Heinz-Joachim List

Expression of the nuclear coactivator AIB1 in normal and malignant breast tissue

The gene of the nuclear receptor coactivator AIB1 (amplified in breast cancer 1) is amplified in breast cancer cell lines as well as in breast tumor tissues. AIB1 mRNA is often highly expressed (>60) in primary breast tumors and it has been shown that AIB1 enhances estrogen and progesterone dependent transcription invitro. Therefore, it has been postulated that AIB1 contributes to the development of breast cancer. However, to date, it has not been shown that AIB1 amplification and overexpression correlates with elevated protein levels in breast cancer tissues. In this study we analyzed protein levels of AIB1 in normal and breast tumor tissues by immunohistochemistry. We compared 41 human breast tumor tissues with 24 normal breast tissue samples and found that AIB1 stained in the nuclei of approximately 46% of the tumors and 30% of the normal tissues. Overall, AIB1 protein levels were significantly higher in tumor tissue than in normal tissue and the highest levels of nuclear staining were found exclusively in breast tumor tissues in 9.8% of the cases. These data suggest that increased AIB1 mRNA expression does not always translate into elevated protein levels and that AIB1 most likely will be relevant to the etiology of a subset of about 10% of breast carcinomas.

Ribozyme Targeting Demonstrates That the Nuclear Receptor Coactivator AIB1 Is a Rate-limiting Factor for Estrogen-dependent Growth of Human MCF-7 Breast Cancer Cells

Human breast tumorigenesis is promoted by the estrogen receptor pathway, and nuclear receptor coactivators are thought to participate in this process. Here we studied whether one of these coactivators, AIB1 (amplified inbreast cancer 1), was rate-limiting for hormone-dependent growth of human MCF-7 breast cancer cells. We developed MCF-7 breast cancer cell lines in which the expression of AIB1 can be modulated by regulatable ribozymes directed against AIB1 mRNA. We found that depletion of endogenous AIB1 levels reduced steroid hormone signaling via the estrogen receptor α or progesterone receptor β on transiently transfected reporter templates. Down-regulation of AIB1 levels in MCF-7 cells did not affect estrogen-stimulated cell cycle progression but reduced estrogen-mediated inhibition of apoptosis and cell growth. Finally, upon reduction of endogenous AIB1 expression, estrogen-dependent colony formation in soft agar and tumor growth of MCF-7 cells in nude mice was decreased. From these findings we conclude that, despite the presence of different estrogen receptor coactivators in breast cancer cells, AIB1 exerts a rate-limiting role for hormone-dependent human breast tumor growth.

The nuclear receptor coactivator AIB1 mediates insulin-like growth factor I-induced phenotypic changes in human breast cancer cells.

The nuclear receptor coactivator AIB1 (amplified in breast cancer 1) is overexpressed in human breast cancers and is required for estrogen signaling. However, the role of AIB1 in breast cancer etiology is not known. Here, we show that AIB1 is rate-limiting for insulin-like growth factor I (IGF-I)-dependent phenotypic changes and gene expression in human breast cancer cells. Reduction of endogenous AIB1 levels by small interfering RNA in MCF-7 breast cancer cells prevented IGF-I–stimulated anchorage-independent growth by reducing IGF-I–dependent anti-anoikis. cDNA array and immunoblot analysis of gene expression revealed that reduction in AIB1 levels led to a significant decrease in the expression of several genes controlling the cell cycle and apoptosis. These AIB1-dependent changes were also observed in the presence of estrogen antagonist and were corroborated in the estrogen receptor-negative cell line MDA MB-231. AIB1 reduction decreased the expression of the IGF-I receptor and IRS-1 in MCF-7 but not in MDA MB-231 cells. IGF-I–stimulated activation of AKT was reduced by AIB1 small interfering RNA treatment, whereas mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) activation by IGF-I was unaffected. We conclude that AIB1 is required for IGF-I–induced proliferation, signaling, cell survival, and gene expression in human breast cancer cells, independent of its role in estrogen receptor signaling.

