Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention AACR Conference on Molecular Diagnostics - 2008
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[Cancer Research 64, 7169-7177, October 1, 2004]
© 2004 American Association for Cancer Research

Endocrinology

Haploid Inactivation of the Amplified-in-Breast Cancer 3 Coactivator Reduces the Inhibitory Effect of Peroxisome Proliferator-Activated Receptor {gamma} and Retinoid X Receptor on Cell Proliferation and Accelerates Polyoma Middle-T Antigen-Induced Mammary Tumorigenesis in Mice

Hao Zhang, Shao-Qing Kuang, Lan Liao, Suoling Zhou and Jianming Xu

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The amplified-in-breast cancer 3 (AIB3) is a nuclear receptor coactivator amplified and overexpressed in human breast cancers. AIB3–/– mice die during gestation, whereas AIB3+/– mice exhibit normal development. Here, we demonstrate that AIB3 protein is mainly located in the nuclei of mammary epithelial cells and tumor cells and its levels are elevated in mammary epithelial cells at middle pregnant stage and in mammary tumor cells. To examine whether AIB3 reduction affects mammary tumorigenesis, we generated wild-type mouse mammary tumor virus/polyoma middle-T (WT/PyMT) and AIB3+/–/PyMT mice. Mammary tumor development in AIB3+/–/PyMT female and male mice was substantially accelerated compared with that in WT/PyMT mice, because of increased cell proliferation in early tumorigenic lesions, including ductal hyperplasia and mammary intraepithelial neoplasia. Tumor formation in nude mice that received premalignant AIB3+/–/PyMT mammary tissue was much faster than in nude mice that received transplants of premalignant WT/PyMT mammary tissue, which indicated that the accelerated tumorigenesis in AIB3+/–/PyMT mammary glands is due to a mammary epithelial autonomous defect. Expression of PyMT, estrogen receptor {alpha} and estrogen receptor {alpha}-regulated genes was unaffected in AIB3+/–/PyMT mammary glands, which suggests that the acceleration of mammary tumor formation in AIB3+/–/PyMT mice was not a consequence of changes in PyMT expression or in estrogen receptor function. Importantly, the inhibitory effects of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and retinoid-X receptor (RXR) ligands on AIB3+/–/PyMT cell proliferation and the transcriptional function of PPAR{gamma} in AIB3+/–/PyMT cells were reduced. Thus, AIB3 haplodeficiency may facilitate PyMT-induced tumorigenesis through a partial impairment of PPAR{gamma} and RXR function. These results suggest that AIB3 may be a tumor suppressor that is required for the inhibition of cell proliferation by PPAR{gamma} and RXR.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In human breast cancers, several chromosomal regions including 20q11–12 and other regions encoding the erbB2, c-myc, and cyclin D1 protooncogenes are commonly amplified and overexpressed (1, 2, 3, 4) . In the region of 20q11–12, three genes, amplified-in-breast cancer 1 (AIB1), AIB3, and AIB4, have been identified (2) . AIB1 [p/CIP, RAC3, ACTR, TRAM1 or steroid receptor coactivator (SRC)-3 is a transcriptional coactivator for nuclear receptors and certain other transcription factors (5 , 6) . Knockdown of AIB1 in breast cancer cells inhibits cell proliferation and knockout of AIB1 in mice strongly suppresses the v-Ha-ras-induced mammary tumorigenesis, which suggests that AIB1 is an oncogene (5 , 7 , 8) . On the other hand, AIB3 was found amplified in 10% of breast cancers, 30% of colon cancers, and 13% of lung cancers, which suggests that altered expression of AIB3 may play a role in tumorigenesis (1 , 2 , 9) . However, thus far, there is no in vivo evidence demonstrating functional roles for AIB3 in tumorigenesis.

Interestingly, although the sequence of AIB3 is not homologous to AIB1, AIB3 (ASC-2, PRIP, RAP250, TRBP, or NRC) also is a potent transcriptional coactivator (9, 10, 11, 12, 13) . AIB3 interacts and coactivates not only many nuclear receptors, including peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), retinoid-X receptor (RXR), retinoic acid receptor (RAR), thyroid hormone receptors (TRs), vitamin D receptor (VDR), glucocorticoid receptor (GR), estrogen receptors (ERs), and liver X receptor (9, 10, 11, 12, 13, 14) , but also a number of other transcription factors including AP-1, nuclear factor-{kappa}B, serum response factor, cAMP response element binding protein, CCAAT/enhancer binding protein {alpha}/EBP{alpha} and E2F-1 (10 , 15, 16, 17) . Recent studies have suggested that AIB3 may enhance transcription through interacting and/or recruiting multiple coactivator complexes, such as SRC-1/cAMP response element-binding protein binding protein complex, activating signal cointegrator 2 complex and TR-associated protein or VDR-interacting protein complex, and thereby facilitating chromatin remodeling and general transcription factor assembly (9, 10, 11, 12, 13 , 18) . Furthermore, AIB3 interacts with the coactivator activator (COAA), and COAA contains an RNA recognition domain and modulates RNA splicing patterns in a nuclear receptor-dependent manner (19 , 20) . AIB3 also interacts with a DNA-dependent protein kinase and regulates its kinase activity. The DNA-dependent protein kinase is a heterotrimeric nuclear phosphatidylinositol 3-kinase that functions in DNA repair, recombination, and transcriptional regulation (21) . These findings suggest that through modulating transcriptional activities of many transcription factors, AIB3 may be involved in multiple signaling pathways that regulate a variety of biological and pathological processes in response to hormones, growth factors, cytokines, and environmental stimuli.

Although AIB3 is widely expressed, its expression levels are tissue- and cell type-specific, which suggests that AIB3 may mediate the function of transcription factors and modulate gene expression in a cell type-specific manner (22) . We and others have shown that inactivation of both of the AIB3alleles in mice results in embryonic lethality due to defects in placental, cardiac, and hepatic development (23, 24, 25) . These phenotypes are similar to those encountered in mice lacking PPAR{gamma} or PPAR{gamma}-binding protein (26 , 27) . These findings suggest that AIB3 is an essential coactivator that may mediate PPAR{gamma} functions in vivo. However, because of the early lethality, the in vivo role of AIB3 in late developmental stages and adulthood is still unknown.

Mouse models are powerful tools to study mechanisms of mammary tumorigenesis. The mouse mammary tumor virus long terminal repeat (MMTV)-directed expression of the polyoma middle-T oncoprotein (PyMT) in the mammary luminal epithelium induces multifocal mammary tumors because of its ability to associate with and activate the Src family kinases, the phosphatidylinosital 3'-OH kinase (PI3K) and the Shc adapter protein (28, 29, 30, 31) . Although the mammary tumorigenesis is rapid in MMTV/PyMT transgenic mice, detailed analysis indicates that the PyMT mammary tumor model recapitulates many characteristic morphologies and typical biomarker expression associated with poor prognosis (32 , 33) .

