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Endocrinology Vol. 141, No. 8 2814-2821
Copyright © 2000 by The Endocrine Society

ARTICLES

Laminin Inhibits Estrogen Action in Human Breast Cancer Cells1

Terry L. Woodward, Haolan Lu and Sandra Z. Haslam

Department of Physiology, Michigan State University, East Lansing, Michigan 48824-1101

Address all correspondence and requests for reprints to: Sandra Z. Haslam, Michigan State University, Department of Physiology, 108 Giltner Hall, East Lansing, Michigan 48824-1101. E-mail: shaslam{at}pilot.msu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Breast tumors that lack estrogen responsiveness have a poor prognosis. Despite the critical importance to breast cancer treatment, little is known about the loss of estrogen responsiveness and the development of antiestrogen resistance. We have examined the regulation of estrogen-induced proliferation, estrogen regulation of progesterone receptor (PR) expression, and estrogen signaling pathways in estrogen receptor (ER) positive (MCF-7 and T47D) breast cancer cell lines by specific extracellular matrix proteins (ECM) under serum-free conditions. Estrogen, supplemented with submaximal concentrations of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF), stimulated DNA synthesis of MCF-7 cells 7- to 10-fold and T47D cells 2-fold on collagen I or fibronectin. However, estrogen-induced proliferation was greatly reduced on laminin. In contrast, IGF-I or EGF, alone, stimulated proliferation of MCF-7 and T47D cells on all ECM. Thus, ER+ breast cancer cells were not refractory to mitogens when cultured on laminin. Similarly, estrogen induction of PR occurred on fibronectin or collagen I, but not on laminin. While ER content was similar on all ECM, estrogen stimulation of estrogen response element (ERE)-luciferase activity was significantly lower in MCF-7 cells cultured on laminin. Therefore, changes in ECM composition that occur in breast cancer may alter estrogen-responsiveness and the effectiveness of antiestrogen therapies in ER+ breast cancer cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WHILE MORE than 60% of breast cancers are ER+, only two-thirds of ER+ tumors respond to endocrine therapy (1). Understanding the mechanisms underlying the loss of estrogen (E) responsiveness in breast cancer may be critical to the treatment of this disease because E-responsive tumors have a much better prognosis than E-nonresponsive tumors (2, 3). Patients with E-nonresponsive tumors are twice as likely to die (22% vs. 12%), have a much shorter survival time, and have a higher risk of recurrence (35% vs. 21%) when compared with patients with E-responsive tumors (4). Furthermore, E-nonresponsive tumors are more likely to be metastatic and when they are metastatic they more frequently invade life-threatening visceral tissues and the brain than E-responsive tumors (4). Unfortunately, long-term treatment of E-responsive breast cancer with antiestrogens has not been effective because all patients with advanced breast cancer eventually experience progression while on treatment, becoming antiestrogen resistant (5). Antiestrogen resistance cannot be solely explained by loss of the ER because more than 30% of antiestrogen-resistant tumors express ER (6). Despite the critical importance to breast cancer treatment, the mechanism(s) leading to the loss of E-responsiveness and antiestrogen resistance are poorly understood.

Unlike many ER+ breast cancer cells, normal mammary epithelial cells in culture do not proliferate in response to E. We have demonstrated that stromal cells in coculture with mammary epithelial cells permit E-induced proliferation of epithelium, in the presence of serum (7). To determine if stromal tissue is required for E-mediated mammary gland development, Cunha et al. (8) performed surgical tissue recombinations of wild-type (ER+/+) or ER knockout (ER-/-) mammary epithelium with ER+/+ or ER-/- mammary stroma, and transplanted them under the kidney capsule of athymic nude mice. Mammary gland ductal elongation and morphogenesis occurred when ER+/+ stroma was combined with either ER-/- or ER+/+ epithelium, but did not occur when ER-/- stroma was combined with either ER-/- or ER+/+ epithelium. These results indicate that ER+/+ mammary stroma is required for E-mediated morphogenesis and proliferation of normal mammary epithelium. These results support the hypothesis that E-induced normal mammary gland development likely occurs by E-binding to ER in the stroma and subsequent release of a growth factor from the stroma which induces proliferation of epithelium. Although the mechanism(s) involved in epithelial-stromal interactions in E-mediated mammary gland development have been extensively studied for nearly 20 yr, it is still unclear what factors are involved (9).

