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Endocrinology |
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas
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.
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Abstract |
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Introduction |
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B. SRC coactivators amplify the transcriptional activities of these transcription factors through recruiting chromatin modification enzymes to the promoter/enhancer regions of their target genes (1–6). These chromatin modification enzymes include acetyltransferases, such as CBP, p300, and P/CAF, and methyltransferases, such as CARM1 and PRMT1 (1, 2). As the cellular levels of these SRC coactivators are usually limiting in normal cells, the changes of their concentrations and activities are thought to play remarkable roles in the regulation of gene expression (2). Multiple lines of evidence have shown that the transcriptional activities of SRC coactivators are regulated by multiple mechanisms involving protein phosphorylation, acetylation, translocalization, and degradation, suggesting that these coactivators can serve as targets for multiple signaling pathways activated by hormones, growth factors, cytokines, ubiquitins, and xenobiotic agents to regulate gene expression (7–10). In the SRC family, elevated expression of SRC-1 and AIB1 has been observed in human breast cancers. In normal human mammary epithelial cells, SRC-1 levels are very low (11). However, SRC-1 protein levels are elevated in 46% of HER-2/neu–positive, but only 6% of HER-2/neu–negative, breast cancers, indicating that overexpression of SRC-1 in breast cancer is associated with HER-2/neu oncogene expression. Furthermore, SRC-1 levels in breast cancers are positively associated with nodal positivity and resistance to endocrine therapy and are inversely associated with disease-free survival (11–13). The AIB1 gene is amplified in 5% to 10% breast cancers and overexpressed in 30% to 60% breast cancers (3, 14, 15). Interestingly, AIB1 amplification and overexpression were detected in both ER positive and negative breast cancers (14, 15). In tamoxifen-treated breast cancer patients, high levels of AIB1 protein were found associated with HER-2/neu expression, tamoxifen resistance, and poor disease-free survival (16). These clinical data suggest that SRC-1 and AIB1 play important roles in breast cancer. Indeed, recent studies have shown that transgenic expression of an active AIB1 isoform in mouse mammary epithelial cells increased mammary epithelial proliferation (17). Overexpression of the full-length AIB1 in mouse mammary epithelial cells led to mammary hyperplasia and the development of malignant mammary tumors, indicating that AIB1 overexpression is sufficient to initiate mammary gland oncogenesis (18).
We showed previously that AIB1 is expressed in the epithelial compartment of the mammary gland (19, 20). Inactivation of AIB1 in mice partially attenuated mammary ductal growth during puberty and lobular alveolar development in response to estrogen and progesterone treatment (19, 21). Recently, we also showed that AIB1 deficiency impairs the insulin-like growth factor-I (IGF-I) signaling pathway, inhibits cell proliferation and migration, and significantly suppresses the mouse mammary tumor virus (MMTV)-v-Ha-ras oncogene-induced mammary tumor initiation and progression (20). These results indicate that AIB1 plays a critical role in the mammary tumorigenesis mediated by pathways involving the membrane-associated tyrosine kinase receptors and their downstream protein kinases. However, the role of AIB1 in mammary carcinogenesis induced by DNA mutagens, such as chemical carcinogens, is unclear.
In this study, we used an established 7,12-dimethylbenz[a]anthracene (DMBA)–induced mouse mammary tumor model to assess the in vivo role of AIB1 in the mammary carcinogenesis triggered by chemical carcinogen exposure. We show that inactivation of AIB1 in mice significantly delayed the latency and reduced the frequency and growth rate of the mammary tumors induced by DMBA. AIB1 deficiency attenuated the up-regulation of several components of the IGF-I/Akt/cyclin D1 signaling pathway, which might be responsible for the suppression of DMBA-induced mammary tumor development in AIB1–/– mice. The fact that inactivation of AIB1 suppresses the carcinogen-induced mammary tumorigenesis in mice suggests that AIB1 is a potential target for prevention and treatment of human breast cancer.
