Helen C. Hurst

Arylamine N‐acetyltransferase 1 expression in breast cancer cell lines: A potential marker in estrogen receptor‐positive tumors

The prognosis for patients with estrogen receptor (ER)‐positive breast cancer has improved significantly with the prescription of selective ER modulators (SERMs) for ER‐positive breast cancer treatment. However, only a proportion of ER‐positive tumors respond to SERMs, and resistance to hormonal therapies is still a major problem. Detailed analysis of published microarray studies revealed a positive correlation between overexpression of the drug metabolizing enzyme arylamine N‐acetyltransferase type 1 (NAT1) and ER positivity, and increasing evidence supports a biological role for NAT1 in breast cancer progression. We have tested a range of ER‐positive and ER‐negative breast cancer cell lines for NAT1 enzyme activity, and monitored promoter and polyadenylation site usage. Amongst ER‐positive lines, NAT1 activities ranged from 202 ± 28 nmol/min/mg cellular protein (ZR‐75‐1) to 1.8 ± 0.4 nmol/min/mg cellular protein (MCF‐7). The highest levels of NAT1 activity could not be attributed to increased NAT1 gene copy number; however, we did detect differences in NAT1 promoter and polyadenylation site usage amongst the breast tumor‐derived lines. Thus, whilst all cell lines tested accumulated transcripts derived from the proximal promoter, the line expressing NAT1 most highly additionally initiated transcripts initiating at a more distal, “tissue”‐specific promoter. These data pave the way for investigating NAT1 transcripts as candidate prognostic markers in ER‐positive breast cancer. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.

Androgen Receptor Is Targeted to Distinct Subcellular Compartments in Response to Different Therapeutic Antiandrogens

Purpose: Antiandrogens are routinely used in the treatment of prostate cancer. Although they are known to prevent activation of the androgen receptor (AR), little is known about the mechanisms involved. This report represents the first study of the localization of wild-type AR following expression at physiologic relevant levels in prostate cells and treatment with androgen and antiandrogens. Experimental Design: We have characterized a cellular model for prostate cancer using in situ cellular fractionation, proteomics, and confocal microscopy and investigated the effect of antiandrogens in clinical use on the subcellular localization of the AR. Results: Different antiandrogens have diverse effects on the subcellular localization of the AR. Treatment with androgen results in translocation from the cytoplasm to the nucleoplasm, whereas the antiandrogens hydroxyflutamide and bicalutamide lead to reversible association with the nuclear matrix. In contrast, treatment with the antiandrogen cyproterone acetate results in AR association with cytoplasmic membranes and irreversible retention within the cytoplasm. In addition, we demonstrate that AR translocation requires ATP and the cytoskeleton, regardless of ligand. Conclusions: These results reveal that not all antiandrogens work via the same mechanism and suggest that an informed sequential treatment regime may benefit prostate cancer patients. The observed subnuclear and subcytoplasmic associations of the AR suggest new areas of study to investigate the role of the AR in the response and resistance of prostate cancer to antiandrogen therapy.

Histone Demethylase KDM5B Collaborates with TFAP2C and Myc To Repress the Cell Cycle Inhibitor p21cip (CDKN1A)

ABSTRACT The TFAP2C transcription factor has been shown to downregulate transcription of the universal cell cycle inhibitor p21cip (CDKN1A). In examining the mechanism of TFAP2C-mediated repression, we have identified a ternary complex at the proximal promoter containing TFAP2C, the oncoprotein Myc, and the trimethylated lysine 4 of histone H3 (H3K4me3) demethylase, KDM5B. We demonstrated that while TFAP2C and Myc can downregulate the CDKN1A promoter independently, KDM5B acts as a corepressor dependent on the other two proteins. All three factors collaborate for optimal CDKN1A repression, which requires the AP-2 binding site at −111/−103 and KDM5B demethylase activity. Silencing of TFAP2C-KDM5B-Myc led to increased H3K4me3 at the endogenous promoter and full induction of CDKN1A expression. Coimmunoprecipitation assays showed that TFAP2C and Myc associate with distinct domains of KDM5B and the TFAP2C C-terminal 270 amino acids (aa) are required for Myc and KDM5B interaction. Overexpression of all three proteins resulted in forced S-phase entry and attenuation of checkpoint activation, even in the presence of chemotherapy drugs. Since each protein has been linked to poor prognosis in breast cancer, our findings suggest that the TFAP2C-Myc-KDM5B complex promotes cell cycle progression via direct CDKN1A repression, thereby contributing to tumorigenesis and therapy failure.

