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Endocrinology Vol. 148, No. 10 4536-4544
Copyright © 2007 by The Endocrine Society

Advanced Rat Mammary Cancers Are Growth Hormone Dependent

Qi Shen, Daniel D. Lantvit, Qing Lin, Yongjun Li, Konstantin Christov, Zhuohua Wang, Terry G. Unterman, Rajendra G. Mehta and Steven M. Swanson

Departments of Medicinal Chemistry and Pharmacognosy (Q.S., D.D.L., Q.L., Y.L., Z.W., S.M.S.), Surgical Oncology (K.C., S.M.S.), and Medicine (T.G.U.), University of Illinois at Chicago, and Department of Veterans Affairs Jesse Brown Medical Center (T.G.U.), Chicago, Illinois 60612; and Illinois Institute of Technology Research Institute (R.G.M.), Chicago, Illinois 60616

Address all correspondence and requests for reprints to: Steven M. Swanson, Ph.D., Department of Medicinal Chemistry and Pharmacognosy, 833 South Wood Street (MC 781), University of Illinois at Chicago, Chicago, Illinois 60612. E-mail: swanson{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Epidemiological studies suggest that the GH/IGF-I axis may promote human cancers. Animal models in which the GH/IGF-I axis can be controlled may be helpful in elucidating the role of these hormones during mammary cancer progression. Beginning at 3 or 5 wk of age, spontaneous dwarf rats (Ghdr/dr), which lack GH and have very low serum IGF-I, were treated with either rat or bovine GH twice daily. Other Ghdr/dr rats received vehicle, and wild-type Sprague Dawley rats (Gh+/+, parent strain to SDR) received vehicle. One week later, all rats were exposed to a single injection of N-methyl-N-nitrosourea. Body weight gain and serum IGF-I levels were similar in Gh+/+ and GH-treated Ghdr/dr rats. Furthermore, mammary tumor incidence, latency, and multiplicity were similar in Gh+/+ and GH-treated Ghdr/dr rats. Vehicle-treated Ghdr/dr rats developed no tumors. Once advanced (≥1 cm3) mammary cancers were established in GH-treated Ghdr/dr rats, GH treatments were halted and nearly all tumors regressed completely within 2 wk. Tumor regression was associated with loss of phospho-signal transducer and activator of transcription-3, but not alterations in IGF-I, IGF-I receptor, or GH receptor. These results demonstrate that Ghdr/dr rats, which are nearly refractory to mammary carcinogenesis, can be made vulnerable by restoring GH and IGF-I. Furthermore, advanced rat mammary cancers are dependent on GH and/or IGF-I for their survival. Therefore, therapeutics that target either GH or IGF-I may be effective at treating even advanced mammary cancers.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AN EARLY FULL-TERM PREGNANCY affords women a significant (up to 2-fold) reduction in lifetime risk for developing breast cancer. The protective effect of early pregnancy is also observed in rats (1, 2). We previously compared the serum titers of GH, prolactin, estradiol, progesterone, corticosterone, and thyroid hormone in parous and age-matched virgin rats and found that only GH was significantly altered (decreased) in parous rats, compared with age-matched virgin animals (2). These findings and laboratory and clinical results from others, summarized below, prompted us to study the role of GH in rat mammary carcinogenesis.

The IGFs, their receptors, and binding proteins have been observed during every stage of normal mammary gland development in all mammalian species examined to date (3). GH binds to GH receptors in the stromal and epithelial compartment of the mammary gland and stimulates IGF-I mRNA expression. Kleinberg (4, 5) has shown that GH acts through locally produced IGF-I, which causes development of terminal end buds, the structures that lead the process of mammary gland development during puberty. In many species including primates, GH plays an important role in normal mammary gland growth and development by supporting duct elongation (6) and alveolar development (7, 8, 9, 10). Also, the presence of GH receptors on mammary epithelial cells suggests that in addition to inducing IGF-I, GH may also have direct effects on mammary tissue (11).

The GH/IGF axis is also implicated in the development of mammary cancer. Epidemiological studies suggest a positive association between elevated serum titers of IGF-I and an increased risk of breast (12) cancer and other deadly forms of cancer (13). Animal studies with genetically engineered mice corroborate the evidence in man. For example, Wu et al. (14) have shown that the liver-specific IGF-I gene-deleted mouse develops mammary cancers at a significantly reduced incidence and latency than control mice when either exposed to chemical carcinogen [7,12-dimethylbenz(a)anthracene, DMBA] or crossed with the C3(1)/T antigen (TAg) mouse. IGF-I is a potent mitogen that promotes proliferation by stimulating cell cycle progression and can suppress apoptosis, including cell death induced by anticancer chemotherapeutic agents. Inhibition of IGF-I causes inhibition of cell proliferation when human mammary cancer cells are propagated either in vitro (15) or as tumor xenografts (16).

GH, the main regulator of IGF-I production, may also be an important determinant of cancer incidence (17, 18, 19). Data are emerging that GH may play a critical role in neoplastic transformation. Lobie and colleagues have shown that overexpression of human GH in the nontumorigenic mammary epithelial cell line MCF-10A transformed these cells into tumorigenic cells. When GH expression was driven by the metallothionein 1a promoter, the cells exhibited increased proliferation, decreased apoptosis, altered cellular morphology and anchorage-independent growth and were able to form tumors in immunocompromised mice (20). Furthermore, these researchers have shown that when GH is overexpressed in MCF-7 cells, these cells assume a much more aggressive phenotype including increased cell motility, elevated activity of specific matrix metalloproteinases, and an acquired ability to invade a reconstituted basement membrane (21). The GH receptor has been identified on tumor specimens and cultured human cancer cells (22, 23). Studying biopsies from cancer patients, Mertani et al. (24) have shown that GH receptors are increased in neoplastic mammary tissue, especially in areas in which tumor invasion was evident, relative to normal breast tissue.

Our laboratories have shown that GH signaling is important in rodent prostate (25) and mammary (26) carcinogenesis. We crossed the GH receptor (GHR) gene-disrupted mouse (Ghr–/–) with the C3(1)/TAg mouse, in which males develop prostatic intraepithelial neoplasia and females develop mammary carcinomas driven by the large TAg. In both sexes, carcinogenesis is known to progress to invasive prostate carcinoma in a manner similar to the process observed in humans (27). Progeny of this cross were genotyped and TAg/Ghr+/+ and TAg/Ghr–/– mice were compared. We observed that in both systems (prostate and mammary), carcinogenesis was significantly slowed in animals harboring TAg but lacking GHR (TAg/Ghr–/–), compared with TAg mice with wild-type GHR (TAg/Ghr+/+).