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Materials and Methods |
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Transplantation of mammary epithelium and examination of mammary glands. Mammary gland epithelial transplantation was done as described.1 Briefly, the entire rudiment mammary ducts in 3-week-old female recipient mice were removed by dissecting the lower part of the inguinal fat pats from the nipple to the proximal mammary lymph node. A small piece (
1.5 mm3) of mammary tissue was taken from the mammary gland region between the nipple and the lymph node of the 3-month-old donor mice and implanted into the cleared fat pads of the recipient mice. The transplanted mammary epithelium was allowed to develop for 10 weeks in the host before it was harvested for morphologic analysis. To test the response of the transplanted mammary epithelium to the hormonal stimulation provided by the pituitary isograft, a pituitary gland was isolated from a male littermate and implanted under the kidney capsule of the female recipient mouse that had received the mammary isografts for 2 weeks. The pituitary transplantation was done as described (22, 23). Mammary gland morphology was examined by whole-mount staining with carmine alum as described (19, 20). Mammary gland X-gal staining also was carried out as described previously (19, 20).
7,12-Dimethylbenz[a]anthracene treatment and examination of tumorigenesis. DMBA was purchased from Sigma (St. Louis, MO) and dissolved in cottonseed oil at a concentration of 5 mg/mL. To induce tumorigenesis in mice without pituitary isografts, 8-week-old female WT, AIB1+/–, and AIB1–/– mice were fed weekly by gastric intubation with 1 mg DMBA in 0.2 mL for 6 consecutive weeks. To induce tumorigenesis in mice under pituitary isograft stimulation, pituitaries from WT siblings were implanted under the right kidney capsules of female WT, AIB1+/–, and AIB1–/– recipient mice at 5 weeks of age. These recipient mice were fed weekly with 1 mg DMBA for 3 consecutive weeks starting at 8 weeks of age. Pituitaries remained in place for the duration of the experiment. All of the pituitary isografts were checked for viability after necropsy as described (23).
After DMBA treatment, mammary glands were examined in a time course to check mammary tumor initiation and progression by whole-mount staining and by sectioning the paraffin-embedded tissues and H&E staining as described previously (20, 24). Palpation for mammary tumors was carried out weekly for each DMBA-treated mouse. Once the initial palpable mammary tumors ( 0.5 mm in diameter) were detected, the tumor length (L) and width (W) were measured once weekly. The tumor volume was calculated by (L x W2) / 2 as described (20, 24, 25). Occurrence of skin tumors also was recorded. Tumor-free curves were generated by running the Prism 3.0 software (GraphPad Software, San Diego, CA) Statistical differences were compared by either log-rank test for tumor-free curves, 
2 test for tumor incidence, or unpaired t test for tumor volume. Significant difference was determined at P 
0.05.
Mice were euthanized when the primary tumor reached 1.5 cm in diameter or when mice became moribund. The number of mammary tumors developed in each mouse was counted and recorded. Mammary tumors were collected for further histologic and molecular analysis. Ovaries were carefully examined for ovarian tumors.
RIA of estradiol and progesterone. Blood samples were collected from DMBA-treated mice bearing pituitary isografts at the time of sacrifice. Serum samples were prepared and stored at –80°C until assayed for 17ß-estradiol and progesterone by using the DSL-4400 and DSL-3900 RIA kits (Diagnostic Systems Laboratories, Webster, TX).
Immunohistochemistry. Immunohistochemistry was done on dewaxed and rehydrated mammary tissue sections prepared after 4% paraformaldehyde fixation and paraffin embedding (20, 26, 27). Primary antibodies against AIB1 (Santa Cruz Biotechnology, Santa Cruz, CA), ER
(Zymed, San Francisco, CA), PR (Santa Cruz Biotechnology), and anti-Ser10-phosphorylated histone H3 (PH3; Upstate, Charlottesville, VA) were applied according to the manufacturer's instructions. Primary antibodies were recognized by secondary antibodies labeled with biotin. The biotinylated secondary antibodies were detected by using the ABC kit containing the diaminobenzidine substrate for horseradish peroxidase (HRP; Vector Lab, Burlingame, CA). After immunostaining, sections were counterstained with methyl green. In addition, the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay was done to detect apoptotic cells as described previously (20).
Immunoblotting analysis. Immunoblotting analysis was done as described previously (20). Tissue lysates were prepared from mammary glands and mammary tumors. Tissue lysate containing 50 µg protein was separated by running SDS-PAGE and blotted onto nitrocellulose membrane. Blots were incubated with primary antibodies and secondary antibodies conjugated with the HRP and developed using the enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ). Primary antibodies against IGF-I receptor ß (IGF-IRß), AIB1, and cyclin D1 were purchased from Santa Cruz Biotechnology. Antibodies against the insulin receptor substrate (IRS)-1 and IRS-2 were from Upstate. Antibodies against Akt and phospho-S473 Akt were from Cell Signaling Technology (Beverly, MA).
