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Breast Cancer Research Laboratory Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA
1Correspondence: Breast Cancer Research Laboratory, Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111, USA. E-mail: j_russo{at}fccc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) negative MCF-10F cell line on the in vitro expression of neoplastic transformation was evaluated. E2 treatment induced the expression of anchorage-independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, and loss of 9p11–13. Only invasive cells that exhibited a 4p15.3–16 deletion were tumorigenic. Tumors were poorly differentiated ER- and progesterone receptor-negative adenocarcinomas that expressed keratins, EMA, and E-cadherin. Tumors and tumor-derived cell lines exhibited loss of chromosome 4, deletions in chromosomes 3p12.3–13, 8p11.1–21, 9p21-qter, and 18q, and gains in 1p, and 5q15-qter. The induction of complete transformation of MCF-10F cells in vitro confirms the carcinogenicity of E2, supporting the concept that this hormone could act as an initiator of breast cancer in women. This model provides a unique system for understanding the genomic changes that intervene for leading normal cells to tumorigenesis and for testing the functional role of specific genomic events taking place during neoplastic transformation.—Russo, J., Fernandez, S. V., Russo, P. A., Fernbaugh, R., Sheriff, F. S., Lareef, H. M., Garber, J., Russo, I. H. 17-Beta-estradiol induces transformation and tumorigenesis in human breast epithelial cells.
Key Words: estrogen • invasiveness • CGH • breast cancer
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and metastatic disease (2)
induced by oophorectomy in premenopausal women. Ulterior correlation of ovarian function with estrogen production (3)
and the isolation of the estrogen receptor protein (4
, 5)
, combined with the greater incidence of estrogen receptor positive tumors observed in postmenopausal women (6
7
8
9
10
11
12
13
14
15
16)
, led to the identification of a strong association of estrogen dose and length of exposure with increased breast cancer risk (6
, 10
, 13
, 14)
. The importance of ovarian steroidogenesis in normal breast development and in the genesis of breast cancer is highlighted by the facts that early menarche and late menopause are associated with greater breast cancer risk, whereas late menarche and early menopause (that occurring before 40 years of age) result in a significant reduction of the same (17
18
19
20)
. Breast development at puberty and during sexual maturity is stimulated by 17ß-estradiol (E2), which is the predominant circulating ovarian steroid and the most biologically active hormone in breast tissue (21
, 22)
. At menopause E2 plasma levels decrease by 90% (17
18
19)
. Despite the markedly different circulating levels of estrogens in pre- and postmenopausal women, the concentrations of E2 in breast cancer tissues do not differ between these two groups of women, an indication that its uptake from the circulation might not contribute significantly to the total content of this hormone in breast tumors, but rather that de novo biosynthesis (i.e., peripheral aromatization of ovarian and adrenal androgens) plays a more significant role (23
, 24)
.
Considerable epidemiological and clinical evidence link cumulative and sustained exposure to estrogens with increased risk of developing breast cancer. However, there is no clear understanding of the mechanisms through which estrogens cause cancer. In experimental animal models it has been demonstrated that E2, 11ß-methoxyethinylestradiol (Moxestrol), and diethylstilbestrol (DES), as well as their 4-hydroxycatechols, induce kidney cancer in castrated male Syrian golden hamsters (25
26
27)
. In rats, continuous administration of supraphysiological doses of estrogens induces a high percentage of mammary adenocarcinomas, whereas low doses given over long periods induce fibroadenomas (28)
. In both models, however, the tumorigenic effects of estrogens are associated with marked hyperprolactinemia and pituitary hyperplasia resulting from an increase in number of hyperplastic prolactin-secreting cells. The dependence on a functional pituitary gland has been further confirmed in hypophysectomized rats in which estrogens are ineffective as carcinogens (29)
. Nevertheless, the most widely acknowledged mechanism of estrogen carcinogenicity is its binding to its specific nuclear receptor alpha (ER-) for exerting a potent stimulus on breast cell proliferation through its direct and/or indirect actions on the enhanced production of growth factors (21
, 22)
. However, the fact that ER- knockout mice expressing the Wnt-1 oncogene (ERKO/Wnt-1) develop mammary tumors provides direct evidence that estrogens may cause breast cancer through a genotoxic, non-ER--mediated mechanism (30
, 31)
