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Molecular Endocrinology, doi:10.1210/me.2004-0106
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Molecular Endocrinology 19 (3): 644-656
Copyright © 2005 by The Endocrine Society

Overexpression of an N-Terminally Truncated Isoform of the Nuclear Receptor Coactivator Amplified in Breast Cancer 1 Leads to Altered Proliferation of Mammary Epithelial Cells in Transgenic Mice

Maddalena T. Tilli1, Ronald Reiter1, Annabell S. Oh, Ralf T. Henke, Kevin McDonnell, G. Ian Gallicano, Priscilla A. Furth2 and Anna Tate Riegel2

Departments of Oncology and Pharmacology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D.C. 20057

Address all correspondence and requests for reprints to: Priscilla A. Furth or Anna Tate Riegel, Georgetown University, Lombardi Cancer Center, Research Building, Room E307, 3970 Reservoir Road, Washington, D.C. 20057. E-mail: paf3{at}georgetown.edu or ariege01{at}georgetown.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Amplified in breast cancer 1 (AIB1, also known as ACTR, SRC-3, RAC-3, TRAM-1, p/CIP) is a member of the p160 nuclear receptor coactivator family involved in transcriptional regulation of genes activated through steroid receptors, such as estrogen receptor {alpha} (ER{alpha}). The AIB1 gene and a more active N-terminally deleted isoform (AIB1-{Delta}3) are overexpressed in breast cancer. To determine the role of AIB1-{Delta}3 in breast cancer pathogenesis, we generated transgenic mice with human cytomegalovirus immediate early gene 1 (hCMVIE1) promoter-driven over-expression of human AIB1/ACTR-{Delta}3 (CMVAIB1/ACTR-{Delta}3 mice). AIB1/ACTR-{Delta}3 transgene mRNA expression was confirmed in CMV-AIB1/ACTR-{Delta}3 mammary glands by in situ hybridization. These mice demonstrated significantly increased mammary epithelial cell proliferation (P < 0.003), cyclin D1 expression (P = 0.002), IGF-I receptor protein expression (P = 0.026), mammary gland mass (P < 0.05), and altered expression of CCAAT/enhancer binding protein isoforms (P = 0.029). At 13 months of age, mammary ductal ectasia was found in CMV-AIB1/ACTR-{Delta}3 mice, but secondary and tertiary branching patterns were normal. There were no changes in the expression patterns of either ER{alpha} or Stat5a, a downstream mediator of prolactin signaling. Serum IGF-I levels were not altered in the transgenic mice. These data indicate that overexpression of the AIB1/ACTR-{Delta}3 isoform resulted in altered mammary epithelial cell growth. The observed changes in cell proliferation and gene expression are consistent with alterations in growth factor signaling that are thought to contribute to either initiation or progression of breast cancer. These results are consistent with the hypothesis that the N-terminally deleted isoform of AIB1 can play a role in breast cancer development and/or progression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
STEROID HORMONES AND their ligand-activated nuclear hormone receptors regulate the transcription of genes, which play significant roles in mammary gland development and tumorigenesis. The p160/SRC (steroid receptor coactivator) family is a family of coactivators involved in the regulation of steroid receptor-mediated transcription (1) and a subset of other transcription factors (2). The family of SRCs function by bridging the gap between nuclear receptors, other coactivators, and the basal transcriptional machinery (1) and may regulate chromatin structure through a histone acetylase domain (3, 4, 5, 6, 7). Three members of the p160 family have been identified: SRC-1 (8), SRC-2 [TIF2 (transcription intermediary factor-2) or GRIP1 (glucocorticoid receptor-interacting protein 1)] (9), and amplified in breast cancer 1 (AIB1) (10), also known as activator of thyroid hormone and retinoid receptors (ACTR), RAC3, TRAM-1, SRC-3, and the mouse homolog p/CIP (3, 11, 12, 13, 14).

AIB1 was identified during a search of a region frequently amplified in human breast cancer on chromosome 20q (15). The AIB1 gene itself is amplified in 5–10% of breast cancers and the mRNA and protein is overexpressed in approximately 30% of breast tumors (10, 16, 17, 18, 19, 20). p/CIP knockout (KO) mice show less mammary gland alveolar development during pregnancy and respond to estrogen and progesterone with only partial duct differentiation (21). In p/CIP KO mice, which also had a mouse mammary tumor virus (MMTV)/v-Ha-ras transgene, the incidence of both mammary gland ductal hyperplasia and mammary tumorigenesis were suppressed (2). These data indicate that AIB1 may play an important role in breast cancer initiation and progression.

AIB1 has been shown to directly interact with the ER in breast cancer cells (22) and functions to enhance ER-dependent transcription (10, 23). AIB1 overexpression, as well as its interaction with ER, suggests that AIB1 may modulate steroid receptor pathway signaling in breast cancer (19). Alternatively, AIB1 has been shown to regulate other nonsteroid receptor pathways that may be involved in tumor progression through AIB1 activity (12, 14, 24, 25). These findings led to the hypothesis that AIB1 contributes to the development of breast cancer through multiple different signal transduction pathways.

A splice variant of AIB1 (AIB1/ACTR-{Delta}3), which results in an N-terminal truncation, is also overexpressed in breast tumor tissue (26). The {Delta}3 designation refers to a truncation located in the third exon relative to the then first known exon. AIB1/ACTR-{Delta}3 has been found to be a more effective coactivator of estrogen, progesterone, and EGF signaling than the wild-type (WT) ER, suggesting a role for AIB1/ACTR-{Delta}3 in hormone and paracrine signaling in breast cancer (26). Overexpression of the AIB1/ACTR-{Delta}3 isoform may play a role in sensitizing breast cancer cells to hormones and selective ER modulators during breast cancer development (24). Because AIB1/ACTR-{Delta}3 has been shown to be a significantly better coactivator of ER{alpha}- and progesterone receptor (PR)-mediated responses, it is hypothesized that tumors expressing high levels of this isoform will be responsive to endogenous estrogenic stimulation of proliferation contributing to the development of neoplasia (24).

