Christopher R. Mueller

Sp1 Is Phosphorylated and Its DNA Binding Activity Down-regulated upon Terminal Differentiation of the Liver

Using nuclear extracts prepared from rat liver it was demonstrated that binding of a transcription factor to site II of the D-site binding protein promoter could be induced by dephosphorylation of these extracts. Competition band shifts and supershift assays reveal this protein to be the general transcription factor Sp1. Phosphorylation of Sp1 appears to occur as a result of terminal differentiation of the liver. Proteins from both 1-day-old rat liver and adult liver undergoing regeneration have less of the phosphorylated form of Sp1 present with consequent increased DNA binding activity. Sp1 is similarly phosphorylated in brain, kidney, and spleen with phosphatase treatment of the extracts significantly increasing the level of DNA binding activity. Dephosphorylation of Sp1 results in a 10-fold increase in the affinity of Sp1 for its cognate site. Two-dimensional gel electrophoresis reveals that approximately 20% of the detectable protein appears to be in the phosphorylated form in adult liver extracts. Another protein with similar characteristics also appears to be present in the liver. Decreasing Sp1 DNA binding activity by phosphorylation may be an important mechanism for regulating gene expression, and possibly bringing about growth arrest during terminal differentiation.

Regulation of BRCA1 expression and its relationship to sporadic breast cancer

Germ-line mutations in the BRCA1 tumour suppressor gene contribute to familial breast tumour formation, but there is no evidence for direct mutation of the BRCA1 gene in the sporadic form of the disease. In contrast, decreased expression of the BRCA1 gene has been shown to be common in sporadic tumours, and the magnitude of the decrease correlates with disease progression. BRCA1 expression is also tightly regulated during normal breast development. Determining how these developmental regulators of BRCA1 expression are co-opted during breast tumourigenesis could lead to a better understanding of sporadic breast cancer aetiology and the generation of novel therapeutic strategies aimed at preventing sporadic breast tumour progression.

Stress and breast cancer: from epidemiology to molecular biology

Stress exposure has been proposed to contribute to the etiology of breast cancer. However, the validity of this assertion and the possible mechanisms involved are not well established. Epidemiologic studies differ in their assessment of the relative contribution of stress to breast cancer risk, while physiological studies propose a clear connection but lack the knowledge of intracellular pathways involved. The present review aims to consolidate the findings from different fields of research (including epidemiology, physiology, and molecular biology) in order to present a comprehensive picture of what we know to date about the role of stress in breast cancer development.

Hydrocortisone down-regulates the tumor suppressor gene BRCA1 in mammary cells: a possible molecular link between stress and breast cancer.

Psychological stress has been correlated with breast cancer development in numerous epidemiological studies. However, physiological and molecular models which may account for this association are not readily available. We have found that the stress hormone hydrocortisone (cortisol) down‐regulates the expression of the breast cancer susceptibility gene BRCA1 in the nonmalignant mouse mammary cell line EPH4. This effect is concentration‐dependent, is reliant on the continuous presence of hydrocortisone, and is not affected by the addition of lactogenic hormones, or growth conditions. Hydrocortisone was also found to negate a known positive effect of estrogen on BRCA1 expression and, therefore, may interfere with estrogen‐related signaling in mammary epithelial cells. The repressive effect of hydrocortisone is diminished or lost in the mouse mammary lines HC‐11 and SP1, respectively, suggesting regulation of the BRCA1 may differ between lines. We have uncovered two promoter regulatory sites, which are involved in BRCA1 regulation by hydrocortisone, namely the RIBS and UP regulatory elements. Binding of the transcription factor GABP to both sites is lost upon hydrocortisone addition, though the levels of these factors are not altered by hydrocortisone treatment. Because BRCA1 activity is important for a number of intracellular pathways involved in prevention of tumorigenesis, its observed down‐regulation may represent a novel molecular mechanism for cortisols involvement in breast cancer development.

A CREB site in the BRCA1 proximal promoter acts as a constitutive transcriptional element

Transcriptional regulation of the BRCA1 proximal promoter has been suggested to play a role in the decreased expression of BRCA1 observed in sporadic breast cancer. Computer analysis of the sequence of the proximal promoter reveals the presence of a potential CREB site. We have identified CREB/ATF-1 as the factor interacting with this site in nuclear extracts from MCF-7 and T-47D cells. This site is shown to be important for the constitutive expression of the promoter in these cells, as well as in Hep G2 cells. Despite the presence of this site, the BRCA1 promoter is not responsive to cAMP induction. It appears that CREB acts as a constitutive positive element for BRCA1 expression and that any mechanism inactivating CREB function would have a dramatic effect on BRCA1 expression.

GA-binding protein alpha/beta is a critical regulator of the BRCA1 promoter

Decreased expression of BRCA1 may play a role in the etiology of sporadic breast cancer. Deletion and point mutant analysis of proximal promoter elements in the BRCA1 1a promoter revealed a 22 bp region which was critical for the expression of the promoter in MCF-7 cells, but had a much reduced effect in T47D cells. The main transcription factor interacting with this site was identified as GABPα/β, and a discrete DNA binding complex was only observed in nuclear extracts from MCF-7 cells. Cotransfection experiments with GABPα and β1 expression vectors produced transactivation of this element in both lines. These results suggest that GABPα/β is a critical activator of BRCA1 expression, and that its activity may differ in human breast cell lines.