One class of stromal proteins, ECM proteins, has been almost completely ignored when considering E-dependent or -independent influences on proliferation of normal or cancer cells in the breast. Keely et al. (10) have recently demonstrated that in vivo, only stromal cells synthesize the major ECM proteins that make up the epithelial basement membrane in the normal mouse mammary gland. In addition to providing an attachment substrate for cells, recent studies have demonstrated that extracellular matrix proteins bind to cell surface receptors, integrins, and can regulate multiple cellular events, including proliferation, differentiation, motility, and apoptosis. In breast cancer, the stromal cells surrounding tumors have increased or altered expression of many ECM proteins that change with breast cancer progression. Elliott et al. (11) have presented evidence that the proliferative responses of SP-1 and SP-3M murine mammary cancer cell lines to basic fibroblast growth factor and platelet-derived growth factor-BB are dependent on specific ECM proteins. More recently, Lee and Streuli (12) have determined that collagen I (Col I) stimulates EGF responses but inhibits insulin action, whereas, conversely, Engelbreth Holm Swarm tumor matrix (matrigel) inhibits EGF responses and stimulates insulin signaling in normal mammary epithelial cells derived from pregnant mice. To date, no studies have examined the effects of ECM proteins on E-action in breast cancer cells.

We report here that LM inhibits E-induced proliferation and PR induction in two ER+ breast cancer cell lines, MCF-7 and T47D. We have also analyzed the mechanism by which laminin (LM) inhibits E-action. Altered E-responsiveness of MCF-7 and T47D cells was not due to changes in cellular ER concentrations or ER binding. Furthermore, culture of cells on LM did not prevent all mitogenic responses, since IGF-I or EGF induced proliferation on LM. Instead, ERE-activity of MCF-7 cells cultured on LM was significantly reduced.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Col I, collagen IV (Col IV), LM, and vitronectin (VN) were purchased from Becton Dickinson and Co. (Bedford, MA) and fibronectin (FN) was purchased from ICN Biomedicals, Inc. (Aurora, OH). The antiestrogen ICI 182,780 was a gift from ICI Pharmaceuticals (Macclesfield, Cheshire, UK). Radioinert R5020 (promogestone), [17ß-methyl-3H]promogestone (R5020, 85 Ci/mmol), and methyl-3H-Estradol-17ß (120 Ci/mmol) were purchased from New England Nuclear Corp. (Boston, MA). Methyl-3H-thymidine (50 Ci/mmol) was purchased from ICN Biomedicals, Inc. Charcoal/dextran-treated (DCC) FBS was from HyClone Laboratories, Inc. (Logan, UT). IGF-I was purchased from GroPep Pty. Ltd. (Adelaide, Australia). Poly-L-lysine (PLL), phenol-red free DMEM/F12 culture media and all other hormones and growth factors were obtained from Sigma (St. Louis, MO). All other culture reagents were obtained from Life Technologies, Inc. (Grand Island, NY) or Corning, Inc. Laboratories (Corning, NY).

Cell lines, culture conditions, plasmids, and ECM coating
The human breast cancer cell lines, MCF-7 (ER+) and MDA MB231 (ER-), were kindly provided by Dr. W. Helferich, (University of Illinois, Urbana, IL) and T47D (ER+) by Dr. B. K. Vonderhaar (NIH, Bethesda, MD). Serum-free media (SFM) contained phenol-red free DMEM/F12 culture media with 125 ng/ml insulin, nonessential amino acids at a 1x concentration (Life Technologies, Inc. catalog number 11140) and gentamycin (50 µg/ml), with or without 5 ng/ml EGF and 25 ng/ml IGF-I, as noted. SFM with 5% DCC FBS was used for all serum-containing experiments. Col I, Col IV, LM, and FN were coated at 6.25 µg/cm2, PLL at 12.5 µg/cm2 and VN at 500 ng/cm2 as previously described (13). ERE-tk109-luc and control tk109-luc plasmids were kindly provided by Dr. Barry Gehm (Northwestern University Medical School Chicago, IL) (14). A ß-galactosidase plasmid, p6RL, with a constitutively active CMV promoter was provided by Dr. Donald Jump (Michigan State University, East Lansing, MI).

DNA content
Cells were plated in 24-well dishes in SFM or in media with 5% DCC as noted. Treatments were applied 2 days later for 4 days and cells harvested for DNA assay (13). Media was changed every 2 days. DNA content was determined by a fluorometric assay using Hoescht 33258 dye (15).

