517-bp promoter II/PGL3-Basic construct containing wild-type -517/-16 bp of P450arom promoter II 5'-flanking DNA was used as a template for site-directed mutagenesis. Briefly, DNA template (0.5 pmol) was denatured and annealed with mutagenic and selection oligonucleotides. Mutant strand was synthesized in the reaction mixture containing 1x synthesis buffer, 5 units of T4 DNA polymerase, and 2 units of T4 DNA ligase at 37°C for 90 min. The mutagenesis reaction mixture was then used to transform BMH 71-18 mutS competent cells. These transformed competent cells were incubated in a medium containing GeneEditor antibiotic selection mix overnight to select the desired mutant plasmids. The plasmids isolated from the BMH 71-18 mutS were transformed into JM109 competent cells. The transformed JM109 competent cells were grown overnight on the LB plates containing ampicillin and GeneEditor antibiotic selection mix to further select the mutated plasmids. The mutation of binding consensus was confirmed by DNA sequencing. Consensus binding sequences for mutation and primers used were depicted in Table 1
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Double-stranded oligonucleotides were obtained through annealing sense and antisense sequences. The double-stranded oligonucleotide probes were end-labeled with [-32P]ATP using T4 kinase. EMSAs were performed as described previously (24)
. Briefly, 5 µg of nuclear extract were incubated with the radiolabeled double-stranded oligonucleotide probe for 15 min at room temperature in a reaction buffer containing 20 mM HEPES (pH 7.6), 75 mM KCl, 0.2 mM EDTA, 20% glycerol, and 2 µg of poly(deoxyinosinic-deoxycytidylic acid) as a nonspecific competitor. Protein-DNA complexes were resolved on 6% nondenaturing polyacrylamide gels. EMSAs were performed after the addition of 0.5 µl of an antibody against C/EBP, C/EBPß, C/EBP, or CREB to the binding reaction, followed by a 30-min incubation on ice before electrophoresis. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). We used the following double-stranded probes. C/EBP binding site probe (5'-GAA GAA GAT TGC CTA AAC AA-3') represents an identical 20-bp-long sequence (-303/-322) in the promoter II regulatory region of the P450arom gene. Mutated C/EBP binding site probe (5'-GAA GAA Gcc cGC CTg gtC AA-3') contains a mutated version of C/EBP binding motif that does not interact with any of the C/EBP isoforms.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To address the specificity of effects of T47D cells on HAFs, we used media conditioned with either the MCF-7 breast cancer cell line or normal breast epithelial cells (NCM) to treat HAFs. Medium conditioned with MCF-7 cells but not with normal epithelial cells induced P450arom transcript levels via promoter II (Fig. 1B)
. Furthermore, other malignant cell lines HepG2 and PC-3 failed to activate P450arom gene transcription via promoter II, which demonstrated that the stimulatory effect produced by T47D and MCF7 cells was specific for breast cancer (Fig. 1C)
. These results demonstrate that malignant breast epithelial cells in culture produce specific factors, which stimulate aromatase expression via promoter II.
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. FBS or FBS plus DEX also decreased cAMP levels, whereas treatment with the adenylate cyclase inducer forskolin (10 µm) gave rise to a striking increase in cAMP levels in HAFs (positive control; Fig. 2A
). On the other hand, addition of the adenylate cyclase inhibitor SQ 22,536 to the culture medium 0.5 h before the treatment with TCM for 48 h did not inhibit promoter II activation (negative control; Fig. 2B
). These results indicate that the activation of P450arom promoter II by TCM is mediated via a cAMP-independent pathway.