AP-2γ promotes proliferation in breast tumour cells by direct repression of the CDKN1A gene

Overexpression of the activator protein (AP)‐2γ transcription factor in breast tumours has been identified as an independent predictor of poor outcome and failure of hormone therapy. To understand further the function of AP‐2γ in breast carcinoma, we have used an RNA interference and gene expression profiling strategy with the MCF‐7 cell line as a model. Gene expression changes between control and silenced cells implicate AP‐2γ in the control of cell cycle progression and developmental signalling. A function for AP‐2γ in cell cycle control was verified using flow cytometry: AP‐2γ silencing led to a partial G1/S arrest and induction of the cyclin‐dependent kinase inhibitor, p21cip/CDKN1A. Reporter and chromatin immunoprecipitation assays demonstrated a direct, functional interaction by AP‐2γ at the CDKN1A proximal promoter. AP‐2γ silencing coincided with acquisition of an active chromatin conformation at the CDKN1A locus and increased gene expression. These data provide a mechanism whereby AP‐2γ overexpression can promote breast epithelial proliferation and, coupled with previously published data, suggest how loss of oestrogen regulation of AP‐2γ may contribute to the failure of hormone therapy in patients.

Alternative TFAP2A isoforms have distinct activities in breast cancer

IntroductionAP-2α is a transcription factor implicated in the regulation of differentiation and proliferation in certain tissues, including the mammary gland. In breast tumours, continued expression of AP-2α has been correlated with a better prognosis, but this is hard to reconcile with a reported role in the upregulation of the ERBB2 oncogene. The existence of TFAP2A isoforms, deriving from alternative first exons and differing in their N-terminal sequence, has been described in some mammals, but their relative abundance and activity has not been investigated in the human breast.MethodsExpression levels of four TFAP2A isoforms were assayed at the level of RNA and protein (via the generation of isoform-specific antibodies) in a panel of breast tumour cell lines and in tissue from normal breast and primary tumour samples. Expression constructs for each isoform were used in reporter assays with synthetic and natural promoters (cyclin D3 and ERBB2) to compare the activation and repression activity of the isoforms.ResultsWe demonstrate that the two isoforms AP-2α 1b and AP-2α 1c, in addition to the originally cloned, AP-2α 1a, are conserved throughout evolution in vertebrates. Moreover, we show that isoform 1c in particular is expressed at levels at least on a par with the 1a isoform in breast epithelial lines and tissues and may be more highly expressed in tamoxifen resistant tumours. The isoforms share a similar transactivation mechanism involving the recruitment of the adaptors CITED2 or 4 and the transactivators p300 or CBP. However, isoform 1b and 1c are stronger transactivators of the ERBB2 promoter than isoform 1a. In contrast, AP-2α 1a is the only isoform able to act as a repressor, an activity that requires an intact sumoylation motif present within the N-terminus of the protein, and which the other two isoforms lack.ConclusionsOur findings suggest that TFAP2A isoforms may be differentially regulated during breast tumourigenesis and this, coupled with differences in their transcriptional activity, may impact on tumour responses to tamoxifen therapy. These data also have implications for the interpretation of tumour studies that seek to correlate outcomes with TFAP2A expression level.

Dual association by TFAP2A during activation of the p21cip/CDKN1A promoter

The cyclin-dependent kinase inhibitor p21cip/CDKN1A is induced to promote growth arrest in response to a variety of stimuli in normal cells and loss of correct regulation of this gene is frequently observed in cancer. In particular, the upregulation of CDKN1A by p53 is considered to be a central mechanism of tumour suppression. Other transcription factors with tumour suppressor activity can also regulate CDKN1A, including the developmentally regulated factor, TFAP2A. Here we identify a novel AP-2 binding site within the proximal promoter of the CDKN1A gene and show this is required for optimal, p53-independent expression of p21cip/CDKN1A. We further describe a non-tumourgenic breast epithelial cell line model to study the role of endogenous TFAP2A and p53 in the control of drug-induced p21cip expression using ChIP. Maximal expression of CDKN1A requires TFAP2A which binds to two regions of the promoter: the proximal region where the AP-2 site lies and upstream near the major p53 binding site. The pattern of binding alters with time post-induction, with the proximal, p53-independent site becoming more important at later stages of p21cip induction. This pattern of promoter interaction by TFAP2A is distinct from that seen for the TFAP2C family member which represses CDKN1A expression.

Functional analysis of a breast cancer-associated FGFR2 single nucleotide polymorphism using zinc finger mediated genome editing.

Genome wide association studies have identified single nucleotide polymorphisms (SNP) within fibroblast growth factor receptor 2 (FGFR2) as one of the highest ranking risk alleles in terms of development of breast cancer. The potential effect of these SNPs, in intron two, was postulated to be due to the differential binding of cis-regulatory elements, such as transcription factors, since all the SNPs in linkage disequilibrium were located in a regulatory DNA region. A Runx2 binding site was reported to be functional only in the minor, disease associated allele of rs2981578, resulting in increased expression of FGFR2 in cancers from patients homozygous for that allele. Moreover, the increased risk conferred by the minor FGFR2 allele associates most strongly in oestrogen receptor alpha positive (ERα) breast tumours, suggesting a potential interaction between ERα and FGFR signalling. Here, we have developed a human cell line model system to study the effect of the putative functional SNP, rs2981578, on cell behaviour. MCF7 cells, an ERα positive breast cancer cell line homozygous for the wild-type allele were edited using a Zinc Finger Nuclease approach. Unexpectedly, the acquisition of a single risk allele in MCF7 clones failed to affect proliferation or cell cycle progression. Binding of Runx2 to the risk allele was not observed. However FOXA1 binding, an important ERα partner, appeared decreased at the rs2981578 locus in the risk allele cells. Differences in allele specific expression (ASE) of FGFR2 were not observed in a panel of 72 ERα positive breast cancer samples. Thus, the apparent increased risk of developing ERα positive breast cancer seems not to be caused by rs2981578 alone. Rather, the observed increased risk of developing breast cancer might be the result of a coordinated effect of multiple SNPs forming a risk haplotype in the second intron of FGFR2.