We have also shown that the spontaneous dwarf rat (SDRs), which lack GH due to a point mutation in the GH gene (Gh), is resistant to chemically induced mammary carcinogenesis (28). This model is significant because the SDR differs from the Sprague Dawley by only this single-point mutation. The Sprague Dawley rat exposed to N-methyl-N-nitrosourea (MNU) (29) is one of the most commonly used, thoroughly characterized models for human breast cancer, particularly with regard to the ability of hormones to regulate tumor growth (30, 31) and is also considered to be an excellent model of human breast cancer (31). The purpose of the current study was to determine whether advanced mammary tumors in this model were dependent on GH for their survival. Here we report that the SDR can be made vulnerable to MNU-induced mammary carcinogenesis by treatment with GH and that once mammary tumors were established, cessation of GH treatments induced rapid and dramatic mammary tumor regression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All experiments involving animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and under a protocol approved by the Institutional Animal Care and Use Committee of the University of Illinois at Chicago. The SDR arose in a colony of Sprague Dawley rats at Morishita Pharmaceutical Co. (Shiga, Japan) in 1977 (32). The phenotype is due to a nonsense point mutation designated dr that results in an unstable mRNA transcript roughly half the size of the normal GH mRNA transcript (33). Pituitary GH mRNA is only 3–6% of control and GH protein is undetectable in serum of the SDR (34), which displays postnatal growth retardation and proportionate dwarfism (35). Also, serum IGF-I titers are only about 20% of control Sprague Dawley rats. No other abnormalities are evident in the SDR; their behavior is indistinguishable from that of their wild-type littermates, and lactation in SDR is adequate to feed their young. Because the dr mutation effectively results in a null mutation in Gh, the symbol Ghdr/dr is used here to represent the Ghdr/dr mutation. Also, because the SDRs and Sprague Dawley rats differ only in the dr mutation, wild-type rats and SDRs are designated Gh+/+ and Ghdr/dr, respectively, in this manuscript. The SDR is not commercially available and were bred at the Biologic Resources Laboratory, University of Illinois at Chicago. Wild-type Sprague-Dawley rats were purchased from Harlan (Indianapolis, IN). All rats were fed a Teklad 8640 diet (Harlan Teklad, Madison, WI), provided water ad libitum and housed in a temperature- and humidity-controlled environment with a regular light-dark illumination cycle. All animals were weighed and palpated weekly. Mammary tumor size and tumor location for each rat were recorded throughout each study. Tumor measurements were made with a digital caliper and tumor volume was calculated (36) using the following formula: tumor volume = ({pi}/6) x (larger diameter) x (smaller diameter)2. All animals were killed by CO2 asphyxiation in accordance with guidelines set forth by the American Medical Veterinary Association (37).

GH
Recombinant rat GH was purchased from the National Hormone and Peptide Program (Torrance, CA) and dissolved in 10 mN NaOH and administered twice daily (at approximately 0900 and 1800 h every day) at a dose of 1.25 mg/kg per injection. Bovine GH was kindly provided by Dr. Gregg Bogosian (Monsanto Co., St. Louis, MO). The material used for the current studies was the zinc salt of recombinant bovine GH and not Posilac, which is a pegylated form of bovine GH specifically formulated for use in the dairy industry. The GH was dissolved in 10 mM NaHCO3, 20 mM NaOH, and 1 mM EDTA (pH 12). Rats were injected twice daily as above with buffer or 150 µg GH. We estimate that the bovine GH dose ranged from about 6 mg/kg per injection at the start of the experiment to 0.6 mg/kg per injection by the end of the 3-month injection period.

Serum IGF-I
Blood was collected for analysis of serum IGF-I 1, 2 or 4 wk after initiation of hormone or vehicle treatments and at the time the animals were killed. Blood was collected from the retroorbital plexus (38) during the studies and from trunk blood at the time the animals were killed. The blood samples were centrifuged at 1500 x g for 20 min, and serum was harvested and stored at –80 C until analysis by RIA using a commercially available kit (Nichols Institute Diagnostics, San Clemente, CA).

Immunohistochemistry
Portions of each tumor were fixed in 10% neutral-buffered formalin and embedded in paraffin. Four-micrometer sections were stained with hematoxylin and eosin for diagnosis of malignancy by a pathologist experienced in rodent mammary carcinogenesis (K.C.). For immunohistochemistry the sections were dewaxed in xylene and rehydrated through graded ethanol. For antigen retrieval, the sections were boiled in 10 mM citrate buffer (pH 6.0) for 10 min and allowed to cool to room temperature for 20 min. For analysis with antiphospho-signal transducer and activator of transcription (STAT)5a/b AX1 (Advantex BioReagents, Conroe, TX), the sections were heated in a pressure cooker with antigen retrieval solution AXAR1 (Advantex BioReagents) and allowed to cool to room temperature for 20 min. Endogenous peroxidase activity was quenched by incubating for 30 min in 3% H2O2 followed by several water rinses. Nonspecific binding was blocked by incubation with goat serum (for antirabbit antibodies) or horse serum (for antimouse antibodies). Sections were then incubated for 2 h with rabbit anti-Ki67 (Abcam, Cambridge, MA; 1:100) and mouse monoclonal antibody antiphosphoSTAT5a/b AX1 (1:1500). Primary antibody was detected using Vectastain Elite ABC kit (supplied with rabbit IgG) according to the manufacturer’s specifications (Vector Laboratories, Burlingame, CA). The signal was developed by addition of diaminobenzidine, and the slides were counterstained with Harris hematoxylin, dehydrated, and coverslips affixed with mounting medium.

Apoptosis was assessed by the terminal deoxynucleotide-transferase-mediated deoxyuridine 5-triphosphate-digoxigenin nick end labeling (TUNEL) assay using the peroxidase in situ apoptosis detection kit from Chemicon (Temecula, CA). Slides were counterstained with methyl green for visualization of tissue morphology. To determine the percent of cells positive for p-STAT5, Ki67, or TUNEL (apoptotic index), at least 1000 cells were scored in three separate fields for each slide read. The pathologist was blinded as to the group association for each slide.

Immunoblotting
Rabbit anti-STAT3 and rabbit antiphosphoSTAT3 were purchased from Cell Signaling Technology (Danvers, MA); mouse anti-STAT5 was purchased from BD Biosciences (Franklin Lakes, NJ); rabbit anti-STAT5a and mouse anti-STAT5b were from Santa Cruz Biotechnology (Santa Cruz, CA); mouse antiphospho-STAT5 was purchased from AdvantexBio Reagents; rabbit anti-Ki67 antibody was purchased from Abcam and mouse anti-ß-actin from Sigma Chemical Co. (St. Louis, MO). Anti-GHRcyt-AL47 is a rabbit serum raised against a bacterially expressed N-terminally His-tagged fusion protein incorporating human GHR residues 271–620 as described previously. Dr. Stuart J. Frank (University of Alabama at Birmingham, Birmingham, AL) kindly provided this antibody (39).

For tissue cell lysis, the T-PER tissue protein extraction reagent (Pierce Biotechnology, Rockford, IL) was used. The reagent uses a proprietary detergent in 25 mM bicine and 150 mM sodium chloride (pH 7.6) and was supplemented with Halt protease inhibitor mixture (Pierce). Tissue debris was removed by centrifugation for 5 min at 10,000 rpm. The supernatant was saved, and protein concentrations were determined by the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA) using BSA as a reference standard.