. This postulate is further supported by the observations that when ovariectomized mice are supplemented with E2 they develop a higher tumor incidence with shorter latency time than controls, even in the presence of the pure antiestrogen ICI-182,780. Experimental studies of estrogen metabolism (32
, 33)
, formation of DNA adducts (34)
, carcinogenicity (35
36
37)
, mutagenicity (38)
, and cell transformation (39
40
41
42)
have supported the hypothesis that reaction of specific estrogen metabolites, namely, catechol estrogen-3,4-quinones (CE-3,4-Q) and to a much lesser extent, CE-2,3-Q, can generate critical DNA mutations that initiate breast, prostate, and other cancers (43)
. To definitively outline the pathways through which estrogens act as carcinogens in the human breast and for assessing whether one or more of the mechanisms described above are responsible of carcinogenic initiation, an experimental system is needed in which E2 by itself or its metabolites induce transformation of human breast epithelial cells (HBEC) in a well-controlled environment, preferentially in vitro. Toward this purpose, we developed an in vitro/in vivo system of cell transformation that fulfills these requirements. Using this model we have demonstrated that E2 and its metabolite 4-hydroxyestradiol (4-OH-E2) induce transformation of MCF-10F, an ER--negative human breast epithelial cell line (39
40
41
42
,44)
. In response to estrogen treatment, the cells form colonies in agar methocel, lose the capacity to differentiate by forming 3-dimensional structures when grown in a collagen matrix, or their ductulogenic capacity, forming instead spherical and solid masses, and exhibit an increase in cell proliferation and in their invasive capabilities in Matrigel (39
40
41
42
,44)
. More important, the expression of these phenotypes indicative of neoplastic transformation was not abrogated by their simultaneous treatment with the anti-estrogen ICI-182,780 (ICI), suggesting that the transformation of MCF-10F cells by these compounds did not require the presence of the ER- (40
, 41)
. The present work describes the novel findings that, in this experimental model, E2-induced transformation of HBEC in vitro increased the invasive potential of the cells. In addition, selection of the most highly invasive cells in the Matrigel chambers identified transformed cells that express phenotypic and genotypic variations that correlate with their tumorigenic potential in a heterologous host, but still maintained their cell lineage characteristics. We also report that the induced tumors exhibit genomic alterations that are similar to those reported in primary breast cancer, as determined by comparative genomic hybridization (CGH).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) -negative human breast epithelial cell line MCF-10F, which was cultured in Dulbecco’s modified Eagle medium (DMEM):F12 medium containing 1.05 mM calcium, antibiotics, antimycotics, hormones, growth factors, and equine serum as described previously (44
, 45)
. MCF-10F cells in their 123rd passage were treated with 70 nM 17ß-estradiol (E2) (Sigma Chemical Co., St. Louis, MO, USA) dissolved in DMSO. This dose was selected because it induced greater colony efficiency in agar methocel, number of solid masses in collagen gel, and invasiveness in the Matrigel invasion chamber than the 0.007 nM dose of E2 previously tested (39
, 40
, 44)
. E2 was added to the culture medium for 24 h at 72 and 120 h postplating. At the end of the first week of treatment, the cells were passaged for administration of two more 24 h periods of E2 treatment (39)
. At the end of each treatment period, the culture medium was replaced with fresh medium. MCF-10F cells treated with DMSO at a final concentration of 0.01% were used as controls. All cells were collected 24 h after the last treatment, and thereafter were maintained in culture for 10 additional passages.
Selection of E2-treated MCF-10F cells by Matrigel invasion assay
Control and E2-treated MCF-10F cells in their 10th passage were trypsinized and seeded in the upper chamber of seven and eight Matrigel invasion chambers, respectively, at a concentration of 2.5 x 104 cells/well each, and incubated at 37°C in a 5% carbon dioxide incubator for 22 h. At the end of this period, the inserts of each chamber were carefully removed from the wells with sterile forceps, then the upper/inner surfaces of the membranes were wiped with cotton tipped applicators for removing all noninvading cells. Each membrane was then cut from the insert using a sterile scalpel blade and individually placed in a well from a 24-well plate, with the lower surface that held the invading cells up, facing the culture medium. The cells were fed with DMEM medium containing 5% horse serum, maintained at 37°C in a 5% CO2 incubator until they reached confluence, then transferred to a 75cc flask. Seven cell lines from control MCF-10F cells were thus selected and labeled A-1 to A-7. Four cell lines were selected from E2-treated MCF-10F cells, which were designated B2, C3, C4, and C5 (Fig. 1
).
