Amplified In Breast Cancer Research Articles

Steroid receptor coactivators, HER-2 and HER-3 expression is stimulated by tamoxifen treatment in DMBA-induced breast cancer

BackgroundSteroid receptor coactivators (SRCs) may modulate estrogen receptor (ER) activity and the response to endocrine treatment in breast cancer, in part through interaction with growth factor receptor signaling pathways. In the present study the effects of tamoxifen treatment on the expression of SRCs and human epidermal growth factor receptors (HERs) were examined in an animal model of ER positive breast cancer.MethodsSprague-Dawley rats with DMBA-induced breast cancer were randomized to 14 days of oral tamoxifen 40 mg/kg bodyweight/day or vehicle only (controls). Tumors were measured throughout the study period. Blood samples and tumor tissue were collected at sacrifice and tamoxifen and its main metabolites were quantified using LC-MS/MS. The gene expression in tumor of SRC-1, SRC-2/transcription intermediary factor-2 (TIF-2), SRC-3/amplified in breast cancer 1 (AIB1), ER, HER-1, -2, -3 and HER-4, as well as the transcription factor Ets-2, was measured by real-time RT-PCR. Protein levels were further assessed by Western blotting.ResultsTamoxifen and its main metabolites were detected at high concentrations in serum and accumulated in tumor tissue in up to tenfolds the concentration in serum. Mean tumor volume/rat decreased in the tamoxifen treated group, but continued to increase in controls. The mRNA expression levels of SRC-1 (P = 0.035), SRC-2/TIF-2 (P = 0.002), HER-2 (P = 0.035) and HER-3 (P = 0.006) were significantly higher in tamoxifen treated tumors compared to controls, and the results were confirmed at the protein level using Western blotting. SRC-3/AIB1 protein was also higher in tamoxifen treated tumors. SRC-1 and SRC-2/TIF-2 mRNA levels were positively correlated with each other and with HER-2 (P ≤ 0.001), and the HER-2 mRNA expression correlated with the levels of the other three HER family members (P < 0.05). Furthermore, SRC-3/AIB1 and HER-4 were positively correlated with each other and Ets-2 (P < 0.001).ConclusionsThe expression of SRCs and HER-2 and -3 is stimulated by tamoxifen treatment in DMBA-induced breast cancer. Stimulation and positive correlation of coactivators and HERs may represent an early response to endocrine treatment. The role of SRCs and HER-2 and -3 should be further studied in order to evaluate their effects on response to long-term tamoxifen treatment.

The interaction between hepatitis B virus X protein and AIB1 oncogene is required for the activation of NFκB signal transduction

We identified the interaction between HBV X (HBx) protein and the oncogene AIB1 (amplified in breast cancer 1). A serine/proline motif (SSPSPS) in HBx was found to be required for the interaction. Two LXD motifs [LLXX(X)L, X means any amino acids], LLRNSL and LLDQLHTLL in AIB1 were also found to be involved in the HBx–AIB1 interaction. The HBx–AIB1 interaction was important for the activation of NFκB signal transduction, the HBx mutant that did not interact with AIB1showed dramatically lower NFκB activation activity than the WT HBx. These findings contribute to the new understanding on signal transduction activation mechanisms of HBx.

Preclinical and clinical studies of estrogen deprivation support the PDGF/Abl pathway as a novel therapeutic target for overcoming endocrine resistance in breast cancer