Immunoblot was used for semiquantitative analysis of specific proteins in samples. Fifty micrograms of each protein sample were loaded per lane of each 7.5 or 10% SDS-PAGE run. After proteins had been transferred to a polyvinyl difluoride membrane, the membranes were blocked with 5% nonfat milk in Tris-buffered saline (TBS) and 0.05% Tween 20 (TBS-T) buffer (for phospho-antibodies, 5% BSA TBS-T buffer was used) for 1 h at room temperature. Mouse anti-STAT5 (1:500), mouse anti-STAT5 p-Tyr694/99 (1:1000), mouse anti-STAT3 (1:2000), or rabbit anti-STAT3 p-Tyr705 (1:1000) antibodies in blocking buffer were incubated with membranes overnight at 4 C. For anti-GHRcyt-AL47 antibody, membranes were blocked for 1 h at room temperature in 2% BSA in TBS (TBS without Tween 20) and then incubated with antibody (1:3000) in 2% BSA TBS-T buffer overnight at 4 C (40). Detection was performed with peroxidase-conjugated antimouse or antirabbit IgG (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) at a dilution 1:25,000 for 1 h at room temperature. Visualization was performed using the enhanced chemiluminescence plus chemiluminescence detection kit (GE Healthcare Bio-Sciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Because the SDR (Ghdr/dr) differs genetically from the Sprague Dawley (Gh+/+) by a point mutation in the Gh gene, the first set of experiments, summarized in Fig. 1AGo, was designed to test the hypothesis that administration of recombinant rat GH would render the SDR as vulnerable to carcinogen-induced mammary carcinogenesis as the well-characterized Sprague Dawley rat. Recombinant rat GH was administered to Ghdr/dr rats by twice-daily sc injections (1.25 mg/kg body weight) beginning at 5 wk of age. There were three groups of five rats each that received either hormone or vehicle treatments beginning at 5 wk of age and continuing for 60 d (1 month before the animals were killed). The Gh+/+ rats received vehicle and served as positive control because the Sprague Dawley rat is a well-established and thoroughly characterized model of mammary cancer (31). The second group comprised of Ghdr/dr rats was treated with recombinant rat GH. The third group of rats were Ghdr/dr rats treated with hormone vehicle and served as the negative control group because the SDR is known to be resistant to chemically induced mammary carcinogenesis (28, 41). One week after hormone or vehicle treatments began (i.e. at 6 wk of age), all rats received a single ip injection of the mammary carcinogen MNU (50 mg/kg). All animals were weighed and palpated for mammary tumors weekly until the animals were killed 3 months after carcinogen exposure.


Figure 1
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  		FIG. 1. Experimental design. For each of the two experiments outlined above, rats were divided into three groups. Group 1 consisted of Gh+/+ rats treated with hormone vehicle. Group 2 was Ghdr/dr rats treated with either recombinant rat GH (A) or recombinant bovine GH (B). Group 3 was Ghdr/dr rats treated with hormone vehicle. Vehicle or GH (A: recombinant rat, 1.25 mg/kg per injection; B: recombinant bovine, 150 µg/rat per injection) was administered at about 0900 and 1800 h every day over the course of the studies as indicated. Injections were ceased for 1 month and followed either by killing the animals (A) or resumption of injections (B). All rats received a single ip injection of MNU (50 µg/kg body weight).

 
Two classical effects of GH action include the stimulation of body growth and induction of IGF-I biosynthesis by the liver, which in turn secretes IGF-I into the circulation. These end points were used as biomarkers for the physiological relevance of the GH dose administered in the present studies. During the first month of injections, recombinant rat GH induced a similar rate of body weight gain in Ghdr/dr rats relative to the Gh+/+ rats (14.8 and 16.5 g/wk, respectively; P > 0.05 by linear regression). Also, the serum titers of IGF-I in Ghdr/dr rats (45 ± 11 ng/ml), which are normally only 10–15% of wild-type controls (456 ± 36 ng/ml), were elevated to wild-type levels by GH treatments (453 ± 21 ng/ml) when sampled 1 month after hormone of vehicle treatments began. These data indicate that the administration protocol of recombinant rat GH used in this experiment is physiologically relevant to Gh+/+ rats. Most importantly, administration of recombinant rat GH rendered the Ghdr/dr rats as vulnerable to MNU-induced mammary cancer as Gh+/+ rats (Table 1Go). By 60 d after carcinogen exposure, both Gh+/+ rats and GH-treated Ghdr/dr rats developed mammary cancer at the same incidence, median latency, and frequency. No tumors developed in the Ghdr/dr rats treated with hormone vehicle. Once mammary tumors were established, GH treatments were halted to evaluate the dependence of these tumors on GH injections. The tumors were monitored for 1 month, at which time all rats were killed. Of the 10 tumors that had arisen in the Gh+/+ rats, one tumor regressed, three did not change substantially (>25%), and six continued to grow in size. In contrast, of the 10 tumors in the Ghdr/dr rats, six regressed completely (no longer palpable), two did not change in volume, and two continued to grow.


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  		TABLE 1. GH-deficient rats can be made vulnerable to MNU-induced mammary tumorigenesis by treatment with recombinant rat GH

 
Having established that administering physiologically relevant amounts of recombinant rat GH to Ghdr/dr rats rendered them similarly susceptible to MNU-induced mammary carcinogenesis as Gh+/+ rats and that most of these tumors appear to be dependent on GH treatments, the purpose of the next experiment was to confirm these findings with bovine GH, which was far more readily available than recombinant rat GH. These experiments are summarized in Fig. 1BGo. As in the first experiment, rats were divided into three groups. The first group was wild-type Gh+/+ rats (n = 15), which received twice-daily injections of GH vehicle only (positive control group). The experimental group consisted of Ghdr/dr rats (n = 16) that received twice-daily injections of GH (150 µg/rat per injection) bovine GH in buffer beginning at 21 d of age. The negative control group was comprised of Ghdr/dr rats (n = 6) that received twice-daily injections of hormone vehicle. Twice-daily injections of hormone or vehicle continued for all rats for approximately 90 d. One week after hormone or vehicle treatments began, all rats received a single ip injection of MNU (50 mg/kg). None of the vehicle-treated Ghdr/dr rats developed any palpable mammary tumors during the study. However, by 3 months after MNU, all of the Gh+/+ rats and the GH-treated Ghdr/dr rats had developed one or more mammary tumors of approximately 1 cm3. At this point, GH treatments were stopped for 10 Ghdr/dr rats to assess the requirement of GH injections to support tumor growth. The day of the last GH injection, the histopathology of all tumors in GH-treated Ghdr/dr rats was assayed by needle biopsy. Three rats were killed while tumors were actively regressing during the first week after GH withdrawal. After 30 ± 3 d on hormone vehicle, the seven remaining rats were put back on twice-daily GH injections (150 µg/rat per injection as before) until the animals were killed 21 d later.