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Evaluation of transformation phenotypes
Control MCF-10F cells and E2-transformed cells at passage 10, and those invasive cell lines isolated from the invasion chamber described above were expanded and evaluated for the expression of the following phenotypes of neoplastic transformation: colony formation in agar-methocel or colony efficiency (CE), ductulogenic capacity in collagen matrix, invasiveness in Matrigel invasion chambers, and tumorigenic assay in severe combined immunodeficient (SCID) mice.
MDA-MB-231, an ER--negative human breast cancer cell lines purchased from the Tissue Culture Collection (Rockville, MD, USA) and BP1-Tras, a tumorigenic cell line derived from benz(a)pyrene (BP)-transformed-c-Ha-ras transfected MCF-10F cells (46)
were used as positive controls.
Colony formation in agar-methocel
Control and treated cells were suspended at a density of 2 x 104 cells/ml in 2 ml of 0.8% methocel (Sigma) dissolved in DMEM:F-12 (1:1) medium containing 20% horse serum. Cells from each treatment group were plated in eight 24-well chambers precoated with 0.5 ml 5% agar base in DMEM: F-12 medium. Cells were fed fresh medium twice a week. To evaluate colony efficiency, the total number of viable cells was counted at 10x magnification in four wells that were stained with neutral red (1:300) after 24 h postplating and in four additional wells after 21 days in culture. In the latter, each colony was measured using a graduated eyepiece fitted in a transmission light microscope at 10x magnification. The number of colonies >50 µ in diameter was counted and results of colony efficiency were expressed as a percentage of the original number of viable cells after 24 h of plating.
Ductulogenic assay
Control and E2-treated MCF-10F cells at their ninth passage post-treatment were suspended at a final density of 2 x 103 cells/ml in 89.3% (Vitrogen100) collagen matrix (Collagen Co., Palo Alto, CA, USA) and plated into four 24-well chambers precoated with 89.3% of collagen base. The cells were fed fresh medium containing 5% horse serum twice a week. The cells were examined under an inverted microscope for 21 days or longer to evaluate the number of duct-like or spherical mass structures, as described in ref 40
. At the end of the observation period the structures were photographed, fixed in 10% neutral buffered formalin, and processed for histological examination.
Invasion assay
The invasion assay was performed using 24-well plate Matrigel invasion chambers (BD Biosciences, Bedford, MA, USA) fitted with cell culture inserts (Falcon Cell Culture Inserts) closed with an 8 µm pore-size PET membrane coated with a uniform Matrigel basement membrane matrix. Chambers were stored at –20°C and brought to room temperature in a laminar flow hood for 2 h, and the insert chambers were hydrated by placing 500 µl of culture medium containing 5% horse serum at 37°C for 2 h in a humidified tissue culture incubator. Then the medium was removed from the inserts and 500 µl of 20% horse serum was added to each well as chemoattractant. E2-treated, and all control cells were trypsinized, and each cell line was seeded in triplicate in the upper chamber at a concentration of 2.5 x 104 cells/well and incubated at 37°C in a 5% carbon dioxide incubator for 22 h. At the end of this period, the membranes of each chamber were fixed with Diff-Quick fixative and stained with Diff Quick Solutions I and II (Sigma), cut out with a sharp scalpel, and mounted onto glass slides. The total number of cells that invaded through the membrane was counted under a light microscope and the invasion index was expressed as the means ± SE of the cells that migrated through the membrane and attached to the lower surface.
Tumorigenic assay
The tumorigenic ability of all the cell lines was tested in 45-day-old female SCID mice that were purchased from Taconic Farms (Germantown, NY, USA). Cells were injected using protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Fox Chase Cancer Center. Trypsinized cells were suspended in PBS and injected in the mammary fat pad of the abdominal region of the mice at a concentration of 10–15 x 106 cells in a volume of 0.1 ml. The animals were palpated twice a week to detect tumor development. Tumors were measured in two dimensions with a Vernier caliper and when they reached a maximal diameter of 1 cm, the animals were euthanized by carbon dioxide inhalation. All of the animals were autopsied and carefully examined for identification of visceral metastases. Tumors were excised under sterile conditions and divided in three fragments; one was fixed in 10% neutral buffered formalin (NBF) and processed for histopathological and immunocytochemical examination. A second fragment was rapidly frozen in liquid nitrogen and stored at –80°C for future use, and a third fragment was used for cell culture. Those animals that did not develop tumors were followed up for 6–10 months postinjection, then euthanized and autopsied, fixing the site of injection in 10% NBF for histopathological analysis.