In this study, we have evaluated the effect of haploid inactivation of AIB3 on PyMT-induced mammary tumorigenesis through generating AIB3+/–/PyMT bitransgenic mice. We demonstrate that AIB3 is highly expressed in mammary epithelial cells and tumor cells. Haploid inactivation of AIB3 substantially accelerated the development of mammary tumors in both female and male mice because of desensitization of mammary epithelial cells and tumor cells to the inhibition of cell proliferation mediated by ligand-activated PPAR{gamma} and RXR.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mouse Breeding and Genotyping.
Animal protocols were approved by the Animal Care and Use Committee for Baylor College of Medicine. AIB3+/– mice with a 129SVEV strain background were generated as described previously (23) . AIB3–/– mice were lethal at middle gestation stages (23) . In this study, AIB3+/– mice were first bred to wild-type FVB mice, and their female offspring were paired with male MMTV/PyMT (PyMT) transgenic mice with a FVB strain background from The Jackson Laboratory (Bar Harbor, ME; ref. 28 ). Female AIB3+/– and male AIB3+/–/PyMT mice were identified by PCR analyses of tail DNA samples, as described previously (23 , 28) , and were randomly paired to generate WT/PyMT and AIB3+/–/PyMT mice for experiments. On average, these mice had a mixed genetic background of 75% FVB and 25% 129SVEV. Whenever it was possible, littermates with both genotypes were used in the same experiment.

Mammary Tumor Detection and Measurement.
Mice were examined twice a week for mammary tumors by eye examination and finger palpation. Tumor length (L) and width (W) were measured twice a week by calipers. Tumor volume was estimated by (L x W2)/2, as described previously (34) . Mice were euthanized at different stages of mammary tumorigenesis, and their mammary glands and tumors were collected for morphologic and biochemical analyses.

Generation of AIB3 Recombinant Proteins and Polyclonal Antibodies.
The cDNA fragments encoding three AIB3 peptides, AIB3-P1 (Gly22–Asp146), AIB3-P2 (Gly903–Gln1029), and AIB3-P3 (Glu1954–Lys2067), were fused to the coding sequence of glutathione S transferase (GST) in pGEX-2T plasmid, as described previously (35) . Fusion proteins were produced from transformed bacteria and were purified with the glutathione affinity column. New Zealand rabbits were primed with Freund’s complete adjuvant after blood samples were collected for the production of preimmune serum. Primed rabbits were immunized by injection of the purified recombinant proteins mixed with Freund’s incomplete adjuvant, as described previously (35) . Antiserum was made from blood samples collected 12 days postinjection and were tested by immunoblotting and immunohistochemistry analysis.

Morphologic Analysis and Immunohistochemistry.
For whole-mount staining, the inguinal mammary fat pads were dissected, fixed in acidic EtOH, and stained with carmine alum (36) . For histologic analysis, tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned in a thickness of 5 µm, and stained with hematoxylin and eosin. Immunohistochemistry was carried out by following the indirect-enzyme labeling procedure (22) . For immunohistochemistry and Western blot, rabbit antiserum against AIB3 was affinity-purified with a column conjugated with AIB3 recombinant protein. Polyclonal antibodies against ER and progesterone receptor (PR) were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Polyclonal antibodies against phospho(S10)-Histone H3 and monoclonal antibody against PyMT were purchased from Upstate (Charlottesvile, VA) and Oncogene Research (San Diego, CA), respectively. For the detection of apoptotic cells, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay kit was purchased from Roche Molecular Biochemicals (Indianapolis, IN) and the assay was carried out as described previously (37) .

Analysis of mRNA.
For RNase protection assay, total RNA was extracted from mammary glands and tumor tissues, and the assay was carried out with a RNase protection assay III kit (Ambion, Austin, TX). Antisense RNA probes for PyMT, keratin 18 (K18), and cyclophilin A were transcribed with a Maxiscript kit containing T7 RNA polymerase (Ambion). The lengths of mRNA protected by PyMT, K18, and cyclophilin A probes were 130, 160, and 103 nucleotides, respectively. The expression level of K18 was used to normalize gene expression levels in mammary epithelial cells. The cyclophilin expression level was used to normalize gene expression in all of the cell types. For semiquantitative reverse transcription-PCR (RT-PCR), 1 µg of total RNA was reverse transcribed into cDNA with the Superscript II kit (Invitrogen). The PCR primer sets for L19 mRNA were 5'-CTGAAGGTCAAAGGGAATGTG and 5'-GGACAGAGTCTTGATCTC. The primer sets for K18 were 5'-CAAGATCATCGAAGACCTGAGGGC and 5'-TGTTCATACTGGGCACGGATGTCC. The primer sets for lipocalin 2 (LCN2; ref. 38 ) were 5'-CTCTTCCTCCTCCAGCACAC and 5'-CACACTCACCACCCATTCAG. PCR analyses were conducted on a Mastercycler (Eppendorf) under the following conditions: 94°C for 50 seconds, 55°C for 40 seconds, 72°C for 90 seconds, 20 cycles for L19 and keratin 18 or 25 cycles for LCN2. Real-time RT-PCR was performed with the One Step Master Mix reagent (Applied Biosystems, Foster City, CA) and the mouse AIB3-specific primer sets and TaqMan probe as described previously (22) .

Mammary Tissue Transplantation.
Transplantation of mammary tissues was conducted as described previously (32) . Briefly, about 1 mm3 of donor tissue was excised from a region between the mammary lymph node and dorsal edge and was implanted into an ectopic site under the dorsal skin and an orthotopic site in the fourth mammary fat pad of the recipient nude mice. The age of donor mice was 6 weeks.

Southern and Western Blot Analyses.
Genomic DNA was extracted from mouse-tail tips or mammary tumors. Southern blot analysis was done, as described previously, to identify wild-type and targeted AIB3 alleles (23) . For Western blot analysis, fresh tissues were frozen in liquid nitrogen and were ground into powders for protein extraction (23) . Tissue lysates containing 10 µg of protein were separated by SDS-PAGE and blotted onto nitrocellulose membranes. The membranes were incubated with a 1:1000 dilution of the rabbit anti-AIB3 IgG as described (10 , 23) . The bond primary antibody was visualized by reacting with a goat antirabbit IgG conjugated with the horseradish peroxidase (1:5000, Bio-Rad, Hercules, CA) and reagents in an Enhanced Chemiluminescence (ECL) Kit (Amersham Biosciences, Piscataway, NJ).