3H-thymidine incorporation
Growth factor assay. Cells were plated in SFM for 48 h and growth factors added for 17 h. Cells were labeled with 3H-thymidine for 2 h and 3H-thymidine incorporation into DNA was determined as previously described (16).

Estrogen response assay. Cells were plated in SFM for 24 h and pretreated with the antiestrogen ICI 182,780 (200 nM) for 2 d. ICI 182,780 was removed and cells then treated with E (10 nM) for 21 h and labeled with 3H-thymidine for 2 h as previously described (15, 17).

Steroid hormone binding assay
Ligand binding assays were used to determine ER and PR content in whole cells as previously described (13). PR content was determined after 3 days E (10 nM) treatment, ER was determined after 3 days of culture in the absence of E.

ERE luciferase and ß-galactosidase assays
Cells were plated in SFM containing EGF (5 ng/ml) and IGF-I (25 ng/ml) for 24 h, and transfected with 2 µg/well ERE-tk109-luc or control tk109-luc and 1.5 µg/well p6RL ß-galactosidase using Superfect transfection reagent (QIAGEN, Inc., Valencia, CA) for 2 h. E (at 10 nM or as described in dose response experiment), no treatment, or ICI 182,780 (200 nM), were added directly following transfection for 24 h, and cells lysates were assayed for luciferase activity and ß-galactosidase content in luciferase reporter lysis buffer (Promega Corp., Madison, WI). Luciferase activity was determined using a Promega Corp. Luciferase Assay System with Reporter Lysis Buffer (Promega Corp.), as per Promega Corp. Technical Bulletin 161 and read on a TD-20e luminometer (Turner Designs, Inc., New York, NY). ß-galactosidase content was determined using chlorophenol red-ß-D-galactopyranoside sodium salt (CPRG, Roche Molecular Biochemicals, Indianapolis, IN) and read at 575 nM.

Statistical analysis
All data were expressed as the mean ± SEM. Differences between means were tested for statistical significance using the Student’s t test or paired Student’s t test where appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Estrogen-induced proliferation on various ECM proteins in the presence of serum
Two ER+ cell lines, MCF-7 and T47D, were used to study the effects of ECM on E-responsiveness, and the MDA MB231 cell line was used as ER- control. In the presence of 5% DCC FBS, attachment of all three cell lines was greater than 90% on all ECM proteins (data not shown). In the absence of E, DNA content/well of MCF-7, T47D or MDA MB231 cells was similar on all ECM proteins, except on LM, where T47D and MDA MB231 cells had significantly lower DNA/well following 4 days of culture (P < 0.05) (Fig. 1Go, A–C). E treatment for 4 days significantly increased DNA/well of MCF-7 and T47D cells by 1.6- to 1.9-fold on plastic (Plas), Col I, Col IV, and FN (P < 0.05) (Fig. 1Go, A and B). MCF-7 and T47D cells plated on LM were not significantly growth stimulated by E (Fig. 1Go, A and B). MDA MB231 cells did not proliferate in response to E on any ECM protein (Fig. 1CGo). ECM proteins, such as FN and VN, are abundantly present in serum (18). In fact, FN was present at 15 µg/ml in our 5% DCC FBS containing media; this concentration is greater than the quantity of any ECM we used to coat the culture wells. Thus, it is noteworthy that LM appears to have a dominant inhibitory effect over FN because basal proliferation and E-induced proliferation were reduced by LM even in the presence of high concentrations of FN in serum. When lower concentrations of LM were used in serum containing cultures, there was less effective inhibition of E-induced proliferation indicating the dose-dependent nature of the LM effect (data not shown). Because we wished to investigate the distinct effects of specific ECM proteins under hormone and growth factor defined conditions, we developed serum-free media (SFM) that support E-responsiveness of the MCF-7 and T47D cells. In all subsequent experiments cells were plated and cultured in SFM.



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  		Figure 1. Effect of various ECM proteins on E-dependent proliferation of breast cancer cells. DNA content was determined for ER+ MCF-7 (A), ER+ T47D (B), and ER- MDA MB231 (C) cells following 4 day culture in control or E (10 nM)-containing media in the presence of 5% DCC FBS. Cells were plated on Plas, Col I, Col IV, LM, FN, or VN. Each value represents the mean ± SEM of three to eight experiments in panels A and B. C, Data from a representative experiment (of three experiments) with error bars of replicates within experiment. *, P < 0.05 that E-treated groups have a higher DNA content than control groups. **, P < 0.05 that LM control groups have a lower DNA content than all other ECM control groups.