IntroductionThe majority of breast tumors at primary diagnosis are estrogen receptor positive (ER+). Estrogen (E) mediates its effects by binding to the ER. Therapies targeting the estrogenic stimulation of tumor growth reduce mortality from ER+ breast cancer. However, resistance remains a major clinical problem.MethodsTo identify molecular mechanisms associated with resistance to E-deprivation, we assessed the temporal changes in global gene expression during adaptation to long-term culture of MCF7 human breast cancer cells in the absence of estradiol (E2), long term estrogen deprived (LTED), that leads to recovery of proliferative status and models resistance to an aromatase inhibitor (AI). The expression levels of proteins were determined by western blotting. Proliferation assays were carried out using the dual platelet derived growth factor receptor (PDGFR)/Abelson tyrosine kinase (Abl) inhibitor nilotinib. Luciferase reporter assays were used to determine effects on ER-mediated transactivation. Changes in recruitment of cofactors to the gene regulated by estrogen in breast cancer 1 (GREB1) promoter were determined by chromatin immunoprecipitation (ChIP). Gene expression data were derived from 81 postmenopausal women with ER+ BC pre-treatment and at two-weeks post-treatment with single agent anastrozole in a neoadjuvant trial.ResultsThe PDGF/Abl canonical pathway was significantly elevated as early as one week post E-deprivation (P = 1.94 E-04) and this became the top adaptive pathway at the point of proliferative recovery (P = 1.15 E-07). Both PDGFRβ and Abl protein levels were elevated in the LTED cells compared to wild type (wt)-MCF7 cells. The PDGF/Abl tyrosine kinase inhibitor nilotinib, suppressed proliferation in LTED cells in the presence or absence of E. Nilotinib also suppressed ER-mediated transcription by destabilizing the ER and reducing recruitment of amplified in breast cancer-1 (AIB1) and the CREB binding protein (CBP) to the promoter of the E-responsive gene GREB1. High PDGFRβ in primary ER+ breast cancer of 81 patients prior to neoadjuvant treatment with an AI was associated with poorer antiproliferative response. Additionally PDGFRβ expression increased after two weeks of AI therapy (1.25 fold, P = 0.003).ConclusionsThese preclinical and clinical data indicate that the PDGF/Abl signaling pathway merits clinical evaluation as a therapeutic target with endocrine therapy in ER+ breast cancer.

Amplified in breast cancer 1 enhances human cholangiocarcinoma growth and chemoresistance by simultaneous activation of Akt and Nrf2 pathways

Transcriptional coactivator amplified in breast cancer 1 (AIB1) plays important roles in the progression of several cancers such as prostate cancer, breast cancer, and hepatocellular carcinoma. However, its role in cholangiocarcinoma (CCA), a chemoresistant bile duct carcinoma with a poor prognosis, remains unclear. In this study we found that AIB1 protein was frequently overexpressed in human CCA specimens and CCA cell lines. Down-regulation of AIB1 induced the G2/M arrest and decreased the expression of mitosis-promoting factors including Cyclin A, Cyclin B, and Cdk1 through suppressing the Akt pathway, which resulted in inhibiting CCA cell proliferation. In addition, AIB1 enhanced the chemoresistance of CCA cells at least in part through up-regulating the expression of antiapoptotic protein Bcl-2. AIB1 regulated the expression of Bcl-2 in CCA cells through activating the Akt pathway as well as suppressing intracellular reactive oxygen species (ROS). AIB1 suppressed ROS by up-regulating antioxidants such as glutathione synthetase and glutathione peroxidase, which are targets of the NF-E2-related factor 2 (Nrf2), a critical transcription factor that regulates antioxidants, detoxification enzymes, and drug efflux proteins. AIB1 also increased the expression of another two Nrf2 targets, ABCC2 and ABCG2, to enhance drug efflux. AIB1 served as an essential coactivator for Nrf2 activation by physically interacting with Nrf2 to enhance its transcriptional activity. AIB1 plays an important role in proliferation and chemoresistance of CCA through simultaneous activation of Akt and Nrf2 pathways, suggesting that AIB1 is a potential molecular target for CCA treatment.

The transcriptional activity of co‐activator AIB1 is regulated by the SUMO E3 Ligase PIAS1

Amplified in breast cancer 1 (AIB1) is a transcriptional coactivator of nuclear receptors and other transcription factors. It is required for animal growth and reproductive development, and has also been implicated in breast carcinogenesis. Although AIB1 is known to be covalently modified by SUMO-1, which serves to regulate its stability and transcriptional activity, the exact SUMO E3 ligase involved in its sumoylation has not been determined. In order to resolve this question, we investigated the interaction between AIB1 and different members of PIAS proteins (all are SUMO E3 ligases) through immunoprecipiation. Among the five different PIAS proteins, only PIAS1 co-immunoprecipitated with AIB1 in extract prepared from breast cancer cells (MCF-7). Over-expression of PIAS1 together with AIB1 in MCF-7 cells led to increased sumoylation of AIB1, resulting in repression of its transcriptional activity. In contrast, the PIAS1 mutant (C350S) lacking E3 ligase activity appeared to have no effect on the sumoylation of AIB1. Through sumoylation of AIB1, PIAS1 also promoted the stability of AIB1 and attenuated its interaction with estrogen receptor α (ERα), resulting in repression of the transactivation activity of ERα. In addition, MCF-7 cells co-transfected with wild-type PIAS1 and AIB1 showed about 40% reduction in cell growth, while cells co-transfected with wild-type PIAS1 and mutant AIB1 resistant to sumoylation showed about 34% increase in cell growth compared to cells transformed with wild-type AIB1 only. Taken together, these results suggested that PIAS1 may play a crucial role in the regulation of AIB1 transcriptional activity through sumoylation.

