Growth and characterization of N-methyl-N-nitrosourea-induced mammary tumors in intact and ovariectomized rats
Gudmundur Thordarson,3,
Adrian V. Lee1,
Meghan McCarty,
Katharine Van Horn,
Oriana Chu,
Yu-Chien Chou2,
Jason Yang2,
Raphael C. Guzman2,
Satyabrata Nandi2 and
Frank Talamantes
Department of Molecular, Cell and Developmental Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064,
1 Baylor College of Medicine, Breast Center, Houston, TX 77030 and
2 Cancer Research Laboratory, University of California, Berkeley, CA 94720, USA
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Abstract
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It is well established that 8590% of chemically induced mammary tumors in rats will disappear or diminish significantly in size after the ovaries are removed from the animal. However, it is less well established whether a high percentage of these mammary tumors will grow back with prolonged time after ovariectomy. It is also not known what changes in gene expression take place in the tumors as they develop an independence from hormones for growth. This study was carried out to investigate this. Virgin, 50-day-old female SpragueDawley rats were injected with N-methyl-N-nitrosourea (MNU) at the dose of 50 mg MNU/kg body wt. When at least one mammary tumor had grown to 1.01.5 cm in one dimension, the animal was bilaterally ovariectomized and reduction and then re-growth of the tumors monitored. Control animals were treated identically except they were not ovariectomized when tumors appeared. Re-growths and new tumors and tumors that developed in the control rats were removed when they reached 1.01.5 cm in diameter and all animals were killed 25 weeks after the MNU injection. All the animals in the study (100%) developed mammary tumors after MNU injection with an average latency of 56.5 days. After ovariectomy, 93% of the tumors showed 50% or more reduction in size and 76% of the tumors could not be detected by palpation. However, in 96% of the animals where tumor reduction or disappearance occurred, a re-growth or new mammary tumor development took place with an average latency period of 52.8 days from the day of ovariectomy. Of these post-ovariectomy tumors, 36% occurred at a location where tumors had developed prior to ovariectomy, but 64% appeared at new locations. The circulating levels of 17ß-estradiol (E2) was undetectable in the ovariectomized (OVX) rats and significant reduction was seen in the serum concentrations of progesterone (P4), prolactin (PRL), growth hormone (GH) and insulin-like growth factor-I (IGF-I). The tumors from the OVX rats showed indications of progression as evident from loss of differentiation and invasive characteristics. Comparison between tumors from OVX and intact rats revealed a significantly increased expression of P450 aromatase and elevated activation of extracellular signal-regulated kinase 1 and 2, but reduced levels of the progesterone receptor and cyclin D1 in OVX rats. However, the estrogen receptor (ER) content remained similar in tumors from both groups, at least at the protein level, and so did the expression of IGF-I, IGF-II, insulin receptor substrate-1 (IRS1), IRS-2 and epidermal growth factor receptor. IGF-I receptor (IGF-IR) and ErbB-2 were expressed, respectively, in 50 and 70% of the tumors from the OVX animals, whereas these genes were expressed in 100% of the tumors from the intact rats. It is concluded that chemically induced rat mammary tumors may still depend on the ER and local syntheses of E2 and growth factors for growth initially after ovariectomy. However, as these tumors progress, they develop a more aggressive phenotype and lose their dependency on the ER and possibly growth factors.
