Molecular mechanisms for aberrant expression of the human breast cancer specific gene 1 in breast cancer cells: control of transcription by DNA methylation and intronic sequences.

Breast cancer specific gene 1 (BCSG1), also referred as synuclein gamma, is the third member of a neuronal protein family synuclein. BCSG1 is not expressed in normal breast tissues but highly expressed in advanced infiltrating breast carcinomas. When over expressed, BCSG1 significantly stimulates breast cancer metastasis. To elucidate the molecular mechanisms underlying the abnormal transcription of BCSG1 in breast cancer cells, in this study, we isolated a 2195 base pair fragment of human BCSG1 gene. This fragment includes 1 kb 5'-flanking region, exon 1, and intron 1. By analysing the promoter activity and the methylation status of the exon 1 region, we show that (1) Intron 1 plays critical roles in the control of BCSG1 gene transcription through cis-regulatory sequences that affect BCSG1 transcription in cell type-specific and cell type-nonspecific manners. (2) The activator protein-1 (AP-1) is functionally involved in BCSG1 transcription in breast cancer cells through its binding to an AP-1 motif located in the intron 1. (3) The exon 1 region of BCSG1 gene contains a CpG island that is unmethylated in BCSG1-positive SKBR-3 and T47D cells but densely methylated in BCSG1-negative MCF-7 cells. (4) Treating MCF-7 cells with a demethylating agent 5-Aza-2'-deoxycytidine specifically activated BCSG1 transcription. Thus, our results suggest that while the cellular content of transcription activators and repressors that interact with the cis-regulatory sequences present in the intron 1 contribute prominently to the tissue-specific expression of BCSG1, demethylation of exon 1 is an important factor responsible for the aberrant expression of BCSG1 in breast carcinomas.

Oncostatin M-induced growth inhibition and morphological changes of MDA-MB231 breast cancer cells are abolished by blocking the MEK/ERK signaling pathway.

Cytokine oncostatin M (OM) has profound effects on proliferation and differentiation of breast cancer cells. OM treated cells show reduced growth rate and differentiated phenotypes. The mechanisms underlying the OM growth-inhibitory activity in breast cancer cells have not been fully elucidated. In this study, we investigated the OM-elicited signaling pathways in breast cancer cell lines MDA-MB231 and MCF-7. We show that OM rapidly activates the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription (STAT) 1 and 3 in both cell lines. Intriguingly, OM-induced growth inhibition and morphological changes in MDA-MB231 cells are completely abolished by inhibitors to ERK upstream kinase MEK (nitrogen/extracellular-regulated protein kinase kinase), but the MEK inhibitors have little effects on OM growth-inhibitory activity in MCF-7 cells. In addition, expressions of the cyclin kinase inhibitors p21 and p27 are strongly induced by OM in MCF-7 cells, but their expression is only slightly increased by OM in MDA-MB231 cells. These data together demonstrate that the growth-inhibitory activity of OM can be mediated by different signaling pathways in a cell line-specific manner. While the MEK/ERK pathway is the predominant signaling pathway that leads to the growth inhibition of MDA-MB231 cells, activation of additional signaling pathways are necessary for OM to exert its growth-inhibitory activity in MCF-7 cells.

Requirement of Sp1 and estrogen receptor alpha interaction in 17beta-estradiol-mediated transcriptional activation of the low density lipoprotein receptor gene expression.

Estrogen is one of the most important physiological regulators of low density lipoprotein receptor (LDLR) expression. Despite many studies conducted in animals and humans showing increased expressions of LDLR messenger RNA by hormone treatment, the molecular basis of the effect of estrogen on LDLR transcription has not been clearly elucidated. By using HepG2 cells that transiently express functional estrogen receptor alpha (ERalpha) and LDLR promoter constructs, we show that the specific interaction of ERalpha with the transcription factor Sp1 bound to the LDLR promoter is responsible for the activation of LDLR transcription by estrogen. We demonstrate that 1) mutations to abrogate the binding of Sp1 to its recognition sequences present in repeat 1 and repeat 3 elements of the LDLR promoter completely abolish the ERalpha-mediated activation of the LDLR promoter activity; 2) mutations that abolish the selective DNA-binding activity or inactivate the C-terminal transcription activation function (AF2) of ERalpha had no effect on the ability of ERalpha to activate LDLR transcription; however, transcriptional activation was completely lost by deletion of the N-terminal transcription activation region (AF1); 3) a subregion of AF1 (amino acids 67-139) was further identified to be important for ERalpha to activate the LDLR promoter; and 4) ERalpha enhanced the formation of Sp1-repeat 3 DNA complexes. We also show that mutation at the sterol-responsive element-1 site diminishes the activity of ERalpha on LDLR transcription, thereby suggesting that the sterol-responsive element-1-binding protein may interact with the Sp1-ERalpha complex to trans-activate LDLR gene transcription. This study for the first time provides a molecular basis for an understanding of the regulation of LDLR transcription by estrogens.