Isolation, Culture, and Analysis of Mammary Tumor Cells.
Primary mammary tumor cells were isolated from mammary tumors in WT/PyMT and AIB3+/–/PyMT female mice and were cultured, as previously described (39) . To reduce fibroblasts and enrich epithelial tumor cells, we treated cells in initial cultures with 0.25% trypsin-EDTA and replated. After 15 minutes, cell suspension was transferred to another dish for 20 minutes before the cells were cultured for growth. Enriched primary tumor cells harvested from the second passage were plated in 60-mm dishes and were treated with 10–5 mol/L troglitazone (Sigma, St. Louis, MO), 10–7 mol/L all-trans-retinoic acid or 10–7 mol/L 9-cis-retinoic acid (9-cis-RA) for 3 days. Treated cells were washed in PBS and fixed in 4% paraformaldehyde for 30 minutes on ice. For immunocytochemistry, the fixed cells were incubated with primary antibodies against phospho(S10)-Histone H3 and secondary antibodies (1:1000) labeled with the Texas Red fluorescein. After being washed with PBS, slides were counterstained with 4',6-deamidine-2-phenylindole (DAPI) and mounted in Vectashield (Vector, Burlingame, CA).

Transfection Assays.
Primary WT/PyMT and AIB3+/–/PyMT tumor cells were seeded at 40% confluence in DMEM containing 10% charcoal-stripped fetal calf serum for 1 day and were harvested by trypsinization. Cells were washed and resuspended in PBS (6 x 106 cells/0.9 ml). Each 0.9 ml of cell suspension was mixed with pSGS-human PPAR{gamma} (hPPAR{gamma}; 5 µg), pSGS-DM1-hRXR{alpha} (5 µg), and pRSV-ßGal (1 µg) expression plasmids and the GS-tk-Luc PPAR{gamma} reporter plasmids (5 µg). Cell and DNA mixtures were electroporated at 100 volts and 200 µF, were cultured for 12 hours, and then treated with troglitazone (10–5 mol/L) and 9-cis-RA (10–7 mol/L) for 24 hours. Cell lysates were prepared and luciferase activity was measured with a luciferase assay kit (Promega). The ß-galactosidase activity in the same amount of cell lysate also was measured. Relative luciferase units were normalized to the ß-galactosidase activity.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of AIB3 Polyclonal Antibodies and Detection of High AIB3 Immunoreactivities in Mammary Epithelial Cells and Mammary Tumor Cells.
To generate AIB3 polyclonal antibodies for immunohistochemistry, we produced and purified three GST-AIB3 fusion proteins, AIB3-P1, AIB3-P2, and AIB3-P3 (Fig. 1ACitation , Lanes 1 and 2; and data not shown). By using these fusion proteins as antigens, we generated rabbit-anti-AIB3 antiserum. All three antisera reacted with GST-AIB3 fusion proteins and GST protein in immunoblotting analysis (Fig. 1ACitation , Lanes 3 and 4; and data not shown). After serum samples were adsorbed through GST columns and were purified with AIB3 recombinant protein affinity columns, the anti-AIB3 polyclonal antibodies reacted specifically with AIB3 recombinant proteins but not with GST (Fig. 1ACitation , Lanes 5 and 6; and data not shown). These antibodies immunoprecipitated and detected the AIB3 protein (~240 kDa) in human ZR-75-1 breast cancer cells and wild-type mouse embryonic fibroblasts but not in the AIB3–/– mouse embryonic fibroblasts lacking AIB3 (Fig. 1ACitation , Lanes 7, 8, and 9), indicating that these antibodies can specifically recognize human and mouse AIB3 protein.



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  		Fig. 1. Generation of AIB3 antibodies and immunochemical analysis of AIB3. A, Lanes 1 and 2, gel image showing Coomassie-stained bacterial lysate (Lane 1) and purified GST-AIB3-P2 fusion protein (P2, Lane 2). Lanes 3 and 4, the AIB3 antiserum reacted with GST (Lane 3) and GSTAIB3-P2 (P2, Lane 4) proteins on an immunoblot. Lanes 5 and 6, affinity-purified AIB3 antibodies specifically reacted with AIB3-P2 protein in an immunoblot. Lanes 7–9, detection of AIB3 protein in ZR-75-1 (Lane 7), wild-type mouse embryonic fibroblasts (Lane 8) but not in AIB3–/– mouse embryonic fibroblasts (Lane 9). AIB3 in cell lysates was immunoprecipitated with AIB3-P2 antiserum and then were assayed by immunoblotting with the affinity-purified AIB3-P2 antibodies. The Immunoglobulin G (IgG) band reflects the added rabbit serum at the immunoprecipitation step. B, immunohistochemistry. a–d, AIB3 immunoreactivity was detected by AIB3-P2 antibodies in the mouse mammary glands at ages of 6 weeks (a), 10 weeks (b), pregnant day 10 (c), and lactation day 8 (d). e, a strong AIB3 immunoreactivity detected by AIB3-P2 antibodies in mammary glands of PyMT transgenic mice. f, the negative control for immunohistochemistry with preimmune serum and a tissue section prepared from mammary glands of PyMT mice. g and h, immunohistochemical staining of ER{alpha} (g) and PR (h) in mammary glands of 10-week-old mice. *, some of the positively stained mammary luminal epithelial cells. Scale bar in f, 50 µm for a–f. Scale bar in h, 50 µm for g–h. (TEB, terminal end bud; MD, mammary duct; AO, mammary alveola or acinus; + and –, places in which mammary luminal epithelial cells are positive (+) or negative (–) to AIB3 immunostaining; Hy, hyperplasia.)

 
High levels of AIB3 immunoreactivity were detected mainly in the nuclei of the body cells and cap cells of the terminal end buds in the mammary glands of young mice and in the mammary and myoepithelial cells in both young and adult mice Fig. 1B–a and –b)Citation . The AIB3 immunoreactivity in the mammary epithelial cells was slightly increased at middle pregnant stage but significantly decreased at lactation stage (Fig. 1B–c and –d)Citation . At lactation day 8, most nuclei of the acinar secretory epithelial cells manifested a relatively weak AIB3 immunoreactivity, and a small proportion of these cells were negative to AIB3 immunostaining (Fig. 1B–d)Citation . Interestingly, as compared with the immunostaining in normal mammary epithelial cells, AIB3 immunoreactivity was significantly stronger in the mammary epithelial proliferative lesions induced by PyMT, including mammary epithelial hyperplasia, mammary intraepithelial neoplasia (MIN), and ductal carcinoma (Fig. 1B–eCitation ; and data now shown). These results demonstrated that AIB3 protein mainly presents in the nuclei of mammary epithelial compartments, and its cellular concentration is much higher in the mammary tumor cells.

In contrast to AIB3 that was detected in all of the mammary epithelial cells of the virgin mammary gland, ER{alpha} and PR proteins were detectable only in about 40% of mammary epithelial cells. These ER{alpha}- and PR-positive mammary epithelial cells were scattered among those mammary epithelial cells negative to ER{alpha} and PR (Fig. 1B–g and –h)Citation . Therefore, we conclude that AIB3 is expressed in mammary epithelial cells both positive and negative with regard to ER{alpha} and PR.

