Catherine Ibarra

Isoform specificity of trimethylamine N-oxygenation by human flavin-containing monooxygenase (FMO) and P450 enzymes Selective catalysis by fmo3

In the present study, we expressed human flavin-containing monooxygenase 1 (FMO1), FMO3, FMO4t (truncated), and FMO5 in the baculovirus expression vector system at levels of 0.6 to 2.4 nmol FMO/mg of membrane protein. These four isoforms, as well as purified rabbit FMO2, and eleven heterologously expressed human P450 isoforms were examined for their capacity to metabolize trimethylamine (TMA) to its N-oxide (TMAO), using a new, specific HPLC method with radiochemical detection. Human FMO3 was by far the most active isoform, exhibiting a turnover number of 30 nmol TMAO/nmol FMO3/min at pH 7.4 and 0.5 mM TMA. None of the other monooxygenases formed TMAO at rates greater than 1 nmol/nmol FMO/min under these conditions. Human fetal liver, adult liver, kidney and intestine microsomes were screened for TMA oxidation, and only human adult liver microsomes provided substantial TMAO-formation (range 2.9 to 9.1 nmol TMAO/mg protein/min, N = 5). Kinetic studies of TMAO formation by recombinant human FMO3, employing three different analytical methods, resulted in a Km of 28 +/- 1 microM and a Vmax of 36.3 +/- 5.7 nmol TMAO/nmol FMO3/min. The Km determined in human liver microsomes ranged from 13.0 to 54.8 microM. Therefore, at physiological pH, human FMO3 is a very specific and efficient TMA N-oxygenase, and is likely responsible for the metabolic clearance of TMA in vivo in humans. In addition, this specificity provides a good in vitro probe for the determination of FMO3-mediated activity in human tissues, by analyzing TMAO formation at pH 7.4 with TMA concentrations not higher than 0.5 mM.

Molecular and Cellular PharmacologyIsoform specificity of trimethylamine N-oxygenation by human flavin-containing monooxygenase (FMO) and P450 enzymes: Selective catalysis by fmo3

In the present study, we expressed human flavin-containing monooxygenase 1 (FMO1), FMO3, FMO4t (truncated), and FMO5 in the baculovirus expression vector system at levels of 0.6 to 2.4 nmol FMO/mg of membrane protein. These four isoforms, as well as purified rabbit FMO2, and eleven heterologously expressed human P450 isoforms were examined for their capacity to metabolize trimethylamine (TMA) to its N-oxide (TMAO), using a new, specific HPLC method with radiochemical detection. Human FMO3 was by far the most active isoform, exhibiting a turnover number of 30 nmol TMAO/nmol FMO3/min at pH 7.4 and 0.5 mM TMA. None of the other monooxygenases formed TMAO at rates greater than 1 nmol/nmol FMO/min under these conditions. Human fetal liver, adult liver, kidney and intestine microsomes were screened for TMA oxidation, and only human adult liver microsomes provided substantial TMAO-formation (range 2.9 to 9.1 nmol TMAO/mg protein/min, N = 5). Kinetic studies of TMAO formation by recombinant human FMO3, employing three different analytical methods, resulted in a Km of 28 +/- 1 microM and a Vmax of 36.3 +/- 5.7 nmol TMAO/nmol FMO3/min. The Km determined in human liver microsomes ranged from 13.0 to 54.8 microM. Therefore, at physiological pH, human FMO3 is a very specific and efficient TMA N-oxygenase, and is likely responsible for the metabolic clearance of TMA in vivo in humans. In addition, this specificity provides a good in vitro probe for the determination of FMO3-mediated activity in human tissues, by analyzing TMAO formation at pH 7.4 with TMA concentrations not higher than 0.5 mM.