Whereas the Ghdr/dr rats displayed their usual slow rate of body weight gain when treated with hormone vehicle, the Ghdr/dr rats supplemented with bovine GH displayed a similar profile of body weight gain as that observed in vehicle-treated Gh+/+ rats (Fig. 2Go). Weight gain was quite sensitive to GH withdrawal: when the GH injections were stopped, the animals began losing weight at a rate similar to that observed during GH-induced weight gain. When the GH injections resumed, the animals quickly regained the weight that they had lost during the month without GH. Similar to body weight gain, the serum IGF-I levels were responsive to GH treatments. The Ghdr/dr rats had very low serum IGF-I, but the treatments of bovine GH raised the serum IGF-I titers to levels similar to levels observed in Gh+/+ rats (Fig. 3Go).


Figure 2
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  		FIG. 2. Effect of bovine GH treatment on body weight gain in GH-deficient (Ghdr/dr), carcinogen-exposed rats. Carcinogen plus either hormone or vehicle treatments were conducted for approximately 3 months as described in Fig. 1BGo. All treatments were suspended for 1 month. Finally, bovine GH or vehicle treatments were resumed for a further 3 wk in rats previously treated with bovine GH or vehicle, respectively.

 

Figure 3
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  		FIG. 3. Serum IGF-I titers in carcinogen-exposed rats. Serum from vehicle-treated wild-type Sprague Dawley (Gh+/+) rats or SDRs treated with either vehicle (Ghdr/dr) or bovine GH (Ghdr/dr + GH) 2 wk after initiation of injections (see Fig. 1BGo). Asterisk indicates significant difference from other two groups (P < 0.05, ANOVA).

 
Bovine GH, like rat GH, was able to support carcinogen-induced mammary carcinogenesis in Ghdr/dr rats. Two months after MNU, tumor incidence and latency were similar between GH-treated Ghdr/dr rats and Gh+/+ rats (Table 2Go). Many mammary tumors were observed in the rats of the GH-treated Ghdr/dr group, albeit fewer than in the Gh+/+ rats. By about 3 months after MNU exposure, all Gh+/+ rats and GH-treated Ghdr/dr rats had developed at least one, and in some cases several, mammary tumors. Needle biopsies confirmed that all tumors were indeed mammary cancers. All tumors were measured with calipers, and the 15 Ghdr/dr rats previously receiving GH injections were processed as follows. Five rats continued to receive GH throughout the study and were killed at the end of the experiment (~135 d after carcinogen exposure). These rats had developed six tumors within 3 months of carcinogen but did not develop any more tumors over the subsequent 7 wk of the study. Injections were stopped for the remaining 10 Ghdr/dr rats that had received GH injections, and all of these tumors began to regress rapidly. Some tumors lost as much as two thirds of their initial volume during the first week without GH injections. The rate of regression was similar to that observed upon ovariectomy of wild-type Sprague Dawley rats bearing MNU-induced mammary tumors (29). All 10 of the tumors carried by these seven Ghdr/dr rats regressed after the GH injections stopped. Eight tumors regressed completely, whereas the two others regressed 37 and 56% respectively, to approximately 0.19 cm3 (Fig. 4AGo).


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  		TABLE 2. GH-deficient rats can be made vulnerable to MNU-induced mammary carcinogenesis by treatment with recombinant bovine GH

 

Figure 4
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  		FIG. 4. Dependence of rat mammary tumors on GH for their survival. A, Change in tumor volume during the 1-month period between cessation and reinitiation of bovine GH or vehicle injections (see Fig. 1BGo for experimental design). Each bar represents the change in volume of a single tumor from the day of the last day of injections to the day before injections resumed 1 month later. Gray bars represent new mammary tumors that arose during this period in Gh+/+ rats. Open bars represent tumors present in Gh+/+ rats at the time injections were stopped. Black bars represent tumors present in Ghdr/dr rats at the time injections were stopped. Note that whereas all but one tumor continued to grow in the Gh+/+ rats, all tumors in the Ghdr/dr rats regressed after GH treatments were suspended, and most of these tumors regressed completely. B, Regeneration of regressed tumors on resumption of GH injections. Bovine GH injections were resumed for 3 wk after a 1-month hiatus. Each bar represents the change in volume of an individual tumor. Three animals were killed after the 1-month withdrawal period leaving seven regressed tumors for further analysis. Note that new tumor growth was established at all original tumor sites and two of the tumors, which had not regressed completely, were resurrected by GH administration. Tumor growth rate was similar to that observed in Gh+/+ rats that developed new tumors during GH withdrawal (see gray bars in A).

 
The other three rats of the 10 in which GH injections were stopped were killed approximately 7 d after cessation of GH injections when the tumors had regressed approximately 50%. Their four tumors were processed and stored for analysis of mammary epithelial cell proliferation, apoptosis, and GH/IGF signaling. As illustrated in Fig. 5Go, proliferation was significantly decreased, whereas apoptosis was significantly increased in the regressing tumors. Initial studies to assess potential mechanisms for regression included analysis of IGF-I at the RNA level and IGF-I receptor (IGF-IR) and GH receptor at both the protein and RNA level. No significant changes in these targets were observed between growing tumors in Gh+/+ rats and regressing tumors in Ghdr/dr rats approximately 7 d after GH withdrawal (data not shown). However, GH injections did stimulate IGF-I mRNA expression more than 20-fold in the livers of Ghdr/dr rats, indicating that these animals are capable of responding to GH in a manner similar to Gh+/+ rats (RT-PCR data not shown; see Fig. 3Go for serum IGF protein levels). STAT3 phosphorylation, abundant in growing tumors of both Gh+/+ rats and Ghdr/dr rats receiving GH, was not detectable in regressing mammary cancers after GH withdrawal (Fig. 6AGo). It was interesting to find phospho-STAT5a/b expression was abundant in normal mammary epithelial cells but was almost undetectable in the mammary tumors (Fig. 6BGo), a phenomenon previously reported by Rui and colleagues (42) in human breast cancer specimens.


Figure 5
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  		FIG. 5. Effect of bovine GH on mammary tumor cell proliferation or apoptosis assessed by immunohistochemical analysis of Ki67 expression (A) or TUNEL (B), respectively. Rats were treated as described in Fig. 1BGo. Tumors were harvested for analysis upon reaching a volume of 1 cm3 (Gh+/+ – GH and Ghdr/dr + GH; 3 months after MNU), when tumors had regressed approximately 50% (~2 wk after cessation of bovine GH treatments; Ghdr/dr +/– GH) or after regressed tumors were revived by reinitiation of GH treatments (Ghdr/dr +/–/+). Asterisks indicate significant difference (P < 0.05; ANOVA).