Generation of AIB3+/–/PyMT Mice.
AIB3–/– mice die at middle gestation stage, but both female and male AIB3+/– mice exhibit normal development and reproductive function (23) . The morphology and extent of mammary gland development were also comparable between wild-type and AIB3+/– mice (data not shown). Because AIB3 is a nuclear receptor coactivator overexpressed in human breast cancers, alteration of AIB3 levels may affect breast tumorigenesis through modulating nuclear receptor functions. To test this hypothesis, we generated AIB3+/–/PyMT and WT/PyMT mice with 75% FVB and 25% 129SVEV strain background. Most of the AIB3+/–/PyMT and WT/PyMT mice that were used in the comparisons were matched littermates. All of the mice were heterozygous to the PyMT transgene. The PyMT breast tumor model has been well established; in the model, expression of the PyMT transgene, driven by MMTV in mammary epithelial cells, induces rapid production of multifocal mammary adenocarcinomas with nearly 100% frequency (28 , 32) .

Mammary Tumorigenesis Was Significantly Accelerated in Both Female and Male AIB3+/–/PyMT Mice.
No tumors were found in wild-type and AIB3+/– mice without PyMT, which indicated that haploid inactivation of AIB3 is not tumorigenic. In the female WT/PyMT group, palpable mammary tumors were detected at the age of 44 days in one mouse and the age of 96 days in all mice. Fifty percent of these mice developed palpable mammary tumors at the age of 72 days. However, in the female AIB3+/–/PyMT group, palpable mammary tumors were developed at 29 days in the first mouse, 48 days in 50% of the mice, and 66 days in all of the mice (Fig. 2A)Citation . These observations demonstrate that haploid disruption of the AIB3 gene significantly accelerates mammary tumorigenesis in the female PyMT mice.



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  		Fig. 2. Acceleration of mammary tumorigenesis in AIB3+/–/PyMT mice. A, mammary tumor-free curves for WT/PyMT (WT; n = 26) and AIB3+/–/PyMT (AIB3+/–; n = 23) female mice. Palpable mammary tumors were detected significantly earlier in AIB3+/–/PyMT female mice (AIB3+/–) than in WT/PyMT female mice (WT; P < 0.005, log-rank test). B, average volume (mm3) of the biggest mammary tumor in each female mouse at the age of 60 days. The average tumor volume is significantly larger in AIB3+/–/PyMT female mice (AIB3+/–; n = 23) than in WT/PyMT (WT; n = 22) female mice (**, P < 0.01, unpaired t test). C, mammary tumor-free curves for WT/PyMT (WT; n = 23) and AIB3+/–/PyMT (AIB3+/–; n = 21) male mice. The mammary tumor latency of male mice is much longer than that of female mice. Palpable mammary tumors in AIB3+/–/PyMT male mice developed significantly faster than in WT/PyMT male mice (P < 0.001, log-rank test). D, average volumes of mammary tumors in WT/PyMT (white columns; n = 20) and AIB3+/–/PyMT (black columns; n = 17) male mice at ages of 24 and 40 weeks. ***, P < 0.001, unpaired t test.

 
The accelerated mammary tumorigenesis in AIB3+/–/PyMT female mice was further reflected by larger volumes of individual tumors at early stages compared with those in age-matched WT/PyMT female mice. At the age of 60 days, the average volume of mammary tumors in AIB3+//PyMT female mice was approximately 2-fold bigger than that in WT/PyMT female mice (Fig. 2B)Citation . When measured at the age of 120 days, average mammary tumor volume was no longer different between WT/PyMT and AIB3+/–/PyMT female mice, which suggests that mammary tumor growth has reached a plateau stage by this age in both groups.

The mammary tumor development in male AIB3+/–/PyMT mice was even more significantly accelerated compared with that in male WT/PyMT mice. Among 23 male WT/PyMT mice analyzed, 50% of them developed mammary tumors in 30 weeks and 100% in 38.5 weeks. However, it only took 22 and 24 weeks for 50% and 100%, respectively, of male AIB3+/–/PyMT mice (n = 21) to develop mammary tumors, which was significantly shorter than the time required for WT/PyMT mice to form mammary tumors (Fig. 2C)Citation . Unlike female mice in which multifocal adenocarcinomas were developed in several mammary tumors, most WT/PyMT and AIB3+/–/PyMT male mice developed only one mammary tumor. The average volumes of mammary tumors in AIB3+/–/PyMT males were 10-fold and 3-fold greater than those in WT/PyMT males when measured at ages of 24 and 40 weeks, respectively (Fig. 2D)Citation . Taken together, these results demonstrate that mammary tumor development is significantly accelerated in both female and male AIB3+//PyMT mice, which suggests that the promotion of PyMT-induced mammary tumorigenesis by AIB3 haplodeficiency is independent of female ovarian hormones.

To gain more insight into the effect of AIB3 haplodeficiency on mammary tumorigenesis, we examined the inguinal mammary gland morphology and histologic structure in female WT/PyMT and AIB3+/–/PyMT mice at different stages of mammary tumorigenesis. At the age of 1 week, the morphology of the primitive mammary gland trees in female WT/PyMT mice was normal, but several nodules were usually observed in female AIB3+/–/PyMT mammary glands (Fig. 3Citation , compare A–b with A–a). Histologic analysis confirmed that these nodules contained multiple layers of mammary epithelial cells, which suggests that, within 1 week after birth, epithelial hyperplastic lesions were already developed in female AIB3+/–/PyMT mammary glands, but not yet in WT/PyMT mammary glands (Fig. 3Citation , compare B-b with B-a). At the age of 3 weeks, some ductal nodules and ductal hyperplasia were observed in whole-mounted WT/PyMT mammary glands and in mammary gland sections (Fig. 3A–c and B–c)Citation . At the same age, the AIB3+/–/PyMT littermates developed multifocal in situ mammary tumors in the area between the nipple and lymph node (Fig. 3A–d)Citation . Histologic examination revealed that some mammary ducts were filled with epithelial tumor cells and some tumor cells had already invaded into the surrounding stromal tissues in AIB3+/–/PyMT mammary glands (Fig. 3B–d)Citation . At the age of 6 weeks, epithelial proliferative lesions progressed in WT/PyMT mammary glands. Many small cystic lesions were distributed along or at the ends of mammary ducts (Fig. 3A–e)Citation . These cystic lesions were filled with fluid and lined with multiple layers of epithelial cells or thickened papillary epithelium (Fig. 3B–e)Citation . Most of these cystic lesions were surrounded by smooth basement membranes, which suggests that these lesions were not invasive at this stage. In some areas, a few small solid mammary tumors were also observed. In contrast, a number of large dense mammary nodules were developed around the lymph nodes of AIB3+//PyMT mammary glands at the age of 6 weeks, although cystic lesions were also observed (Fig. 3A–f)Citation . Analysis of histologic sections revealed that many solid mammary tumors were formed in AIB3+/–/PyMT mammary glands at this stage. Some of these tumors were highly invasive (Fig. 3B–f)Citation . At the age of 9 weeks, mammary tumors in AIB3+/–/PyMT female mice were much larger than those in WT/PyMT female mice (Fig. 3Citation , compare A-g with A-h). At this stage, tumors of both genotypes were composed of poorly differentiated nests or cords of tumor cells that were usually separated by dense connective tissues (Fig. 3B–g and –h)Citation . Although extensive mammary adenocarcinomas were developed in mice with both genotypes at this stage, the stromal tissues in AIB3+/–/PyMT mammary glands were much less abundant than in WT/PyMT glands. The tumor cell mass in AIB3+/–/PyMT mammary glands was much higher than in WT/PyMT mammary glands (Fig. 3B–g and –h)Citation . These results demonstrate that mammary tumorigenesis is clearly accelerated in AIB3+/–/PyMT mice.