RNase protection assays. Total RNA was extracted from mouse inguinal mammary glands and tumors using the Trizol reagent (Invitrogen, Carlsbad, CA). The mouse mCYC-1 multiprobe template set for detecting mRNAs of cyclins A2, B1, C, D1, D2, D3, A1, and B2 and control mRNAs of L32 and the glyceraldehyde-3-phosphate dehydrogenase was purchased from BD PharMingen (San Jose, CA). Antisense RNA probes were labeled using T7 RNA polymerase and [32P]UTP according to the Maxiscript kit protocol (Ambion, Austin, TX). RNase protection assay (RPA) was done according to the manufacturer's instruction using the RPAIII kit (Ambion).
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Results |
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99% FVB strain background were intercrossed to generate WT, AIB1+/–, and AIB1–/– mice that were analyzed in the present study. Staining of whole-mounted mammary glands isolated from 6- and 8-week-old females with FVB strain background revealed that the mammary ductal growth was significantly delayed in AIB1–/– mice compared with WT mice (Fig. 1A, data not shown). Because the reduced mammary ductal morphogenesis was observed in AIB1–/– mice with either FVB or 129SvEv/C57BL/6J strain backgrounds, we conclude that the role of AIB1 in mammary ductal growth under natural physiologic conditions is independent of genetic background.
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To examine the response of the mammary ducts from the transplanted WT and AIB1–/– mammary epithelium to the elevated hormonal stimulation in the same WT recipients, we isolated pituitaries from their male siblings and implanted one pituitary into the kidney capsule of each female WT recipient at 2 weeks after receiving the mammary epithelial transplants. It is known that the pituitary isograft in the kidney capsule significantly increases the circulating levels of progesterone and prolactin and moderately increases the circulating level of estrogen (28–30). Interestingly, when the reconstituted mammary glands in the recipient mice were examined at 10 weeks after transplantation or 6 weeks after receiving the pituitary isografts, extensive mammary ductal side branches and numerous lobuloalveolars were developed along the mammary ducts derived from the transplanted WT and AIB1–/– mammary epithelium, and no appreciable differences in the mammary gland morphology could be observed between the transplanted WT and AIB1–/– mammary glands (Fig. 1B, e and f). These results suggest that the highly enhanced hormonal stimulation provided by the pituitary isograft can rescue the delayed mammary ductal morphogenesis caused by AIB1 deficiency in the mammary epithelium.
Amplified in breast cancer 1 deficiency selectively suppresses 7,12-dimethylbenz[a]anthracene–induced mammary carcinogenesis. DMBA is a known chemical carcinogen that causes DNA damage and induces tumorigenesis in the mammary gland, ovary, and epidermis and leukemia (23, 29, 30). To examine the role of AIB1 in chemical carcinogen–induced neoplasia, we treated 8-week-old female WT, AIB1+/–, and AIB1–/– mice with DMBA once weekly for 6 consecutive weeks and recorded the DMBA-induced tumorigenesis. Mammary tumors were developed in 45% (5 of 11), 44% (11 of 25), and 11% (2 of 19) of DMBA-treated WT, AIB1+/–, and AIB1–/– mice, respectively, indicating that the frequency of mammary tumor formation in AIB1–/– mice is significantly lower than in WT and AIB1+/– mice (P < 0.05, 2 test). The mammary tumor latency after the initial DMBA treatment also was significantly extended in AIB1–/– mice compared with WT and AIB1+/– mice (P < 0.05, log-rank test; Fig. 2A). In contrast, DMBA-induced skin tumors were observed in 45% (5 of 11), 48% (12 of 25), and 39% (7 of 18) of WT, AIB1+/–, and AIB1–/– mice, respectively. There were no significant differences (P > 0.05, 2 test) in the skin tumor frequencies and latencies among these three groups (Fig. 2B). The skin tumor occurrence also was not associated with the mammary tumor occurrence. Ovarian tumors were only observed in one of the DMBA-treated AIB1+/– mice. Taken together, these results indicate that inactivation of AIB1 selectively protects the mammary glands from DMBA-induced carcinogenesis under natural ovarian hormonal conditions.