Development of cell lines from tumors
C5 cells injected to 10 mice gave origin to nine tumors from which four tumors were used for developing cell lines. The tumors were dissected in small fragments of 0.5 to 1 mM in thickness and incubated in a petri dish with culture medium until confluent, at 
5 days after plating. Explants were passed twice and maintained in DMEM culture medium with a 0.001 mM Ca2+ concentration until confluent. The tumoral cell lines derived from C5 were designated C5-A1-T1, C5-A4-T4, C5-A6-T6, and C5-A8-T8. These cells were used at passage three for fingerprint and tumorigenic assay analyses (Fig. 1)
.
Histopathological and immunocytochemical analyses
Tissues fixed in formalin, dehydrated, and embedded in paraffin were cut at 5 µm thickness and stained with hematoxylin and eosin for histopathological analysis. For immunocytochemical analysis, tissue sections were mounted on aminoalkylsilane-coated or positively charged slides, deparaffinized, rehydrated, and incubated in 2% hydrogen peroxide at room temperature for 15 min for quenching endogenous peroxidase activity. The sections were sequentially incubated in two changes of Target Retrieval Solution at 98°C for 5 min each. All tissue sections were incubated in diluted normal blocking serum for 20 min. Excess serum was blotted from the slides and the sections were incubated with the following mouse monoclonal antibodies: AE1, anti-human low MW cytokeratin, AE3, anti-human high MW cytokeratin, progesterone receptor clone PR88 (Biogenex, San Ramon, CA, USA), and anti-ER- clone 1D5 (DakoCytomation Colorado Inc., Fort Collins, CO, USA). The polyclonal antibodies rabbit anti-human ERß (Biogenex, San Ramon, CA, USA), cell adhesion molecule 5.2, cytokeratin peptides 7 and 8 (48 kDa and 52 kDa, respectively), and E-cadherin (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA), epithelial membrane antigen (EMA) clone E29, and vimentin (DakoCytomation Colorado Inc.) were also tested. After incubation in a humidity chamber at 4°C overnight, sections were washed in buffer and incubated with horse biotinylated secondary antibody (Vector Laboratories, Inc., Burlingame, CA, USA) at room temperature for 30 min, followed by a 30 min incubation with Vectastain Elite avidin-biotin complex kit (Vector Laboratories), washed in PBS buffer, and incubated in peroxidase substrate solution containing hydrogen peroxide and 3, 3'-diaminobenzidine-HCl for 2 min. Sections incubated with nonimmune serum were used as negative controls. All sections were lightly counterstained with hematoxylin. Immunostaining was evaluated by examination of slides under a bright-field microscope and graded according to the intensity of the brown staining.
Verification of cell lineage by DNA fingerprint analysis
Cell lineage was verified in the tumors and all the cell lines by fingerprint analysis using capillary electrophoresis and employing the markers listed in Table 1
. The human breast epithelial cells MDA-MB-231 cells and BP1-Tras were used as negative and positive controls, respectively. For fingerprint analysis DNA was extracted from frozen tumors and from 70–80% confluent cells in culture (Table 2
). Tissues and cells were treated with lysis buffer containing 100 mM NaCl, 20 mM Tris-Cl pH 8.0, 25 mM EDTA pH 8.0, 0.5% SDS, and 200 µg/ml proteinase K and incubated at 65°C for 15 min with gentle agitation. The samples were cooled down on ice and treated with 100 µg/ml RNase at 37°C for 30 min. The DNA was purified with a phenol extraction (pH=8.0) followed by chloroform: isoamyl alcohol (24:1). The aqueous layer was adjusted to 0.75M with ammonium acetate and the DNA was precipitated with 100% ethanol. The samples were centrifuged, dried, and dissolved in distilled water. For fingerprint analysis, polymerase chain reaction (PCR) was carried out in a final volume of 10 µl of 1x PCR buffer (Invitrogen, San Diego, CA, USA) containing 1.5 mM MgCl2, 0.5 pmol of each primer, 100 µM dNTPs, 0.25U TaqPlatinum (Invitrogen), and 60–90 ng DNA. Six markers, CSF1PO, TPOX, THO1, VWA, F13AO1, and FESFPS (Table 1)
were used in this analysis. Commercially available fluorescently labeled forward primers were used in each PCR reaction. The PCR conditions consisted of a denaturation step (3 min at 94°C), followed by 35 cycles at 94°C for 30 s, annealing temperature for 45 s and 72°C for 30 s, with an extension step at 72°C for 5 min. The fluorescent PCR was mixed with an internal standard size marker and fractionated using CEQ8000 (Beckman Coulter, Fullerton, CA, USA). The size of the different alleles determined in the number of base pairs were compared among tumors and cell lines derived from MCF-10F with the cell line MDA-MB-231.