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  		Fig. 3. Early initiation of mammary tumorigenesis in AIB3+/–/PyMT mice. A, a–f, carmine alum-stained whole-mount inguinal mammary glands isolated from 1-, 3-, and 6-week-old (1W, 3W, 6W) WT/PyMT and AIB3+/–/PyMT female mice. Some of the mammary gland ductal nodules are circulated. The sizes of the nodules or tumors in AIB3+/–/PyMT mice are bigger than in WT/PyMT mice. A, g–h, in situ macroscopic morphologies of mammary tumors (arrows) observed in 9-week-old WT/PyMT and AIB3+/–/PyMT female mice. All of the tumors in A-h are bigger than those in A-g. B, hematoxylin and eosin-stained mammary gland and tumor sections prepared from the inguinal tumors of 1-, 3-, 6-, and 9-week-old (1W, 3W, 6W, 9W) WT/PyMT and AIB3+/–/PyMT female mice. The progression of mammary tumorigenesis is more advanced in AIB3+/–/PyMT mice than in WT/PyMT mice at the same ages. Arrows in B-b, necrotic cells; arrowhead in B-d, a mammary tumor-invasive spot. Scale bars: A-a–f, 1 mm; A-g and -h, 1 cm; B-a and -b, 20 µm; B-c and -d, 50 µm; B-e–h, 100 µm. (LN, lymph node.)

 
Accelerated Mammary Tumor Development in AIB3+/–/PyMT Mice Is a Mammary Epithelium-Autonomous Phenotype.
To investigate whether the accelerated mammary tumorigenesis in AIB3+/–/PyMT mice was due to a primary defect in the mammary epithelium or a consequence of possible secondary effects on systemic circulating factors, we transplanted mammary epithelial cells from 6-week-old WT/PyMT and AIB3+/–/PyMT mice to nude mice. At the age of 6 weeks, mammary epithelial malignant lesions in the PyMT transgenic mice were restricted in the fat pad region between the nipple and the lymph node (zone 1 and 2). The mammary ducts beyond the lymph node (zone 3) contain premalignant mammary epithelial cells (32) . At this age, the mammary duct has not extended into the dorsal region (zone 4) of the fat pad. Small pieces (1 mm3) of mammary tissue containing mammary epithelial cells were taken from zone 3 of mammary glands in WT/PyMT or AIB3+/–/PyMT donor mice and implanted into two sites per female mouse, one under the dorsal skin and the other into an inguinal mammary fat pad. All 12 dorsal subcutaneous sites receiving WT/PyMT (n = 6) or AIB3+/–/PyMT (n = 6) mammary tissues failed to develop any palpable tumors in a 20-week observation period, which suggests that both WT/PyMT and AIB3+/–/PyMT premalignant mammary epithelial cells in zone 3 were not able to form tumor in an ectopic site under the skin (data not shown). In contrast, all six sites in the nude mouse mammary fat pads implanted with WT/PyMT mammary tissues developed palpable tumors in an average period of 12 weeks, and the average volume of individual tumors reached ~10 mm3 in a 15-week observation period (Fig. 4A and B)Citation . Importantly, at those mammary sites receiving AIB3+/–/PyMT mammary tissues, palpable mammary tumors formed in ~5 weeks, which was significantly faster than tumors developed from WT/PyMT mammary tissues (Fig. 4A)Citation . Furthermore, the AIB3+/–/PyMT tumors also were 10 times bigger than WT/PyMT tumors in the fat pads of nude mice at 15 weeks postimplantation (Fig. 4B)Citation . These results indicate that the accelerated mammary tumor development in AIB3+/–/PyMT mice is a mammary epithelium-autonomous feature but not a consequence of any endocrine alterations.



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  		Fig. 4. A, tumor latencies of transplanted premalignant WT/PyMT (n = 6) and AIB3+/–/PyMT (n = 6) mammary tissues. The tumor appearance time is the days required to develop palpable mammary tumor from a small piece of transplanted premalignant mammary tissue with the indicated genotype. B, average tumor volumes, measured 15 weeks posttransplantation of the premalignant mammary tissues. The AIB3+/–/PyMT tumors (n = 6) are significantly larger than the WT/PyMT tumors (n = 6; P < 0.001, unpaired t test). C, quantitative analysis of proliferative cell population in the hyperplasia (Hy), MIN, and adenocarcinoma (AC) in the mammary glands of 4-, 6-, and 9-week-old WT/PyMT (white columns) and AIB3+/–/PyMT (hatched columns) female mice. Proliferative cells were detected by immunohistochemistry with anti-phospho(S10)-histone H3 antibody. A total of 1,000 cells on sections prepared from each mammary gland were examined, and phospho(S10)-histone H3-positive cells were counted. Mammary glands isolated from three to five female mice were analyzed for each age and genotype group. *, P < 0.001, unpaired t test.

 
Cell Proliferation Is Increased in AIB3+/–/PyMT Mammary Ductal Hyperplastic and Intraepithelial Neoplastic Lesions.
To seek cellular mechanisms responsible for the faster mammary tumor development in AIB3+//PyMT mice, we examined cell proliferation rate at different stages of mammary tumorigenesis by detecting cells with immunoreactivity of the phospho(S10)-histone H3, a specific cell proliferation marker, in female mammary glands (40) . The ratio of phospho(S10)-histone H3-positive mammary epithelial cells to total epithelial cells was ~2-fold higher in the lesions of mammary ductal hyperplasia and MIN of AIB3+/–/PyMT mammary glands than in those same lesions of WT/PyMT mammary glands (Fig. 4C)Citation . However, no substantial differences in cell proliferation were observed between large AIB3+/–/PyMT and WT/PyMT mammary adenocarcinomas (Fig. 4C)Citation . In addition, apoptotic cells detected by TUNEL assays were rare and had no differences between AIB3+/–/PyMT and WT/PyMT mammary glands (data not shown). These results suggest that AIB3 haplodeficiency promotes cell proliferation at relatively early stages of mammary tumor development, which is consistent with our observation demonstrating that AIB3+/–/PyMT tumors were 2-fold larger than WT/PyMT tumors at 60 days but not at 120 days (Fig. 2BCitation ; and data not shown).

