Ligand and Linkage Isomers of Bis(ethylthiocarbamato) Copper Complexes with Cyclic C6H8 Backbone Substituents: Synthesis, Characterization, and Antiproliferation Activity.

A series of isomeric bis(alkylthiocarbamate) copper complexes have been synthesized, characterized, and evaluated for antiproliferation activity. The complexes were derived from ligand isomers with 3-methylpentyl (H2L2) and cyclohexyl (H2L3) backbone substituents, which each yield a pair of linkage isomers. The thermodynamic products CuL2a/3a have two imino N and two S donors resulting in three five-member chelate rings (555 isomers). The kinetic isomers CuL2b/3b have one imino and one hydrazino N donor and two S donors resulting in four-, six-, and five-member rings (465 isomers). The 555 isomers have more accessible CuII/I potentials (E1/2 = -811/-768 mV vs. ferrocenium/ferrocene) and lower energy charge transfer bands than their 465 counterparts (E1/2 = -923/-854 mV). Antiproliferation activities were evaluated against the lung adenocarcinoma cell line (A549) and nonmalignant lung fibroblast cell line (IMR-90) using the MTT assay. CuL2a was potent (A549EC50 = 0.080 µM) and selective (IMR-90EC50/A549EC50 = 25) for A549. Its linkage isomer CuL2b had equivalent A549 activity, but lower selectivity (IMR-90EC50/A549EC50 = 12.5). The isomers CuL3a and CuL3b were less potent with A549EC50 values of 1.9 and 0.19 µM and less selective with IMR-90EC50/A549EC50 ratios of 2.3 and 2.65, respectively. There was no correlation between reduction potential and A549 antiproliferation activity/selectivity.

Investigations of Bis(alkylthiocarbamato)copper Linkage Isomers.

Linkage isomers are coordination compounds with the same composition but different donor atoms, resulting in distinct physical and electronic structures. A pair of linkage isomers, CuL555 and CuL465, derived from phenylglyoxal bis(ethylthiocarbamate) were synthesized, isolated, and characterized by structural, electrochemical, and spectroscopic methods. The isomers are stable in solution under ambient conditions, but CuL465 converts to CuL555 in acid, consistent with quantum-chemical calculations. The complexes were screened against a lung adenocarcinoma cell line (A549) and a nonmalignant lung fibroblast cell line (IMR-90) to evaluate the antiproliferation activity. CuL555 and CuL465 possessed EC50 values of 0.113 ± 0.030 and 0.115 ± 0.038 µM for A549 and 1.87 ± 0.29 and 0.77 ± 0.22 µM for IMR-90, respectively.

Synthesis, Characterization, and Biological Activity of Hybrid Thiosemicarbazone-Alkylthiocarbamate Metal Complexes.

A series of hybrid ligands (H2L1-H2L3) derived from 4-methyl-3-thiosemicarbazide and hydrazinecarbothioic acid O-alkyl esters were synthesized and characterized by NMR. The ligands were chelated with copper (4-6), nickel (7-9), and zinc (10-12) and characterized by spectroscopy, electrochemistry, and single crystal X-ray crystallography. The chelated metals displayed substantial anodic shifts in the CuII/I reduction potential of ∼160 mV relative to their bis(thiosemicarbazone) analogues. The metal chelates 4-12 were evaluated for potential anticancer activity by MTT assays, and selected results were confirmed by clonogenic and trypan blue assays. The copper derivatives 4 and 6 were found to have potent and cancer-selective antiproliferative effects, with GI50 values less than 100 nM in A549 lung adenocarcinoma cells compared with at least 20-fold less activity in IMR90 nonmalignant lung fibroblasts. In comparison, the nickel complexes were much less active and had little cancer-selectivity. Varying by ligand, the zinc complexes were less potent or had comparable activity compared to that of the corresponding copper complex. UV-visible spectroscopy indicated that zinc complex 10 was transmetalated in the presence of equimolar copper, whereas nickel complex 7 was not. Copper complexes 4 and 6 were also assessed in the NCI60 screen and were found to have cytotoxic activity against most solid tumor cell lines. In MTT assays, 4 and 6 were substantially more active against A549 cancer cells than Cu(ATSM) and were more cancer-selective (for A549 compared to IMR-90) than Cu(GTSM). Our results suggest that hybrid thiosemicarbazone-alkylthiocarbamate copper complexes have potential for development as new anticancer agents.

Interaction between smoking history and gene expression levels impacts survival of breast cancer patients.

In contrast to studies focused on cigarette smoking and risk of breast cancer occurrence, this study explored the influence of smoking on breast cancer recurrence and progression. The goal was to evaluate the interaction between smoking history and gene expression levels on recurrence and overall survival of breast cancer patients. Multivariable Cox proportional hazards models were fitted for 48 cigarette smokers, 50 non-smokers, and the total population separately to determine which gene expressions and gene expression/cigarette usage interaction terms were significant in predicting overall and disease-free survival in breast cancer patients. Using methods similar to Andres et al. (BMC Cancer 13:326, 2013a; Horm Cancer 4:208-221, 2013b), multivariable analyses revealed CENPN, CETN1, CYP1A1, IRF2, LECT2, and NCOA1 to be important predictors for both breast carcinoma recurrence and mortality among smokers. Additionally, COMT was important for recurrence, and NAT1 and RIPK1 were important for mortality. In contrast, only IRF2, CETN1, and CYP1A1 were significant for disease recurrence and mortality among non-smokers, with NAT2 additionally significant for survival. Analysis of interaction between smoking status and gene expression values using the combined samples revealed significant interactions between smoking status and CYP1A1, LECT2, and CETN1. Signatures consisting of 7-8 genes were highly predictive for breast cancer recurrence and overall survival among smokers, with median C-index values of 0.8 and 0.73 for overall survival and recurrence, respectively. In contrast, median C-index values for non-smokers was only 0.59. Hence, significant interactions between gene expression and smoking status can play a key role in predicting breast cancer patient outcomes.