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To quantify the incidence and latency of the pituitary isograft-promoted and DMBA-induced mammary tumor development, we recorded the time required for formation of palpable mammary tumors. Palpable mammary tumors were first detected in 9 and 7 weeks after DMBA treatment in WT and AIB1+/– mice, respectively. Palpable mammary tumors were observed in 50% of WT and AIB1+/– mice with pituitary isografts by 16 and 18 weeks after DMBA treatment (Fig. 4A). Sixty-two percent (23 of 31) WT and 86% (25 of 29) AIB1+/– mice developed palpable mammary tumors by 39 weeks after DMBA treatment. Other mice died of DMBA-induced leukemia or other types of cancers (see next paragraph). There were no statistical differences in mammary tumor incidences and latencies between WT and AIB1+/– mice. Importantly, palpable mammary tumor in AIB1–/– mice with the pituitary isografts was first detected at 17 weeks after DMBA treatment, and 50% of AIB1–/– mice did not develop mammary tumors until 31 weeks after DMBA treatment (Fig. 4A). Palpable mammary tumors were observed in 16 of 22 (73%) AIB1–/– mice by 52 weeks after DMBA treatment. Although the incidence of the pituitary isograft-stimulated and DMBA-induced mammary tumors in AIB1–/– mice was similar to that observed in WT and AIB1+/– mice, these results indicate that the mammary tumor latency of AIB1–/– mice was significantly longer than that of WT and AIB1+/– mice (P < 0.0001, log-rank test).
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AIB1–/– mammary tumors grow slower than wild-type mammary tumors. The growth rate of the primary mammary tumor in each mouse was measured by estimating its volume once weekly after it was detected by palpation. The average growth rate of mammary tumors in WT mice with pituitary isografts was similar to that in AIB1+/– mice with pituitary isografts. In contrast, the average volume of mammary tumors in AIB1–/– mice was significantly smaller than in WT mice by 4 weeks after palpable mammary tumors were detected (P < 0.05, unpaired and two-sided t test; Fig. 4B). These results showed that DMBA-induced mammary tumors grow much slower in the absence of AIB1.
The number of breast tumors per tumor-bearing mouse was comparable among WT, AIB1+/–, and AIB1–/– mice at the experimental end point (P > 0.05, two-sided F test), which were 2.7, 2.6, and 2.3 tumors per mouse, respectively. Histopathologic analysis of mammary tumors developed in WT, AIB1+/–, and AIB1–/– mice did not reveal obvious histologic differences. Most mammary tumors examined in the pituitary isografted mice were squamous adenocarcinomas and type B mammary adenocarcinomas, whereas those mammary tumors in mice without pituitary isografts were predominantly type B mammary adenocarcinomas.
Amplified in breast cancer 1 deficiency reduces cell proliferation in the mammary epithelium and neoplasia lesions induced by pituitary isograft and 7,12-dimethylbenz[a]anthracene treatment. To search for cellular mechanisms responsible for the delayed mammary tumor initiation and growth, we analyzed normal mammary epithelial and DMBA-transformed mammary epithelial proliferation by immunostaining PH3, a specific marker of cell mitosis (24). The fractions of PH3-positive mammary epithelial cells in 8-week-old WT and AIB1–/– mice were similar, 6% of total mammary luminal epithelial cells. On pituitary isograft stimulation for 3 weeks, the PH3-positive epithelial cells in WT and AIB1–/– mammary glands increased to 16% and 13%, respectively, suggesting that the pituitary isograft stimulated a lesser increase in proliferation of AIB1–/– mammary epithelial cells compared with WT mammary epithelial cells (P < 0.05, unpaired and two-sided t test). The 16% and 20% cell proliferation rates observed in DMBA-induced AIB1–/– MINs and invasive adenocarcinomas, respectively, were much lower than the 25% and 26% cell proliferation rates observed in the same types of neoplasia lesions in WT mice (P < 0.01, unpaired and two-sided t tests; Fig. 4C). We also did TUNEL assays to analyze mammary epithelial and tumor cell apoptosis and detected 1% to 2% programmed cell death in both WT and AIB1–/– mammary epithelium and DMBA-induced neoplasia lesions (data not shown). These results indicate that the effects of AIB1 deficiency are due to the inhibition of mammary epithelial and tumor cell proliferation and not to the promotion of cell apoptosis.