Antiproliferative effects of selective cyclooxygenase-2 inhibitor modulated by nimotuzumab in estrogen-dependent breast cancer cells

Breast cancer is the most common malignancy in women, and many breast cancer patients fail conventional treatment strategies of chemotherapy, radiation, and antiestrogen therapy. Research into the molecular pathways and biomarkers involved in the development of breast cancer should yield information that will guide therapeutic decisions. Epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) are involved in the carcinogenesis of breast cancer and exist tight crosstalk with estrogen receptor (ER) pathway. Combination of EGFR and COX-2 inhibitors, therefore, could be an effective strategy for reducing cell growth in estrogen-dependent breast cancer. In order to verify the effects of EGFR and COX-2 inhibitors, breast cancer cells MCF-7 and SKBR-3 were characterized for receptors status and then treated with respective inhibitors (nimotuzumab and celecoxib) alone and in combination. Both cell lines were sensitive to celecoxib, but not to nimotuzumab. However, combination of two drugs demonstrated synergistic effects on cell killing. Moreover, association of two drugs resulted in SKBR-3 cells, a further G0/G1 phase arrest than one drug alone. Downregulation of p-EGFR, p-Akt, p-mTOR, and amplified in breast cancer 1 (AIB1) were observed in both cell lines, and upregulation of E-cadherin was only found in MCF-7, after treatment with single agent or in combination. These studies suggest that nimotuzumab and celecoxib exert synergistic antiproliferation effects in breast cancer, which partly correlates with ER status. Due to Akt/mTOR, EMT and AIB1 pathways participate in this process, therefore, E-cadherin and AIB1 may be considered as possible biomarkers to predict response in ER-positive breast cancer cells treated with EGFR and COX-2 inhibitors.

Igf1r is not required for AIB1-induced mammary hyperplasia and ductal branching

The oncogene AIB1 (amplified in breast cancer 1) is a transcriptional coactivator which is up-regulated in many types of tumors including breast cancer. Studies with cell lines and animal models reveal that AIB1 interacts with the IGF-I signaling pathway at different molecular levels. To determine whether AIB1-dependent cell growth requires IGF-I signaling, we deleted the Igf1r gene specifically in the mammary gland of transgenic mice which overexpress AIB1 and are characterized by the development of epithelial hyperplasia, a pre-neoplastic change in breast tissue. Loss of Igf1r alone reduced cell proliferation, ductal branching and fat pad occupancy in comparison with wild-type glands. However, in the transgenic mice that overexpress moderate levels of AIB1, the absence of Igf1r had a minimal effect on epithelial hyperplasia and ductal branching in the mammary gland. Thus, our results confirm the essential role of Igf1r in mammary gland morphogenesis and demonstrate that overexpression of AIB1 circumvents the requirement for the Igf1r pathway in promoting epithelial growth during mammary development.

Assessing estrogen signaling aberrations in breast cancer risk using genetically engineered mouse models

Aberrations in estrogen signaling increase breast cancer risk. Molecular mechanisms that impact breast cancer initiation, promotion, and progression can be investigated using genetically engineered mouse models. Increasing estrogen receptor alpha (ERα) expression levels twofold is sufficient to initiate and promote breast cancer progression. Initiation and promotion can be increased by p53 haploinsufficiency and by coexpressing the nuclear coactivators amplified in breast cancer 1 (AIB1) or the splice variant AIB1Δ3. Progression to invasive cancer is found with coexpression of these nuclear coactivators as well as following a single dose of 7,12-dimethylbenz(a)anthracene. Loss of signal transducer and activator of transcription 5a reduces the prevalence of initiation and promotion but does not protect from invasive cancer development. Cyclin D1 loss completely interrupts mammary epithelial proliferation and survival when ERα is overexpressed. Loss of breast cancer gene 1 increases estrogen signaling and cooperates with ERα overexpression in initiation, promotion, and progression of mammary cancer.