Haploid Inactivation of AIB3 Reduces AIB3 but Has No Effect on the Expression of PyMT, ER{alpha}, and Estrogen-Regulated Genes in AIB3+/–/PyMT Mammary Glands.
AIB3 mRNA and protein were analyzed by real time RT-PCR and Western blot with RNA samples and tissue lysates prepared from mammary tumors. The AIB3 mRNA in WT/PyMT tumors was higher than that in AIB3+/–/PyMT tumors, but their difference did not reach statistical significance because of considerable standard variations (Fig. 5A)Citation . The AIB3 protein levels were substantially higher in WT/PyMT tumors than in AIB3+/–/PyMT tumors (Fig. 5B)Citation . The AIB3 immunoreactivity detected by immunohistochemistry with the anti-AIB3-P2 antibodies was also higher in WT/PyMT tumors than in AIB3+/–/PyMT tumors (data not shown). Therefore, it is clear that haploid inactivation of AIB3 indeed reduces AIB3 protein in AIB3+/–/PyMT tumors.



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  		Fig. 5. Analysis of AIB3 and PyMT expression. A, measurement of AIB3 mRNA by real-time RT-PCR. Total RNA was prepared from WT/PyMT (n = 4) and AIB3+/–/PyMT (n = 3) mammary tumors, and real-time RT-PCR was performed in triplicate for each mammary tumor. The relative AIB3 mRNA concentrations were normalized to 18S internal controls. B, analysis of AIB3 protein by immunoblotting. Tissue lysates were prepared from WT/PyMT (Lanes 1 and 3) and AIB3+//PyMT (Lanes 2 and 4) mammary tumors and were separated by 6% SDS-PAGE. The blot was stained with Ponceau S to show protein amounts (bottom panel) before it was incubated with the anti-AIB3 antibodies. C, analysis of PyMT mRNA expression by RNase protection assay. Total RNA was extracted from mammary glands (M. glands) of wild-type (WT) and AIB3+/– female mice (n = 3) and from WT/PyMT and AIB3+/–/PyMT mammary tumors (M. Tumors; n = 3) as indicated. RNase protection assay was performed with 10 µg of RNA. RNase protection assay analysis for K18 and cyclophilin A (Cyc) served as internal controls for mammary epithelial cell population and total cell population, respectively. The PyMT probe protected two major PyMT-specific bands that appeared only in PyMT transgenic mice (Lanes 7–12).

 
The MMTV promoter that directs PyMT expression in the mammary epithelial cells contains hormonally regulated elements for binding nuclear receptors such as GR and PR. Because alteration of AIB3 levels might modulate PyMT expression, we analyzed PyMT mRNA by RNase protection assay. As expected, PyMT mRNA was absent in the mammary glands of wild-type and AIB3+/– mice without PyMT (Fig. 5C)Citation . In WT/PyMT mice, PyMT mRNA was detected in both mammary glands and mammary tumors (Fig. 5CCitation ; and data not shown). Consistent with previous report (32) , strong PyMT immunoreactivity was identified in most tumor cells, whereas it was either undetectable or detected at low levels in most normal mammary luminal epithelial cells in the same gland (data not shown). In all AIB3+/–/PyMT mice examined, both PyMT mRNA and protein were detected in glands and tumors with similar amounts and distribution patterns as those in WT/PyMT mammary glands and tumors (Fig. 5CCitation ; and data not shown). These results indicate that AIB3 haplodeficiency does not change the temporal and spatial expression pattern and levels of the PyMT transgene in the mammary gland. Therefore, the faster development of breast tumors in AIB3+/–/PyMT mice is not due to any alteration of PyMT expression.

Because estrogen and ER{alpha} play an important role in hormonal promotion of mammary tumorigenesis (41 , 42) and AIB3 cellular concentrations have been shown to affect ER transcriptional activity in cultured cells (11, 12, 13) , we compared ER{alpha} and its target gene expression in WT/PyMT and AIB3+/–/PyMT glands. Immunohistochemistry with ER{alpha}-specific antibodies revealed no differences in ER{alpha} distribution pattern and immunoreactivity intensity between WT/PyMT and AIB3+/–/PyMT glands, ductal hyperplasia, MIN, and tumors. Consistent with previous reports (32 , 33) , scattered ER{alpha} positive cells were detected mainly in normal mammary luminal epithelial cells and ductal hyperplastic and MIN locations. The transformed epithelial cells in bigger adenocarcinomas were basically negative to ER{alpha} (data not shown). PR expression is regulated by ER{alpha}. Immunohistochemistry with PR-specific antibodies also revealed a spatial distribution pattern of PR similar to that of ER{alpha}, and no differences in spatial distribution and cellular immunostaining intensity of PR were observed in AIB3+/–/PyMT and WT/PyMT mammary glands and tumors (data not shown). Furthermore, the mRNA of another ER{alpha} target gene, lipocalin 2 (38) , was also equally detected by semiquantitative RT-PCR in WT/PyMT and AIB3+/–/PyMT mammary glands and tumors (data not shown). These results suggest that AIB3 haplodeficiency does not promote or inhibit ER{alpha} function in gland, which is consistent with the normal development of mammary gland in AIB3+/– mice.

We also examined the AIB3 gene locus by Southern blot analysis because the chromosomal region of the AIB3 gene is unstable and amplified in certain human breast tumors (2) . Among 12 WT/PyMT mammary tumors, only the wild-type AIB3 allele was detected, and the intensity of hybridization signals was comparable when an identical amount of DNA samples was loaded. In all of the 12 AIB3+/–/PyMT mammary tumors examined, both wild-type and targeted AIB3 alleles were detected and their hybridization signals were at a 1 to 1 ratio. When an identical DNA amount was loaded, the hybridization signal of the hemizygous wild-type allele in AIB3+/–/PyMT tumors was reduced to one half, compared with the signal of homozygous wild-type alleles in WT/PyMT tumors (data not shown). These results indicate that the faster development of breast tumors in AIB3+/–/PyMT mice is not caused by AIB3 amplification or loss of heterozygosity.

AIB3 Haplodeficiency Desensitizes AIB3+/–/PyMT Mammary Tumor Cells to PPAR{gamma} and RXR-Mediated Inhibition of Cell Proliferation.
Several nuclear receptors including PPAR{gamma}, RAR, and their heterodimeric partner RXR have been recognized as negative regulators for breast and colon cancers, and their ligands have been demonstrated as potential chemopreventative agents for breast and colon carcinogenesis (43, 44, 45, 46, 47, 48, 49) . AIB3 interacts with and coactivates PPAR{gamma}, RAR, and RXR in the presence of their active ligands (9 , 11, 12, 13) . We also previously demonstrated that mice lacking AIB3 exhibit several developmental defects similar to those observed in PPAR{gamma} null mice and the transcriptional capability of PPAR{gamma} is significantly reduced in AIB3–/– mouse embryonic fibroblasts (23) . Therefore, we reasoned that haploid inactivation of AIB3 may partially attenuate the function of these nuclear receptors and desensitize the inhibition of cell proliferation by their ligands.