The role of the breast cancer susceptibility gene 1 (BRCA1) in sporadic epithelial ovarian cancer

Mutations within the BRCA1 tumor suppressor gene occur frequently in familial epithelial ovarian carcinomas but they are a rare event in the much more prevalent sporadic form of the disease. However, decreased BRCA1 expression occurs frequently in sporadic tumors, and the magnitude of this decrease has been correlated with increased disease progression. The near absence of somatic mutations consequently suggests that there are alternative mechanisms that may contribute to the observed loss of BRCA1 in sporadic tumors. Indeed, both allelic loss at the BRCA1 locus and epigenetic hypermethylation of the BRCA1 promoter play an important role in BRCA1 down-regulation; yet these mechanisms alone or in combination do not always account for the reduced BRCA1 expression. Alternatively, misregulation of specific upstream factors that control BRCA1 transcription may be a crucial means by which BRCA1 is lost. Therefore, determining how regulators of BRCA1 expression may be co-opted during sporadic ovarian tumorigenesis will lead to a better understanding of ovarian cancer etiology and it may help foster the future development of novel therapeutic strategies aimed at halting ovarian tumor progression.

The unliganded glucocorticoid receptor positively regulates the tumor suppressor gene BRCA1 through GABP beta.

Loss of BRCA1 tumor suppressor function is a critical event in breast tumorigenesis. We have previously identified the stress hormone hydrocortisone as a negative regulator of BRCA1 expression in nonmalignant mammary cells. Here, we have identified a direct role for the unliganded glucocorticoid receptor (GR) in BRCA1 upregulation in the absence of hydrocortisone. The positive regulatory effect of GR is lost upon the addition of hydrocortisone. We have shown that GR interacts with the BRCA1 promoter only in the absence of hydrocortisone, and that this interaction is mediated through the β-subunit of the ets transcription factor GA-binding protein (GABP) at the RIBS promoter element. GR and GABPβ interact in both coimmunoprecipitation and mammalian two-hybrid assays, and this interaction involves the N-terminal to central regions of both proteins. This work presents the first evidence of a ligand-independent role for GR as a positive regulator of gene expression, and loss of GR from the BRCA1 promoter in response to stress hormones leads to decreased BRCA1 expression. Because low levels of BRCA1 have been implicated in the development of sporadic breast cancer, this may represent a novel mechanism through which prolonged stress signaling increases breast cancer risk. Mol Cancer Res; 10(4); 558–69. ©2012 AACR.

Binding of the Ets factor GA-binding protein to an upstream site in the factor IX promoter is a critical event in transactivation.

Factor IX is an essential vitamin K-dependent serine protease that participates in the intrinsic pathway of coagulation. The protein is expressed exclusively in the liver. The rare Leyden form of hemophilia B (inherited factor IX deficiency) results from point mutations in three proximal promoter elements that decrease factor IX expression. Recovery of expression occurs following puberty, with factor IX protein levels rising into the normal range. We have previously implicated the PAR domain D-site-binding protein (DBP) as well as an upstream element, site 5, as playing important roles in the phenotypic recovery of hemophilia B Leyden. Here we demonstrate that site 5 binds both the CCAAT/enhancer-binding protein (C/EBPalpha) and the ubiquitous Ets factor GA-binding protein (GABPalpha/beta). Transactivation of the factor IX promoter by the PAR proteins DBP and hepatic leukemia factor (HLF) is dependent on the binding of GABPalpha/beta to site 5, and coexpression of these two factors is required for optimal activation of this promoter. The binding of C/EBPalpha to site 5 also augments the activity of GABPalpha/beta. Analysis of the developmental regulation of site 5-binding proteins in rat liver has shown that C/EBPalpha and the GABPbeta subunit increase markedly in the 2 weeks after birth. These observations establish a functional association between the Ets factor GABPalpha/beta and C/EBPalpha and indicate that the two PAR proteins, DBP and HLF, may play complementary roles in factor IX activation. Given the developmental changes exhibited by these proteins, it is likely that they play a role in regulation of the normal factor IX promoter as well as promoters carrying hemophilia B Leyden mutations.

Ovarian Cancers Harboring Inactivating Mutations in CDK12 Display a Distinct Genomic Instability Pattern Characterized by Large Tandem Duplications.

CDK12 is a recurrently mutated gene in serous ovarian carcinoma, whose downregulation is associated with impaired expression of DNA damage repair genes and subsequent hypersensitivity to DNA-damaging agents and PARP1/2 inhibitors. In this study, we investigated the genomic landscape associated with CDK12 inactivation in patients with serous ovarian carcinoma. We show that CDK12 loss was consistently associated with a particular genomic instability pattern characterized by hundreds of tandem duplications of up to 10 megabases (Mb) in size. Tandem duplications were characterized by a bimodal (∼0.3 and ∼3 Mb) size distribution and overlapping microhomology at the breakpoints. This genomic instability, denoted as the CDK12 TD-plus phenotype, is remarkably distinct from other alteration patterns described in breast and ovarian cancers. The CDK12 TD-plus phenotype was associated with a greater than 10% gain in genomic content and occurred at a 3% to 4% rate in The Cancer Genome Atlas-derived and in-house cohorts of patients with serous ovarian carcinoma. Moreover, CDK12-inactivating mutations together with the TD-plus phenotype were also observed in prostate cancers. Our finding provides new insight toward deciphering the function of CDK12 in genome maintenance and oncogenesis. Cancer Res; 76(7); 1882-91. ©2016 AACR.