Abbreviations:
-lac,
-lactalbumin; E2, 17ß-estradoil; EGF, epidermal growth factor; EGFR, EGF receptor; ER
(-ß), estrogen receptor-
(-ß); ERK-1 (-2), extracellular signal-regulated kinase-1 (-2); GH, growth hormone; HDT, hormone-dependent tumor; HIT, hormone-independent tumor; IGF-I (-II), insulin-like growth factor-I (-II); IGF-IR, IGF-I receptor; IRS-1 (-2), insulin receptor substrate-1 (-2); MAP kinase, mitogen-activated protein kinase; MNU, N-methyl-N-nitrosourea; OVX, ovariectomized; P4, progesterone; P450arom, P450 aromatase; PR, progesterone receptor; PRL, prolactin; RIA, radioimmunoassay; RPA, ribonuclease protection assay; TGF-
, transforming growth factor-
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Introduction
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Mammary cancers are traditionally categorized as hormone dependent tumors (HDT) or hormone independent tumors (HIT) on the bases of their hormonal requirement for growth (1). Approximately 4050% of human breast cancers at diagnosis are HDTs and regress upon hormone therapy (2,3). However, although the tumors respond initially to hormonal treatments, the benefits are temporary and relapse will occur usually within months or a few years of treatment (4). Chemically induced rat mammary cancers show similarities to that of human breast cancers in that ~8090% of these tumors regress after ovariectomy of the animals, i.e. they are hormone dependent for growth (5,6). But after the initial regression, a high percentage of the regressed tumors grow back and tumors may develop where no were detectable before (7). Human breast cancers that develop resistance to antiestrogen therapy after initially responding to treatment continue to express the estrogen receptor-
(ER
) and genes regulated by the ER, such as the progesterone receptor (PR) and pS2 genes, are also expressed after resistance to antiestrogen has been developed (8,9). Some expression of the ER has been reported in chemically induced rat mammary cancers that actively proliferate after ovariectomy of the animal (10). Therefore, the ER in mammary cancers may be active in the absence of ovarian estrogen in the rat and in human breast cancers after the development of resistance to antiestrogens. Biosynthesis of estrogen within the mammary cancers could be a possibility, as aromatase activity is not uncommon in estrogen-dependent breast cancer specimens (11). The sensitivity of the ER to its ligand could also be increased in an environment of low estrogen as has been shown to occur in estrogen-starved MCF-7 breast cancer cells (12). Mutated form of the ER with increased sensitivity to estrogen has been described in hyperplastic lesions of the human breast (13), supporting this notion. In addition, the ER could be activated through other routes than binding to its `natural ligand' estrogen. It is now well established that the ER can be activated by a number of agents other than estrogen itself. For example, it has been shown that growth factors such as insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF) and transforming growth factor-
(TGF-
) are all capable of activating the ER (14).
This study was carried out to determine the extent of chemically-induced mammary tumor growth after ovariectomy in the rat, and to begin to elucidate the changes in the mammary tumors associated with the loss of requirement for ovarian hormones for their growth.
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Materials and methods
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Animals
Virgin SpragueDawley rats were purchased from Harlan SpragueDawley (San Diego, CA). The animals were brought to the vivarium at University of California, Santa Cruz at least 1 week prior to any experimentation. The rats were kept on a 14 h light and 10 h dark cycle, at a constant temperature (2022°C) and humidity (7678%) and with unrestricted access to food (5020 Purina Diet; PMI Nutrition, Brentwood, MO) and water. At 5060 days of age, the animals were anesthetized with a ketamine (Aveco, Fort Dodge, IO) and xylazine (Mobay, Shawnee, KA) mixture (30 mg ketamene:6 mg xylazine/kg body wt) and given a single i.p. injection of N-methyl-N-nitrosourea (MNU) at the dose of 50 mg/kg body wt. The animals were then palpated once weekly for the detection of mammary tumors. When at least one tumor had grown to 1.01.5 cm in diameter, the animals were bilaterally ovariectomized (OVX) and the regression and then regrowth of these tumors monitored using a caliper. Tumors that showed regrowth and tumors that developed at new sites after ovariectomy were collected when they had grown to 1.01.5 cm in diameter. Control animals were treated with MNU as described above, but these animals were left intact and mammary tumors collected when grown to 1.01.5 cm in diameter. For the tumor collections, the animals were anesthetized as described above and the tumors excised. All animals were killed by CO2 inhalation no later than 6 months after the MNU injection, quickly decapitated and whole blood collected from the trunk by drainage. The blood was centrifuged at 1000 g for 20 min, the serum was harvested, stored at 80°C and later used for measuring circulating concentrations of various hormones. All the remaining mammary tumors were also collected.
Tissue collection
At the time of tumor collection, a sample was excised from each tumor and fixed in Tellyesniczky's fixative for regular histological preparations. Another sample was obtained and fixed in 4% paraformaldehyde for 3 h for immunocytochemistry. The remainder of the tumors were snap frozen in liquid nitrogen and stored at 80°C until they were used for extraction of 17ß-estradiol (E2), IGF-I, ER,
-lactalbumin (
-lac), total proteins and RNA.