The critical role of the PE21 element in oncostatin M-mediated transcriptional repression of the p53 tumor suppressor gene in breast cancer cells.

Cytokine oncostatin M (OM) exerts growth-inhibitory and differentiative effects on breast cancer cells. Previously we showed that the transcription from the p53 gene in breast cancer cells was down regulated by OM. To elucidate the molecular mechanisms underlying the OM effect on p53 transcription, in this study, we dissected the p53 promoter region and analysed the p53 promoter activity in breast tumor cells. We showed that treatment of MCF-7 cells with OM induced a dose- and time-dependent suppression of p53 promoter activity. The p53 promoter activity was decreased to 35% of control at 24 h and further decreased to 20% at 48 h by OM at concentrations of 5 ng/ml and higher. Deletion of the 5'-flanking region of the p53 promoter from -426 to -97 did not affect the OM effect. However, further deletion to -40 completely abolished the repressive effect of OM. The p53 promoter region -96 to -41 contains NF-kappaB and c-myc binding sites, and a newly identified UV-inducible element PE21. Mutations to disrupt NF-kappaB binding or c-myc binding to the p53 promoter decreased the basal promoter activity without affecting the OM-mediated suppression, whereas mutation at the PE21 motif totally abolished the OM effect. We further demonstrated that insertion of PE21 element upstream of the thymidine kinase minimal promoter generated an OM response analogous to that of the p53 promoter. Finally, we detected the specific binding of a nuclear protein with a molecular mass of 87 kDa to the PE21 motif. Taken together, we demonstrate that OM inhibits the transcription of the p53 gene through the PE21 element. Thus, the PE21 element is functionally involved in p53 transcription regulated by UV-induction and OM suppression.

Identification of a novel sterol-independent regulatory element in the human low density lipoprotein receptor promoter.

The cytokine oncostatin M (OM) activates human low density lipoprotein receptor (LDLR) gene transcription through a sterol-independent mechanism. Previous studies conducted in our laboratory have narrowed the OM-responsive element to promoter region -52 to +13, which contains the repeat 3 and two TATA-like sequences. We now identify LDLR promoter region -17 to -1 as a sterol-independent regulatory element (SIRE) that is critically involved in OM-, transcription factor CCAAT/enhancer-binding protein (C/EBP)-, and second messenger cAMP-mediated activation of LDLR transcription. The SIRE sequence overlaps the previously described TATA-like element and consists of an active C/EBP-binding site (-17 to -9) and a functional cAMP-responsive element (CRE) (-8 to -1). We demonstrate that (a) mutations within either the C/EBP or CRE site have no impact on basal or cholesterol-mediated repression of LDLR transcription, but they completely abolish OM-mediated activation of LDLR transcription; (b) replacing the repeat 3 sequence that contains the Sp1-binding site with a yeast transcription factor GAL4-binding site in the LDLR promoter construct does not affect OM inducibility, thereby demonstrating that OM induction is mediated through the SIRE sequence in conjunction with a strong activator bound to the repeat 3 sequence; (c) electrophoretic mobility shift and supershift assays confirm the specific binding of transcription factors C/EBP and cAMP-responsive element-binding protein to the SIRE; (d) cotransfection of a human C/EBPbeta expression vector (pEF-NFIL6) with the LDLR promoter construct pLDLR234 increases LDLR promoter activity; and (e) OM and dibutyryl cAMP synergistically activate LDLR transcription through this regulatory element. This study identifies, for the first time, a cis-acting regulatory element in the LDLR promoter that is responsible for sterol-independent regulation of LDLR transcription.