Proteomic analysis of pathways involved in estrogen-induced growth and apoptosis of breast cancer cells.

BackgroundEstrogen is a known growth promoter for estrogen receptor (ER)-positive breast cancer cells. Paradoxically, in breast cancer cells that have been chronically deprived of estrogen stimulation, re-introduction of the hormone can induce apoptosis.Methodology/Principal FindingsHere, we sought to identify signaling networks that are triggered by estradiol (E2) in isogenic MCF-7 breast cancer cells that undergo apoptosis (MCF-7:5C) versus cells that proliferate upon exposure to E2 (MCF-7). The nuclear receptor co-activator AIB1 (Amplified in Breast Cancer-1) is known to be rate-limiting for E2-induced cell survival responses in MCF-7 cells and was found here to also be required for the induction of apoptosis by E2 in the MCF-7:5C cells. Proteins that interact with AIB1 as well as complexes that contain tyrosine phosphorylated proteins were isolated by immunoprecipitation and identified by mass spectrometry (MS) at baseline and after a brief exposure to E2 for two hours. Bioinformatic network analyses of the identified protein interactions were then used to analyze E2 signaling pathways that trigger apoptosis versus survival. Comparison of MS data with a computationally-predicted AIB1 interaction network showed that 26 proteins identified in this study are within this network, and are involved in signal transduction, transcription, cell cycle regulation and protein degradation.ConclusionsG-protein-coupled receptors, PI3 kinase, Wnt and Notch signaling pathways were most strongly associated with E2-induced proliferation or apoptosis and are integrated here into a global AIB1 signaling network that controls qualitatively distinct responses to estrogen.

Abstract 3115: Role of nuclear coactivator AIB1 in endothelial cell function and angiogenesis

Abstract The nuclear receptor coactivator oncogene AIB1 (Amplified In Breast Cancer 1) has a clearly defined role in steroid and growth factor signaling in epithelial cells of cancers. Recently, we have demonstrated that AIB1 is functionally required for HER2-mediated mammary tumorigenesis in MMTV/Neu mouse model for breast cancer through regulation of HER2 phosphorylation and signaling. Generation of neu/HER2 transgenic mice with either loss of one or both copies of the AIB1 gene resulted in reduced or complete abolition of mammary tumor development, respectively. One very interesting aspect of this study was that the tumors that arose in HER2 AIB1 HET (+/-) mice were phenotypically different from those in HER2 AIB1 WT (+/+) mice in that they were smaller, more necrotic and significantly less vascularized. This data indicated to us the possibility that the role of AIB1 was not confined to the mammary epithelium proliferation but also had major effects on the surrounding stromal cells. In addition, recent expression array data indicated that AIB1 is also overexpressed in human breast cancer stroma which includes the endothelium. In here, we examined the role of AIB1 in endothelial cell function. We demonstrate that reduction of AIB1 levels in endothelial cells cultured in vitro prevents their ability to form endothelial tubes, in conjuction with reduced proliferation and motility. Furthermore, in ECIS (Electric Cell-substrate Impedance Sensing) assay, HUVEC cells with low AIB1 expression loss the ability to form a monolayer on the surface of gold-film electrodes. Consistent with the reduced proliferation that we observed, cDNA array analysis that compared gene expression patterns of HUVEC cells +/- AIB1 shRNA indicates that a number of key cell cycle regulatory genes and cytokines were regulated by reduction in AIB1 levels. Overall these data describe a new role for nuclear receptor coactivator AIB1 in the endothelium. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3115. doi:10.1158/1538-7445.AM2011-3115

AIB1 shows variation in interaction with ERβTAD and expression as a function of age in mouse brain