In this study, we sought to determine the role of increased levels of AIB1-{Delta}3 in mammary oncogenesis in normal mammary epithelial cells in an in vivo model. To this end, a transgenic mouse model with overexpression of a nuclear receptor coactivator, AIB1/ACTR-{Delta}3, has been developed. We have determined that simple overexpression of AIB1/ACTR-{Delta}3 alone in these mice leads to proliferation of mammary epithelial cells and alterations in growth factor signaling pathways with important implications for breast cancer initiation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of CMV-AIB1/ACTR-{Delta}3 Transgenic Mice and Characterization of Transgene Expression in the Mammary Gland
To examine the role of the AIB1/ACTR-{Delta}3 isoform in vivo, transgenic mice that overexpress human AIB1/ACTR-{Delta}3, a splice variant of the human AIB1 sequence previously shown to be overexpressed in breast cancer tissues and cell lines (26) were generated (CMV-AIB1/ACTR-{Delta}3 mice). The CMV–AIB1/ACTR-{Delta}3 transgene construct included the splice variant under the control of the human CMV immediate early gene 1 (hCMVIE1) promoter and a bovine GH (BGH) polyadenylation site, as shown in Fig. 1AGo. Expression of the CMV-AIB1/ACTR-{Delta}3 transgene in the mammary glands of transgenic mice was confirmed by RT-PCR using transgene-specific primers and verified by in situ hybridization (Fig. 1Go, B and C). Western blot analysis, with an antibody that recognizes both mouse and human AIB1, of the transgenic and WT mammary glands, reproducibly showed the endogenous form (p/CIP) in all mice (right, thin arrow, Fig. 1DGo) and an additional higher mobility form only in the CMV-AIB1/ACTR-{Delta}3 mice (right, thick arrow, Fig. 1DGo). The higher mobility of the second form found in the transgenic mice is consistent with a posttranslational modification such as phosphorylation. Indeed, a significant phosphorylation mobility shift has been shown previously for AIB1 (27). Furthermore, the removal of the N terminus of AIB1 causes a significant increases in the MAPK phosphorylation of AIB1 (28). The predicted size and mobility of the AIB1/ACTR-{Delta}3 protein translated after transfection into primate COS cells was smaller than full-length AIB1 (left, open triangle, Fig. 1DGo; and Ref. 26) and smaller than the endogenous p/CIP identified in mouse 32D cells and the two forms found in the transgenic mammary gland tissue. The full-length form of AIB1 translated after transfection into COS cells (left, closed triangle, Fig. 1DGo) was larger than either form identified in the transgenic mice. Thus, the apparent molecular weight of AIB1 and its isoform are highly dependent on the species or tissue in which they are expressed. Unfortunately, without an antibody that can unequivocally distinguish between the mouse and human protein, we cannot positively identify the higher mobility form as being translated from the significant levels of AIB1-{Delta}3 mRNA that is expressed (Fig. 1Go, B and C). By all measures, the total increase in expression levels by AIB1/ACTR-{Delta}3 overexpres-sion did not appear to be more than 2-fold in the transgenic mice. However, it is known that AIB1/ACTR-{Delta}3 is a highly potent coactivator, and even in relatively low amounts, compared with endogenous it shows significant potentiation of steroid-induced transcription (24, 26).



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  		Fig. 1. Detection of Transgene Expression in Mammary Gland Tissue

A, Structure of the CMV-AIB1/ACTR-{Delta}3 construct used to generate the CMV-AIB1/ACTR-{Delta}3 transgenic mice. Indicated are the locations of the exon 3 deletion, the PCR primers for genotyping (PCR sense and antisense), and the AIB1/ACTR RT-PCR probe and primer. B, mRNA expression of the transgene-specific transcript for human AIB1/ACTR-{Delta}3 was detected by in situ hybridization. Sense riboprobe was hybridized to the CMV-AIB1/ACTR-{Delta}3 and WT mammary gland slides as a control for DNA signal. Arrow indicates mammary ductal ectasia. Digital photographs were taken at x20. C, expression of the CMV-AIB1/ACTR-{Delta}3 transgene (184 bp) in a mammary gland from a CMV-AIB1/ACTR-{Delta}3 mouse was detected by RT-PCR. A mammary gland from a WT mouse was included as a negative control. To exclude contamination by genomic DNA, reverse transcriptase (RT enzyme) was omitted from the reaction as indicated (–). D, Representative Western blot showing relative levels of endogenous mouse AIB1 protein in WT and transgenic mice compared with the AIB1/ACTR-{Delta}3 protein expression in the transgenic animals (AIB1 = 150–160 kDa). Protein extract from the 32D mouse hematopoetic cell line is included as mouse AIB1 protein control. Protein extract from COS cells transfected with human full-length AIB1 (left, closed arrow) and AIB1/ACTR-{Delta}3 (left, open arrow) were also included as controls. Thin arrows indicate endogenous mouse form (p/CIP). Thick arrow on right indicates a higher mobility form only in the CMV-AIB1/ACTR-{Delta}3 mice.

 
CMV-AIB1/ACTR-{Delta}3 Mice Display Increased Mammary Gland Weight
Size, growth, mating, pregnancy, gestation, litter size, and lactation were normal in the CMV-AIB1/ACTR-{Delta}3 transgenic mice. However, upon examination of the transgenic animals at necropsy, it was found that the mammary glands of the CMV-AIB1/ACTR-{Delta}3 mice were larger in appearance than the mammary glands of their WT litter mates. Mammary glands from 7-wk-old and 10- to 13-month-old transgenic and WT animals were harvested and the weights of each gland were measured. A significant increase mammary gland weight in the CMV-AIB1/ACTR-{Delta}3 transgenic mice was observed at both the 7 wk (P < 0.05) and 10- to 13-month (P < 0.05) time points when compared with WT controls (Fig. 2AGo). To determine whether the increased mammary gland weight in the CMV-AIB1/ACTR-{Delta}3 animals was simply due to increased total body mass, body weight was measured for the mice before harvesting the mammary glands. Mean body weights for CMV-AIB1/ACTR-{Delta}3 transgenic animals did not differ from their WT litter mate controls regardless of age (Fig. 2BGo). Serum IGF-I levels were measured and compared in the transgenic and WT litter mates because loss of AIB1/SRC-3 expression has been associated with lower serum IGF-I levels (21). Serum IGF-I levels were normal in the CMV-AIB1/ACTR-{Delta}3 transgenic mice [WT (n =14): 872.9 ± 88.8 ng/ml, CMV-AIB1/ACTR-{Delta}3 (n =15): 807.5 ± 61.38 ng/ml; P = 0.5447]. Whole mouse necropsies were performed to examine for signs of systemic disease. Mammary gland, uterine, and ovarian tissue was taken for histological analyses. No tumors were found on necropsy, and although enlarged mammary glands were documented in the transgenic mice, the ovaries and uteri were normal sized. Hematoxylin and eosin (H&E)-stained sections of ovaries and uteri from the transgenic mice were examined for the appearance of abnormalities and compared with the normal WT controls, but no pathology was found in either of these organs. Interestingly, while this manuscript was under revision, a report appeared in the literature in which the introduction of a higher expressing MMTV-AIB1 transgene was found to significantly increase IGF-I serum levels in association with the appearance of tumors in multiple tissues (29). The absence of tumors in multiple tissues in the mice described here may be due to the lower transgene expression levels, the lack of an elevated systemic IGF-I level, and/or the different AIB1 isoform expressed.