 
Cell attachment and morphology in serum-free culture on various ECM proteins
An important function of ECM is to mediate cellular adhesion. MCF-7 and T47D cell attachment in SFM was not different among ECM proteins, ranging from 70–90% (data not shown). Morphology of ER+ breast cancer cells was altered by attachment to different ECM proteins. MCF-7 cells attached quickly and had a flattened cuboidal morphology on Col I, but cells were more elongated and took longer to flatten when cultured on FN or LM (Fig. 2Go). T47D cells also had a flattened cuboidal morphology on Col I, but formed small aggregates of cells that also flattened more slowly on LM, FN, VN, or Col IV (data not shown). MDA MB231 cells attached, flattened well and were elongated in appearance on all ECM proteins (data not shown).



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  		Figure 2. Phase contrast photomicrographs of MCF-7 on various ECM proteins in SFM. MCF-7 cells were plated in 24-well plates on Col I, LM or FN. Cells were photographed after 24 h in culture. Magnification, x100. Bar, 50 µM.

 
Estrogen and growth factor-induced proliferation on various ECM proteins in the absence of serum
In the absence of serum and growth factors, we did not obtain significant proliferation in response to E on any ECM protein. The minimal conditions necessary for E-induction of proliferation have not been previously defined. Although some researchers have reported that serum is necessary for E-induced proliferation, others have found that cells can be plated in serum and switched to serum-free conditions and achieve E-induced proliferation (19, 20, 21, 22). No studies have reported E-induced proliferation of MCF-7 and T47D cells when plated and cultured in serum-free media. Our studies required, however, that the cells be plated and cultured in SFM, to avoid exposure to ECM proteins in serum. Because Katzenellenbogen and Norman (23) have shown that either insulin or IGF-I was required for E-induction of PR in MCF-7 cells, we tested if growth factors would permit E-induced proliferation. We determined the minimal conditions required to obtain significant E-induced proliferation when the breast cancer cells were plated and cultured in serum-free conditions. In SFM, IGF-I, or EGF or IGF-I + EGF were able to support significant E-induced proliferation. Dose response studies were performed to determine the minimum concentrations of IGF-I and/or EGF that were necessary to obtain an E response. The minimum concentrations of EGF and IGF-I required to demonstrate E stimulated proliferation were 5 ng/ml and 25 ng/ml, respectively. We used these submaximal concentrations to obtain E-induced proliferation because maximal concentrations of growth factors masked the mitogenic effect of E (data not shown). At these concentrations, E caused a significant increase in the DNA content of both MCF-7 and T47D cells on Col I (Fig. 3Go, A and B).



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  		Figure 3. Effect of IGF-I and EGF on E-mediated proliferation in breast cancer cells in SFM on Col I. DNA content of MCF-7 (A) and T47D (B) cells were determined after 4 days of treatment with EGF (5 ng/ml) + IGF-I (25 ng/ml) with or without E (10 nM) in SFM. Each value represents the mean ± SEM from at least three experiments *, P < 0.05 that IGF-I and IGF-I+EGF had a higher DNA content than no treatment. **, P < 0.05 that E+IGF-I+EGF group is greater than IGF-I+EGF groups.

 
Although E-induced proliferation of MCF-7 and T47D cells plated and cultured in SFM occurred on specific ECM proteins, the amount of stimulation was small. Cell cycle analysis of MCF-7 cells revealed that about 30% of cells were in S-phase when cultured in the absence of E, serum and growth factors on Col I. Treatment of the cells with the antiestrogen ICI 182,780 reduced the percentage of S-phase cells to 10%, indicating that ER activity occurs in the absence of E. To increase the sensitivity of our proliferation assay, basal proliferation was reduced by the antiestrogen ICI 182,780 before E treatment, and 3H-thymidine incorporation was measured instead of total DNA (17). E-treatment of MCF-7 cells on Col I or FN increased 3H-thymidine incorporation 7- and 10-fold on Col I and FN, respectively, but only 2-fold on LM (Fig. 4AGo). In T47D cells, E increased thymidine incorporation 1.5- to 2-fold on FN or Col I, respectively (Fig. 4BGo). No stimulation by E was observed in T47D cells cultured on LM. Because treatment with ICI 182,780 has previously been shown to reduce strength of attachment of T47D cells to LM, we repeated this experiment in the presence of 5% DCC FBS containing media, which results in increased attachment. In the presence of serum, T47D cell proliferation was significantly increased by E treatment on Col I and FN, but not on LM (Fig. 4CGo). These results indicate that LM inhibition of E-induced proliferation occurs independent of strength of attachment. Moreover, the inhibition of proliferation by LM occurs in the presence of serum which contains a significant concentration of FN (18). MDA MB231 cells did not exhibit E-inducible proliferation on any ECM protein examined (Fig. 4D).