Estrogen mediates its multiple functions in the brain through the recruitment of a number of interacting proteins. In this paper, we report the identification of 160kD interacting nuclear protein of estrogen receptor (ER)β-transactivation domain (TAD) as amplified in breast cancer 1(AIB1) by pull down assay, immunoblotting, far-western analysis and immunoprecipitation. Further we show the age dependent interaction and expression of AIB1 in the brain of young (6weeks), adult (25weeks) and old (70weeks) AKR strain mice of both sexes. The immunoprecipitation data revealed higher interaction of AIB1 in young than adult and old male mice. In contrast, the interaction was low in young, increased in adult but decreased in old female. However, immunoblotting showed age related increase in the expression of AIB1 in both male and female mice. Further, the level of interaction of AIB1 with ERβTAD in young and old male was significantly higher than female of same age, whereas the expression of AIB1 in adult and old female was significantly higher than male of same age. These data suggest that such age dependent variation in the interaction of AIB1 with ERβTAD and its expression may be helpful to regulate estrogen-mediated gene functions during aging and neurodegenerative diseases.

Altered AIB1 or AIB1Δ3 Expression Impacts ERα Effects on Mammary Gland Stromal and Epithelial Content

Amplified in breast cancer 1 (AIB1) (also known as steroid receptor coactivator-3) is a nuclear receptor coactivator enhancing estrogen receptor (ER)α and progesterone receptor (PR)-dependent transcription in breast cancer. The splice variant AIB1Δ3 demonstrates increased ability to promote ERα and PR-dependent transcription. Both are implicated in breast cancer risk and antihormone resistance. Conditional transgenic mice tested the in vivo impact of AIB1Δ3 overexpression compared with AIB1 on histological features of increased breast cancer risk and growth response to estrogen and progesterone in the mammary gland. Combining expression of either AIB1 or AIB1Δ3 with ERα overexpression, we investigated in vivo cooperativity. AIB1 and AIB1Δ3 overexpression equivalently increased the prevalence of hyperplastic alveolar nodules but not ductal hyperplasia or collagen content. When AIB1 or AIB1Δ3 overexpression was combined with ERα, both stromal collagen content and ductal hyperplasia prevalence were significantly increased and adenocarcinomas appeared. Overexpression of AIB1Δ3, especially combined with overexpressed ERα, led to an abnormal response to estrogen and progesterone with significant increases in stromal collagen content and development of a multilayered mammary epithelium. AIB1Δ3 overexpression was associated with a significant increase in PR expression and PR downstream signaling genes. AIB1 overexpression produced less marked growth abnormalities and no significant change in PR expression. In summary, AIB1Δ3 overexpression was more potent than AIB1 overexpression in increasing stromal collagen content, inducing abnormal mammary epithelial growth, altering PR expression levels, and mediating the response to estrogen and progesterone. Combining ERα overexpression with either AIB1 or AIB1Δ3 overexpression augmented abnormal growth responses in both epithelial and stromal compartments.

Overexpression of AIB1 negatively affects survival of surgically resected non-small-cell lung cancer patients

BackgroundThe amplified in breast cancer 1 (AIB1) gene has been considered to play an oncogenic role in human cancers, but its clinical/prognostic significance in non-small-cell lung cancer (NSCLC) is still unclear. Patients and methodsThe methods of immunohistochemistry and FISH were utilized to examine protein expression and amplification of AIB1 in 230 informative surgically resected NSCLCs and in 30 samples of normal lung tissues. ResultsOverexpression and amplification of AIB1 were found in 48.3% and 8.2% of NSCLCs, respectively. AIB1 overexpression was associated with AIB1 gene amplification and cell proliferation but not related to estrogen receptor (ER)-α, ER-β, progesterone receptor or androgen receptor status. A positive correlation between AIB1 overexpression and an ascending pathologic node stage in lung adenocarcinoma (ADC) was observed (P = 0.043). Univariate survival analysis demonstrated a significant association of AIB1 overexpression with shortened patient survival, especially for those with stage III disease (P < 0.001). Importantly, AIB1 expression was evaluated as the most significant predictor for survival in multivariate analysis (hazards ratio = 2.069, P < 0.001). ConclusionOverexpression of AIB1 might provide a selective advantage for lymph node metastasis of lung ADC and serve as a useful biomarker for poor prognosis for NSCLC patients.