The expression of p53 tumor suppressor gene in breast cancer cells is down-regulated by cytokine oncostatin M.

Previously (J. Liu, et al., Cell Growth Differ., 8: 667-676, 1997), we showed that oncostatin M (OM), a cytokine produced by activated T cells and macrophages, inhibited the proliferation of breast cancer cells derived from solid tumors and malignant effusions. OM-treated cells showed reduced growth rates and differentiated phenotypes. Because the p53 tumor suppressor protein plays an important role in cellular proliferation, we examined p53 protein expression in three OM-responsive breast cancer cell lines, MCF-7, MDA-MB231, and H3922. Western blot analysis showed that p53 protein levels in all three of the cell lines were decreased by OM treatment. Reduction of p53 protein was detected after 1 day of OM treatment and reached maximal suppression of 10-20% of control after 3 days in H3922 and 40% of control after 4 days in MCF-7 cells. A comparison of p53 mRNA in OM-treated cells versus untreated control cells showed that exposure to OM reduced the steady-state levels of p53 mRNA transcripts to an extent similar to that of the p53 protein levels. This observation suggests that the effect of OM on p53 protein expression does not occur at the posttranslational level. Nuclear run-on assays verified that OM decreased the number of actively transcribed p53 mRNAs, which suggests a transcriptional regulatory mechanism. The effect of OM on p53 expression seems to be mediated through the extracellular signal-regulated kinase (ERK) pathway, inasmuch as the inhibition of ERK activation with a specific inhibitor (PD98059) to the ERK upstream kinase mitogen/extracellular-regulated protein kinase kinase abrogated the OM inhibitory activity on p53 expression in a dose-dependent manner. In addition to OM, we showed that the p53 protein expression in MCF-7 cells was also decreased by phorbol 12-myristate 13-acetate treatment (PMA). Because both OM and PMA induce MCF-7 cells to differentiate, our data suggest that p53 expression in breast cancer cells is down-regulated during the differentiation process.

Induction of low density lipoprotein receptor (LDLR) transcription by oncostatin M is mediated by the extracellular signal-regulated kinase signaling pathway and the repeat 3 element of the LDLR promoter.

Oncostatin M (OM) activates the transcription of the human low density lipoprotein receptor (LDLR) in HepG2 cells through a sterol-independent mechanism. Our previous studies showed that mutations within the repeat 3 sequence of the LDLR promoter significantly decreased OM activity on LDLR promoter luciferase reporter constructs that contain the sterol responsive element-1 (repeat 2) and Sp1 binding sites (repeats 1 and 3). In this study, we investigated the signal transduction pathways that are involved in OM-induced LDLR transcription. In HepG2 cells, OM induced a rapid increase in LDLR mRNA expression, with increases detected at 30 min and maximal induction at 1 h. This OM effect was not blocked by protein synthesis inhibitors, inhibitors of p38 kinase, phosphatidylinositol 3-kinase, or c-Jun N-terminal kinase, but OM activity was completely abolished by pretreating cells with inhibitors of the extracellular signal-regulated kinase (ERK) kinase (mitogen/ERK kinase (MEK)). To investigate whether the repeat 3 sequence of the LDLR promoter is the OM-responsive element that converts ERK activation at the promoter level, three luciferase reporters, pLDLR-TATA containing only the TATA-like elements of the promoter, pLDLR-R3 containing repeat 3 and the TATA-like elements, and pLDLR-234 containing repeats 1, 2, 3 and the TATA-like elements were constructed and transiently transfected into HepG2 cells. OM had no effect on the basal promoter construct pLDLR-TATA; however, including a single copy of repeat 3 sequence in the TATA vector (pLDLR-R3) resulted in a full OM response. The activity of OM on pLDLR-R3 was identical to that of pLDLR-234. Importantly, the ability of OM to increase luciferase activities in both pLDLR-R3- and pLDLR-234-transfected cells was blocked in a dose-dependent manner by inhibition of MEK. These results demonstrate that the mitogen-activated protein kinase MEK/ERK cascade is the essential signaling pathway by which OM activates LDLR gene transcription and provide the first evidence that the repeat 3 element is a new downstream target of ERK activation.