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  		Figure 4. Estrogen-induced 3H-thymidine incorporation into DNA in breast cancer cells on various ECM proteins in SFM. Cells were cultured with EGF (5 ng/ml) and IGF-I (25 ng/ml). MCF-7 (A), T47D (B), or MDA MB231 (D) cells were pretreated with 200 nM ICI 182,780 or T47D cells were pretreated with 200 nM ICI 182,780 in 5% DCC (C) for 48 h, followed by no treatment (control) or E (10 nM) treatment for 21 h. All values were normalized to DNA content/well. Each value represents the mean ± SEM of three separate experiments. *, P < 0.05 that E-treated groups are greater than control treated groups.

 
Because basal proliferation in the absence of E was lowest on LM for all three cell lines, it was important to determine if LM nonspecifically inhibited mitogenesis. To address this question, we performed dose response studies with IGF-I and EGF, two growth factors known to be mitogenic for MCF-7 and T47D cells. In MCF-7 cells, EGF or IGF-I significantly increased thymidine incorporation into DNA 2-fold and 3-fold, respectively, on Col I, LM, and FN (Fig. 5Go). Stimulation of proliferation by EGF or IGF-I was not different on Col I, LM, or FN. These data demonstrate that LM does not block all mitogenic pathways.



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  		Figure 5. EGF- and IGF-I-induced 3H-thymidine incorporation into DNA in breast cancer cells on various ECM proteins in SFM. MCF-7 cells were treated with EGF (A) or IGF-I (B) at the indicated concentrations for 17 h, and 3H-thymidine incorporation measured. All values were normalized to DNA content/well. Each value represents the mean of three separate experiments.

 
It has been previously reported that IGF-I and EGF responses can be mediated through ligand-independent activation of ER. In this regard, ICI 182,780 also decreased thymidine incorporation in MCF-7 cells treated with IGF-I or EGF by 45% and 35%, respectively. However, even in the presence of antiestrogen IGF-I or EGF still increased proliferation significantly (data not shown). Therefore, it appears that ER activity may increase basal cell proliferation and mitogen activated cell proliferation, but the growth factors are not dependent on ER for mitogenesis.

Estrogen-induction of progesterone receptor
To determine if LM inhibited other E-dependent responses, an endogenous E-regulated protein, PR, was examined in MCF-7 cells. T47D cells were not used in these experiments because they have high levels of PR that are not regulated by E (24). E increased PR levels on all ECM proteins except LM in the absence of serum (Fig. 6Go). Similar to previous studies with E-induction of PR in the MCF-7 cells (23), E-induction of PR required submaximal concentrations of insulin, IGF-I or EGF (data not shown), therefore, we added IGF-I and EGF to these cultures as we did for the proliferation experiments. In the presence of 5% DCC FBS, E significantly increased PR levels 1.7- to 1.9-fold on Col I or FN, but did not increase PR levels at all on LM (data not shown). Thus, LM also has a dominant inhibitory effect on E-induction of PR even in the presence of FN in serum.



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  		Figure 6. Effect of various ECM proteins on E regulation of PR levels in MCF-7 cells in SFM. Specific 3H-R5020 binding was determined in MCF-7 cells cultured in SFM containing EGF (5 ng/ml) and IGF-I (25 ng/ml). Cells were cultured in the presence of E (10 nM) for 3 days. PR was determined by specific 3H-R5020 binding and normalized to DNA content. Each value represents the mean ± SEM of three separate experiments. *, P < 0.05 that E-treated groups are greater than control groups.

 
Estrogen receptor status and activation of ERE
Because LM reduced the proliferation response to E, we hypothesized that ER levels might be down-regulated when cells were plated on LM. ER content, determined by ligand binding, was lower in T47D cells than in MCF-7 cells (Fig. 7AGo) as has been previously reported (24). However, ER levels were similar on all ECM proteins for both cell lines (Fig. 7AGo). Therefore, the decrease in E-responsiveness of MCF-7 and T47D cells cultured on LM was not due to a decrease in ER concentration. MDA MB231 cells were ER negative on all ECM proteins (data not shown).