Estrogen-Dependent and Estrogen-Independent Mechanisms Contribute to AIB1-Mediated Tumor Formation

We have previously reported the oncogenic properties of the gene amplified in breast cancer 1 (AIB1), a member of the p160 family of hormone receptor coactivators. In a transgenic mouse model, AIB1 overexpression resulted in a high incidence of tumors in various tissues, including mammary gland, uterus, lung, and pituitary. To determine whether the AIB1 oncogenicity in this model depended on its function as an estrogen receptor (ER) coactivator, we abolished ER signaling through two independent approaches, by performing ovariectomy on AIB1 transgenic (AIB1-tg) mice to prevent gonadal estrogen production and by crossing AIB1-tg mice with ERalpha-null mutant mice. Ovariectomized (ovx) mice, but not AIB1 x ERalpha-/- mice, still developed mammary gland hyperplasia and ductal carcinoma in situ. Both approaches, however, completely prevented the development of invasive mammary tumors, indicating that invasive mammary tumor formation is strictly estrogen dependent. Once developed, AIB1-induced mammary tumors can subsequently lose their dependence on estrogen: Injection of ERalpha(+) tumor cell lines derived from such tumors into ovx or untreated wild-type mice resulted in a similar rate of tumor growth in both groups. Surprisingly, however, ovx mice had an approximately 4-fold higher rate of metastasis formation, suggesting that estrogen provided some protection from metastasis formation. Lastly, our experiments identified oncogenic functions of AIB1 that are independent of its ER coactivation, as both approaches, ovariectomy and ER-/- crosses, still resulted in a high incidence of tumors in the lung and pituitary. We therefore conclude that AIB1 can exert its oncogenicity through tissue-specific estrogen-dependent and estrogen-independent functions.

The AIB1 gene polyglutamine repeat length polymorphism and the risk of breast cancer development

Carriers of BRCA1/2 mutations are at high lifetime risk of breast cancer (BC); however, the BC onset broadly vary in individual patients. Recently, polyglutamine (poly-Q) repeat length polymorphism of the amplified in breast cancer 1 (AIB1) gene was analyzed as a risk factor influencing BC onset in BRCA1/2 mutation carriers with contradictory results. We genotyped AIB1 poly-Q repeat in 243 BRCA1/2 mutation carriers, 61 patients with familial BC (negatively tested for the presence of BRCA1/2 mutation), 221 patients with sporadic BC, and 176 non-cancer controls using denaturing high-performance liquid chromatography and statistically evaluated the effect of AIB1 poly-Q repeat length polymorphism on BC onset. Having used previously published statistical analyses of AIB1 poly-Q repeat length (≥28 and ≥29 repeat cutpoints or analysis of AIB1 poly-Q repeat length as continuous variable), we did not find any association between AIB1 poly-Q repeat length and BC development in analyzed BC groups. However, the analysis of individual genotypes revealed that AIB1 genotype consisting of 28/28 glutamine repeats served as a protective factor in BRCA1 mutation carriers (HR=0.64; 95% CI 0.41-0.99; P=0.045) and as a risk factor in carriers of mutation in exon 11 of the BRCA2 gene (HR=3.50; 95% CI 1.25-9.78; P=0.017). Our results confirm that AIB1 poly-Q repeat length polymorphism does not influence the BC risk in general but suggest that the specific AIB1 genotypes should be considered in patients with BC carrying mutation in the BRCA1/2 genes.

Effect of Low-Dose Tamoxifen on Steroid Receptor Coactivator 3/Amplified in Breast Cancer 1 in Normal and Malignant Human Breast Tissue