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  		Fig. 2. Transgenic Mice Exhibit Increased Mammary Gland Weight without Concurrent Body Weight Increase

Mammary gland weight (A) and body weight (B) were determined for WT and CMV-AIB1/ACTR-{Delta}3 mice at 7 wk (8 WT, 11 CMV-AIB1/ACTR-{Delta}3) and 10/13 months (12 WT, 12 CMV-AIB1/ACTR-{Delta}3) of age. Bars indicate mean ± SEM. *, P < 0.05 when compared with WT mammary gland weights.

 
CMV-AIB1/ACTR-{Delta}3 Overexpression Increases Mammary Epithelial Cell Proliferation and Alters Mammary Epithelial Cell Growth Pathways
Immunohistochemistry for proliferating cell nuclear antigen (PCNA) and phospho-histone H3 (serine 10), a mitosis marker, were performed to determine whether the increased mammary gland size was due to an alteration in proliferation of mammary epithelial cells in the CMV-AIB1/ACTR-{Delta}3 mammary glands (Fig. 3AGo). The percentage of PCNA-positive cells was significantly greater (P = 0.003) in mammary glands from CMV-AIB1/ACTR-{Delta}3 transgenic mice (28 ± 4%) when compared with PCNA positivity in WT controls (11 ± 3%) (Fig. 3AGo, top panels). Similarly, the percentage of phospho-histone H3-positive cells was significantly greater (P = 0.0001) in mammary glands from CMV-AIB1/ACTR-{Delta}3 transgenic mice (21 ± 1%) when compared with phospho-histone H3 positivity in WT controls (6 ± 1%) (Fig. 3AGo, second row of panels). These data indicate that an increase in the proliferative rates of the mammary epithelial cell compartment in the CMV-AIB1/ACTR-{Delta}3 mice were at least one factor responsible for the increased mammary gland weight.



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  		Fig. 3. CMV-AIB1/ACTR-{Delta}3 Transgenic Mice Exhibit Alterations in Growth Pathways

A, Immunohistochemistry was performed to assess proliferation of mammary epithelial cells by PCNA and phospho-histone H3, as well as to visualize differences in cyclin D1 and ER{alpha} protein expression in mammary glands from WT (left panels) and CMV-AIB1/ACTR-{Delta}3 transgenic (right panels) mice. Arrows indicate representative mammary epithelial cells with nuclear-localized PCNA, phospho-histone H3, cyclin D1, or ER{alpha} staining, respectively. To the right is a graphical representation of the mean percentage of positively staining PCNA, phospho-histone H3, or cyclin D1 nuclei in WT (n =12) and CMV-AIB1/ACTR-{Delta}3 (n = 12) animals. ER{alpha} protein expression was unchanged in CMV-AIB1/ACTR-{Delta}3 mice as compared with WT controls. Digital photographs taken at x40 (PCNA, cyclin D1, ER{alpha}) and x60 (phospho-histone H3). B, IGF-IR protein expression was altered in extracts from mammary glands of CMV-AIB1/ACTR-{Delta}3 mice (n = 12) as compared with WT controls (n =12). Actin is included as a loading control. Quantification of IGF-IR expression levels is indicated on the right. IGF-IR: 90 kDa. *, Statistical significance when compared with WT, PCNA: P = 0.003, phospho-histone H3: P = 0.0001; cyclin D1: P = 0.002, and IGF-IR: P = 0.026. Bars indicate mean ± SEM.

 
In an effort to determine the genetic pathways that may be responsible for the increased proliferation, protein expression of genes that have been previously shown to be connected with or controlled by AIB1 were examined, namely cyclin D1 (30), ER{alpha} (22, 23), and IGF-I receptor (IGF-IR) (14, 25). The percentage of mammary epithelial cells demonstrating nuclear-localized cyclin D1 was significantly greater (P = 0.002) in mammary glands from CMV-AIB1/ACTR-{Delta}3 transgenic mice (15 ± 1%) when compared with WT controls (10 ± 1%) as determined by immunohistochemistry (Fig. 3AGo). Additionally, relative IGF-IR expression levels was significantly greater (P = 0.026) in mammary glands from CMV-AIB1/ACTR-{Delta}3 transgenic mice (2.66 ± 0.63) when compared with WT controls (1.00 ± 0.29) as determined by Western blot analysis (Fig. 3BGo).