To test our hypothesis, mammary tumor cells were isolated from WT/PyMT and AIB3+/–/PyMT tumors and treated with troglitazone (PPAR{gamma} ligand), all-trans-retinoic acid (RAR ligand), or 9-cis-RA (RAR and RXR ligand). After 3 days of treatment, the ratio of proliferating cells to total cells was determined by counting phospho(S10)-histone H3 positive cells detected by immunofluorescence labeling. As expected, the proliferation rates of WT/PyMT cells were dramatically reduced after treatment with troglitazone, all-trans-retinoic acid, or 9-cis-RA (Fig. 6A)Citation . However, after same treatments, AIB3+/–/PyMT cells exhibited differential responses to these ligands. All-trans-retinoic acid inhibited the proliferation of AIB3+//PyMT cells to a similar degree as that of WT/PyMT cells, which suggests that AIB3 haplodeficiency has little effect on RAR-mediated suppression of cell proliferation (Fig. 6A)Citation . In contrast, the proliferation of AIB3+/–/PyMT cells was only slightly decreased after troglitazone or 9-cis-RA treatment, and the differences in cell proliferation rates between vehicle and troglitazone or 9-cis-RA-treated AIB3+/–/PyMT cells were not statistically significant (Fig. 6A)Citation . In agreement with the insensitivity of AIB3+/–/PyMT cells to the inhibition of cell proliferation by troglitazone and 9-cis-RA, transfectional assays also confirmed that the transcriptional capabilities of PPAR{gamma} and RXR were significantly reduced in AIB3+/–/PyMT cells compared with that in WT/PyMT cells (Fig. 6B)Citation . These results suggest that haploid inactivation of AIB3 in AIB3+/–/PyMT cells causes insensitive inhibition of cell proliferation mediated by PPAR{gamma} and RXR.



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  		Fig. 6. A, quantitative analysis of cell proliferation rates after WT/PyMT (white columns) and AIB3+/–/PyMT (gray columns) tumor cells were treated with vehicle (–) or with troglitazone (TGZ), all-trans-retinoic acid (ATRA), or 9-cis-RA (+). Mitotic cells were analyzed by immunocytofluorescence labeling with anti-phospho(S10)histone H3 antibodies. Data of three experiments are presented as mean ± SD. All cells (~1,000) in 10 random-view fields with x40 magnification were examined, and the cell proliferation index was calculated by (phospho(S10)-histone H3-positive cell number/total cell number) x 100 (%). *, P < 0.05 and **, P < 0.01 by unpaired t tests. B, transfection assays. WT/PyMT (white columns) and AIB3+/–/PyMT (gray columns) cells were transfected with pSGS-hPPAR{gamma} and GS-tk-Luc plasmids by electroporation and treated with troglitazone (labeled as PPAR group), or were transfected with pSGS-hPPAR{gamma}, pSGS-DM1-hRXR{alpha}, and GS-tk-Luc and treated with troglitazone and 9-cis-RA (labeled as PPAR/RXR group). Luciferase activity was normalized to cotransfected ß-galactosidase and was presented as an average percentage of WT/PyMT cells. Data represent three experiments with triplicate samples in each experiment. *, P < 0.05, unpaired t test.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that AIB3 protein was mainly located in the nuclei of mammary luminal and myoepithelial cells and tumor cells. AIB3 protein was elevated in the mammary luminal epithelial cells at middle pregnant stage and in PyMT-induced mammary tumor cells. The PyMT-induced mammary tumorigenesis in AIB3+/–/PyMT female and male mice was significantly accelerated compared with age- and sex-matched WT/PyMT mice. Morphologic and histologic analyses demonstrated that the acceleration of mammary tumor development in AIB3+/–/PyMT mice was mainly accredited to the early initiation of ductal hyperplasia and rapid formation of MIN in their mammary glands. The marked acceleration of mammary tumorigenesis by AIB3 haplodeficiency was further confirmed by the transplantation experiments showing that the tumor latency was substantially shorter for AIB3+//PyMT premalignant mammary tissues than for WT/PyMT premalignant mammary tissues to develop palpable tumors in the mammary fat pads of recipient nude mice. These results clearly demonstrate that genetic reduction of AIB3 in the mammary epithelial cells accelerates oncogene-induced mammary tumorigenesis.

The mammary tumorigenic latency of male PyMT transgenic mice was much longer than that in female PyMT mice because of two major differences in the mammary glands of male and female PyMT mice. First, the mammary epithelial cell population is very small in the male mammary gland; therefore, the probability of mammary tumorigenesis is relatively low. Second, the PyMT may be expressed at a lower level because the MMTV promoter is less active in the male mammary epithelial cells because of the absence of ovarian hormones. Interestingly, the acceleration extent of mammary tumorigenic latency in male AIB3+/–/PyMT versus male WT/PyMT mice was much greater than that in female AIB3+/–/PyMT versus female WT/PyMT mice. These results suggest that reduction of AIB3 in the male mammary epithelial cells more dramatically promotes PyMT-induced mammary tumorigenesis.

The results from transplantation experiments also indicate that the favorable effect of AIB3 haplodeficiency on PyMT-induced tumorigenesis is not due to any alterations of systemic factors such as circulating hormones. Because these mammary tumors were developed from small pieces of transplanted premalignant mammary tissues in the stromal environment of the recipient mammary fat pads and AIB3 is highly expressed in the mammary epithelial compartment and PyMT-induced mammary tumors, the inhibitory role of AIB3 in PyMT-induced mammary tumorigenesis likely reflects its autonomous function in the mammary epithelial cells.

Several lines of evidence suggest that the acceleration of mammary gland tumorigenesis in AIB3+//PyMT mice was not caused by the mouse strain hybrids. First, our closed colony was randomly bred to ensure that independently segregating genes would be randomly assorted to each genotype (50) . Second, our sorted data indicate that AIB3+/–/PyMT female mice in 13 of 14 litters developed mammary gland tumors earlier than their littermates with closer genetic background to each other. Third, another study with similar breeding strategy in our laboratory showed a comparable mammary gland tumorigenesis in steroid receptor coactivator-1–/–/PyMT and WT/PyMT mice (data not shown). Fourth, PPAR{gamma}+/–/PyMT and WT/PyMT mice, generated by crossing mice with C57BL/129SvJ and FVB strain background, also showed no difference in mammary gland tumorigenesis (51) .