Ribonuclease protection assays (RPAs) and northern analysis
The mammary tumors were pulverized in liquid nitrogen. Approximately 30 mg of each tumor was homogenized in guanidine isothiocyanate using QIAshredder columns (Qiagen, Valencia, CA) and the RNeasy kit (Qiagen) was used to isolate total RNA. Total RNA was then used for both northern analysis and RPAs. Ribonuclease protection assays were carried out using the RPA II kit (Ambion, Austin, TX) according to the instructions provided by the manufacturer. Briefly, specific antisense riboprobes (ERß, PR, IGF-IR, EGFR, ErbB-2, pS2 and ß-actin, all of rat origin) were generated by in vitro transcription, gel purified and hybridized to 20 mg total RNA. The template for generating ß-actin (internal standard) was obtained from Ambion. Hybridization was carried out overnight at 45°C followed by incubation with an RNase-A/T1 mixture for 30 min at 37°C. The RNases were then inactivated and the protected RNA fragments precipitated. RNA hybrids were denatured for 5 min at 80°C in loading buffer and protected fragments identified on 5% acrylamide8 M urea gels. The gels were vacuum dried and RNA probe corresponding to the protected mRNA fragments quantitated by phosphor imaging (PhosphorImager; Molecular Dynamics, Sunnyvale, CA). Total RNA from rat tissue known to have a high or low level of the mRNA of interest were used, respectively, as positive and negative controls in the assays. The integrity of the probe was determined by omitting the RNase digestion reaction. To confirm that the probe was present in excess concentration in the reaction mixture, the amount of sample RNA was doubled, which doubled the intensity of the signal.
For northern analyses, the RNA was size-fractionated on a 1.0% agarose/6% formaldehyde gel and transferred onto a Magnacharge nylon membrane (MSI, Westborough, MA). The RNA was then cross-linked to the membrane by UV irradiation and then hybridized to cDNA probes detecting P450 aromatase (P450arom), IGF-I, IGF-II, ER
and the L7 mRNAs (internal standard).
Probes for RPAs
A fragment of the rat PR cDNA cloned into pGEM 4Z (Promega, Madison, WI), approximately encoding for amino acids 745927 of PR, was linearized with StuI, a unique site within this PR cDNA fragment. In vitro transcription of this linearized template from the T7 RNA polymerase site generated an antisense riboprobe of 270 nucleotides that was used in RPA.
For the EGFR RPA, a riboprobe was generated from an approximately 344 nucleotide PCR product corresponding to nucleotides 30903434 of the rat EGFR coding region (15). The PCR product was cloned into pT7 Blue vector (Novagen, Madison, WI) and transcribed in vitro with T7 RNA polymerase.
Plasmid containing a fragment of the IGF-IR cDNA suitable for generating a RNA probe was transcribed in vitro as previously described (16).
To generate an ErbB-2 probe, an approximately 1200 nucleotide fragment of the original rat ErbB-2 (neu) cDNA clone (17) was subcloned into pBluescript II KS (Stratagene, La Jolla, CA) by restriction digestion with XbaI followed by ligation. This subclone spans from nucleotides 2337 to 3534 of the original ErbB-2 cDNA clone (17). After determining the orientation of this fragment, it was digested with BglII to yield a 284 nucleotide riboprobe when in vitro transcribed with T3 RNA polymerase.
The RNA probe for ERß was constructed by first using SacI to excise an approximately 600 nucleotide fragment from the full-length rat ERß cDNA (18) spanning from nucleotides 1246 to 1851. This fragment was subcloned into pBluescript II KS and when linearized with MscI and transcribed in vitro with T3 RNA polymerase, generated a 200 nucleotide riboprobe.
A probe for pS2 was generated from a 292 nucleotide PCR product cloned into the pCR II vector (Invitrogen, Carlsbad, CA). When this plasmid was linearized with BamHI and transcribed in vitro with T7, a 292 nucleotide riboprobe was generated.
Probes for northern blot analysis
The full-length rat IGF-I cDNA (19) was used as a template for generating 32P-labeled DNA probe for northern analysis. A probe for the northern analysis of P450arom was generated using an approximately 1500 nucleotide fragment of the mouse P450arom cDNA as a template. This fragment contains most of the coding sequence of mouse P450arom (20). The IGF-II mRNA was hybridized to a probe generated from exon 4 of the rat IGF-II gene as the template. A mouse ER
cDNA clone of 1884 nucleotides, constituting the entire coding region of ER
(21) was used as a template for generating radioactive probe to detect ER
mRNA. The mRNA of the ribosomal protein gene L7 probed with 32P-labeled human L7 DNA (22) was used as an internal standard for all the northern analyses. Rediprime II/Randomprime labeling system (Amersham Pharmacia Biotech, Piscataway, NJ) was used for generating all the 32P-labeled probes in the northern analyses.