Neither ER{alpha} (Fig. 3AGo, bottom panels) nor PR (data not shown) expression levels were increased in the mammary epithelial cells of CMV-AIB1/ACTR-{Delta}3 transgenic mice when compared with WT controls. There were no significant differences found in the ductal branching patterns of the CMV-AIB1/ACTR-{Delta}3 transgenic mice as compared with WT mice (Fig. 4Go). However, by 13 months of age a percentage (40%, n = 10) of the transgenic mice demonstrated ductal ectasia (Fig. 1BGo, arrow). Neither alveolar growth, differentiation, nor the development of secretory mammary epithelial cells were found in the CMV-AIB1/ACTR-{Delta}3 transgenic mice indicating that neither prolactin nor progesterone signaling pathways were activated by the levels of transgene expression found in this model. As a second test to examine for evidence of increased prolactin signaling, Stat5a immunohistochemistry was performed. Stat5a is a signaling protein that is activated and nuclear-localized in response to prolactin stimulation (31). In nonpregnant mice, Stat5a is localized in both the cytoplasm and nucleus (32). When prolactin levels are increased in nonpregnant mice, cytoplasmic Stat5a is translocated to the nucleus of mammary epithelial cells resulting in primarily nuclear localization (33). The proportion of nuclear and cytoplasmic localization of Stat5a was the same in both transgenic and WT mice, indicating that there was no abnormal activation of prolactin or Stat5a signaling by AIB1/ACTR-{Delta}3 overexpression in the transgenic mice (Fig. 4Go). No secretory mammary epithelial cells were found on examination of H&E-stained sections (Fig. 4CGo). Similarly, there was no evidence that the levels of AIB1/ACTR-{Delta}3 overexpression altered either local or systemic progesterone signaling because there were no increases in tertiary branching or development of alveoli in the mammary gland and uterine weights and appearance were normal in the transgenic mice.



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  		Fig. 4. Transgenic Mice Demonstrate Normal Mammary Gland Morphology at Younger Ages with the Appearance of Ductal Ectasia with Age

With the exception of the appearance of ectasia in older mice, mammary gland morphology with normal primary, secondary and tertiary branching was unchanged by transgene expression. Localization of Stat5a was not altered by transgene expression. Representative mammary gland whole mount analysis of WT and CMV-AIB1/ACTR-{Delta}3 transgenic mice at 8 months of age (A) and at 13 months of age (B). Digital photographs taken for top panels at x0.5 and for bottom panels at x4. C, Immunohistochemical detection of Stat5a protein expression in mammary glands from WT (left panel) and CMV-AIB1/ACTR-{Delta}3 transgenic (right panel) mice. Digital photographs taken at x40. Arrows indicate cytoplasmic (wide) and nuclear (narrow) Stat5a localization.

 
Expression of CCAAT/Enhancer Binding Protein (C/EBPß) Liver-Enriched Inhibitory Protein (LIP) Isoform Is Altered in the Mammary Glands of CMV-AIB1/ACTR-{Delta}3 Transgenic Mice
C/EBPß, a transcription factor that binds to DNA at specific sequences as dimers to regulate transcription of genes involved in proliferation and differentiation, has been shown to be a regulator of mammary epithelial cell fate during pregnancy (34) and cancer (35). C/EBPß KO mice display defects in mammary gland growth, indicating that it is crucial for normal development of the mammary gland (36, 37) and differentiation of mammary epithelial cells (38, 39). C/EBPß was examined in the CMV-AIB1/ACTR-{Delta}3 mice to determine whether this pathway was contributing to the increased mammary gland growth seen in the transgenic mice. C/EBPß has two isoforms that have opposing effects on gene activation: LIP and liver-enriched activating protein (LAP). A dominant-negative function for LIP is indicated by the inhibition of transcriptional activation by LAP of genes involved in mammary gland differentiation (40). This inhibition by LIP expression results in an increase in proliferation of mammary epithelial cells (34). The ratio of LAP to LIP, rather than the absolute expression levels of each, indicates whether C/EBPß will activate or inhibit transcription. For example, during pregnancy, LIP levels increase more than LAP levels and the LAP:LIP ratio is less than 5, resulting in an increase in proliferation, whereas during lactation LIP levels are undetectable and the LAP:LIP ratio is greater than 100, resulting in differentiation (38, 41, 42). To determine the transcriptional activation potential of C/EBPß in the CMV-AIB1/ACTR-{Delta}3 transgenic mammary glands, LAP and LIP expression in CMV-AIB1/ACTR-{Delta}3 transgenic mice and WT control mice were measured by Western blot analysis (Fig. 5Go). The LAP:LIP ratio was found to be significantly decreased (P = 0.029) in CMV-AIB1/ACTR-{Delta}3 animals (8.51 ± 1.05) when compared with WT animals (21.14 ± 7.04). Subsequent analysis of LAP levels showed no significant difference between WT and transgenic animals, indicating that the change in ratio was due to an increase in LIP levels in the CMV-AIB1/ACTR-{Delta}3 mice. Interestingly, we also observed ductal ectasia in 40% (n = 10) of the CMV-AIB1/ACTR-{Delta}3 mice (see Fig. 1DGo), which is consistent with the phenotype seen previously in LIP overexpressing mice (34).



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  		Fig. 5. CMV-AIB1/ACTR-{Delta}3 Transgenic Mice Exhibit an Altered LAP/LIP C/EBPß Ratio

A, Representative Western blot of mammary glands extracts from CMV-AIB1/ACTR-{Delta}3 and WT animals. B, Ratios of LAP to LIP in CMV-AIB1/ACTR-{Delta}3 (n = 12) and WT (n = 12) animals. Asterisks indicate statistical significance when compared with WT; P = 0.014. C, Quantification of LAP expression. Bars indicate mean ± SEM.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
AIB1 is overexpressed in steroid hormone targeted tumors [breast (10) and prostate (43) cancers] in correlation with ER, PR (16), and Her2/neu expression (19). Interestingly, AIB1 is also overexpressed in nonsteroid hormone-targeted tumors [meningiomas (44), gastric (45), pancreatic (46), and liver (47) cancers]. These latter data suggest that AIB1 acts by changing other pathways in addition to steroid receptor signaling pathways that lead to increased cell proliferation and carcinogenesis (25, 48). The data presented in this study confirms this general hypothesis for AIB1/ACTR-{Delta}3. We have also shown that overexpression of AIB1/ACTR-{Delta}3 caused increases in critical molecules in growth factor pathways, independent of changes in ER{alpha} expression levels and PR signaling, leading normal mammary epithelial cells to proliferate. Because AIB1-{Delta}3 is increased in human breast tumors, data from the CMV-AIB1/ACTR-{Delta}3 mouse model suggests that ER{alpha}-independent pathways also are relevant for proproliferative changes in the mammary gland.