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. Use of serial deletion mutants of promoter II fused to luciferase reporter gene demonstrated that the -517/-278-bp region contained critical stimulatory elements (Fig. 3)
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).4
The -278/-100-bp region contains two SF-1 sites and a CRE. One of these SF-1 binding sites (-136/-124 bp) and CRE (-211/-197 bp) were shown previously to be critical for cAMP-induced promoter II activity in ovarian granulosa cells and endometriosis-derived stromal cells (24
, 25) . We determined the effect of TCM on the activity of the -517-bp promoter II/luciferase construct, because this construct showed the highest baseline activity (Fig. 3)
. Treatment with TCM for 48 h induced the activity of the -517-bp construct by 5.7-fold (Fig. 5)
. Site-directed mutagenesis of five potentially important cis-acting elements demonstrated that CRE (-211/-197 bp) and a C/EBP binding site (-317/-304 bp) were essential for TCM induction of promoter II activity. In particular, mutation of the -317/-304-bp C/EBP binding site completely abolished both baseline and TCM-induced activities. Mutation of the two SF-1 sites or the -350/-337-bp C/EBP binding site did not effect TCM induction of promoter II activity (Fig. 5)
. We found that NCM did not change the activity of the -517-bp construct in comparison with incubation with DMEM/F12 only (data not shown). Therefore, promoter II activity in NCM-treated HAFs is similar to the baseline level. In this particular experiment illustrated in Fig. 5
, we determined the TCM fold induction of promoter in comparison with NCM treatment.
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depicts the effects of the adipogenic transcription factors, C/EBP, C/EBPß, and C/EBP, on the activity of the -517-bp promoter II construct in HAFs. Ectopic expressions of C/EBPß (3.5-fold) and C/EBP (2.5-fold) stimulated promoter II activity, whereas C/EBP did not have any significant effect (Fig. 6)
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and C/EBPß significantly stimulated the -517-bp promoter II construct (Fig. 6)
. To determine whether C/EBP or C/EBPß mediates TCM induction of promoter II, EMSA was used using an oligonucleotide probe (-322/-303 bp) containing the -317/-304-bp C/EBP binding site, nuclear extracts from HAFs incubated with or without TCM, and supershifting antibodies against C/EBP, C/EBPß, and C/EBP. This C/EBP binding site (-317/-304 bp) was chosen to be included in the probe, because this element was found to be critical for TCM activation of promoter II (Fig. 5)
. We identified two specific complexes (1 and 2) as verified by wild-type and mutated cold competitors in TCM-treated HAFs (Fig. 7)
. Antibodies against both C/EBPß and C/EBP supershifted complex 1, indicating the presence of C/EBPß and C/EBP. On the other hand, antibodies against C/EBP or CREB did not eliminate or supershift any of these complexes. To further investigate whether the activation of P450arom promoter II is mediated by C/EBPß, we demonstrated that the effects of TCM and C/EBPß were not additive. TCM stimulated the -517 construct by 6-fold, whereas the addition of C/EBPß to TCM did not further increase this induction, which was suggestive that the effects of TCM on promoter II were, at least in part, mediated by C/EBPß (Fig. 7B)
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or C/EBP, because C/EBP does not bind to the regulatory element at -317/-304 bp, which is critical for TCM stimulation of promoter II. Although C/EBP binds to this site, ectopic expression of C/EBP does not increase promoter II activity. To confirm this conclusion, we determined the effects of TCM on the mRNA levels of C/EBP isoforms in HAFs. Treatments with TCM or NCM did not change the mRNA levels of C/EBP or C/EBP. On the other hand, only TCM induced C/EBPß expression in HAFs strikingly (Fig. 8)
. Thus, we conclude that TCM induction of P450arom promoter II in HAF is mediated, at least in part, by the induction of the expression of C/EBPß, which binds to the -317/-304 bp region in this promoter.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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but persistent expression of C/EBPß and C/EBP proximal to malignant cells (20)
. On the basis of these data, we suggest the following model. Breast cancer cells secrete cytokines that selectively down-regulate essential adipogenic factors, which inhibit the differentiation of fibroblasts to mature adipocytes. Estrogen production in these fibroblasts maintained in the undifferentiated state by malignant cells is further enhanced by tumor-derived factors, which exist in the T47D or MCF-7 cell conditioned media. These factors act via a cAMP-independent pathway to increase C/EBPß expression in adipose fibroblasts and enhance the binding of C/EBPß to a specific promoter II regulatory sequence. The end result is increased local estrogen concentration in the breast tumor.