Hormone assays
The serum concentrations of prolactin (PRL) and growth hormone (GH) were measured by radioimmunoassay (RIA) using reagents obtained from the Hormone Distribution Program NIDDK. E2 concentrations in serum and tumor extracts and progesterone (P4) in the circulation were measured using RIA kits (Diagnostic Products, Los Angeles, CA) that had been modified for the use in rats (23). IGF-I concentrations, both in the circulation and in mammary tumor extracts, were assessed by RIA kit from Diagnostic Systems (Webster, TX).
Protein and DNA assays
The protein concentrations of extracted mammary tumor samples were measured by BCA Protein Assay kit (Pierce, Rockford, IL) using BSA as a reference standard. The total DNA content of the tumor homogenates was assessed by fluorometric DNA assay (24) using calf thymus DNA as reference standards. Immunoblotting of cyclin D1, IRS-1, IRS-2 and IGF-IR was carried out as previously described, as was the assessment of phosphorylation levels for extracellular signal-regulated kinase (ERK)-1 and ERK-2 (25).
Immunocytochemistry
Estrogen receptor-
was localized in the mammary tumors using immunocytochemistry. Briefly, after paraformaldehyde fixation, the tissues were imbedded in paraffin and sectioned to 5 mm. The sections were deparaffinized, rehydrated and then treated with 2% glycine and 0.3% hydrogen peroxide to block endogenous aldehydes and peroxidase activity, respectively. Non-specific binding was blocked by incubating the tissues with 2% normal goat serum and 3% dried fat-free milk in PBS. The tissue sections were incubated overnight in antiserum to ER
(MC-20; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:250. After extensive rinsing, the slides were incubated in secondary antibody followed by treatment with the ABC reagent and diaminobenzidine (Vector Laboratories, Burlkingame, CA).
Mammary gland differentiation
The content of
-lactalbumin (
-lac) in the tumors was used as an indicator of differentiation of the mammary cancer cells. The tissues were ground to a fine powder in liquid nitrogen with pestle and mortar and then homogenized on ice with a Polytron homogenizer in 2 vol (w/v) 50 mM TrisHCl, 5 mM MgCl2 buffer, pH 7.5, containing 1 mM Pefabloc (Roche, Indianapolis, IN) and 1 mM pepstatin A (Sigma, St Louis, MO). When the tissues had been homogenized, a 20 µl sample was obtained and used for measuring the total DNA content of the preparation, but the remainder of the samples were extracted for 1 h and then centrifuged at 20 000 g for 30 min, both steps carried out at 4°C. The supernatant was collected and used to measure total protein and
-lac concentration. An RIA, specific for rat
-lac, was developed. Rat
-lac was purified from rat milk according to a previously established method (24). Antiserum generated against rat
-lac was generously provided by Dr Kurt E.Ebner at the University of Kansas Medical Center. The within and between coefficient variation of the assay was 3.0 and 18.4%, respectively.
Extraction of mammary tumors for measuring E2, ER and IGF-I content
17ß-Estradiol was extracted from the mammary tumor according to a previously established procedure (26). Briefly, the tissue was pulverized in liquid nitrogen as described above. Subsequent steps were carried out at 04°C. The pulverized tissue was homogenized in 4 vol (w/v) Tris buffer (10 mM TrisHCl, 1 mM EDTA, 3 mM NaN3, pH 7.4) with a Polytron homogenizer. The homogenate was centrifuged at 100 000 g for 30 min and both the supernatant (cytosol) and pellet (crude nuclear fraction) kept for the assessment of E2 content. The cytosol was extracted in 5 vol (v/v) anhydrous ethyl ether, the ether was poured off after freezing the aqueous phase, evaporated and the precipitate reconstituted in RIA buffer and assayed for E2. The nuclear fraction was resuspended in 10 vol (w/v) 100% ethanol, vortexed and centrifuged at 3000 g for 10 min and the supernatant collected. The pellet was washed once with 10 vol (w/v) 100% ethanol and the supernatants combined and the ethanol evaporated. The precipitate was resuspended in Tris buffer and extracted in ether. After separation of the aqueous and organic phase, the ether was evaporated, the precipitate reconstituted in RIA buffer and assayed.
For measuring the IGF-I content in the mammary tumors, tissues were pulverized as described above and then extracted as previously reported (27). Briefly, the pulverized tissue was homogenized in 5 vol (w/v) 1 M acetic acid. The homogenate was extracted for 4 h on ice and mixed several times during the extraction period followed by centrifugation at 600 g for 10 min. The supernatant was collected and the pellet washed once in acetic acid, centrifuged and the two supernatants combined, aliquoted into appropriate portions for the assay, lyophilized and stored at 20°C until assayed. Samples were resuspended in RIA buffer prior to assay.