As well as being implicated in breast carcinogenesis, AIB1 has also been shown in p/CIP KO mice to play a role in pathways regulating somatic growth (14). The p/CIP KO mice display impaired growth phenotypes, namely dwarfism due to decreased IGF-I levels and impaired IGF-I signaling pathways, delayed puberty, reduced female reproductive function, and blunted mammary gland development (14, 21). Therefore, it was predicted that overexpression of AIB1-{Delta}3 might well increase IGF signaling pathways. Like the p/CIP KO mice, the IGF-IR KO mice exhibit decreased prenatal growth (49). IGF-IR has been shown to be important in mammary gland development because it is required for terminal end bud proliferation in normal mouse mammary gland development (50) and loss of IGF-IR expression inhibits mammary gland ductal development (51). In vitro reduction of AIB1 selectively decreases expression of the IGF-IR gene (25). This interdependence of AIB1 and IGF-IR, also seen in the CMV-AIB1/ACTR-{Delta}3 mice, occurs independent of changes in ER{alpha} expression levels.

IGF-I increases cyclin D1 expression (52). Cyclin D1 is amplified and overexpressed in breast cancer and is hypothesized to be one of the first steps in breast cancer progression (30). In vitro breast cancer cell culture experiments have shown that AIB1 can be recruited to the estrogen-responsive cyclin D1 promoter to enhance cyclin D1 expression (30). Our data provide an in vivo validation of activation by AIB1 of IGF-IR and cyclin D1 signaling pathways as predicted by in vitro studies. Additionally, overexpression of cyclin D1 in MMTV-cyclin D1 transgenic mice also leads to deregulation of cell proliferation in mammary epithelial cells (53), indicating that cyclin D1 is playing a role in the increased proliferation of the mammary glands from the CMV-AIB1/ACTR-{Delta}3 mice. Interestingly, a recent study demonstrated that AIB1 was a coactivator of E2F that is a major inducer of cyclin D1 expression (54). There was no mammary adenocarcinoma development in the CMV-AIB1/ACTR-{Delta}3 mice by 13 months of age, suggesting that time-dependent changes and/or systemic factors may be required for cancer progression when AIB1/ACTR-{Delta}3 is overexpressed at relatively low levels as found in this model. Consistent with this notion, while this manuscript was under revision, a report appeared describing a line of MMTV-AIB1 mice that demonstrate 7.6-fold overexpression of AIB1 in mammary gland tissue in conjunction with elevated serum IGF-I levels (29). These mice begin to develop mammary and other tumors before 1 yr of age. The higher levels of overexpression found in these MMTV-AIB1 transgenic mice also precipitate the development of hyperplasia and accelerated differentiation in the mammary gland, changes that were not found in the lower expressing CMV-AIB1/ACTR-{Delta}3 mice described here. Interestingly both models demonstrated AIB1 or AIB1/ACTR-{Delta}3 mediated increases in mammary epithelial cell proliferation and ductal ectasia. The MMTV-AIB1 mice demonstrate a 50% increase in the fraction of mammary epithelial cells proliferating at 2 wk of pregnancy using bromodeoxyuridine labeling, whereas the CMV-AIB1/ACTR-{Delta}3 mice described here demonstrated an approximately 3-fold increase in the fraction of mammary epithelial cells proliferating in virgin mice using immunohistochemistry for PCNA and phospho-histone H3. Significantly, neither model demonstrated any transgene-mediated changes in prolactin signaling.

The C/EBPß family of transcription factors has been implicated in the regulation of proliferation and differentiation in the mammary gland (41), as well as playing a role in breast carcinogenesis (55). We examined C/EBPß isoforms because transgenic mice with altered levels of the LIP isoform demonstrated mammary gland proliferative phenotypes similar to those we observed in the CMV-AIB1/ACTR-{Delta}3 mice (34, 56). Here, we have shown that overexpression of AIB1/ACTR-{Delta}3 leads to an increase in expression of the inhibitory C/EBPß isoform, LIP, in the CMV-AIB1/ACTR-{Delta}3 mice. Increased LIP expression was found in rodent mammary tumors (57). Overexpression of LIP in a transgenic mice mouse model has been shown to induce mammary epithelial cell proliferation and mammary hyperplasia development by stimulating a growth cascade (34). In addition, increased LIP expression has been found in human breast cancer and correlated with ER negativity and increased proliferation (35, 58). Our data confirm the hypothesis (34) that increases in LIP levels inhibit terminal differen-tiation and lead to proliferation of mammary ducts, which may be an important step in the initiation of carcinogenesis.

Our data show that increased expression of AIB1/ACTR-{Delta}3 did not change ER{alpha} or PR expression levels but did increase expression levels of cyclin D1 and IGF-IR in the proliferating mammary epithelial cells in vivo, consistent with the activation of alternative growth pathways being accessed by AIB1/ACTR-{Delta}3. Consistent with our data, a report appeared while we were preparing this manuscript in which AIB1 contributes to ras oncogenesis in the mammary gland (59). Although ras mutations are not found frequently in many human breast cancers, the data nevertheless indicate an important role for AIB1 in different oncogenic pathways.