Nuclear receptor coactivator expression and activity may partly explain the complex agonist/antagonist effects of tamoxifen at clinical level. In a preoperative trial, dose reduction from 20 to 1 mg tamoxifen was associated with retained antiproliferative effect on breast cancer. Here, we assessed the gene expression of the steroid receptor coactivators SRC-1, SRC-2/transcription intermediary factor 2, and SRC-3/amplified in breast cancer 1 (AIB1) and the growth factor receptor HER-2/neu under three tamoxifen dose regimens. Surgical specimens from estrogen receptor-positive breast cancer and adjacent normal breast tissue from 64 patients treated 4 weeks preoperatively with 20, 5, or 1 mg/d tamoxifen and 28 nontreated breast cancer controls were analyzed for coactivator and HER-2/neu mRNA expression using real-time reverse transcription-PCR. The gene expression levels were related to immunohistochemical expression of Ki67, serum levels of insulin-like growth factor I and sex hormone binding globulin, other prognostic factors, and clinical outcome. The coactivators and HER-2/neu mRNA levels were higher in malignant compared with normal tissue (P < 0.001). Tamoxifen significantly increased the expression of coactivators in normal and malignant tissue irrespective of dose, especially for SRC-3/AIB1 (P < 0.001 tamoxifen-treated versus nontreated subjects). SRC-3/AIB1 and HER-2/neu mRNA levels were positively correlated (P = 0.016), but the coactivators could not explain the variability of Ki67, insulin-like growth factor I, and sex hormone binding. Although not significant, SRC-3/AIB1 tended to be higher in subjects with poor clinical outcome and unfavorable prognostic factors. Increased coactivator mRNA levels seem to be an early response to tamoxifen without dose-response relationship in the 1- to 20-mg range. Clinical and molecular effects of low-dose tamoxifen should be further explored.

Overexpression of transcriptional coactivator AIB1 promotes hepatocellular carcinoma progression by enhancing cell proliferation and invasiveness

Amplified in breast cancer 1 (AIB1) is a transcriptional coactivator for nuclear receptors and other transcription factors. AIB1 has an important role in malignancy of several cancers such as breast and prostate cancers. However, its involvement in human hepatocellular carcinoma (HCC) progression remains unclear. Here, we found that AIB1 protein was overexpressed in 23 of 34 human HCC specimens (68%). Down-regulation of AIB1 reduced HCC cell proliferation, migration, invasion, colony formation ability and tumorigenic potential in nude mice. These phenotypic changes caused by AIB1 knockdown correlated with increased expression of the cell cycle inhibitor p21(Cip1/Waf1) and decreased Akt activation and the expression of proliferating cell nuclear antigen (PCNA) and matrix metallopeptidase MMP-9. In agreement with these findings, clinical AIB1-positive HCC expressed higher levels of PCNA than AIB1-negative HCC. A positive correlation was established between the levels of AIB1 protein and PCNA protein in HCC, suggesting that AIB1 may contribute to HCC cell proliferation. In addition, MMP-9 expression in AIB1-postive HCC was significantly higher than that in AIB1-negative HCC, suggesting that AIB1-postive HCC may be more invasive. Collectively, our results show that overexpression of AIB1 promotes human HCC progression by enhancing cell proliferation and invasiveness. Therefore, AIB1 is a master regulator of human HCC growth and might be a useful molecular target for HCC prognosis and treatment.

Prepubertal genistein exposure affects erbB2/Akt signal and reduces rat mammary tumorigenesis

Breast cancer is the most common malignancy in women worldwide and pharmaceutical agents have therapeutic and preventive effects in breast cancer. The human epidermal growth factor receptor 2/neu is one of the most important oncogenes in human breast cancer. Prepubertal exposure to endogenous estradiol and a phytoestrogen, genistein (Gen), has been shown to reduce future breast cancer risk. Gen downregulates tyrosine kinase regulated protein expression and reduces prostate cancer. In this study, the effects of prepubertal exposure to Gen on rat mammary carcinogenesis and the erbB2/Akt signal pathway were investigated. Prepubertal female Sprague-Dawley rats were daily exposed to Gen at 125 mg/kg (Gen-1) and 500 mg/kg (Gen-5) from postnatal days 22-28. Subsequently, the rats were given a single dose of 100 mg/kg 7.12-dimethylbenz [a] anthracene on postnatal day 42 to induce mammary tumor. The mRNA expression of erbB2 and amplified in breast cancer 1 (AIB1) was detected by reverse transcription-polymerase chain reaction. The protein levels of proliferating cell nuclear antigen (PCNA), erbB2, phosphotyrosine protein, Akt, and p-Akt were detected by immunohistochemistry and Western blotting. The activity of protein tyrosine kinase (PTK) was detected by liquid scintillation counting. The percentage of rats with mammary tumors in breast cancer model (BCM), Gen-1, and Gen-5 was 71.43, 52.38, and 33.34%, respectively. The incidence of 7.12-dimethylbenz [a] anthracene-induced mammary tumors significantly decreased in Gen-5 compared with that in BCM. The mRNA levels of AIB1 and erbB2 and the protein levels of erbB2, p-Akt, and PCNA protein expression were downregulated for a long time in the mammary tumors in Gen-5 groups. The activity of PTK was also decreased for a long time. However, the total Akt protein expression did not change significantly among BCM, Gen-1, and Gen-5. Prepubertal exposure of Sprague-Dawley female rats to 500 mg/kg Gen can reduce later breast cancer risk and its protective effect is associated with persistent downregulation of the expression of erbB2, p-Akt, AIB1, and PCNA and with low PTK activity in the mammary tumor. Our results suggest that erbB2/Akt signaling plays a role in tumor formation and targeting erbB2/Akt signaling with prepubertal exposure to Gen may provide greater efficacy to the current therapies used to treat tumors.