The concentrations of ER in the mammary tumors were measured as described before (23), using the Abbott ER-EIA assay (Abbott, North Chicago, IL).
Statistics
Differences in the concentrations of the various hormones in serum and the different end-points measured in the mammary tumors were analyzed by ANOVA using Fisher's Protected Least Significant Difference test. Differences between groups were considered significant when a value of P < 0.05 was obtained.
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Results
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Tumor incidence
The tumor incidence in all the animals injected with MNU was 100% and the average latency period was 56.3 days. After ovariectomy, 93% of the mammary tumors showed >50% reduction in size and 76% of the tumors could not be detected by palpation after on average 23.25 ± 1.85 days (mean ± SEM). However, in 96% of the animals where tumor reduction or disappearance occurred, the mammary tumors showed re-growth or new tumors appeared where no tumors were palpable before. At least one re-growth (tumor developed at a location where tumors had been detected before ovariectomy) was detected in 61% of the animals after ovariectomy and 78% of the OVX rats developed at least one tumor at a new location. Of all the tumors that developed after ovariectomy, 36% were re-growths but 64% developed at new locations. The average latency for re-growths and new mammary tumor development after ovariectomy was 52.8 days (Table I
).
Histological characterization of mammary tumors
Histological examination of all the mammary tumors revealed that 100% of tumors that developed in the intact, control animals were carcinomas (ductal carcinomas, adenocarcinomas, papillary carcinomas). Of the tumors that grew after ovariectomy, 93% were carcinomas (ductal carcinomas, adenocarcinomas, papillary carcinomas) and 7% fibroadenomas. A proportion of the cancers that developed in the OVX rats appeared to be of a more aggressive type then those in the intact rats, as evident from their invasion into adjacent muscles (Figure 1
).

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Fig. 1. Photomicrograph of a mammary tumors from OVX rat. The tissue was prepared as described in Materials and methods and stained with hematoxylin and eosin. Note the invasive characteristics of the epithelial cancer into the adjacent muscle tissue.
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Hormonal concentration in serum of intact and OVX rats
The circulating concentrations of E2 in the intact rats was 7.3 ± 1.4 pg/ml (mean ± SEM) but E2 was undetectable in all (n = 28) of the OVX rats (<0.67 pg/ml) with the exception of five animals where E2 was measurable. These five animals were excluded from the study as they may have been incompletely ovariectomized. As shown in Table II
, the concentrations of GH, PRL, P4 and IGF-I were all significantly reduced in the OVX animals.
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Table II. Hormone concentrations (mean ± SEM) in serum of intact rats (n = 20) and ovariectomized (OVX) rats (n = 23)
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Measurement of different end-points in mammary cancers from intact and OVX rats
State of differentiation of mammary tumors
The level of differentiation of the mammary tumors was assessed by their content of
-lac. Some
-lac synthesis was detected in all the tumors from both intact and OVX rats. However,
-lac was significantly higher in tumors from intact animals as compared with OVX rats (Figure 2
), indicating a loss of differentiation as the tumors progress from a hormone-dependent to hormone-independent state.

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Fig. 2. The -lactalbumin content of the mammary tumors of intact and OVX rats measured with a specific RIA as described in the Materials and methods. Each bar represents the mean ± SEM, n = 1215. *Significantly lower than that in tumors from intact rats (P < 0.05).
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E2 content and P450arom expression in mammary cancer extracts
As shown in Table III
, E2 was detectable in only 36% of tumors from OVX rats and only in the nuclear fraction of these tumors, but was undetectable in the cytosol of tumors from OVX rats. In the intact animals, E2 was detected in 100% of the tumors and was present in both the cytosol and the nuclear fractions, although the nuclei contained significantly higher levels than the cytosol. The nuclear fraction from the tumors of intact rats contained on average 3-fold higher E2 levels then those tumors of OVX animals where E2 was detectable (Table III
). Despite the significant reduction in the E2 content of tumors of OVX rats, these tumors showed significant increase in their capacity of E2 synthesis, as assessed by their increase in P450arom expression, when compared with tumors from intact rats (Figure 3
).