Analysis of amplified in breast cancer 1, estrogen receptor , and progesterone receptor expression and ovarian hormones. Relatively weak AIB1 immunoreactivity was detected in the mammary epithelial cells of adult WT mice (Fig. 5A, a). The AIB1 immunoreactivity was significantly increased in the mammary epithelial cells of the pituitary isograft-stimulated WT mice and in the DMBA-induced WT mammary tumor cells (Fig. 5A, b and c). No AIB1 immunoreactivity was detected by the same AIB1 antibody in the pituitary isograft-stimulated AIB1–/– mammary glands (Fig. 5A, d), indicating that the AIB1 immunoreactivity detected in WT mice was specific. These results show that AIB1 expression is elevated after pituitary isograft stimulation and during DMBA-induced mammary tumorigenesis in virgin WT mice.
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Next, we compared the expression patterns and relative levels of ER and PR in WT and AIB1–/– mammary glands and DMBA-induced mammary tumors by immunohistochemistry. ER and PR immunoreactivities were detected only in a proportion of the mammary epithelial cells as reported previously (20, 31, 32). The relative intensity of ER and PR immunoreactivities and the number of ER- and PR-positive cells were similar in the WT and AIB1–/– mammary epithelium and in the pituitary isograft-induced WT and AIB1–/– mammary alveoli (Fig. 5, a-d in B and C). The number of ER- and PR-positive cells was significantly decreased in the hyperplastic lesions induced by DMBA and was undetectable in the advanced breast tumors in both WT and AIB1–/– mammary glands (Fig. 5, e and f in B and C).
Finally, we measured SRC-1 and TIF2 (SRC-2) mRNA levels to check whether AIB1 (SRC-3) deficiency would result in a compensatory up-regulation of SRC-1 and TIF2 expression. Quantitative real-time reverse transcription-PCR assays revealed that SRC-1 and TIF2 expression levels were quite different among individual DMBA-induced mammary tumors and not correlated with the presence or absence of AIB1. The average ratios of SRC-1 and TIF2 mRNA concentrations to the 18S RNA concentration in DMBA-induced WT and AIB1–/– mammary tumors were not significantly different (P > 0.05, unpaired t test). These results suggest that the change of SRC-1 and TIF2 expression in the DMBA-induced mammary tumors is not influenced by AIB1 deficiency.
Amplified in breast cancer 1 deficiency down-regulates the insulin-like growth factor-I/phosphatidylinositol 3-kinase/Akt/cyclin D1 pathway in 7,12-dimethylbenz[a]anthracene–induced mammary tumors. The IGF-I/phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays a critical role in breast cancer initiation and progression (20, 33). To address the question of whether inactivation of AIB1 suppresses DMBA-induced mammary tumorigenesis through alteration of the IGF-I/PI3K/Akt pathway, we examined several essential components of this signaling pathway. Low levels of IGF-IRß were detected in the mammary gland extracts and much higher levels of IGF-IRß were found in the extracts of DMBA-induced mammary tumors from both WT and AIB1–/– mice, suggesting that IGF-IRß is overexpressed in DMBA-induced mammary tumor cells regardless of AIB1 deficiency. IRS-1 and IRS-2 proteins were undetectable on immunoblots of the mammary gland extracts from WT and AIB1–/– virgin mice. Interestingly, robust levels of IRS-1 and IRS-2 proteins were identified in DMBA-induced WT mammary tumors but not in DMBA-induced AIB1–/– mammary tumors (Fig. 6A). Total Akt protein remained unchanged in WT and AIB1–/– mammary glands and tumors. However, much more of the activated form of Akt, phosphorylated Akt S473, was observed in DMBA-induced WT mammary tumors compared with AIB1–/– mammary tumors (Fig. 6B). These results suggest that AIB1 deficiency can suppress IRS-1 and IRS-2 up-regulation and partially inhibits Akt activation in the DMBA-induced mammary tumors.