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  		Figure 7. ER concentrations in MCF-7 and T47D cells and ERE-luciferase reporter activation by E in MCF-7 cells on various ECM proteins in SFM. A, ER in MCF-7 and T47D cells in SFM containing EGF (5 ng/ml) and IGF-I (25 ng/ml) cultured on Col I, LM, or FN was measured by specific 3H-E ligand binding. Cells were cultured 2 days before assay. 3H-E binding was normalized to DNA content. Each value represents the mean ± SEM of three separate experiments. B, MCF-7 cells were transfected with ERE-tk109-luc or a control plasmid not containing an ERE (tk109-luc) and ß-galactosidase plasmids. Luciferase activity was measured 24 h after ICI 182,780 (200 nM), control, or E (10 nM) treatment. Luciferase activity was normalized to transfection efficiency as measured by ß-galactosidase expression/well. Each value represents the mean ± SEM of three separate experiments *, P < 0.05 that E-treated groups are greater than control groups. **, P < 0.05 that the E-treated LM group is less than E-treated Col I or FN groups. Inset, Luciferase activity of MCF-7 cells transfected with tk109-luc plasmid cultured on various ECM proteins. C, ERE-luciferase reporter activation was carried out in transfected MCF-7 cells cultured in the presence of 0.1–100 nM E. Experimental conditions are the same as in 7 (B).

 
Because ER content was similar on all ECM proteins, we considered the possibility that ER function was altered. To test this, we transfected MCF-7 cells with an ERE-tk109-luc reporter construct, plated cells on different ECM proteins, and analyzed E-induction of ERE activity. In the absence of IGF-I and EGF, E-induction of ERE-luciferase activity was significantly reduced on all ECM proteins (data not shown). In the presence of submaximal concentrations of IGF-I and EGF, ERE-luciferase activity increased 8.1- to 8.3-fold after E treatment of cells plated on Col I or FN (Fig. 7BGo). MCF-7 cells plated on LM had a significantly lower, 2.6-fold, induction of ERE-luciferase activity. Expression of ß-galactosidase from a cytomegalovirus promoter was similar on Col I, FN, and LM (data not shown). Additionally, luciferase activity of control tk109-luc plasmid (which does not contain an ERE) transfected MCF-7 cells was not different on different ECM proteins (Fig. 7BGo, inset). This indicates that the transfection efficiency and general transcriptional activity were not influenced by these specific ECM proteins. Thus, LM appears to specifically reduce ERE-mediated transcription. To determine if the reduced activity of ER in MCF-7 cells cultured on LM was due to a reduced binding affinity of E to the ER, an E dose response (0.1 nM to 100 nM) experiment for ERE activity was conducted. On all 3 ECM proteins, maximal luciferase activity was reached by 1 nM; on LM and FN luciferase activity was highest at 1 nM and Col I at 0.1 nM or 1 nM (Fig. 7CGo). These data indicate that cells cultured on LM or FN have the same sensitivity to E and on Col I the cells may have a slightly increased sensitivity. Furthermore, at peak doses, E increased luciferase expression 2.4-fold on LM, compared with control, but increased luciferase expression 7.0 and 11.3-fold on Col I or FN, respectively (Fig. 7CGo). Thus, LM reduces E-responsiveness by decreasing the efficacy of E in activating ERE-mediated gene transcription. As previously reported, T47D cells showed very weak stimulation of ERE activity, even when cotransfected with ER, preventing analysis on various ECM proteins (25).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that the ECM protein, LM, can reduce E-responsiveness in breast cancer cells. We found that MCF-7 and T47D breast cancer cells cultured on PLL, Col I, Col IV, FN, and VN proliferated in response to E when cells were cultured in SFM supplemented with IGF-I and EGF. However, on LM these cells exhibited little or no E-induced proliferation in SFM. LM also inhibited E-induced proliferation in the presence of serum, indicating that the effects of LM are dominant to other ECM proteins because serum contains FN (18). Several reports have suggested that in fibroblasts, myoblasts, or endothelial cells LM causes induction of differentiation or apoptosis and blocks all other mitogenic pathways (26, 27, 28). In our studies with ER+ breast cancer cells, LM did not inhibit all mitogenic pathways since EGF or IGF-I stimulated cell proliferation on LM, but instead specifically blocked/inhibited E-signaling pathways. These data suggest that the tumor extracellular microenvironment can influence E-dependent control of growth in ER+ breast cancer cells and may be critical for the success of antiestrogen breast cancer therapies.