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We have also used variable number of tandem repeat (VNTR) analysis for confirming the MCF-10F cells lineage. For this purpose, DNA from the different cell lines was digested with HinfI and used in Southern blot analysis. The VNTR probes D2S44 and D14S13 for their corresponding markers on chromosomes 2q21.3-q22 and 14q32.1-q32.3, respectively, were used.
Screening for E2-induced genetic changes by comparative genomic hybridization
For determining whether the transformation of MCF-10F cells by E2 treatment resulted in DNA losses and/or gains at chromosomal and subchromosomal levels we analyzed by comparative genomic hybridization (CGH) (47)
, which detects gains or losses of 5–15Mb, DNA obtained from control and E2-treated MCF-10F cells and the invasive and tumor-derived cells. Protocols for DNA labeling and hybridization were performed as described previously (42)
. Gray-level images of fluorescence were captured with a Zeiss microscope (Thorndale, NY, USA) connected to a cooled, charge-coupled-device camera (Photometrics, Tucson, AZ, USA). Digital image analysis was performed using the Quips software (Vysis, Downers Grove, IL, USA). The threshold was set at 0.8 and 1.2 for losses and gains, respectively. The mean values of individual ratio profiles were calculated from at least 10 metaphase spreads. Averaged values were plotted as profiles alongside individual chromosome ideograms.
Statistical analysis
All the assays for testing colony efficiency, ductulogenesis, solid mass formation, and invasion were run in triplicate and expressed as means ± SE. The size of the tumors induced in the SCID mice was expressed as the mean of the maximum tumor diameter ± SD. Results were evaluated by Student’s test for assessing the significance of a difference.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-negative human breast epithelial cell line MCF-10F with 70 nM E2 twice a week for 2 wk formed colonies in agar methocel and the colony efficiency increased from 0 in controls to 12.0 ± 1 in the treated cells (Fig. 2
). The positive control cells BP1-Tras and MDA-MB231 cells had a moderately (P<0.02) and significantly (P<0.001) higher colony efficiency than E2-transformed cells, respectively (Fig. 2)
.
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This treatment also affected the ductulogenic pattern of cells grown in collagen gel, which was quantitatively evaluated by counting the total number of ductules and spherical masses formed by 10,000 cells plated in collagen. Control MCF-10F cells formed an average of 110 ductular structures (Fig. 3
), but did not form solid masses (Fig. 4
and Fig. 5
). After treatment with E2, MCF-10F cells almost completely lost their ductulogenic capacity (Figs. 3
and 5)
while acquiring the ability to form spherical solid masses (Figs. 4
, 5)
. BP1-Tras and MDA-MB 231 exhibited a complete absence of ductule formation (Fig. 3)
, forming instead solid masses in collagen gel whose values were not significantly different from those formed by E2-treated cells (Fig. 4)
. The differences were highly significant (P<0.0001).
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The ability of cells to invade a Matrigel membrane in vitro is a widely accepted criterion of cell transformation. Control MCF-10F cells exhibited a low invasive capacity, averaging 10 ± 2 cells, whereas the invasive capacity of E2-transformed cells at their 9th passage was significantly higher (80±11 cells) (Fig. 6
). BP1-Tras and MDA-MB231 cells had an invasive index significantly higher than that of MCF-10F control and E2 transformed cells (P<0.001).