 

Figure 6
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  		FIG. 6. Protein analysis of STAT3 and STAT5a/b phosphorylation in mammary tissues. A, Immunoblot illustrating the loss of STAT3 phosphorylation in regressing mammary cancers. See Fig. 1BGo for experimental design overview. Mammary cancers were harvested approximately the time of cessation of bovine GH injections (Stop GH in Fig. 1BGo). Lanes 1–3, Growing mammary cancers harvested from vehicle-treated Gh+/+ rats; lanes 4–6, regressing mammary cancers from Ghdr/dr rats approximately 7 d after cessation of bovine GH injections; lanes 7–9, growing mammary cancers from Ghdr/dr rats that continued to receive GH injections up to the day of harvest. B, Immunohistochemistry of normal mammary gland (left) and mammary and a tumor from a Gh+/+ rat. An antibody that recognizes STATs 5a and 5b was used. Similar results were observed for normal mammary glands and both growing and regressing mammary tumors from Ghdr/dr rats during GH injections or after injections had been halted.

 
In stark contrast to the mammary tumors of the Ghdr/dr rats, all but one of the 34 mammary cancers in the Gh+/+ rats continued to grow (Fig. 4AGo) during the month when GH injections had ceased. Growth of these tumors was robust as expected and 21 new tumors arose in the Gh+/+ rats during this period (Fig. 4AGo). However, no new tumors arose in any of the Ghdr/dr rats in the absence of GH injections. When GH injections were resumed for 3 wk (see Fig 1BGo), seven tumors emerged at the same location of original, regressed tumors (Fig. 4BGo), suggesting that these original tumors were not completely extirpated by the lack of GH.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Women who deliver their first child early in life, especially before the age of 20 yr, have about half the lifetime risk of developing breast cancer relative to nulliparous women (43). The Sprague Dawley rat is a good model for the role of pregnancy in preventing mammary cancer because uniparous rats are less susceptible to chemically induced mammary cancer than age matched nulliparous controls (1, 2). One mechanism by which pregnancy may protect from mammary cancer is by permanently altering the hormonal milieu of the parous individual. We previously reported that among all mammotropic hormones studied, only serum GH titers were significantly lowered in parous rats resistant to cancer relative to age-matched virgins susceptible to cancer (2). These findings prompted us to further explore the role of GH in mammary carcinogenesis.

We have reported that the GH-deficient SDR (Ghdr/dr) is resistant to mammary cancer induced by either MNU or DMBA (28). In the present studies, we demonstrate that replenishing the SDR with physiologically relevant levels of GH renders the SDR vulnerable to MNU-induced mammary carcinogenesis. Our findings corroborate those of Thordarson et al. (41), who reported that SDRs treated with Posilac, a proprietary slow-release formulation of recombinant bovine GH used in the dairy industry, become susceptible to MNU-induced mammary cancer. We extended these findings to demonstrate that MNU-induced rat mammary cancers are dependent on GH because cessation of GH injections caused complete regression of most well-established mammary tumors. The rate of regression after GH withdrawal was similar to that observed upon ovariectomy (29).

In the current experiments, the dose of GH administered was judged physiological based on two well-established effects of GH activity: serum IGF-I levels and body weight gain. As illustrated in Fig. 3Go, serum IGF-I in the SDR is only about 20% that observed in wild-type Sprague Dawley rats. Twice-daily injections of GH yielded SDR serum IGF-I levels comparable with those observed in the wild-type rats. Also, the rate of body weight gain in GH-treated SDRs paralleled that observed in the wild-type rats (Fig. 2Go). When GH injections were halted, there was a rapid and precipitous decline in body weight observed in the SDRs so treated (Fig. 2Go). Nevertheless, a caveat of the current experiments is that GH was administered as twice-daily injections, which differs from the two dozen or so daily pulses of GH normally observed in wild-type female rats (44).

Given the significant indirect effects of GH that are mediated by IGF-I, an important question is the extent to which mammary carcinogenesis is dependent on IGF-I rather than GH. This question is particularly difficult to approach because both GH (45) and IGF-I (46) have been shown to work through many different signaling pathways in various systems. Cross talk between these two sets of pathways has been observed through STAT3 (see below) and direct physical and functional interaction of the GHR with IGF-IR (47). Another example is the stimulation of the MAPK members ERK1/2, which are stimulated by GH administration to Gh–/– rats (41) and IGF-IR stimulation (48).

Whereas studies have implicated IGF-I as an important determinant in a variety of human and experimental cancers (49), more recent data indicate that the problem is complex and that further research is necessary to clarify the role of IGF-I in breast cancer (50). In our experimental system, it is difficult to separate the direct effects of GH from the indirect effects of GH mediated by IGF-I. Thordarson et al. (41) compared MNU-exposed SDRs treated with either GH or IGF-I and found that GH was significantly more effective at supporting mammary carcinogenesis than IGF-I alone as judged by tumor incidence, latency, or multiplicity. However, GH induces IGF-binding protein-3, the major serum IGF-I binding protein (51). As noted by Thordarson et al., in the absence of GH, IGF-binding protein-3 levels are likely to be low, shortening the serum half-life and efficacy of exogenously administered IGF-I.

We have begun to evaluate the mechanism by which GH withdrawal induces tumor regression in this model. We first compared GHR, IGF-IR, and IGF-I levels in the actively growing mammary cancers of Gh+/+ rats with the regressing cancers of Ghdr/dr rats. Using quantitative PCR and immunoblot, we found no significant difference in expression of either GHR or IGF-IR in the growing mammary cancers of Gh+/+ rats, compared with the regressing tumors of Gh–/– rats after GH injections had been halted. We also observed no change in tumor IGF-I mRNA expression as a function of GH administration, suggesting that GH may not be the major regulator of IGF-I in mammary tumors in this system.

We next examined STAT5 and STAT3 because these transcription factors play important roles in mammary gland biology and neoplasia. STAT5 is a key factor for mammary gland development. Inactivation of Stat5a and Stat5b prevent pregnancy-induced alveolar differentiation (52, 53). Rui and colleagues (42) reported a gradual loss of activation of STAT5 that corresponded with increasingly aggressive human breast cancer specimens. In the present studies, we found plentiful phospho-STAT5 in normal rat mammary gland but none detectable in any of the tumors (Fig. 6Go), which parallels the observations of the human tissues. Activation of STAT3 appeared to be dependent on GH with robust phospo-STAT3 observed in tumors of Gh+/+ rats and Gh–/– rats that received GH. However, phospho-STAT3 was undetectable in the regressing tumors of Gh–/– rats after GH administration was stopped. This is significant because STAT3 activation is associated with a variety of human cancers (54) and is known to be activated by both GH (55) and IGF-I (56).

Whereas we have yet to dissect the effects of GH from those of IGF-I in modulating carcinogenesis, the SDR does allow us to study the effects of GH independent of other pituitary hormones. In the past, the only means to remove GH from the circulation was by hypophysectomy. This approach has been used to demonstrate that mammary cancers in rats are highly dependent on the pituitary (57). Furthermore, breast cancer patients with advanced mammary cancers have benefited from hypophysectomy. The majority of patients that have shown an objective response had previously responded to endocrine therapy (e.g. ovariectomy and/or adrenalectomy). However, hypophysectomy was also shown to be an effective therapy for the treatment of advanced breast cancer with objective response rates ranging from 33 to 50% (58). Whereas the rationale for performing hypophysectomy was to eliminate LH and ACTH), thereby suppressing steroid biosynthesis by the glands responsible for elevated serum estrogens (59), some patients who did not respond to either ovariectomy or adrenalectomy also benefited from hypophysectomy (60). Therefore, pituitary hormones other than LH or ACTH may contribute significantly to mammary carcinogenesis.