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2- to 3-fold higher than that in AIB1–/– mammary glands and tumors (Fig. 6C). Accordingly, the cyclin D1 protein also was much higher in DMBA-induced WT mammary tumors than in AIB1–/– mammary tumors (Fig. 6D). These results suggest that AIB1 is required for IGF-I/PI3K/Akt signaling pathway–stimulated up-regulation of cyclin D1 during the DMBA-induced breast tumor initiation and progression. |
Discussion |
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Interestingly, both endogenous and transplanted WT and AIB1–/– mammary glands exhibited similar development of side branches and lobular alveoli when they were stimulated by the elevated hormones derived from the pituitary isografts, suggesting that enhanced hormonal stimulation can compensate for the developmental deficit caused by AIB1 deficiency. Although the exact mechanisms responsible for this compensation is not clear, the combination of elevated progesterone and prolactin or the partially redundant function among SRC family members may contribute to this functional compensation on significantly enhanced hormonal stimulations (1, 34, 35). In agreement with the interpretation that the persistently enhanced hormonal stimulation is able to improve certain initial deficits in the mammary gland development, other studies also have shown that multiple cycles of pregnancy and lactation were able to restore mammary gland morphology and function in certain mutant mice with partially compromised mammary gland development (36, 37).
In WT and AIB1+/– mice that did not receive the pituitary isografts, DMBA treatment mainly induced mammary tumors and skin tumors. The frequency and latency of DMBA-induced skin tumors in AIB1–/– mice were unaltered compared with WT and AIB1+/– mice. However, the susceptibility of AIB1–/– mice to DMBA-induced mammary tumorigenesis was significantly reduced, manifesting much lower mammary tumor incidence and much longer mammary tumor latency compared with DMBA-treated WT and AIB1+/– mice. These results indicate that inactivation of AIB1 selectively protects the mammary gland from tumorigenesis induced by chemical carcinogens, such as DMBA. This tissue-selective role of AIB1 in carcinogenesis may be partially accredited to its high expression level in the mammary epithelium and low expression level in the skin (19), to its regulatory function in the expression of the IGF-I signaling pathway components (discussed below; refs. 7, 18, 20, 38), and to its transcriptional coactivation function for ER and PR in the mammary gland (1, 16, 23, 39). Although the relative contribution of AIB1 to specific pathway-mediated tumorigenesis is not clear at this stage, our results show that suppression of AIB1 function increases the resistance of the mammary gland to the chemical carcinogen–induced mammary tumorigenesis.
In WT and AIB1 mutant mice that received the pituitary isografts, DMBA treatment predominantly induced mammary gland tumors, and skin tumors were rare. It likely was because the elevated hormones that were directly and indirectly derived from the pituitary isografts selectively promoted the susceptibility of the mammary epithelial cells to the carcinogen by stimulating the mammary epithelial proliferation and increasing the mammary epithelial population (23, 28–30). Consequently, the latency of DMBA-induced mammary tumors in the mice with pituitary isografts was much shorter than the latency in the mice without pituitary isografts. Most mice developed mammary tumors before they developed the skin tumors. Interestingly, the enhanced hormonal conditions caused by the pituitary isografts accelerated the DMBA-induced mammary tumorigenesis in both WT and AIB1 mutant mice, suggesting that AIB1 is not absolutely essential for hormonal promotion of chemical carcinogen–induced breast cancer. This conclusion is consistent with our previous findings showing that AIB1 was not essential for ER-mediated gene expression and ductal morphogenesis of the mammary gland (19–21).
Most importantly, our results showed that AIB1 deficiency significantly delayed the DMBA-induced mammary carcinogenesis in mice with or without the pituitary isografts. Further analyses revealed that AIB1 expression was elevated during the DMBA-induced mammary tumorigenesis, recapitulating the situation in human breast cancer where AIB1 was found to be overexpressed (3, 16). AIB1 deficiency mainly suppressed the proliferation of mammary epithelial cells and DMBA-induced mammary tumor cells and the DMBA-induced mammary tumor growth. Consistent results also were observed in another mouse model of human breast cancer where mammary tumors were induced by the MMTV-v-Ha-ras oncogene (20). Because the DMBA and the ras use different molecular mechanisms to induce mammary tumorigenesis and AIB1 deficiency suppresses both the DMBA-induced and the ras-induced mammary tumorigenesis, the molecular mechanism by which AIB1 promotes breast cancer risk may involve a common pathway, such as the IGF-I signaling pathway, which can modulate the susceptibility of the mammary epithelium to the tumorigenesis induced by both DMBA and the ras oncogene.