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Tumorigenic response
MCF-10F cells between passages 130–132 and E2-treated cells between their passages 7 and 9 were injected to 10 SCID mice each to test their tumorigenic capabilities. Neither control nor E2-treated cells formed tumors after a 6 month follow-up period. Instead, BP1-Tras and MDA-MB231 cells were highly tumorigenic with a short latency period (Table 3
). Because the tumorigenic response of these two cell lines was associated with a highly invasive phenotype, we tested whether selection of more invasive cells among E2-transformed MCF-10F cells would allow them to express the tumorigenic phenotype and to determine whether this phenotype was exclusively induced by estrogen, and not the result of the selection of more invasive control cells. For this purpose, MCF-10F cells in their 133rd passage and E2-treated MCF-10F cells in their 10th passage were trypsinized and seeded in the upper chamber of seven and four matrigel invasion chambers, respectively. Those cells that at 22 h postseeding had crossed the Matrigel membrane were cultured, giving origin to seven MCF-10F cell lines labeled A1 to A7. From the E2-treated cells, four lines were obtained and designated B2, C3, C4, and C5 (Fig. 1)
. Injection of A1 to A7 cells to SCID mice did not induce a tumorigenic response even after 6 months of follow-up (Table 3)
. After injection of the E2-transformed cells B2, C3, C4, and C5 to SCID mice, only C3 and C5 were tumorigenic in 2/12 and 9/10 animals injected, respectively. The clone C5 produced tumors larger than the ones produced by C3 (Table 3)
. From the nine tumors obtained from C5 cells, four tumoral cell lines designated C5-A1-T1, C5-A4-T4, C5-A6-T6, and C5-A8-T8 were derived. These cells were subsequently injected to another set of five SCID mice per cell line for testing their tumorigenic capabilities. All these cell lines formed palpable tumors, C5-A8-T8 being the fastest growing tumor (Fig. 1
, Table 3
). Cell lines B2 and C4 did not induce tumors even after a 9 month follow-up.
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Histopathological analysis revealed that all the E2 70 nM-C5 cells formed tumors, and those tumors formed by their derived cells were poorly differentiated adenocarcinomas (Fig. 7
). They invaded the mammary fat pad (Figs. 7a, b)
and the skeletal muscle of the abdominal wall (Fig. 7c
). Tumors formed by E2 70 nM-C3 cells were also poorly differentiated adenocarcinomas; they were smaller and more circumscribed than E2 70 nM-C5 formed tumors. BP1-Tras and MDA-MB231 cells also formed undifferentiated adenocarcinomas that were less invasive than those generated by E2 70 nM-C5-derived cells.
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The immunocytochemical reactivity of the E2-induced tumors in SCID mice was compared with the reactivity of normal breast tissues, primary breast cancer, control MCF-10F cells, and with tumors formed by BP1-Tras and MDA-MB231 cells in SCID mice (Table 4
). AE1 and AE3, human low and high MW cytokeratins were expressed in the cytoplasm of the neoplastic cells in all E2-induced tumors (Figs. 7e, g)
in a pattern similar to those observed in normal breast tissues, in primary invasive ductal carcinomas of the breast (Figs. 7d, f
) and in MCF-10Fcells (Table 4)
. The cytokeratin peptide 7 and 8 (CAM5.2) diffusely stained the cytoplasm of neoplastic cells with greater variations in the degree of intensity (Fig. 7i
) than in the invasive ductal carcinoma of the breast used as positive control (Fig. 7h
). E-cadherin was positive in all E2-induced carcinomas, exhibiting a diffuse and moderate reactivity (Fig. 7k
), less intense than that observed in the invasive ductal carcinoma used as a positive control (Fig. 7j
). Epithelial membrane antigen (EMA) had similar level of reactivity in E2-induced tumors than in primary breast cancer, in normal breast tissues and in MCF-10F cells, but less intense that in tumors formed by BP1-Tras and MDA-MB231 in SCID mice (Table 4)
. The latter expressed high reactivity for vimentin (Table 4)
. Estrogen receptor alpha (ER-), which was positive in normal breast tissues and in primary breast cancer, was negative in MCF-10F cells and in all E2-induced tumors in SCID mice. The same pattern of reactivity was observed for progesterone receptor (Table 4)
.
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Fingerprint analysis
MCF-10F cells transformed with 70 nM E2, and all the tumors and cells derived from them were used for fingerprint analysis, which was performed using the six markers indicated in Table 1
. All the tumors and cell lines derivate from MCF-10F showed the same sizes for the different markers (Table 2)
. The cell line MDA-MB-231 showed different sizes compared to MCF-10F and its derivates for five out of six markers tested (Table 2)
. These results indicated that all the tumors and cell lines tested, except MDA-MD231, originated from MCF-10F cells. These data were also confirmed using variable number of tandem repeat (VNTR) analysis. The Southern blot showed that the DNA profile of all of the cell lines have the same HinfI restriction pattern as MCF-10F cells, and different from MDA-MB-231 cells (data not shown).