1.3-Å Resolution structure of human glutathione S-transferase with S-hexyl glutathione bound reveals possible extended ligandin binding site

Cytosolic glutathione S‐transferases (GSTs) play a critical role in xenobiotic binding and metabolism, as well as in modulation of oxidative stress. Here, the high‐resolution X‐ray crystal structures of homodimeric human GSTA1‐1 in the apo form and in complex with S‐hexyl glutathione (two data sets) are reported at 1.8, 1.5, and 1.3Å respectively. At this level of resolution, distinct conformations of the alkyl chain of S‐hexyl glutathione are observed, reflecting the nonspecific nature of the hydrophobic substrate binding site (H‐site). Also, an extensive network of ordered water, including 75 discrete solvent molecules, traverses the open subunit–subunit interface and connects the glutathione binding sites in each subunit. In the highest‐resolution structure, three glycerol moieties lie within this network and directly connect the amino termini of the glutathione molecules. A search for ligand binding sites with the docking program Molecular Operating Environment identified the ordered water network binding site, lined mainly with hydrophobic residues, suggesting an extended ligand binding surface for nonsubstrate ligands, the so‐called ligandin site. Finally, detailed comparison of the structures reported here with previously published X‐ray structures reveal a possible reaction coordinate for ligand‐dependent conformational changes in the active site and the C‐terminus. Proteins 2002;48:618–627.

Morphologically normal-appearing mammary epithelial cells obtained from high-risk women exhibit methylation silencing of INK4a/ARF.

Purpose: p16(INK4a) has been appreciated as a key regulator of cell cycle progression and senescence. Cultured human mammary epithelial cells that lack p16(INK4a) activity have been shown to exhibit premalignant phenotypes, such as telomeric dysfunction, centrosomal dysfunction, a sustained stress response, and, most recently, a dysregulation of chromatin remodeling and DNA methylation. These data suggest that cells that lack p16(INK4a) activity would be at high risk for breast cancer development and may exhibit an increased frequency of DNA methylation events in early cancer. Experimental Design: To test this hypothesis, the frequencies of INK4a/ARF promoter hypermethylation, as well as four additional selected loci, were tested in the initial random periareolar fine needle aspiration samples from 86 asymptomatic women at high risk for development of breast cancer, stratified using the Masood cytology index. Results:INK4a/ARF promoter hypermethylation was observed throughout all early stages of intraepithelial neoplasia and, importantly, in morphologically normal-appearing mammary epithelial cells; 29 of 86 subjects showed INK4a/ARF promoter hypermethylation in at least one breast. Importantly, INK4a/ARF promoter hypermethylation was not associated with atypia, and the frequency of hypermethylation did not increase with increasing Masood cytology score. The frequency of INK4a/ARF promoter hypermethylation was associated with the combined frequency of promoter hypermethylation of retinoic acid receptor-β2, estrogen receptor-α, and breast cancer-associated 1 genes (P = 0.001). Conclusions: Because INK4a/ARF promoter hypermethylation does not increase with age but increases with the frequency of other methylation events, we predict that INK4a/ARF promoter hypermethylation may serve as a marker of global methylation dysregulation.

IRF-1 Promotes Apoptosis in p53-damaged Basal-type Human Mammary Epithelial Cells: A Model for Early Basal-type Mammary Carcinogenesis

Mammary gland homeostasis is regulated by both endogenous and exogenous signals, creating a balance between proliferation and apoptosis. It is thought that breast cancer develops from the acquisition of multiple genetic changes. The function of tumor suppressor p53 is fequently lost in cancers; however, not all cells that lose p53 progress to become invasive cancer. We have developed a model of early mammary carcinogenesis to investigate some of the internal and external signaling pathways that target the elimination ot normal basal-type human mammary epithelial cells (HMECs) that acutely acquire p53-damage. Here, we show that both tamoxifen (Tam) and three-dimensional prepared extracellular matrix culture (3-D rECM) induce apoptosis in HMEC cells with acute loss of p53 [*p53(-) HMECs] through induction of interferon regulatory factor-1 (IRF-1). Tam and rECM signaling in *p53(-) HMECs (1) promotes the recruitment of a STAT1/ CBP complex to the IRF-1 promoter, (2) upregulates IRF-1, (3) activates caspase-1 and -3, and (4) induces apoptosis. Suppression of IRF-1 with siRNA oligos inhibited both Tam- and rECM-induced apoptosis. These observations demonstrate that IRF-1 plays a critical role in eliminating p53-damaged cells, and may play a more global role in mammary gland homeostasis.