Indeed, we found that AIB1 deficiency inhibited the up-regulation of IRS-1 and IRS-2 in DMBA-induced mammary tumor cells, which in turn suppressed the overactivation of the downstream protein kinase Akt and the overexpression of cyclin D1 in the pituitary-stimulated mammary gland and in the DMBA-induced mammary tumor cells. In agreement with this finding, our previous work showed that inactivation of AIB1 suppressed the up-regulation of IRS-1 and IRS-2 expression in the ras-induced mammary tumor cells and resulted in partial IGF-I resistance in these cells (20). Inversely, other studies showed that overexpression of AIB1 could enhance Akt activity and cyclin D1 expression in cultured cells and in the mammary tumors induced by the MMTV-AIB1 transgene (18, 38). Therefore, the overexpression of AIB1 may cause up-regulation of IRS-1 and IRS-2 and overactivation of the downstream IGF-I signaling components to promote cell proliferation and mammary tumorigenesis.
A lesser increase in the estrogen level was observed in the pituitary isograft-stimulated AIB1–/– mice compared with the pituitary isograft-stimulated WT mice. However, this difference in estrogen level was unlikely responsible for the suppression of DMBA-induced mammary tumorigenesis in AIB1–/– mice based on the following observations. First, the estrogen level was still significantly increased in AIB1–/– mice after stimulated by the pituitary isografts. Second, the AIB1–/– mammary glands showed a similar response to the pituitary isograft stimulation as WT mammary glands in terms of ductal side branching and lobular alveolar development. Finally, the expression level of PR, an ER target gene in the mammary epithelial cells, was comparable in AIB1–/– and WT mammary glands on pituitary isograft stimulation.
In summary, our study showed that AIB1 plays a mammary epithelial autonomous role in the regulation of mammary ductal growth and tumorigenesis. Inactivation of AIB1 function selectively protects the mammary gland over the skin from DMBA-induced tumorigenesis and inhibits mammary tumor growth. The suppression of IRS-1 and IRS-2 elevation during the DMBA-induced mammary tumorigenesis by AIB1 deficiency may be responsible, at least in part, for preventing the PI3K/Akt/cyclin D1 pathway from overactivation and the target cells from overproliferation. These findings suggest that AIB1 may serve as a potential target for prevention of breast cancer initiation as well as for treatment of breast cancer progression.
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Acknowledgments |
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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.
We thank Frances S. Kittrell and Suoling Zhou for technical assistance.
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Footnotes |
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Received 4/ 5/05. Revised 6/16/05. Accepted 6/21/05.
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References |
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B-mediated transactivations. J Biol Chem 1998;273:10831–4.B kinase. Mol Cell Biol 2002;22:3549–61.
and retinoid X receptor on cell proliferation and accelerates polyoma middle-T antigen-induced mammary tumorigenesis in mice. Cancer Res 2004;64:7169–77.. Endocrinology 2002;143:2357–65.. Cancer Res 1999;59:1869–76.This article has been cited by other articles:
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  B. W. O'Malley and R. Kumar Nuclear Receptor Coregulators in Cancer Biology Cancer Res., November 1, 2009; 69(21): 8217 - 8222. [Abstract] [Full Text] [PDF]
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 S. Alkner, P.-O. Bendahl, D. Grabau, K. Lovgren, O. Stal, L. Ryden, M. Ferno, and on behalf of the South Swedish and South-East Swed AIB1 is a predictive factor for tamoxifen response in premenopausal women Ann. Onc., July 23, 2009; (2009) mdp293v1. [Abstract] [Full Text] [PDF]
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 A. M. Redmond, F. T. Bane, A. T. Stafford, M. McIlroy, M. F. Dillon, T. B. Crotty, A. D. Hill, and L. S. Young Coassociation of Estrogen Receptor and p160 Proteins Predicts Resistance to Endocrine Treatment; SRC-1 is an Independent Predictor of Breast Cancer Recurrence Clin. Cancer Res., March 15, 2009; 15(6): 2098 - 2106. [Abstract] [Full Text] [PDF]