Abstract 1537: Role of S100PBP in pancreatic adenocarcinoma

We have recently discovered a 37kDa novel S100P-binding protein, S100PBP which exhibits no homology to any currently known protein. We have therefore explored its expression in a wide range of normal and cancer tissues, and performed several in vitro functional assays to define its putative roles. In order to determine S100PBP localization in human samples immunohistochemistry was performed on a multi-organ tissue array (Pantomics) and on an in-house made pancreatic tissue array including normal pancreas, primary cancer and metastatic lesions. To determine S100PBP functions cells with both silenced and stably over-expressing S100PBP were established. Affymetrix gene expression arrays and a battery of functional assays including adhesion, migration and invasion were performed using these cells. We show that S100PBP is ubiquitously expressed in different normal and tumor specimens in a cell and tissue type dependent manor. In normal pancreas S100PBP is widely expressed in both exocrine and endocrine compartments while in pancreatic adenocarcinoma it is present in early precursor lesions, PanINs, and retained in some cancer cells, but with significantly lower intensity. We further show that in pancreatic cancer cells S100PBP binds, in addition to S100P, several other S100 proteins, namely S100A2, S100A4 and S100A6 in a calcium-dependant fashion. Over-expression of S100PBP also causes changes in the expression levels of S100P and S100A9, which were both down-regulated, while the levels of the S100A4 transcript increased. Furthermore, manipulation of S100PBP expression leads to phenotypic changes affecting the adhesive, migratory and invasive capacity of pancreatic cancer cells, which varied depending on the cellular context and constellation of presence of different S100 proteins. Both over-expression and silencing of S100PBP also induced an increase/decrease in Cathepsin Z levels, respectively. This protease appears to be critically important for inducing the observed changes in cellular adhesion. In summary, S100PBP is ubiquitously expressed and modulates the function of several S100 proteins, as well as Cathepsin Z, and appears to play an important role in the adhesive, migratory and invasive capabilities of pancreatic cancer cells. 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 1537. doi:10.1158/1538-7445.AM2011-1537

Abstract 5288: S100PBP is ubiquitously expressed and modulates the function of S100 calcium-binding proteins in pancreatic cancer

We have recently discovered a 37kDa novel S100P-binding protein, S100PBP, which shows no homology to any currently known protein; we have therefore explored its expression in a wide range of normal and cancer tissues, its cellular localization and performed several in vitro functional assays to define its putative roles. In order to determine S100PBP localization in human samples immunohistochemistry was performed on a multi-organ tissue array (Pantomics) and on an in-house made pancreatic ductal adenocarcinoma tissue array including normal, cancer and metastatic lesions. To determine S100PBP function cells with both silenced and stably over-expressing S100PBP were established and investigated by Affymetrix gene expression arrays and a battery of functional assays including adhesion, migration and invasion. We show that this protein is almost ubiquitously expressed, in varying levels, between normal and tumor specimens, which is both cancer- and tissue-type dependent. In normal pancreas it is widely expressed in both exocrine and endocrine compartments while in pancreatic adenocarcinoma its expression is absent in the stroma and retained in some of the cancer cells but with significantly lower intensity. We further show that in addition to S100P, in pancreatic cancer cells S100PBP binds, in a calcium-dependant fashion, several other S100 proteins, namely S100A2, S100A4 and S100A6. Over-expression of S100PBP led to changes in the expression levels of S100P and S100A9, which were down-regulated, and S100A4, which was up-regulated. Furthermore, manipulation of S100PBP expression led to phenotypic changes affecting the adhesive, migratory and invasive capacity of pancreatic cancer cells, that varied depending on the cellular context and constellation of the different S100 proteins present. Both over-expression and silencing of S100PBP also led to an increase/decrease in Cathepsin Z levels, respectively. In summary, S100PBP is ubiquitously expressed and is deregulated in many different cancer types, and appears to play a role in the adhesive, migratory and invasive capabilities of pancreatic cancer cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5288.

Transcriptional regulation of cyclin-dependent kinase inhibitor 1A (P21) by the transcription factor AP-2γ

AP-2 transcription factors constitute a family of sequence-specific DNA-binding proteins encoded by five highly homologous yet functionally distinct genes, AP-2α to AP-2e. AP-2γ appears to play a major role in breast cancer, being expressed in a large proportion of primary tumours. In this study we have analysed in more detail the mechanism of transcriptional regulation of the p21/cyclin-dependent kinase inhibitor 1A (p21/CDKN1A) gene by AP-2γ.