Our data show that overexpression of AIB1/ACTR-{Delta}3 by the CMV promoter in these mice resulted in increased proliferation of mammary epithelial cells. Expression of the CMV-driven transgene was detected in the mammary gland, consistent with possible direct effects. Although contributions from possible indirect effects due to expression of AIB1/ACTR-{Delta}3 expression in other tissues cannot be excluded, the transgene did not increase IGF-I serum levels, and there was no evidence of altered prolactin signaling. AIB1/ACTR-{Delta}3 simultaneously activates IGF and cyclin D1 pathways, as well as increasing expression of the LIP isoform of C/EBPß in this model, suggesting that AIB1/ACTR-{Delta}3 works through multiple pathways and different growth factors. The proliferative changes found may also be attributable to potential interaction of AIB1/ACTR-{Delta}3 with other signaling pathways. Interestingly, our data indicate that overexpression of AIB1/ACTR-{Delta}3 alone, although producing a proliferative environment in the mammary gland, an essential first step in the multistep hypothesis of cancer progression, is not sufficient to induce mammary carcinogenesis by 13 months of age. Other genetic manipulations must contribute to the AIB1-{Delta}3 proliferation phenotype in the latter steps of cancer progression to further the development to cancer. This model may be useful in examining events occurring in human tumors that overexpress AIB1-{Delta}3 and in determining the mechanisms leading from AIB1-{Delta}3 to cancer progression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of CMV-AIB1/ACTR-{Delta}3 Transgenic Mice
The expression plasmid for AIB1/ACTR-{Delta}3 was described previously (26). Briefly, nucleotides 267–439 of the full-length AIB1/ACTR are missing, resulting in the loss of the basic helix-loop-helix and per-arnt-sim (PAS) A region of the AIB1/ACTR protein. Expression of AIB1/ACTR-{Delta}3 is directed by the human cytomegalovirus immediate early gene 1 (hCMVIE1) promoter. The vector contains a BGH polyadenylation site. Before introduction into embryos, bacterial sequences contained in the plasmid were removed by digestion with NruI and HaeII (Fig. 1AGo). After gel purification, DNA fragments were injected into the pronucleus of one cell stage FVB inbred mouse embryos (Taconic, Germantown, NY). Embryos were reimplanted into the oviducts of pseudo-pregnant foster mothers and allowed to develop to term. To identify founder animals, tail DNA was screened by Southern blot analysis for incorporation of the transgene. The mice were housed and bred in a specific pathogen-free facility, maintained on a 12-h light, 12-h dark schedule, and food and water were provided ad libitum. All procedures involving animals were performed in accordance with current federal and university guidelines and were reviewed and approved by the Georgetown University Institutional Animal Use and Care Committee.

DNA Isolation, Southern Blot Analysis, Genotyping, and Breeding of CMV-AIB1/ACTR-{Delta}3 Mice
DNA was isolated from tails by overnight digestion with proteinase K at 55 C (15 µg/ml proteinase K, 10 nM Tris-HCl, 100 mM NaCl, 50 nM EDTA, 1% sodium dodecyl sulfate) and extracted by phenol/chloroform and precipitated by ethanol. For Southern blot analysis, genomic DNA was digested with KpnI and DraIII before they were processed following standard laboratory procedures. For specific detection of the transgene, the AIB1/ACTR probe was prepared by random priming of a 678-bp EcoRI fragment from ACTR. After the founder mouse had been identified, subsequent analysis for the presence of the transgene was performed by PCR with primers designed against the T7 promoter (PCR sense primer: 5'-TAATACGACTCACTATAGGG-3') and the AIB1 sequence (PCR antisense primer: 5'-ATGTTTCCGTCTCGATTCACC-3') (Fig. 1Go). The PCR conditions were 95 C for 5 min followed by 35 cycles of 95 C for 30 sec, 52 C for 30 sec, and 72 C for 1 min. Transgenic mice were propagated in a breeding colony under continuous observation. Male and female mice bred normally. There was no evidence of infertility in either male or female mice. Female mice delivered pups and lactated normally.

Analysis of Transgene Expression by RT-PCR and in Situ Hybridization
Mammary glands from 7-wk-old WT and CMV-AIB1/ACTR-{Delta}3 transgenic mice were snap-frozen in liquid nitrogen at the time of necropsy for RNA and protein analyses. Total RNA from mammary glands was isolated with the RNAeasy kit after the manufacturer’s instructions (QIAGEN, Valencia, CA). To prevent contamination by genomic DNA, samples were treated with deoxyribonuclease (QIAGEN). RT-PCR was performed using the thermoscript one-step RT-PCR kit (Invitrogen Life Technologies, Carlsbad, CA). To ensure absence of genomic DNA, samples were run in parallel without reverse transcriptase. For specific detection of the transgene, one oligonucleotide primer was designed against the T7 promoter (5'-TAATACGACTCACTATAGGG-3'), and the downstream primer was directed against the AIB1 sequence (5'-GAGTCCACCATCCAGCAAGTATT-3'). The RT-PCR conditions were 55 C for 30 min and 95 C for 5 min, followed by 40 cycles of 95 C for 30 sec, 55 C for 30 sec, and 72 C for 30 sec. The PCR products were separated by electrophoresis on a 1% agarose gel, transferred to a polyvinylidene difluoride membrane, hybridized with a 32P-labeled oligonucleotide probe (5'-GAAACTCGCCGCTCAGCC-3') internal to the primers, and visualized by autoradiograghy on standard x-ray film. The mRNA-specific amplification yielded a 184-bp product. To confirm mRNA expression of the specific transgene transcript for human AIB1/ACTR-{Delta}3 in the mammary epithelial cells, in situ hybridization with digoxigenin-labeled riboprobes was performed. The probe sequence consisted of 300 bp: 140 bp of the human AIB1/ACTR-{Delta}3 comprising of nucleotide sequences not present in the mouse genome and 160 nucleotides that include the BGH polyadenylation site. The 300-bp sequence was amplified by PCR using the AIB1/ACTR-{Delta}3/pcDNA3 plasmid as template. Two separate PCRs were performed for the antisense and sense template. For usage in the consecutive reverse transcription, either the forward or reverse primer included a T7 promoter sequence 5' to the template-specific sequence. Digoxigenin-labeled sense and antisense riboprobes were made from the two resulting DNA templates using the DIG RNA labeling kit (Roche Diagnostics Corp., Indianapolis, IN) according to the protocol provided. Tissue slides were stained by in situ hybridization using the constructed riboprobes and a previously described protocol (60) was used with modifications. In brief, slides were deparaffinized, tissue proteins were digested with proteinase K and acetylated. The RNA probe was mixed with hybridization solution (Sigma, St. Louis, MO) (1.5 ng probe/1.0-µl solution) and incubated with the tissue for 16–18 h at 42 C. Slides were washed and digested with RNase A (Roche). Refixation and cross-linking were performed by treating with formamide/2x sodium chloride-sodium citrate (SSC) solution (1:1) for 10 min at 52 C followed by two 5-min washout steps at 52 C in 1x SSC and 0.5x SSC. After blocking, a solution of alkaline phosphatase-tagged antidigoxigenin antibody fragments (Roche) in buffer I [100 mM Tris-HCl (pH 7.5) and 150 mM NaCl] (1:250) was applied and incubated with the tissue for 16–18 h at 4 C. After washing with buffer I, a staining solution was applied, consisting of 0.375 mg/ml nitroblue tetrazolium (Roche) and 0.175 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (Roche) diluted in buffer II [100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl2]. Once sufficient staining was observed, the reaction was terminated by rinsing with buffer III [10 mM Tris-HCl (pH 8.0) and 1 mM EDTA] for 10 min. Slides were washed in 0.5% Tween 20 and water for 5 min each and then allowed to dry completely before mounting with sealing solution and cover slips. Image capturing was performed with a x20 objective (Zeiss, Oberkochen, Germany) and a 4-megapixel digital camera (Scion Corp., Frederick, MD) mounted on a microscope (Zeiss). mRNA expression was then evaluated in breast epithelial cells, always in conjunction with the sense (control) probe. Results were categorized as follows: 1) negative (–)—antisense probe signals comparable to sense probe in intensity; 2) positive (+)—strong antisense probe signals, exceeding sense probe intensity in more than 50% of the cells.