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Table III. The content of epidermal growth factor receptor (EGFR), ErbB-2, insulin-like growth factor-I (IGF-I) and 17ß-estradiol (E2) in mammary tumors from intact and ovariectomized rats
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Fig. 3. Northern blot analysis showing the expression of P450 aromatase (P450arom) in mammary tumors from intact and ovariectomized rats. (A) Phosphor images of P450arom and L7 mRNAs and (B) the analyzed data. Each bar represents the mean ± SEM of six observations. *P450arom significantly higher than that in tumors of intact rats.
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Expression of ER
, ERß, PR and pS2 in mammary tumors
The ER
protein did not appear to differ significantly between tumors from intact and OVX animals when measured by the specific immunoassay (Figure 4A
). These results were further confirmed with immunostaining where no obvious difference was seen in the ER
staining between the two groups (Figure 4B
). However, there was a significant reduction in ER
mRNA levels in tumors from OVX animals as compared with intact rats (Figure 4C
). The mRNA levels for PR was also measured in tumors from the two animal groups. There a significant reduction was seen in PR mRNA levels of the tumors from the OVX rats (Figure 5
). No expression of either ERß or pS2 was detectable in any of the mammary tumors from either intact or OVX rats (data not shown).

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Fig. 5. Ribonuclease protection assay measuring the progesterone receptor (PR) expression in mammary tumors from intact and ovariectomized rats. (A) Phosphor images of the PR and ß-actin mRNAs and (B) the analyzed data. Each bar represents the mean ± SEM of six observations. *PR significantly lower than in tumors from intact rats.
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Expression of IGF-I, IGF-II, IGF-IR, IRS-1 and IRS-2 in mammary tumors
Although the circulating levels of IGF-I was significantly reduced in the OVX animals, there was no significant difference in the IGF-I content of the mammary tumors obtained from intact and OVX rats. Similarly, the levels of IGF-I mRNA were not different in mammary tumors obtained from intact and OVX rats (Table III
).
The expression of IGF-IR was measured both by western blotting and RPA. IRS-1 and IRS-2 were assessed by western blotting, and northern blotting was used to assess the expression of IGF-II. These four genes were expressed at a similar level in mammary tumors from both intact and OVX rats when they were detectable (data not shown). However, not all the tumors that were examined did express all four genes. IGF-II expression was the most erratic and expression was detectable only in ~2030% of the tumors from both groups. IGF-IR was expressed in all tumors from the intact animals, whereas only 50% of tumors from OVX animals expressed IGF-IR. IRS-1 and IRS-2 were expressed at similar levels in tumors from both groups of animals (data not shown).
EGFR and ErbB-2
Some EGFR expression was found in all tumors from both intact and OVX rats and there was no significant difference in the level of expression between the two groups (Table III
). ErbB-2 expression, on the other hand, was found in 70% of tumors from OVX rats, whereas 100% of the tumors from intact rats expressed ErbB-2. However, in tumors from the OVX animals that expressed ErbB-2, the levels were not different from those of intact rats (Table III
).
Mitogen-activated protein kinase (MAP) kinase phosphorylation and cyclin D1 levels
Phosphorylation of the MAP kinase ERK1 and ERK2 was not detectable in mammary tumors from the intact rats. However, substantial phosphorylation of both forms was found in tumors from the OVX animals (Figure 6
). Conversely, cyclin D1 levels were substantially reduced in tumors from the OVX rats as compared with tumors from the intact animals (Figure 7
).

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Fig. 6. Western blot analysis showing the phosphorylation of the extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2). Note the absence of phosphorylated ERK1 and ERK2 in tumors from intact rats.
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Fig. 7. Western blot analysis detecting the levels of cyclin D1 in tumor extracts from intact and ovariectomized rats. Note the reduced levels of cyclin D1 in tumors from ovariectomized rats.