Comparative genomic hybridization (CGH) analysis
Estrogen treatment of MCF-10F cells resulted in losses and gains of genetic material that CGH showed to be progressive at different stages of the tumorigenic process. In E2-treated cells, the first loss detected was in 9p11–13. The same loss was also maintained in E2 70 nM-C5 cells, in the tumors formed by these cells in SCID mice, and in all the cell lines derived from these tumors (Table 5
). E2 70 nM-C5 cells also exhibited loss of 4p, which expanded to the loss of the complete chromosome in the tumors derived from these cells as well as in the cell lines derived from the tumors (Table 5)
. Four additional losses appeared in all the tumors and in their derived cells that included 3p12.3–13, 8p11.1–21, 18q, and 9p21-pter, whereas the loss of 9p11–13 observed in previous cell lines was no longer evident (Table 5)
.
|
Gains in 1p and 5q15-qter were observed in the four tumors formed by C5 cells in SCID mice (An1, An 4, An 6, and An 8) and the cell lines derived from them (C5-A1-T1, C5-A4-T4, C5-A6-T6, and C5-A8-T8) (Table 5)
. In the cell line E2 70 nM-C5 that gave origin to the different tumors, the gain of 1p, 5q, and loss of chromosome 4 did not reach threshold values for being considered as gains or losses for xenografting, but in the tumors the C5 clone with these chromosomal alterations probably had a selective advantage; therefore these changes were very distinct.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our previous work has demonstrated that treatment of the immortalized ER--negative human breast epithelial cell line MCF-10F with E2 and its metabolites 2- and 4-hydroxyestradiol induce anchorage-independent growth, loss of ductulogenic pattern, and invasiveness in a Matrigel basement membrane (39
40
41
42
43
, 48)
. The transforming capabilities of estrogens have been confirmed in MCF-10A, another ER--negative immortalized human breast epithelial cell line in which E2 and estrogenic substances, such as Zeranol (Ralgro, Calgary, Alberta, Canada), a nonsteroidal agent with estrogenic activity used as a growth promoter in the U.S. beef and veal industry (49)
, and 4-hydroxyequilenin (50)
induce anchorage-independent growth. These observations support the concept that estrogens induce neoplastic transformation through nonreceptor alpha-mediated mechanisms, exerting direct genotoxic effects, as previously suggested (32
33
34
35
36
37
38)
. Our findings of specific mutations in p53, and loss of heterozygosity (LOH) in chromosomes 11 and 13 further support this concept (40
, 51)
.
Using CGH, a molecular cytogenetic method for screening gains and losses at chromosomal and subchromosomal levels, we have detected that MCF-10F cells transformed by E2 had lost 9p11–13, a loss that persisted in the invasive cell line E2-70 nM-C5. This locus contains the serine protease family member PRSS3 (trypsinogen-IV), a putative tumor suppressor gene (52)
in which an allelic imbalance has been reported in hepatocellular carcinoma (53)
, carcinoma in situ of the bladder (54)
, and renal cell carcinoma (55)
. The loss of 9p11–13 was not detected by CGH technique in the tumors and tumor-derived cell lines, probably because the change did not reach the threshold for detection or because the cell population was heterogeneous. However, losses in 9p21-pter were clearly evident in the tumor and tumor cell lines. Losses of chromosome 9 regions are frequently reported in bladder carcinoma, especially in premalignant lesions such as hyperplasia and carcinoma in situ (CIS). Simultaneous losses in 9p11-q12 and in 9p21 have been reported in CIS of the bladder (56)
. Losses in this locus have also been reported in peripheral T cell lymphoma (57)
, melanoma cell lines (58)
, malignant fibrous histiocytoma (59)
, and parathyroid adenomas (60)
. The 9p21-pter region includes both the p16 and p15 genes. These observations indicate that loss of these tumor suppressor genes on 9p contribute to the progression of the invasive to the tumorigenic phenotypes in the natural progression of the disease.