Mammary Gland Measurements, Whole Mount Analysis, Necropsy and Pathology Studies
Female mice were euthanized and necropsies performed when they reached 7 wk of age (8 WT, 11 CMV-AIB1/ACTR-{Delta}3) or 10 or 13 months (12 WT, 12 CMV-AIB1/ACTR-{Delta}3). After determining the whole body weight, both no. 4 mammary glands were removed and weighed separately. One no. 4 mammary gland from each mouse was whole mounted by fixing in Carnoy’s solution and staining in carmine alum as previously published (61). The other no. 4 mammary gland was fixed in 10% formaldehyde and embedded in paraffin using standard techniques. Sections (5 µm) were cut for H&E staining and histological analyses. A full necropsy was performed to examine mice for the presence of lesions in the female reproductive tract including ovaries, uterus, and fallopian tubes, the lungs, liver, kidney, lymph nodes, and skin. Ovaries, fallopian tubes, and uteri were fixed in 10% formaldehyde and embedded in paraffin using standard techniques. Sections (5 µm) were cut for H&E staining and histological analyses. Sections of ovarian and uterine tissue were microscopically examined to determine whether they exhibited any abnormal histological changes. Mammary gland whole mounts were used to examine for primary, secondary and tertiary ductal branching patterns, ductal ectasia, and for the presence of alveolar differentiation. H&E-stained slides were examined for the presence of normal ductal structures, ductal ectasia, alveolar differentiation and secretory mammary epithelial cells. On whole mounts, normal mammary gland development is indicated by a normal ductal branching pattern triggered by estrogen stimulation during puberty. On whole mounts, activation of progesterone signaling is suggested by an increase in tertiary branching and alveolar differentiation. On whole mounts, activation of prolactin signaling is indicated by alveolar differentiation. On H&E-stained sections, activation of prolactin signaling is indicated by the appearance of secretory mammary epithelial cells. Image capturing of mammary gland whole mounts was performed with 0.5x (whole mammary gland) and 4x (close-up) objectives (Nikon Instruments, Inc., Melville, NY) and a DXM1200F digital camera (Nikon) mounted on a Nikon Eclipse E800M microscope. Image capturing of H&E-stained sections was performed with a x40 objective (Nikon) using a DXM1200F digital camera (Nikon) mounted on a Nikon Eclipse E800M microscope.

PCNA, Phospho-Histone H3, Cyclin D1, ER{alpha}, PR, and Stat5a Immunohistochemical Analyses
Immunohistochemical detection of PCNA expression in mammary epithelial cells was performed as a relative measurement of cellular proliferation as previously described (61) on WT (n =12) and CMV-AIB1/ACTR-{Delta}3 (n =14) mice. Briefly, for PCNA analyses, paraffin-embedded mammary gland tissue sections were deparaffinized, rehydrated, quenched with 3% hydrogen peroxide, exposed to two drops of the enhanced polymer system (EPOS) PCNA immunostaining system (U7032, DAKO, Carpinteria, CA) for 1 h at room temperature, stained with the diaminobenzidine peroxidase substrate kit (SK-4100, Vector Laboratories, Inc., Burlingame, CA) for 5 min, counterstained with hematoxylin, and mounted with glycerol vinyl alcohol mount.

Tissue sections from 6-month-old animals (2 WT, 1 CMV-AIB1/ACTR-{Delta}3), 8-month-old animals (8 WT, 1 CMV-AIB1/ACTR-{Delta}3), and 13-month-old animals (2 WT, 12 CMV-AIB1/ACTR-{Delta}3) for other immunohistochemical analyses were deparaffinized, rehydrated and antigens exposed with high-pH target retrieval solution (S3307, DAKO) and high temperature (cyclin D1 and phospho-histone H3) or with a Decloaking Chamber (Biocare Medical, Walnut Creek, CA) (ER{alpha} and Stat5a). Detection of cyclin D1, ER{alpha}, and PR protein expression in mammary epithelial cells was accomplished using the Mouse on Mouse (M.O.M.) peroxidase kit (PK-2200; Vector) using a 1:50 dilution of the mouse monoclonal cyclin D1 antibody (sc8396; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or a 1:50 dilution of the mouse monoclonal ER{alpha} antibody (IM2133; Beckman Coulter Immunotech, Miami, FL) according to the manufacturers’ directions and as previously published (61). Detection of phospho-histone H3 (serine 10) mitosis marker and Stat5a in mammary epithelial cells were accomplished using the rabbit IgG peroxidase kit (PK-6101; Vector) according to the manufacturer’s directions using a 1:10,000 dilution of the rabbit polyclonal phospho-histone H3 (Ser10), Mitosis Marker antibody (06–570; Upstate Biotechnology, Waltham, MA) or a 1:125 dilution of the rabbit polyclonal Stat5a antibody (sc-1081, Santa Cruz). Digital photographs of immunohistochemistry were taken using the Nikon Eclipse E800M microscope setup with Nikon DMX1200 camera and software (Nikon). The percentage of positive PCNA, phospho-histone H3, or cells with nuclear-localized cyclin D1 within a minimum of 1000 mammary epithelial cells was determined.