AIB3 is expressed in the mammary luminal epithelial cells both positive and negative to ER{alpha}. Multiple in vitro experiments have showed that AIB3 enhances the transcriptional activity of ER{alpha} in transfected cells (9, 10, 11 , 13) . Furthermore, ovarian steroids and ER{alpha} are known to play an important role in hormonal promotion of mammary carcinogenesis (8 , 52, 53, 54) . On the basis of these observations, one would ask why haploid inactivation of AIB3 promotes rather than suppresses PyMT-induced mammary tumorigenesis. There are several possible explanations. First, the PyMT-induced mammary proliferative lesions including hyperplasia and MIN develop before the increase of ovarian steroids during pubertal development. Therefore, the early initiation and progression of PyMT-induced mammary tumorigenesis may be independent of hormonal promotion. Second, the PyMT-induced adenocarcinomas were basically negative to ER{alpha} and PR, and, therefore, they were not able to respond to ovarian steroids at the advanced malignant stage. Third, ER{alpha} and PR may not require high concentrations of AIB3 to function in the mammary luminal epithelial cells, and, thereby, haploid inactivation of AIB3 is not stringent enough to affect ER{alpha} and PR functions in the mammary gland. This notion is supported by our observations demonstrating that the mammary gland development in AIB3+/– female mice was normal, the expression of ER{alpha}-regulated genes was not obviously affected in AIB3+/–/PyMT mammary glands, and the PyMT expression directed by the MMTV hormone responsive promoter was comparable between WT/PyMT and AIB3+/–/PyMT mammary tumors. Therefore, the promotion by AIB3 haplodeficiency of PyMT-induced mammary tumorigenesis observed in this study does not exclude the possible role of AIB3 in hormonal promotion of mammary tumorigenesis mediated by ER{alpha} and PR. Additional mouse models with hormonal promotion of mammary carcinogenesis and with mammary epithelium-specific knockout of both AIB3 alleles are required for further investigation of the role of AIB3 in ER{alpha}- and PR-mediated hormonal promotion of mammary carcinogenesis.

Cell proliferation analysis revealed that AIB3 haplodeficiency accelerates PyMT-induced mammary tumorigenesis, although it enhances cell proliferation in the mammary ductal hyperplasia and MIN lesions. Although the cell proliferation rate was similar between advanced WT/PyMT and AIB3+/–/PyMT adenocarcinomas, cultured AIB3+/–/PyMT cells exhibited a much higher proliferation rate than WT/PyMT cells. AIB3 is a transcriptional coactivator for PPAR{gamma}, RAR, and RXR. The ligands of these nuclear receptors have been shown to inhibit cancer cell growth, induce cell apoptosis, promote terminal differentiation of breast cancer cells, and even reverse malignant changes of certain cancers (43 , 55, 56, 57) . Haploid inactivation of PPAR{gamma} alleles promotes colon carcinogenesis in mice treated with PPAR{gamma} ligand (48) . Retinoids can affect multiple signal pathways, suppress tumorigenesis in rodents, and inhibit the growth of transplanted breast tumors (57) . Our analysis demonstrates that the inhibitory sensitivity of troglitazone (a PPAR{gamma} ligand) and 9-cis- RA (a ligand for RAR and RXR) to cell proliferation was attenuated in AIB3+/–/PyMT cells compared with WT/PyMT cells. It is presently unclear why all-trans-retinoic acid was still fully effective to inhibit the proliferation of AIB3+/–/PyMT cells because all-trans-retinoic acid also should activate RXR at the concentration used. It is possible that all-trans-retinoic acid-bound RXR might have higher affinity than 9-cis-RA-bound RXR to interact with AIB3. Overall, these results suggest that AIB3 serves as a coactivator for PPAR{gamma} and RXR to mediate the inhibitory role of troglitazone and 9-cis-RA in mammary epithelial cell proliferation. This notion is further supported by our observations showing that the defects of placental development in AIB3–/– mice was similar to those encountered in mice lacking PPAR{gamma} and PPAR{gamma}-binding protein and the transcriptional capability of PPAR{gamma} was significantly impaired in AIB3–/– mouse embryonic fibroblasts (23) and AIB3+/–/PyMT cells (Fig. 6B)Citation . Thus, reduction of AIB3 in AIB3+/–/PyMT mammary epithelial cells likely dampens the function of PPAR{gamma}/RXR and thereby increases cell proliferation and accelerates mammary tumorigenesis.

One recent study showed comparable mammary gland tumorigenesis in PPAR{gamma}+/–/PyMT and WT/PyMT mice without ligand treatment (51) , which suggests that AIB3 concentration may be more critical than PPAR{gamma} concentration for PPAR{gamma}/RXR-regulated cell proliferation under physiologic conditions. Intriguingly, the same study also showed that overproduction of a constitutively active Vp16/PPAR{gamma} fusion transcription factor in mouse mammary epithelial cells exacerbates PyMT-induced tumorigenesis (51) . Although Vp16/PPAR{gamma} overexpression may significantly alter the balance of cellular concentrations of coactivators and corepressors available for other transcriptional regulators, this study suggests that the mechanisms by which PPAR{gamma} regulates mammary gland tumorigenesis are extremely complex.

The interactive molecular mechanisms between PyMT-stimulated signaling pathways and PPAR{gamma}/RXR-mediated inhibition of cell proliferation are complex and are not fully understood at the current time. It is known that PyMT activates the ras pathway that stimulates mitogen-activated protein (MAP) kinase (58) . Several studies have shown that MAP kinase directly phosphorylates PPAR{gamma}, and the phosphorylated PPAR{gamma} has greatly reduced transcriptional activity and ability to promote cell differentiation (43 , 59 , 60) . A combined effect of the MAP kinase activation by PyMT and AIB3 haplodeficiency on the reduction of PPAR{gamma} transcriptional activity may additively alter the function of PPAR{gamma} on cell differentiation and promote PyMT-induced mammary tumorigenesis.

In summary, AIB3 is highly expressed in the mammary epithelial cells and tumor cells. Haploid inactivation of AIB3 desensitizes the PPAR{gamma}-and RXR-mediated inhibition of cell proliferation and significantly accelerates mammary tumorigenesis in both male and female PyMT transgenic mice. Because of the early initiation and rapid progression of the PyMT-induced mammary tumorigenesis as well as the negative expression of ER{alpha} and PR in these tumors, additional models manifesting hormonal promotion of mammary tumorigenesis and harboring mammary epithelial cell- specific inactivation of both AIB3 alleles will be required to evaluate the role of AIB3 in ER{alpha} and PR-mediated functions in the mammary gland.


   ACKNOWLEDGMENTS
 
We thank Drs. Daniel Medina, Cory Brayton, Neil McKenna, Ming Zhang, Sudit S. Mukhopadhyay, and Steven S. Chua in Baylor College of Medicine, Houston, TX and Lan Ko in Medical College of Georgia, Augusta, GA for valuable discussion and help.


   FOOTNOTES
 
Grant support: This work was supported by the NIH grant DK58242 (J. Xu), the United States Army Medical Research and Material Command Breast Cancer Research Awards DAMD17-00-1-0148 (J. Xu), and the postdoctoral fellowship award DAMD17-02-1-0293 (H. Zhang).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Jianming Xu, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-6199; Fax: (713) 798-3017; E-mail: jxu{at}bcm.tmc.edu

Received 4/ 3/04. Revised 7/12/04. Accepted 8/ 6/04.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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