In the present work we further demonstrate that E2 induces, in addition to the expression of early phenotypes of neoplastic transformation, tumorigenesis in a heterologous host. This phenomenon became possible only after the selection of invasive cells that exhibit specific changes, such as the deletion of chromosome 4p15.3–16, the first one detected. Injection of these cells to SCID mice resulted in the formation of tumors in which the entire chromosome 4 was deleted, a change that became a permanent feature of all tumors and tumor-derived cell lines. Allelic losses at one or both arms of chromosome 4 have been frequently reported in several tumor types, including breast cancers, either sporadic or occurring in BRCA1 and BRCA2 germline mutation carriers (61
, 62)
. Regions that have been frequently reported to be deleted are 4p16.3 (50%), 4p15.1–15.3 (57%), 4q25–26 (63%), and 4q33–34 (76%) (63)
. The tumors induced by E2-transformed cells in SCID mice are fast growing and ER-negative, being similar to the tumors exhibiting similar deletions and diagnosed in young women, in whom tumors are large at the time of diagnosis, having a high percentage of cells in S-phase and being negative for estrogen receptors (61
, 62)
. Chromosome 4 contains numerous genes of potential interest in cancer development, among them is Slit2, a gene located at 4p15.2 that encodes a protein that inhibits leukocyte chemotaxis and is a putative ligand for the ROBO receptors gene (64)
. SLIT2 is primarily a secreted protein that in conditioned medium suppresses the growth of several breast cancer lines (64)
. Therefore, the loss of the 4p15.3–16 region in E2-70 nM C5 cells could be the event that triggers a cascade that select tumorigenic cell population.
Additional losses initially detected in the tumors and maintained in the tumor-derived cell lines were in chromosomes 3 p12.3–13, 8 p11–21, and 18q. The region lost in chromosome 3 (p12.3–13) has been reported to exhibit imbalances in MCF-7 cells developing resistance to tamoxifen (65)
; region 8p11–21 encodes the frizzled-related gene FRP1/FRZB, which is turned off in 78% of breast carcinomas (66)
and associated with androgen in prostate cancer (67)
. The loss of chromosome arm 18q is a common event in primary breast cancers (68
69
70
71
72)
, ductal hyperplasia (73)
, and breast cancer cell lines (74)
, and is often interpreted as representing loss of one or more tumor-suppressor genes. The relevance of these losses in estrogen-induced cell transformation is that among the genes located in the q arm of chromosome 18 are two independent tumor suppressor loci in segment 18q21.1: one at SMAD4 and the other potentially at an enhancer of DCC or an unrelated novel gene (68
, 72)
.
Treatment of MCF-10F cells with E2-induced genomic gains in 1p and 5q15-qter, both of which became evident in tumors and remained at the same level of expression in all tumor-derived cells. Amplification of 1p has already been reported in primary breast cancer (69
, 9)
and in established breast cancer cell lines (47)
. Gain in 5q15-qter has not been found often in breast cancer (76)
, but it has been reported in immortalized human ovarian surface epithelial (HOSE) cells using HPV16E6E7 ORFs (80)
and in the cell lines SW480 and SW620 derived from different stages of colon carcinoma in the same patient (81)
. Although at the present time the role played by these gains in 1p and 5q15-qter in the process of estrogen-induced tumorigenesis is not known, a likely explanation is that the gains resulted from amplifications of smaller chromosomal segments that probably arose through real DNA amplification processes, suggesting that many genes present in these chromosomal loci are potential targets for the carcinogenic effect of 17-ß-estradiol (82)
.
Altogether our data indicate that 17-ß-estradiol is able to induce complete neoplastic transformation of human breast epithelial cells, as proven by the formation of tumors in SCID mice. This model demonstrates a sequence of chromosomal changes that correlates with specific stages of neoplastic progression. Our data also support the concept that 17-ß-estradiol can act as a carcinogenic agent without the need of the ER, although we cannot rule out the possibility that receptors such as ERß or other mechanisms could play a role in the transformation of human breast epithelial cells. These are areas of active research in our laboratory. The knowledge that breast cancer in women is associated with prolonged exposure to high levels of estrogens gives relevance to this model of estrogen-induced carcinogenesis (6
, 8
9
10
, 15
, 16)
. For this reason, this model is extremely valuable for enhancing our understanding of estrogen-induced carcinogenicity.
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ACKNOWLEDGMENTS |
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Received for publication January 5, 2006. Accepted for publication March 31, 2006.
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