AIB1 is a predictive factor for tamoxifen response in premenopausal women

BackgroundClinical trials implicate the estrogen receptor (ER) coactivator amplified in breast cancer 1 (AIB1) to be a prognostic and a treatment-predictive factor, although results are not unanimous. We have further investigated this using a controlled randomised trial of tamoxifen versus control. Materials and methodsA total of 564 premenopausal women were entered into a randomised study independent of ER status. Using a tissue microarray, AIB1 and ER were analysed by immunohistochemistry. ResultsAIB1 scores were obtained from 349 women. High AIB1 correlated to factors of worse prognosis (human epidermal growth factor receptor 2, Nottingham histological grade 3, and lymph node metastases) and to ER negativity. In the control arm, high AIB1 was a negative prognostic factor for recurrence-free survival (RFS) (P = 0.02). However, ER-positive patients with high AIB1 responded significantly to tamoxifen treatment (P = 0.002), increasing RFS to the same level as for systemically untreated patients with low AIB1. Although ER-positive patients with low AIB1 had a better RFS from the beginning, this was not further improved by tamoxifen (P = 0.8). ConclusionsIn the control group, high AIB1 was a negative prognostic factor. However, ER-positive patients with high AIB1 responded significantly to tamoxifen. This implicates high AIB1 to be an independent predictive factor of improved response to tamoxifen and not, as has previously been discussed, a factor predicting tamoxifen resistance.

Predictors of response to exemestane as primary endocrine therapy in estrogen receptor–positive breast cancer

Endocrine therapy is the most important treatment of choice for estrogen receptor (ER)-positive breast cancer. Potential mechanisms for resistance to endocrine therapy involve ER-coregulatory proteins and cross-talk between ER and other growth factor-signaling networks. However, the factors and pathways responsible for endocrine therapy resistance, particularly resistance to aromatase inhibitors, have not been clearly established. Sixteen postmenopausal patients with ERalpha-positive primary breast cancer were treated daily with 25 mg of exemestane (an aromatase inhibitor) for 6 months. Expressions of ERalpha, ERbeta, progesterone receptor (PgR), androgen receptor (AR), amplified in breast cancer 1 (AIB1), aromatase, epidermal growth factor receptor, human epidermal growth factor receptor type 2, Ki67, cyclin D1, p53, Bcl2, signal transducer and activator of transcription 5 (Stat5), and insulin-like growth factor binding protein 5 (IGFBP5), and phosphorylations of ERalpha serine (Ser) 118, ERalpha Ser167, Akt Ser473, and p44/42 MAPK threonine (Thr) 202/tyrosine (Tyr) 204, were examined by immunohistochemistry on pretreatment tumor biopsies and post-treatment surgical specimens. Analyses were made to test for correlations with response to exemestane. Of the 16 patients, seven responded and nine retained stable disease. High-level expression of AIB1 and phosphorylation of Akt Ser473 were significantly associated with a better response to exemestane, suggesting that these factors could be considered as predictors of exemestane response. Expressions of ERalpha, ERbeta, PgR, aromatase, Ki67, cyclin D1, and p53, and phosphorylations of ERalpha Ser118, ERalpha Ser167, and p44/42 MAPK Thr202/Tyr204, were decreased, whereas expressions of Stat5 and IGFBP5 were increased in post-treatment specimens compared to the values in pretreatment biopsies. Thus, the analysis of factors involved in the estrogen-dependent growth-signaling pathways may be useful in identifying patients responsive to exemestane.