The results of the current studies add to a growing literature indicating that interruption of GH signaling may be an effective means of treating breast cancer. For example, Pollak and colleagues (61) reported that breast cancers derived from MCF-7 cells grew significantly more slowly in immunodeficient little (Ghrhrlit/lit) mice relative to control immunodeficient mice (Ghrhrlit/±). In another paper, Pollak et al. (62) reported significantly lower incidence of dimethylbenz[a]anthracene-induced mammary cancers in mice transgenic for the bovine GH antagonist (63) relative to nontransgenic littermates. Schally and colleagues (64) published studies recently that demonstrate an inhibitory effect of GHRH antagonists on the growth of estrogen-independent human mammary xenografts in nude mice. GHRH antagonists decreased IGF-I levels both in serum and tumors of treated animals (64). Schally and colleagues (65) reported that the GHRH antagonists JV-1–36 and JV-1–38 inhibited the growth of and enhanced apoptosis in estrogen-independent MTX mouse mammary cancers. In recent years, two independent groups have found that the GH antagonist pegvisomant can block the growth of human colon (66) and breast (67) tumors in immunodeficient mice. Thus, the preclinical findings presented here could be rapidly translated by testing pegvisomant, which is approved by the Food and Drug Administration for treatment of acromegaly, for its efficacy against breast cancer.

In summary, we report that the GH-deficient SDR can be made vulnerable to mammary carcinogenesis by GH supplementation. Furthermore, even advanced mammary cancers are dependent on GH injections for their survival in Ghdr/dr rats because tumors approximately 1 cm in diameter regressed on cessation of GH injections at a rate similar to that observed in Gh+/+ rats on ovariectomy. Whereas STAT3 was phosphorylated in growing cancers, no phospho-STAT3 was detectable in the tumors induced to regress by halting GH injections. Also, carcinogenesis in this model is associated with the loss of STAT5 phosphorylation, a finding that parallels human breast cancer. This model system should prove useful for studying the role of GH in mammary carcinogenesis. The effects of the GH/IGF-I axis on mammary epithelial cell proliferation, differentiation, and apoptosis as well as its association with human cancer risk implicate this pathway as a potential candidate for the targeting of novel therapeutic strategies.


   Acknowledgments
 
The authors thank Dr. Gregg Bogosian (Monsanto Co.) for kindly providing recombinant bovine GH. The authors also thank Dr. Stuart J. Frank for generously providing anti-GHRcyt-AL47.


   Footnotes
 
This work was supported by Grant R01-CA099904 from the National Institutes of Health.

Disclosure Summary: The authors have nothing to disclose.

First Published Online June 21, 2007

Abbreviations: DMBA, 7,12-dimethylbenz(a)anthracene; GHR, GH receptor; IGF-IR, IGF-I receptor; MNU, N-methyl-N-nitrosourea; SDR, spontaneous dwarf rat; STAT, signal transducer and activator of transcription; TAg, T antigen; TBS, Tris-buffered saline; TBS-T, TBS and Tween 20; TUNEL, terminal deoxynucleotide-transferase-mediated deoxyuridine 5-triphosphate-digoxigenin nick end labeling.

Received April 23, 2007.