Estrogen-induced proliferation in the normal mammary gland requires undefined stromal signaling, but these signals are greatly altered during tumorigenesis (9, 13, 29, 30). Despite the fact that these observations are nearly 20 yr old, little is known about 1) how epithelial cells become E-responsive, 2) what are the minimal conditions required for E-induced proliferation of normal or tumorigenic breast epithelial cells, 3) what factor(s) can repress or alter this activity, and 4) how ER+ breast cancer cells become antiestrogen resistant. This paper has addressed several of these long-standing questions. First, we found that low levels of IGF-I or EGF were necessary for E-responsiveness in SFM. Many studies have demonstrated that IGF-I or EGF can stimulate the ER in the absence of ligand and that IGF-I or EGF can synergize with E to promote proliferation, but this is the first study to determine that IGF-I or EGF are necessary for and potentiate E-induced proliferation. These studies have also shown that ECM proteins, which can be significantly and progressively altered during breast tumorigenesis (31, 32), can act as regulators of E action in breast cancer cells. LM was shown to regulate E action in breast cancer cells even in the presence of other ECM proteins, indicating that ECM should be examined as a potential regulator of E action in the normal mammary gland, especially in light of other recent observations: namely that the major components of the basement membrane are secreted by stromal cells and stromal-epithelial interactions are critical for E action in the normal mammary gland.

This is the first report to demonstrate that specific ECM proteins regulate estrogen action in either normal breast cells or in breast cancer cells. LM has, however, been previously reported to be required for differentiation of normal mammary epithelium in vitro (33). For example, an LM-rich basement membrane induces transcription from the ß-casein promoter by activating a transcriptional enhancer, BCE-1 (34). Culture of normal mammary epithelial cells on purified LM, but not Col I, Col IV, or FN, dramatically increases transcription and synthesis of ß-casein in vitro in the presence of lactogenic hormones (35). The increase in ß-casein expression on LM was inhibited by anti-LM antibodies, demonstrating LM specificity. These in vitro data strongly support the role of LM in the differentiation of normal mammary epithelium. Interestingly, during lactation in the mouse, when mammary epithelial cells are fully differentiated, E is unable to stimulate PR induction or proliferation despite the presence of ER (36, 37). Thus, lactational differentiation in vivo is associated with a lack of E-responsiveness in normal cells. This leads us to speculate that LM and its receptors may also play a role in the loss of E-responsiveness in normal mammary cells during lactation.

Our data demonstrate that culture of MCF-7 cells on LM inhibits ER-mediated transcription pathways, including proliferation, induction of PR, and ERE-mediated transcriptional activation. In all cases, LM inhibited E action, however, the degree of inhibition varies among transcriptional activity, proliferation, and progesterone receptor induction. These differences can be explained, at least partially, by differences in assay sensitivity. For instance, we routinely achieve 100-fold increases in ERE luciferase expression, when comparing ICI treated vs. E-treated MCF-7 cells on Col I. However, when looking at thymidine incorporation, these treatment differences are only 10- to 25-fold, while PR induction is about 2-fold. Luciferase assays for gene transcription are extremely sensitive, as much as 100-fold more sensitive than traditional CAT assays, thereby permitting and amplifying the detection of even minimal changes in transcription. Proliferation is likely a downstream event to both protein production and transcription, and levels of transcripts/protein may need to reach threshold levels to permit proliferation. Finally, other events such as interactions with other intracellular pathways and differential regulation of transcript stability or translation may affect ERE activity, PR and proliferation differently.

Our results indicate that the inhibition of E action by LM is a postreceptor, postbinding event because ER concentration/cell and ER binding are not altered by LM, but E induction of ERE-mediated transcription is inhibited. The effectiveness of E to stimulate gene transcription mediated by the ER is dependent on ER interactions with coactivator and corepressor proteins. Recently several coactivators have been identified that greatly increase ER-dependent transcription, including SRC-1 (steroid receptor coactivator-1), AIB1, and ERAP-140 (estrogen receptor associating protein 140 (26, 27, 38). Similarly, several corepressors including REA (repressor of estrogen receptor activity), NcoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoic acid and thyroid hormone receptor) inhibit ER mediated gene transcription (27, 28, 39). Therefore, the regulation of the concentration and activity of coactivators and corepressors of ER mediated gene transcription may be a postreceptor target for LM inhibition of E action.