CREB-binding protein regulates apoptosis and growth of HMECs grown in reconstituted ECM via laminin-5

Interactions between normal mammary epithelial cells and extracellular matrix (ECM) are important for mammary gland homeostasis. Loss of interactions between ECM and normal mammary epithelial cells are thought to be an early event in mammary carcinogenesis. CREB-binding protein (CBP) is an important regulator of proliferation and apoptosis but the role of CBP in ECM signaling is poorly characterized. CBP was suppressed in basal-cytokeratin-positive HMECs (CK5/6+, CK14+, CK8–, CK18–, CK19–). Suppression of CBP resulted in loss of reconstituted ECM-mediated growth control and apoptosis and loss of laminin-5 α3-chain expression. Suppression of CBP in normal human mammary epithelial cells (HMECs) resulted in loss of CBP occupancy of the LAMA3A promoter and decreased LAMA3A promoter activity and laminin-5 α-3 chain expression. Exogenous expression of CBP in CBP-negative HMECs that have lost reconstituted ECM-mediated growth regulation and apoptosis resulted in (1) CBP occupancy of the LAMA3A promoter, (2) increased LAMA3A activity and laminin-5 α3-chain expression, and (3) enhancement of reconstituted ECM-mediated growth regulation and apoptosis. Similarly, suppression of laminin-5 α3-chain expression in HMECs resulted in loss of reconstituted ECM-mediated growth control and apoptosis. These observations suggest that loss of CBP in basal-cytokeratin-positive HMECs results in loss of reconstituted ECM-mediated growth control and apoptosis through loss of LAMA3A activity and laminin-5 α3-chain expression. Results in these studies may provide insight into early events in basal-type mammary carcinogenesis.

Interferon regulatory factor-1 regulates reconstituted extracellular matrix (rECM)-mediated apoptosis in human mammary epithelial cells.

Interactions between extracellular matrix (ECM) and mammary epithelial cells are critical for mammary gland homeostasis and apoptotic signaling. Interferon regulatory factor-1 (IRF-1) is a transcriptional regulator that promotes apoptosis during mammary gland involution and p53-independent apoptosis. We have recently shown that rapid cell surface tamoxifen (Tam) signaling promotes apoptosis in normal human mammary epithelial cells that were acutely damaged by expression of human papillomavirus type-16 E6 protein (*HMEC-E6). Apoptosis was mediated by recruitment of CREB-binding protein (CBP) to the γ-activating sequence (GAS) element of the IRF-1 promoter, induction of IRF-1 and caspase-1/-3 activation. Here, we show that growth factor-depleted, reconstituted ECM (rECM), similar to Tam, promotes apoptosis in *HMEC-E6 cells through induction of IRF-1. Apoptosis was temporally associated with recruitment of CBP to the GAS element of the IRF-1 promoter, induction of IRF-1 expression and caspase-1/-3 activation. Small interfering RNA-mediated suppression of IRF-1 protein expression in *HMEC-E6 cells blocked (1) induction of IRF-1, (2) caspase-1/-3 activation and (3) apoptosis. These observations demonstrate that IRF-1 promotes rECM-mediated apoptosis and provide evidence that both rECM and rapid Tam signaling transcriptionally activate IRF-1 through recruitment of CBP to the IRF-1 GAS promoter complex.

Abstract A15: Proteomic profiling of early mammary carcinogenesis: Targeting dysregulated protein pathways with tailored therapies