Accepted for publication June 13, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Moon RC 1969 Relationship between previous reproductive history and chemically induced mammary cancer in rats. Int J Cancer 4:312–317[Medline]
  2. Thordarson G, Jin E, Guzman RC, Swanson SM, Nandi S, Talamantes F 1995 Refractoriness to mammary tumorigenesis in parous rats: is it caused by persistent changes in the hormonal environment or permanent biochemical alterations in the mammary epithelia? Carcinogenesis 16:2847–2853[Abstract/Free Full Text]
  3. Wood TL, Yee D 2000 Introduction: IGFs and IGFBPs in the normal mammary gland and in breast cancer. J Mammary Gland Biol Neoplasia 5:1–5[CrossRef][Medline]
  4. Kleinberg DL 1997 Early mammary development: growth hormone and IGF-1. J Mammary Gland Biol Neoplasia 2:49–57[CrossRef][Medline]
  5. Kleinberg DL2000 IGF-I: an essential factor in terminal end bud formation and ductal morphogenesis. J Mammary Gland Biol Neoplasia 5:7–17
  6. Kelly PA, Bachelot A, Kedzia C, Hennighausen L, Ormandy CJ, Kopchick JJ, Binart N 2002 The role of prolactin and growth hormone in mammary gland development. Mol Cell Endocrinol 197:127–131[CrossRef][Medline]
  7. Lyons WR, Li CH, Johnson RE 1958 The hormonal control of mammary growth and lactation. Recent Prog Horm Res 14:219–248; discussion 248–254[Medline]
  8. Nandi S 1958 Role of somatotropin in mammogenesis and lactogenesis in C3H/He CRGL mice. Science 128:772–774[Free Full Text]
  9. Ng ST, Zhou J, Adesanya OO, Wang J, LeRoith D, Bondy CA 1997 Growth hormone treatment induces mammary gland hyperplasia in aging primates. Nat Med 3:1141–1144[CrossRef][Medline]
  10. Plaut K, Ikeda M, Vonderhaar BK 1993 Role of growth hormone and insulin-like growth factor-I in mammary development. Endocrinology 133:1843–1848[Abstract/Free Full Text]
  11. Pearson OH, Llerena O, Llerena L, Molina A, Butler T 1969 Prolactin-dependent rat mammary cancer: a model for man? Trans Assoc Am Physicians 82:225–238[Medline]
  12. Hankinson SE, Willett WC, Colditz GA, Hunter DJ, Michaud DS, Deroo B, Rosner B, Speizer FE, Pollak M 1998 Circulating concentrations of insulin-like growth factor-I and risk of breast cancer. Lancet 351:1393–1396[CrossRef][Medline]
  13. LeRoith D, Roberts Jr CT2003 The insulin-like growth factor system and cancer. Cancer Lett 195:127–137
  14. Wu Y, Cui K, Miyoshi K, Hennighausen L, Green JE, Setser J, LeRoith D, Yakar S 2003 Reduced circulating insulin-like growth factor I levels delay the onset of chemically and genetically induced mammary tumors. Cancer Res 63:4384–4388[Abstract/Free Full Text]
  15. Arteaga CL, Osborne CK 1989 Growth inhibition of human breast cancer cells in vitro with an antibody against the type I somatomedin receptor. Cancer Res 49:6237–6241[Abstract/Free Full Text]
  16. Arteaga CL, Kitten LJ, Coronado EB, Jacobs S, Kull Jr FC, Allred DC, Osborne CK 1989 Blockade of the type I somatomedin receptor inhibits growth of human breast cancer cells in athymic mice. J Clin Invest 84:1418–1423[Medline]
  17. Holly JM, Gunnell DJ, Davey Smith G 1999 Growth hormone, IGF-I and cancer. Less intervention to avoid cancer? More intervention to prevent cancer? J Endocrinol 162:321–330[Abstract]
  18. Waters MJ, Conway-Campbell BL 2004 The oncogenic potential of autocrine human growth hormone in breast cancer. Proc Natl Acad Sci USA 101:14992–14993[Free Full Text]
  19. Canzian F, McKay JD, Cleveland RJ, Dossus L, Biessy C, Boillot C, Rinaldi S, Llewellyn M, Chajes V, Clavel-Chapelon F, Tehard B, Chang-Claude J, Linseisen J, Lahmann PH, Pischon T, Trichopoulos D, Trichopoulou A, Zilis D, Palli D, Tumino R, Vineis P, Berrino F, Bueno-de-Mesquita HB, van Gils CH, Peeters PH, Pera G, Barricarte A, Chirlaque MD, Quiros JR, Larranaga N, Martinez-Garcia C, Allen NE, Key TJ, Bingham SA, Khaw KT, Slimani N, Norat T, Riboli E, Kaaks R 2005 Genetic variation in the growth hormone synthesis pathway in relation to circulating insulin-like growth factor-I, insulin-like growth factor binding protein-3, and breast cancer risk: results from the European prospective investigation into cancer and nutrition study. Cancer Epidemiol Biomarkers Prev 14:2316–2325[Abstract/Free Full Text]
  20. Zhu T, Starling-Emerald B, Zhang X, Lee KO, Gluckman PD, Mertani HC, Lobie PE 2005 Oncogenic transformation of human mammary epithelial cells by autocrine human growth hormone. Cancer Res 65:317–324[Abstract/Free Full Text]
  21. Mukhina S, Mertani HC, Guo K, Lee KO, Gluckman PD, Lobie PE 2004 Phenotypic conversion of human mammary carcinoma cells by autocrine human growth hormone. Proc Natl Acad Sci USA 101:15166–15171[Abstract/Free Full Text]
  22. Decouvelaere C, Peyrat JP, Bonneterre J, Djiane J, Jammes H 1995 Presence of the two growth hormone receptor messenger RNA isoforms in human breast cancer. Cell Growth Differ 6:477–483[Abstract]
  23. Lincoln DT, Sinowatz F, Temmim-Baker L, Baker HI, Kolle S, Waters MJ 1998 Growth hormone receptor expression in the nucleus and cytoplasm of normal and neoplastic cells. Histochem Cell Biol 109:141–159[CrossRef][Medline]
  24. Mertani HC, Garcia-Caballero T, Lambert A, Gerard F, Palayer C, Boutin JM, Vonderhaar BK, Waters MJ, Lobie PE, Morel G 1998 Cellular expression of growth hormone and prolactin receptors in human breast disorders. Int J Cancer 79:202–211[CrossRef][Medline]
  25. Wang Z, Prins GS, Coschigano KT, Kopchick JJ, Green JE, Ray VH, Hedayat S, Christov KT, Unterman TG, Swanson SM 2005 Disruption of growth hormone signaling retards early stages of prostate carcinogenesis in the C3(1)/T antigen mouse. Endocrinology 146:5188–5196[Abstract/Free Full Text]
  26. Zhang X, Mehta RG, Lantvit DD, Coschigano KT, Kopchick JJ, Green JE, Hedayat S, Christov KT, Ray VH, Unterman TG, Swanson SM 2007 Inhibition of estrogen-independent mammary carcinogenesis by disruption of growth hormone signaling. Carcinogenesis 28:143–150[Abstract/Free Full Text]
  27. Shibata MA, Jorcyk CL, Liu ML, Yoshidome K, Gold LG, Green JE 1998 The C3(1)/SV40 T antigen transgenic mouse model of prostate and mammary cancer. Toxicol Pathol 26:177–182[Free Full Text]
  28. Swanson SM, Unterman TG 2002 The growth hormone-deficient Spontaneous Dwarf rat is resistant to chemically induced mammary carcinogenesis. Carcinogenesis 23:977–982[Abstract/Free Full Text]
  29. Gullino PM, Pettigrew HM, Grantham FH 1975 N-nitrosomethylurea as mammary gland carcinogen in rats. J Natl Cancer Inst 54:401–414[Medline]
  30. Pearson OH, Manni A 1978 Hormonal control of breast cancer growth in women and rats. Curr Top Exp Endocrinol 3:75–92[Medline]
  31. Russo J, Gusterson BA, Rogers AE, Russo IH, Wellings SR, van Zwieten MJ 1990 Comparative study of human and rat mammary tumorigenesis. Lab Invest 62:244–278[Medline]
  32. Okuma S 1980 Spontaneous dwarf rat. Exp Anim 29:301–305
  33. Takeuchi T, Suzuki H, Sakurai S, Nogami H, Okuma S, Ishikawa H 1990 Molecular mechanism of growth hormone (GH) deficiency in the spontaneous dwarf rat: detection of abnormal splicing of GH messenger ribonucleic acid by the polymerase chain reaction. Endocrinology 126:31–38[Abstract/Free Full Text]