Others have shown that several cell types can shift from a differentiated to a proliferative state or vice-versa as a result of culture on specific classes of ECM. For example, in fibroblasts, endothelial and muscle cells, LM has been reported to induce differentiation or apoptosis and prevent proliferation, while FN or VN block differentiation and induce proliferation in the same cell types (40, 41, 42). We also found that LM, but not Col I or FN, generally decreased serum-stimulated cell proliferation in the absence of E in MCF-7, T47D, and MDA MB231 cells. We hypothesize from our data that LM may inhibit basal proliferation slightly by regulating cofactors that are used in multiple proliferation pathways, but are critical to estrogen signaling. Alternatively, LM may have multiple mechanisms of action, regulating the activity of several cofactors in different proliferation pathways that act independently of each other. However, dose response studies with IGF-I and EGF in MCF-7 and T47D cells on different ECM proteins revealed that proliferation on Col I, FN, and LM occurred at similar growth factor concentrations and to a similar extent. These data demonstrate that culture of ER+ breast cancer cells on LM does not inhibit all mitogenic responses. To determine if LM induces apoptosis, cell cycle analysis of MCF-7 cells cultured on Col I and LM was performed on a flow cytometer using propidium iodine to stain nuclear DNA. Culture on LM did not increase the percentage of hypodiploid cells, which are indicative of apoptosis (Haslam and Woodward, unpublished observations).

The regulation of E action by ECM proteins is relevant to breast cancer. The basement membrane is frequently degraded during breast cancer progression by increased metalloproteinase expression, but the expression of certain ECM proteins may increase during breast cancer progression. Breast carcinogenesis is frequently accompanied by pronounced changes in stromal tissue, which accounts for as much as 90% of the tumor (43). In fact, the most common type of breast cancer, infiltrating ductal carcinoma, exhibits a pronounced degree of desmoplasia. Several ECM proteins including LM are extensively expressed throughout the interstitial desmoplastic stroma of invasive breast cancers (31). Additionally, LM expression in tumor cells and in the serum increases with tumor progression and metastasis (44, 45). Metastatic tumor cells also have increased expression of two major LM specific integrins, {alpha}6ß1 and {alpha}6ß4 and the nonintegrin 67-kDa laminin receptor (46). The expression of LM and LM receptors by tumor cells has been reported to increase attachment to Col IV in the basement membrane of blood vessels and subsequently promote metastasis (47). Collectively, these studies demonstrate that LM and LM receptor expression may be significantly and substantially increased during breast cancer progression in desmoplastic stroma, metastatic breast cancer cells, and in the serum of women with metastatic breast cancer. E-responsiveness is a diagnostic marker for breast cancer progression because less differentiated, metastatic tumors are frequently refractory to E and antiestrogen therapies. The increased expression of LM and its receptors with advanced breast cancer correlates with decreased E-responsiveness. Consistent with these studies, our results indicate that LM may be involved in blocking E-responsiveness in these tumors.

In summary, this study demonstrates, for the first time, that LM reduces E-responsiveness by decreasing the efficacy of E in activating ERE gene transcription in ER+ breast cancer cells. Because stromal cells are the major source of epithelial basement membrane proteins in the normal gland (9) and this pattern is dramatically altered during carcinogenesis, expression of different ECM proteins may be a major pathway by which stromal cells influence tumor cell behavior. The tumor microenvironment may influence epithelial cell responsiveness to hormones and may therefore lead to hormone insensitivity without the loss of hormone receptors. Absence of hormone responsiveness in breast cancer is associated with a poor prognosis and substantially limits treatment options. However, it is noteworthy that the present studies indicate that these ER+ cells may still be highly responsive to the mitogenic effects of growth factors and this provides a plausible explanation for the growth of ER+ breast cancer cells that are E-independent and/or antiestrogen resistant. The effect of ECM proteins on E regulated tumor growth in vivo are currently under investigation. Advancing our understanding of the mechanism(s) by which ECM proteins influence hormone responsiveness and tumor growth may lead to novel therapeutic approaches to the treatment of breast cancer.


   Acknowledgments
 
We thank Jessica Bennett for excellent technical assistance, Drs. William Helferich and Barbara Vonderhaar for providing the cell lines, Drs. Barry Gehm and Donald Jump for providing the plasmids, and Drs. Susan Conrad and Richard Miksicek for their helpful advice.


   Footnotes
 
1 This work was supported by a U.S. Army Medical Research and Materiel Command Breast Cancer Research Program Grant DAMD 17–96-1–6026 (to T.L.W.) and NIH Grant R01-CA-40104 (to S.Z.H.). Back

Received January 14, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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