Over the last 50 years, the number of cancer related deaths has decreased by only 2%. One of the most promising approaches to reduce breast cancer mortality is to develop tools for early detection and early intervention of breast cancers. Normal mammary gland homeostasis requires the coordinated regulation of signaling networks; whereas, dysregulation of signaling networks occurs during breast cancer initiation. Reverse-phase protein microarray (RPPM) is a high-throughput proteomic tool, developed to test for dysregulation of protein signaling networks in human biopsy specimens. For example, ErbB2/HER2/neu, Erb3, EGFR, Akt, Erk1/2 receptor tyrosine kinases all play a role in cancer cell growth and survival, and developing targeted therapies against these pathways is a promising tailored approach to breast cancer treatment. To test which tyrosine kinases are activated in early mammary carcinogenesis, reverse phase protein microarray analysis (RPPM) was performed on 31 random periareolar fine needle aspirates (RPFNA) obtained from a cohort of asymptomatic high-risk women, testing 59 antibody endpoints related to cell growth and survival pathways, including ER, EGFR, Her2, Erb3, Akt and Erk1/2, to test for patterns of differential protein expression. RPFNA allows for serial sampling of breast cytology and subsequent histology from asymptomatic women at high risk for developing breast cancer and also provides the ability to monitor response to preventative treatments. Epithelial cell clusters from RPFNA samples were isolated using AutoPix automated laser capture microdissection. RPPM uses denatured lysate so antigen retrieval, which is a limitation for tissue arrays, is not a problem. In addition, each sample is printed in serial dilution, providing an internal standard, and RPPM does not require direct labeling of the sample, thus improving reproducibility, sensitivity, and robustness. An interrogation of phosphorylated versus non-phosphorylated state of proteins was performed using a Wilcoxon rank sum test. Spearman rank correlation coefficients were used to estimate the association between RPPM total- and phospho-protein expression as a function of cytology; a 2-sided p value Four distinct clusters of phospho-proteins were identified amongst the 31 RPFNA samples. Varying cell lines, including 15hTert, MDA231, T47D, were then treated with inhibitors based on the protein pathways identified in these clusters. Fluoxetine, a common antidepressant, was shown to affect cancer cell viability as well as the phosphorylation of Erk1/2 pathway in a concentration and time dependent manner. Diagnosing cancers based on proteomic signatures in addition to histopathology will allow for individualized selection of therapeutic combinations that can best target the entire disease-specific errant protein network of a patient. This proteomic signature has great potential for daily clinical practice considering the RPPM technology is commercially available through Theranostics Health and could be used every time a woman undergoes a routine biopsy. In addition, understanding a key protein pathway and how it is wired could have a profound effect on both functional biology and on the understanding of cancer mechanisms as a whole. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A15.

Abstract A20: Random periareolar fine‐needle aspiration is highly reproducible in a CALGB multi‐institutional cross‐sectional study

Background: Biomarkers that vary with risk and response to prevention interventions are referred to as “surrogate endpoint biomarkers”. As outlined by Fabian et al., surrogate endpoint biomarkers should be 1) biologically and statistically significantly associated with cancer development, 2) present in a reasonable proportion of at‐risk individuals, 3) obtainable by minimally invasive procedures, and 4) reversible with prevention interventions that have been validated to decrease cancer incidence. Many modalities have been suggested as potential surrogate endpoint biomarkers for breast cancer, including mammographic density, serum biomarkers, and breast tissue biomarkers. Currently, there is no consensus as to the optimal surrogate endpoint biomarker. Random Periareolar Fine Needle Aspiration (RPFNA) is a research technique developed to assess short‐term breast cancer risk in women at increased risk for breast cancer. While there is increasing acceptance of RPFNA, neither the reproducibility nor the inter‐institutional compatibility of RPFNA has been established. To address these key limitations, the Cancer and Leukemia Group B (CALGB) Prevention Group tested the reproducibility of RPFNA in a multi‐institutional cross‐sectional study. Methods: Sixty‐three high‐risk women from five CALGB institutions (Duke, Ohio State, Roswell Park, Dana‐Farber, and Vermont) underwent RPFNA from July 1, 2007 to June 30, 2008. Duplicate bilateral RPFNA was performed on each woman by a single investigator on a single day. Masood Cytology Index score was assessed by a single blinded cytopathologist. Results: There was a high degree of statistical agreement in the Masood Cytology Index scores of duplicate RPFNA samples from the same breast, with a Spearman correlation coefficient of 0.8312 (p Conclusions: This multi‐institutional study demonstrates that RPFNA is a highly reproducible measure of breast cytology in a cooperative group cross‐sectional trial. RPFNA did not demonstrate a high degree of agreement between breasts, suggesting that breast cancer risk and progression may occur at different rates in individual breasts from a single woman. These data provide important validation of the reproducibility of RPFNA in a multi‐institutional cross‐sectional study that included cohorts that varied in demographic composition. Important future directions will include a larger RPFNA cohort study and testing for the reproducibility of RPFNA samples with atypia before and after administration of chemoprevention agents. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A20.