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 S. Wang, Y. Yuan, L. Liao, S.-Q. Kuang, J. C.-Y. Tien, B. W. O'Malley, and J. Xu Disruption of the SRC-1 gene in mice suppresses breast cancer metastasis without affecting primary tumor formation PNAS, January 6, 2009; 106(1): 151 - 156. [Abstract] [Full Text] [PDF]
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 A. S. Oh, J. T. Lahusen, C. D. Chien, M. P. Fereshteh, X. Zhang, S. Dakshanamurthy, J. Xu, B. L. Kagan, A. Wellstein, and A. T. Riegel Tyrosine Phosphorylation of the Nuclear Receptor Coactivator AIB1/SRC-3 Is Enhanced by Abl Kinase and Is Required for Its Activity in Cancer Cells Mol. Cell. Biol., November 1, 2008; 28(21): 6580 - 6593. [Abstract] [Full Text] [PDF]
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L. Qin, L. Liao, A. Redmond, L. Young, Y. Yuan, H. Chen, B. W. O'Malley, and J. Xu The AIB1 Oncogene Promotes Breast Cancer Metastasis by Activation of PEA3-Mediated Matrix Metalloproteinase 2 (MMP2) and MMP9 Expression Mol. Cell. Biol., October 1, 2008; 28(19): 5937 - 5950. [Abstract] [Full Text] [PDF]
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L. Liao, X. Chen, S. Wang, A. F. Parlow, and J. Xu Steroid Receptor Coactivator 3 Maintains Circulating Insulin-Like Growth Factor I (IGF-I) by Controlling IGF-Binding Protein 3 Expression Mol. Cell. Biol., April 1, 2008; 28(7): 2460 - 2469. [Abstract] [Full Text] [PDF]
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L. Amazit, L. Pasini, A. T. Szafran, V. Berno, R.-C. Wu, M. Mielke, E. D. Jones, M. G. Mancini, C. A. Hinojos, B. W. O'Malley, et al. Regulation of SRC-3 Intercompartmental Dynamics by Estrogen Receptor and Phosphorylation Mol. Cell. Biol., October 1, 2007; 27(19): 6913 - 6932. [Abstract] [Full Text] [PDF]
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Y. Yuan, L. Qin, D. Liu, R.-C. Wu, P. Mussi, S. Zhou, Z. Songyang, and J. Xu Genetic Screening Reveals an Essential Role of p27kip1 in Restriction of Breast Cancer Progression Cancer Res., September 1, 2007; 67(17): 8032 - 8042. [Abstract] [Full Text] [PDF]
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 Y. Yuan and J. Xu Loss-of-Function Deletion of the Steroid Receptor Coactivator-1 Gene in Mice Reduces Estrogen Effect on the Vascular Injury Response Arterioscler Thromb Vasc Biol, July 1, 2007; 27(7): 1521 - 1527. [Abstract] [Full Text] [PDF]
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A. C-K. Chung, S. Zhou, L. Liao, J. C.-Y. Tien, N. M. Greenberg, and J. Xu Genetic Ablation of the Amplified-in-Breast Cancer 1 Inhibits Spontaneous Prostate Cancer Progression in Mice Cancer Res., June 15, 2007; 67(12): 5965 - 5975. [Abstract] [Full Text] [PDF]
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 P. Mussi, C. Yu, B. W. O'Malley, and J. Xu Stimulation of Steroid Receptor Coactivator-3 (SRC-3) Gene Overexpression by a Positive Regulatory Loop of E2F1 and SRC-3 Mol. Endocrinol., December 1, 2006; 20(12): 3105 - 3119. [Abstract] [Full Text] [PDF]
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J. Yan, C.-T. Yu, M. Ozen, M. Ittmann, S. Y. Tsai, and M.-J. Tsai Steroid Receptor Coactivator-3 and Activator Protein-1 Coordinately Regulate the Transcription of Components of the Insulin-Like Growth Factor/AKT Signaling Pathway. Cancer Res., November 15, 2006; 66(22): 11039 - 11046. [Abstract] [Full Text] [PDF]
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P. Mussi, L. Liao, S.-E. Park, P. Ciana, A. Maggi, B. S. Katzenellenbogen, J. Xu, and B. W. O'Malley Haploinsufficiency of the corepressor of estrogen receptor activity (REA) enhances estrogen receptor function in the mammary gland PNAS, November 7, 2006; 103(45): 16716 - 16721. [Abstract] [Full Text] [PDF]
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A. Mukherjee, S. M. Soyal, R. Fernandez-Valdivia, M. Gehin, P. Chambon, F. J. DeMayo, J. P. Lydon, and B. W. O'Malley Steroid Receptor Coactivator 2 Is Critical for Progesterone-Dependent Uterine Function and Mammary Morphogenesis in the Mouse. Mol. Cell. Biol., September 1, 2006; 26(17): 6571 - 6583. [Abstract] [Full Text] [PDF]
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