A role for TGF-β in estrogen and retinoid mediated regulation of the nuclear receptor coactivator AIB1 in MCF-7 breast cancer cells

AIB1 (amplified in breast cancer 1) is a nuclear receptor coactivator gene amplified and overexpressed in breast cancer. However, the mechanisms by which AIB1 is regulated are unclear. Here we show that 17β-estradiol represses AIB1 mRNA and protein expression in MCF-7 human breast cancer cells primarily by suppressing AIB1 gene transcription. Estrogen levels present in fetal calf serum are sufficient to maintain AIB1 mRNA and protein at low basal levels, and this repression is reversed by the addition of antiestrogens or all-trans retinoic acid. Interestingly, cycloheximide inhibition experiments revealed that secondary protein synthesis was necessary to induce AIB1 expression by antiestrogens and retinoids. Experiments with TGF-β and TGF-β blocking antibodies demonstrated that this growth factor modulates AIB1 expression and showed that the antiestrogen and retinoid induction of AIB1 gene expression is mediated at least in part through TGF-β. These data reveal a mechanism of estrogen-induced down-modulation of the overall hormone sensitivity of cells through feedback inhibition of coactivator gene expression. These data also suggest that antiestrogens can shift the sensitivity of cells to non-estrogenic proliferative signaling by increasing cellular levels of AIB1. This effect may play a role in breast cancer progression and resistance to drug treatment.

Heregulin-β1 regulates the estrogen receptor-α gene expression and activity via the ErbB2/PI 3-K/akt pathway

This study examines whether the serine/threonine protein kinase, Akt, is involved in the crosstalk between the ErbB2 and estrogen receptor-α (ER-α) pathways. Treatment of MCF-7 cells with 10−9 M heregulin-β1 (HRG-β1) resulted in a rapid phosphorylation of Akt and a 15-fold increase in Akt activity. Akt phosphorylation was blocked by inhibitors of phosphatidylinositol 3-kinase (PI 3-K), by antiestrogens, the protein tyrosine kinase inhibitor, genistein, and by AG825, a selective ErbB2 inhibitor; but not by AG30, a selective EGFR inhibitor. Akt phosphorylation by HRG-β1 was abrogated by an arginine to cysteine mutation (R25C) in the pleckstrin homology (PH) domain of Akt, and HRG-β1 did not induce Akt phosphorylation in the ER-negative variant of MCF-7, MCF-7/ADR. Transient transfection of ER-α into these cells restored Akt phosphorylation by HRG-β1, suggesting the requirement of ER-α. HRG-β1 did not activate Akt in MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme, further confirming a role for ErbB2. Stable transfection of the cells with a dominant negative Akt or with the R25C-Akt mutant, as well as PI 3-K inhibitors, blocked the effect of HRG-β1 on ER-α expression and activity and on the growth of MCF-7 cells. Stable transfection of MCF-7 cells with a constitutively active Akt mimicked the effect of HRG-β1. Experiments employing selective ErbB inhibitors demonstrate that the effect of HRG-β1 on ER-α expression and activity is also mediated by ErbB2 and not by EGFR, demonstrating that ErbB2 is the primary mediator of the effects of HRG-β1 on ER-α regulation. Taken together, our data suggest that HRG-β1, bound to the ErbB2 ErbB3 heterodimer, in the presence of membrane ER-α, interacts with and activates PI 3-K/Akt. Akt leads to nuclear ER-α phosphorylation, thereby altering its expression and transcriptional activity.

Influence of the human endogenous retrovirus-like element HERV-E.PTN on the expression of growth factor pleiotrophin: a critical role of a retroviral Sp1-binding site.

Germ line insertion of a human endogenous retrovirus-like element (HERV-E.PTN) into the growth factor pleiotrophin (PTN) gene generated a phylogenetically new promoter driving the expression of functional HERV-PTN fusion transcripts. Here we show by in situ hybridization, that HERV-PTN fusion transcripts are expressed in malignant trophoblasts (i.e. choriocarcinoma) and in the proliferative and in the invasive trophoblasts of gestational trophoblastic tissue. Additionally, a 1.9 kb fragment of the HERV-derived PTN promoter was analysed which has strong activity when transiently transfected into choriocarcinoma JEG-3 cells in contrast to HeLa cells. Deletion of the retrovirally-derived promoter portion abolished its activity and an enhancer (+443 to +486) was identified in this region. Electrophoretic mobility shift and supershift experiments identified a Sp1 binding site in this enhancer and site specific mutation of this site abolished its activity in choriocarcinoma cells. Sp1 overexpression in Drosophila SL2 cells showed that the enhancer activity is mediated via Sp1 binding in vivo. Furthermore, mutation of the Sp1 binding site reduced the activity of a promoter test fragment in choriocarcinoma cells by 80%. Our result shows that a retroviral Sp1 binding site in the PTN promoter is important for the expression of growth factor pleiotrophin in human choriocarcinoma cells.