We do not know yet the identities of the unknown factors in TCM that increase C/EBPß expression and P450arom promoter II activity in adipose fibroblasts. Cytokines such as TNF- and IL-6 were shown to increase the transcriptional activity of C/EBPß (28
, 29)
. It is not clear, however, whether these cytokines increase C/EBPß expression or promoter II activity. Our preliminary findings and previous publications demonstrated that these cytokines (IL-11, IL-6, and TNF) do not activate P450arom promoter II, which is up-regulated in vivo in breast tumors. Instead, these substances activate promoter I.4, which is not up-regulated in tumors (17
, 30, 31, 32)
.5
Therefore, these cytokines by themselves probably do not account for the in vivo up-regulation of aromatase expression in breast tumors. Our efforts will continue to identify these unknown factors originating from malignant cells to induce aromatase expression in the adipose fibroblast via promoter II.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by United States Army Medical Research and Materiel Command Grant DAMD17-97-l-7025 and National Cancer Institute Grant CA67167 (to S. E. B.). 
2 To whom requests for reprints should be addressed, at Departments of Obstetrics and Gynecology and Molecular Genetics, University of Illinois at Chicago, 820 South Wood Street, M/C 808, Chicago, IL 60612. Phone: (312) 996-8197; Fax: (312) 996-4238; E-mail: sbulun{at}uic.edu
3 The abbreviations used are: P450arom, aromatase P450; HAF, human breast adipose fibroblast; cAMP, cyclic AMP; C/EBP, CCAAT/enhancer binding protein; PPAR, peroxisome proliferator-activated receptor; TCM, T47D cell conditioned medium; Bt2cAMP, dibutyryl cAMP; PDA, phorbol diacetate; DEX, dexamethasone; FBS, fetal bovine serum; NCM, normal mammary epithelial cell conditioned medium; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SF, steroidogenic factor; CREB, cAMP response element binding protein; EMSA, electrophoresis mobility shift analysis; TNF, tumor necrosis factor; IL, interleukin; CMV, cytomegalovirus.
4 Internet address: http://www.blast.genome.ad.jp/sit/TFSEARCH.
5 J. Zhou, B. Gurates, S. Yang, S. Sebastian, and S. E. Bulun, unpublished observations.
Received 11/29/99. Accepted 12/28/00.
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and interleukin-11 secreted by malignant breast epithelial cells inhibit adipocyte differentiation by selectively downregulating C/EBP and PPAR: mechanism of desmoplastic reaction. Cancer Res., 61: 2250-2255, 2001. and C/EBP. Proc. Natl. Acad. Sci. USA, 92: 9856-9860, 1995. gene expression contributes to the antiadipogenic effects of tumor necrosis factor-. Mol. Endocrinol., 10: 1457-1466, 1996. promotes nuclear localization of cytokine-inducible CCAAT/enhancer binding protein isoforms in hepatocytes. J. Biol. Chem., 271: 17974-17978, 1996. stimulates aromatase gene expression in human adipose stromal cells through use of an activating protein-1 binding site upstream of promoter I.4. Mol. Endocrinol., 10: 1350-1357, 1996.This article has been cited by other articles:
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E. R. Simpson and S. R. Davis Minireview: Aromatase and the Regulation of Estrogen Biosynthesis--Some New Perspectives Endocrinology, November 1, 2001; 142(11): 4589 - 4594. [Abstract] [Full Text] [PDF]
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L. Meng, J. Zhou, H. Sasano, T. Suzuki, K. M. Zeitoun, and S. E. Bulun Tumor Necrosis Factor {{alpha}} and Interleukin 11 Secreted by Malignant Breast Epithelial Cells Inhibit Adipocyte Differentiation by Selectively Down-Regulating CCAAT/Enhancer Binding Protein {{alpha}} and Peroxisome Proliferator-activated Receptor {{gamma}}: Mechanism of Desmoplastic Reaction Cancer Res., March 1, 2001; 61(5): 2250 - 2255. [Abstract] [Full Text]
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