  34. Nogami H, Takeuchi T, Suzuki K, Okuma S, Ishikawa H 1989 Studies on prolactin and growth hormone gene expression in the pituitary gland of spontaneous dwarf rats. Endocrinology 125:964–970[Abstract/Free Full Text]
  35. Okuma S 1984 Study of growth hormone in spontaneous dwarf rat. Nippon Naibunpi Gakkai Zasshi 60:1005–1014[Medline]
  36. Steel GG 1977 Growth rate of tumours. In: Growth kinetics of tumours. Oxford, UK: Clarendon Press; 5–55
  37. Beaver BV, Reed W, Leary S, McKiernan B, Bain F, Schultz R, Bennett BT, Pascoe P, Shull E, Cork LC, Francis-Floyd R, Amass KD, Johnson R, Schmidt RH, Underwood W, Thornton GW, Kohn B 2001 2000 Report of the AMVA Panel on Euthanasia. J Am Vet Med Assoc 218:669–696[CrossRef][Medline]
  38. Van Herck H, Baumans V, Brandt CJ, Boere HA, Hesp AP, van Lith HA, Schurink M, Beynen AC 2001 Blood sampling from the retro-orbital plexus, the saphenous vein and the tail vein in rats: comparative effects on selected behavioural and blood variables. Lab Anim 35:131–139[Medline]
  39. Zhang Y, Guan R, Jiang J, Kopchick JJ, Black RA, Baumann G, Frank SJ 2001 Growth hormone (GH)-induced dimerization inhibits phorbol ester-stimulated GH receptor proteolysis. J Biol Chem 276:24565–24573[Abstract/Free Full Text]
  40. Frank SJ, Gilliland G, Van Epps C 1994 Treatment of IM-9 cells with human growth hormone (GH) promotes rapid disulfide linkage of the GH receptor. Endocrinology 135:148–156[Abstract]
  41. Thordarson G, Semaan S, Low C, Ochoa D, Leong H, Rajkumar L, Guzman RC, Nandi S, Talamantes F 2004 Mammary tumorigenesis in growth hormone deficient spontaneous dwarf rats; effects of hormonal treatments. Breast Cancer Res Treat 87:277–290[CrossRef][Medline]
  42. Nevalainen MT, Xie J, Torhorst J, Bubendorf L, Haas P, Kononen J, Sauter G, Rui H 2004 Signal transducer and activator of transcription-5 activation and breast cancer prognosis. J Clin Oncol 22:2053–2060[Abstract/Free Full Text]
  43. MacMahon B, Cole P, Lin TM, Lowe CR, Mirra AP, Ravnihar B, Salber EJ, Valaoras VG, Yuasa S 1970 Age at first birth and breast cancer risk. Bull World Health Organ 43:209–221[Medline]
  44. Jansson JO, Ekberg S, Isaksson OG, Eden S 1984 Influence of gonadal steroids on age- and sex-related secretory patterns of growth hormone in the rat. Endocrinology 114:1287–1294[Abstract/Free Full Text]
  45. Zhu T, Goh EL, Graichen R, Ling L, Lobie PE 2001 Signal transduction via the growth hormone receptor. Cell Signal 13:599–616[CrossRef][Medline]
  46. Butler AA, Yakar S, Gewolb IH, Karas M, Okubo Y, LeRoith D 1998 Insulin-like growth factor-I receptor signal transduction: at the interface between physiology and cell biology. Comp Biochem Physiol B Biochem Mol Biol 121:19–26[CrossRef][Medline]
  47. Huang Y, Kim SO, Yang N, Jiang J, Frank SJ 2004 Physical and functional interaction of growth hormone and insulin-like growth factor-I signaling elements. Mol Endocrinol 18:1471–1485[Abstract/Free Full Text]
  48. Alexia C, Fallot G, Lasfer M, Schweizer-Groyer G, Groyer A 2004 An evaluation of the role of insulin-like growth factors (IGF) and of type-I IGF receptor signalling in hepatocarcinogenesis and in the resistance of hepatocarcinoma cells against drug-induced apoptosis. Biochem Pharmacol 68:1003–1015[CrossRef][Medline]
  49. Pollak MN, Schernhammer ES, Hankinson SE 2004 Insulin-like growth factors and neoplasia. Nat Rev Cancer 4:505–518[CrossRef][Medline]
  50. Renehan AG, Harvie M, Howell A 2006 Insulin-like growth factor (IGF)-I, IGF binding protein-3, and breast cancer risk: eight years on. Endocr Relat Cancer 13:273–278[Abstract/Free Full Text]
  51. De W, Breant B, Czernichow P, Asfari M 1995 Growth hormone (GH) and prolactin (PRL) regulate IGFBP-3 gene expression in rat ß-cells. Mol Cell Endocrinol 114:43–50[CrossRef][Medline]
  52. Miyoshi K, Shillingford JM, Smith GH, Grimm SL, Wagner KU, Oka T, Rosen JM, Robinson GW, Hennighausen L 2001 Signal transducer and activator of transcription (Stat) 5 controls the proliferation and differentiation of mammary alveolar epithelium. J Cell Biol 155:531–542[Abstract/Free Full Text]
  53. Cui Y, Riedlinger G, Miyoshi K, Tang W, Li C, Deng CX, Robinson GW, Hennighausen L 2004 Inactivation of Stat5 in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation. Mol Cell Biol 24:8037–8047[Abstract/Free Full Text]
  54. Jing N, Tweardy DJ 2005 Targeting Stat3 in cancer therapy. Anticancer Drugs 16:601–607[CrossRef][Medline]
  55. Campbell GS, Meyer DJ, Raz R, Levy DE, Schwartz J, Carter-Su C 1995 Activation of acute phase response factor (APRF)/Stat3 transcription factor by growth hormone. J Biol Chem 270:3974–3979[Abstract/Free Full Text]
  56. Zong CS, Chan J, Levy DE, Horvath C, Sadowski HB, Wang LH 2000 Mechanism of STAT3 activation by insulin-like growth factor I receptor. J Biol Chem 275:15099–15105[Abstract/Free Full Text]
  57. Huggins C, Briziarelli G, Sutton H 1959 Rapid induction of mammary carcinoma in the rat and the influence of hormones on the tumors. J Exp Med 109:25–54[Abstract]
  58. Kennedy BJ 1974 Hormonal therapies in breast cancer. Semin Oncol 1:119–130[Medline]
  59. Luft R, Olivecrona H 1957 Hypophysectomy in treatment of malignant tumors. Cancer 10:789–794[CrossRef][Medline]
  60. Hayward JL, Atkins HJ, Falconer MA, MacLean KS, Salmon LF, Schurr PH, Shaheen CH 1970 Clinical trials comparing transfrontal hypophysectomy with adrenalectomy and with transethmoidal hypophysectomy. In: Gleave EN, Joslin CA, eds. The clinical management of advanced breast cancer. Cardiff, Wales: Alpha Omega Alpha Publishing Co.; 50–53
  61. Yang XF, Beamer WG, Huynh H, Pollak M 1996 Reduced growth of human breast cancer xenografts in hosts homozygous for the lit mutation. Cancer Res 56:1509–1511[Abstract/Free Full Text]
  62. Pollak M, Blouin MJ, Zhang JC, Kopchick JJ 2001 Reduced mammary gland carcinogenesis in transgenic mice expressing a growth hormone antagonist. Br J Cancer 85:428–430[CrossRef][Medline]
  63. Chen WY, Wight DC, Wagner TE, Kopchick JJ 1990 Expression of a mutated bovine growth hormone gene suppresses growth of transgenic mice. Proc Natl Acad Sci USA 87:5061–5065[Abstract/Free Full Text]
  64. Kahan Z, Varga JL, Schally AV, Rekasi Z, Armatis P, Chatzistamou L, Czompoly T, Halmos G 2000 Antagonists of growth hormone-releasing hormone arrest the growth of MDA-MB-468 estrogen-independent human breast cancers in nude mice. Breast Cancer Res Treat 60:71–79[CrossRef][Medline]
  65. Szepeshazi K, Schally AV, Armatis P, Groot K, Hebert F, Feil A, Varga JL, Halmos G 2001 Antagonists of GHRH decrease production of Gh and IGF-I in mxt mouse mammary cancers and inhibit tumor growth. Endocrinology 142:4371–4378[Abstract/Free Full Text]
  66. Dagnaes-Hansen F, Duan H, Rasmussen LM, Friend KE, Flyvbjerg A 2004 Growth hormone receptor antagonist administration inhibits growth of human colorectal carcinoma in nude mice. Anticancer Res 24:3735–3742[Abstract/Free Full Text]
  67. Divisova J, Kuiatse I, Lazard Z, Weiss H, Vreeland F, Hadsell DL, Schiff R, Osborne CK, Lee AV 2006 The growth hormone receptor antagonist pegvisomant blocks both mammary gland development and MCF-7 breast cancer xenograft growth. Breast Cancer Res Treat 98:315–327[CrossRef][Medline]



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