AIB1, IGF-IR, and C/EBPß Immunoblot Analysis
Tissue lysates from 10- and 13-month-old WT and CMV-AIB1/ACTR-{Delta}3 mice were obtained by disrupting approximately 0.1 g of snap-frozen mammary gland tissue in 1 ml of Nonidet P-40 containing lysis buffer with a homogenizer. Protein concentrations were determined by the Bradford procedure and equal portions were resolved on denaturing 4–20% polyacrylamide gradient Tris-glycine gels for IGF-IR and C/EBPß blots. Eighty micrograms of mammary gland samples for the AIB1 blot were resolved on precast 4% gels. Equal loading of samples was verified by Coomassie staining (data not shown). The separated proteins were transferred to a polyvinylidene difluoride membrane and then subjected to primary and secondary antibodies: AIB1 mouse monoclonal antibody (611105, BD Transduction Laboratories, San Diego, CA) (1:500 dilution), SRC3 rabbit polyclonal antibody (ab10310, Abcam Inc., Cambridge, MA) (1:1000 dilution), ACTR rabbit polyclonal antibody (sc-13066, Santa Cruz) (1:500 dilution), insulin-like growth factor 1 receptor (IGF-IR) antibody (3022, Cell Signaling Technology, Inc., Beverly, MA) (1:1000 dilution), or a polyclonal antibody specific for the carboxy terminus of C/EBPß (C-19; Santa Cruz) (1:200 dilution) and horseradish peroxidase-conjugated secondary antibody at a 1:5000 dilution for AIB1 or a 1:7500 dilution for IGF-IR and C/EBPß. Blots were incubated with an enhanced chemiluminescence reagent (Promega, Madison, WI) and exposed to hyperfilm ECL. Three different AIB1-specific antibodies (AIB1, SRC3, and ACTR above) were used to evaluate the relative levels of endogenous mouse AIB1 protein in WT and transgenic mice in comparison to the AIB1/ACTR-{Delta}3 protein expression in the transgenic animals. Protein extracts from the 32D mouse hematopoetic cell line and cell lines transfected with CMV-AIB1/ACTR-{Delta}3 and CMV-AIB1/ACTR plasmid vectors were used as controls. Signal intensity was measured by densitometry for IGF-IR and C/EBPß. After quantification using densitometry, the blot was stripped and reprobed with an actin antibody (MAB1501R, Chemicon, Temecula, CA) at a 1:3000 dilution. Relative levels of IGF-IR were obtained by dividing densitometry readings from IGF-IR by the corresponding actin value and adjusting the mean of this ratio to 1 for WT animals. Levels of LAP and LIP were quantified from Western blot analyses using densitometry to give a LAP to LIP expression ratio. Relative levels of LAP were obtained by dividing densitometry readings from LAP by the corresponding actin value and adjusting the mean of this ratio to 1 for WT animals. To ensure interassay comparability, portions of a pooled whole cell lysate of 32D cells were loaded on each gel and analyzed in parallel.

Serum IGF ELISA
Blood was obtained from WT (n =14) and CMV-AIB1/ACTR-{Delta}3 (n =15) 7-wk-old mice, by decapitation at the time of necropsy, allowed to clot and spun down at 3000 rpm for 20 min at 4 C. Serum was then removed and stored at –20 C until further analysis. IGF-I levels were measured using a commercially available enzyme immunoassay kit (ELISA) provided by Diagnostic Systems Laboratories (Webster, TX) according to the manufacturer’s protocol. A standard curve was included with each assay, and absorbance was measured at dual wavelengths for background correction.

Statistical Analysis
Means and SEM were calculated using SPSS, version 11.0.0 (SPSS, Inc., Chicago, IL). Student’s t tests were used to compare mammary gland and body weights, immunohistochemical analyses, and immunoblot analyses (SPSS, version 11.0.0). Mann-Whitney U t test was used to compare LAP to LIP ratios (SPSS, version 11.0.0).


   ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Angera Kuo (Georgetown University, Washington, DC) for the mouse 32D cell protein extracts. We acknowledge the assistance of the Transgenic, Animal Research, and Histopathology Shared Resources at the Lombardi Cancer Center.


   FOOTNOTES
 
This work was supported by grants from the Breast Cancer Research Program of the Department of Defense awards (DAMD17-99-1-9203, to A.T.R.; DAMD17-01-1-0310, to P.A.F.; and DAMD17-02-10394, to A.S.O.) and a Breast Cancer Alliance Grant (to A.T.R. and P.A.F.). M.T.T. was supported by the National Institutes of Health T32 grant to the Lombardi Cancer Center at Georgetown University. R.T.H. was partially funded by a grant from the German Department of Education and Research. These resources are supported in part by a Cancer Center Support Grant from the National Cancer Institute (P30-CA51008).

First Published Online November 18, 2004

1 M.T.T. and R.R. contributed equally to this paper. Back

2 P.A.F. and A.T.R. contributed equally as senior authors on this paper. Back

Abbreviations: ACTR, Activator of thyroid hormone and retinoid receptors; AIB1, amplified in breast cancer 1; BGH, bovine GH; C/EBPß, CCAAT/enhancer binding protein; CMV, cytomegalovirus; ER{alpha}, estrogen receptor {alpha}; GRIP1, glucocorticoid receptor-interacting protein 1; hCMVIE1, human CMV immediate early gene 1; H&E, hematoxylin and eosin; IGF-IR, IGF-I receptor; KO, knockout; LAP, liver-enriched activating protein; LIP, liver-enriched inhibitory protein;MMTV, mouse mammary tumor virus; PCNA, proliferating cell nuclear antigen; p/CIP, p300/CBP (cAMP response element binding protein-binding protein)/cointegrator-associated protein; PR, progesterone receptor; RAC3, receptor-associated coactivator 3; SRC, steroid receptor coactivator; SSC, sodium chloride-sodium citrate; TIF2, transcription intermediary factor 2; TRAM-1, thyroid hormone receptor molecule-1; WT, wild-type.

Received for publication March 12, 2004. Accepted for publication November 12, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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