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Discussion
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There is little doubt that the initiation of most if not all mammary tumors is dependent on hormonal stimulation. However, after the initial mutations are fixed, the tumorous cells may or may not be dependent on hormones for progression, giving rise, respectively, to hormone-dependent or hormone-independent tumors (1). Breast cancers that respond to hormonal therapy at the time of detection will almost inevitably progress to develop a hormone-independent phenotype (4). The changes occurring as mammary tumors progress from a hormone-dependent to hormone-independent state are not well characterized. We report here the development of an animal model to investigate the progression of mammary tumors from a hormone-dependent to hormone-independent state. Similar to previous observations (7), we found that MNU-induced rat mammary cancers regress rapidly (on average within approximately 23 days) after ovariectomy. However, quite a large percentage of these tumors show renewed growth independent of circulating hormones after a relatively short period (on average 52.8 days) from removal of the ovaries, and a number of tumors developed where no palpable tumors were detectable before ovariectomy. How do tumors initially dependent on hormones for their growth rapidly acquire hormonal independence? This is not known, but it is known that tumors that progress from a hormone-dependent to hormone-independent state maintain some of their characteristics in terms of their gene expression. For example, human breast cancers that develop resistance to antiestrogen therapy after initially responding to treatment continue to express the ER (8) and genes regulated by the ER, such as the PR and pS2 genes, are also expressed after resistance to antiestrogen has been developed (8,9). In the study reported here, we found that ER levels were largely maintained in mammary tumors that developed after ovariectomy of the animals. To assess if the ER was functional in OVX animals, we also measured the expression of genes known to be regulated by ER, such as PR, IRS-1 and IRS-2. All the mammary tumors from the OVX rats showed some expression of the PR. However, this PR expression was significantly lower than that in tumors from intact animals. In contrast, the expressions of both IRS-1 and IRS-2 were maintained in tumors from OVX animals as compared with tumors of intact rats. These are noteworthy findings as the expression of both these genes is known to be stimulated by estrogen (25). Taken together, these results show that at least some ER activity is maintained in these mammary tumors after ovariectomy. None of the OVX animals had detectable circulating levels of E2. This could indicate a local synthesis of E2 in the tumors themselves, as is frequently seen in human breast cancers (11). Indeed, we found a significant increase of P450arom expression in tumors from OVX rats as compared with tumors from intact animals, indicating an increase in the local activity of P450arom in the absence of the ovaries. However, this increase in P450arom expression was not sufficient to maintain E2 levels in the tumors of OVX rats similar to that found in intact animals.
Alternatively, the ER could be activated through means other than binding to estrogen. It is now well established that growth factors are capable of activating the ER (14,28). Of particular interest in this context are the activities of IGF-I, IGF-II, EGF and TGF-
, all of which growth factors normally expressed in the mammary gland (29,30). They are all frequently expressed at high levels in mammary cancer (31,32), and have been all shown to activate the ER. It is also known that the growth factor activation of the ER requires growth factor/growth factor receptor binding that results in the activation of their signal transduction cascade, ultimately leading to the phosphorylation of the ER and stimulation of gene expression (reviewed in refs 14 and 28).
We found here that the expression of IGF-I was maintained at high levels in the mammary tumors after ovariectomy of the animals. Furthermore, IGF-I protein content in mammary carcinomas of intact animals was high and remained the same or slightly elevated after ovariectomy, even though circulating levels of IGF-I were significantly reduced in OVX rats, as compared with intact animals. Similarly, the mRNA of both EGFR and ErbB-2 were kept at similar levels in tumors of intact and OVX rats.
In summary, these results may indicate that the tumors from OVX rats still rely, to some extent, on the ER for their growth. Although the tumors may be dependent on the ER and an increase in the activity of locally synthesized growth factors to maintain their growth shortly after ovariectomy, they will most likely later on develop a more aggressive phenotype that is without any dependence on the ER, as is the case with human breast cancers (4) and the dependence on local growth factors may also change (33). Indeed, we obtained results that certainly indicate that these tumors are progressing and acquiring of ovarian independence of ER activation. Most significantly, the expression of the PR was greatly reduced in tumors from OVX rats demonstrating a reduction in the ER activity. We also found a significant increase in MAP kinase activity (phosphorylation) in the tumors of OVX rats, whereas phosphorylation of MAP kinase was undetectable in the tumors of intact animals. The MAP kinase system is widely used in the signal transduction cascade initiated by many extracellular stimulireceptor interactions and is closely associated with regulation of cell growth (34). For example, both IGF-I and EGF/TGF-
rely on MAP kinase for many of their biological functions (35,36). This could be a very important step in the acquiring of ovarian independence by tumors, as an increase in MAP kinase activity is associated with tumor progression. It has, for example, been shown that overexpression of MAP kinase in fibroblasts causes rapid tumor formation after transplantation of the cells into nude mice (37) and, more importantly, loss of estrogen-dependency for growth is associated with an increase in MAP kinase activation in breast cancer cells (38). Furthermore, primary breast cancers and breast cancers metastasized to the lymph node express elevated levels of MAP kinase as compared with benign and normal breast tissues, where MAP kinase expression was frequently very low or not detectable (39). We do not know what caused the increase in MAP kinase activity observed in the rat mammary tumors after ovariectomy. It may indicate an increase in growth factor activity within the tumors, increased sensitivity to external stimuli or acquisition of constitutive activation. Clearly more work is needed to elucidate how MAP kinase activation is increased after ovariectomy and the importance of this activation for the development of hormone-independent growth.
Another interesting observation associated with tumor growth after ovariectomy was the substantial reduction in the expression of cyclin D1 as compared with the expression levels in tumors from intact animals. It is known that cyclin D1 is important for normal mammary gland development, as cyclin D1 knockout mice have impaired mammary gland growth and differentiation (40,41). The role of cyclin D1 in mammary carcinogenesis is complex and its role there not fully understood. Cyclin D1 is frequently overexpressed in breast cancer. However, this overexpression is associated with ER-positive cancers with good prognosis. On the other hand, ER-negative or antiestrogen resistant breast cancers with poor prognosis show low levels of cyclin D1 expression (42). Our results here corroborate, therefore, with these clinical findings in that high cyclin D1 was found in ovarian-dependent mammary tumors of intact rats, whereas cyclin D1 expression was greatly reduced in the tumors from OVX rats, an additional indication of tumor progression. The significance of this finding is not known. The reduced activity of the ER in OVX rats may have caused a reduction in cyclin D1 expression, as estrogen is an important stimulator of cyclin D1 (43). Perhaps more importantly, this reduced level of cyclin D1 after ovariectomy may be an indication of a reduction in the expression of the retinoblastoma susceptibility gene (RB1), because cyclin D1 expression is directly stimulated by pRB (44). Therefore, the low level of cyclin D1 in tumors of OVX rats could indicate a disturbance in the regulation of genes essential for the progression of the cell through the cell cycle (45). More work is obviously needed here to determine the significance of this reduction in cyclin D1 associated with hormone-independent growth of the tumors.
Further evidence for a more aggressive phenotype associated with tumors from OVX rats was indicated by the finding that these tumors showed a very significant reduction in differentiation and increased evidence of invasive behavior, characteristics closely associated with more aggressive phenotype of mammary cancers (4648). Still another indication of cancer progression in OVX rats was the fact that tumors from these animals did not show consistent expression of some of the growth factor systems. In particular, IGF-IR was expressed only in 50% of tumors from OVX rats, but was expressed without exception in tumors from intact rats. However, when IGF-IR was expressed in the tumors of OVX animals, the levels were comparable in both groups. This loss of IGF-IR expression in some of the tumors of OVX rats may indicate the development of a more aggressive phenotype, as low expression of this gene has been associated with progressing mammary cancers. For example, ER-negative human breast cancer cell lines that show a high degree of metastasizing behavior express low levels of IGF-IR (49,50).
In summary, MNU-induced rat mammary cancers regress rapidly after ovariectomy of the host. However, with prolonged time after ovariectomy, quite a high percentage of these tumors show renewed growth and new tumors develop where no tumors were detectable before. The tumors of OVX rats still expressed high levels of the ER and may, at least initially, rely on ER activation for growth. However, changes in gene expression indicating cancer progression and loss of ER dependency were also evident in cancers from OVX rats.
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Notes
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3 To whom correspondence should be addressedEmail: gummi{at}biology.ucsc.edu 
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Acknowledgments
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We thank Nevada Wilson for excellent assistance with palpating the animals, measuring tumors and collecting tissues. We are also indebted to the following people for their generosity in providing different reagents used in this study: Dr Steven L.Carroll for the EGFR probe, Dr Kurt E.Ebner for the antiserum to rat
-lactalbumin, Dr Jan-Ake Gustafsson for the ERß probe, Dr Derek LeRoith for providing us with the IGF-IR probe, Dr Kelly E.Mayo for the PR probe, Dr Masami Muramatsu for supplying us with the rat ER
probe, Dr Fukumi Nakamura for the pS2 probe, Dr Peter S. Rotwein for probes to measure rat IGF-I and IGF-II, Dr Yutaka Shizuta for providing the AromP450 probe and Dr Robert A.Weinberg for the ErbB-2 probe. We also thank the NIDDK Hormone Distribution Program for supplying the RIA reagents for measuring PRL and GH. This study was supported by USPHS grants CA-71590 and CA-72598 awarded by the National Cancer Institutes. A.V.L. is a recipient of a Susan G.Komen Breast Cancer Foundation Award.
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Received July 12, 2001;
revised August 29, 2001;
accepted September 14, 2001.