Interrogating differences in expression of targeted gene sets to predict breast cancer outcome.

BACKGROUND: Genomics provides opportunities to develop precise tests for diagnostics, therapy selection and monitoring. From analyses of our studies and those of published results, 32 candidate genes were identified, whose expression appears related to clinical outcome of breast cancer. Expression of these genes was validated by qPCR and correlated with clinical follow-up to identify a gene subset for development of a prognostic test. METHODS: RNA was isolated from 225 frozen invasive ductal carcinomas,and qRT-PCR was performed. Univariate hazard ratios and 95% confidence intervals for breast cancer mortality and recurrence were calculated for each of the 32 candidate genes. A multivariable gene expression model for predicting each outcome was determined using the LASSO, with 1000 splits of the data into training and testing sets to determine predictive accuracy based on the C-index. Models with gene expression data were compared to models with standard clinical covariates and models with both gene expression and clinical covariates. RESULTS: Univariate analyses revealed over-expression of RABEP1, PGR, NAT1, PTP4A2, SLC39A6, ESR1, EVL, TBC1D9, FUT8, and SCUBE2 were all associated with reduced time to disease-related mortality (HR between 0.8 and 0.91, adjusted p < 0.05), while RABEP1, PGR, SLC39A6, and FUT8 were also associated with reduced recurrence times. Multivariable analyses using the LASSO revealed PGR, ESR1, NAT1, GABRP, TBC1D9, SLC39A6, and LRBA to be the most important predictors for both disease mortality and recurrence. Median C-indexes on test data sets for the gene expression, clinical, and combined models were 0.65, 0.63, and 0.65 for disease mortality and 0.64, 0.63, and 0.66 for disease recurrence, respectively. CONCLUSIONS: Molecular signatures consisting of five genes (PGR, GABRP, TBC1D9, SLC39A6 and LRBA) for disease mortality and of six genes (PGR, ESR1, GABRP, TBC1D9, SLC39A6 and LRBA) for disease recurrence were identified. These signatures were as effective as standard clinical parameters in predicting recurrence/mortality, and when combined, offered some improvement relative to clinical information alone for disease recurrence (median difference in C-values of 0.03, 95% CI of -0.08 to 0.13). Collectively, results suggest that these genes form the basis for a clinical laboratory test to predict clinical outcome of breast cancer.

Epigenetic analysis of laser capture microdissected fetal epithelia.

Laser capture microdissection (LCM) is a superior method for nondestructive collection of specific cell populations from tissue sections. Although DNA, RNA, and protein have been analyzed from LCM-procured samples, epigenetic analyses, particularly of fetal, highly hydrated tissue, have not been attempted. A standardized protocol with quality assurance measures was established to procure cells by LCM of the medial edge epithelia (MEE) of the fetal palatal processes for isolation of intact microRNA for expression analyses and genomic DNA (gDNA) for CpG methylation analyses. MicroRNA preparations, obtained using the RNAqueous Micro kit (Life Technologies), exhibited better yields and higher quality than those obtained using the Arcturus PicoPure RNA Isolation kit (Life Technologies). The approach was validated using real-time polymerase chain reaction (PCR) to determine expression of selected microRNAs (miR-99a and miR-200b) and pyrosequencing to determine CpG methylation status of selected genes (Aph1a and Dkk4) in the MEE. These studies describe an optimized approach for employing LCM of epithelial cells from fresh frozen fetal tissue that enables quantitative analyses of microRNA expression levels and CpG methylation.

Assessment of phytoestrogen and mycoestrogen recognition by recombinant human estrogen receptor-α using ligand titration arrays.

INTRODUCTION: Exposure to phytoestrogens and mycoestrogens has emerged as a public health issue due to their potentially endocrine disruption activities resulting from direct interaction with sex-steroid hormone receptors. There is a significant requirement for comprehensive, reproducible methods to determine the extent of estrogen mimicry by compounds encountered in the environment to estimate risk:benefit ratios, particularly in humans. OBJECTIVE: To develop a systematic approach for assessing recognition of chemically diverse compounds by human estrogen receptor proteins to aid in their assessment as endocrine disruptor compounds (EDCs). METHODS: Recombinant human estrogen receptor-α protein (rhERα) was expressed in Saccharomyces cervisiae as an ubiquitin fusion under control of a CUP1 promoter and partially purified with heparin affinity chromatography in the unliganded state. A novel radio-ligand binding array was developed to evaluate structurally diverse compounds, both naturally occurring and synthetic, for estrogen binding activity and affinity. RESULTS: Binding affinities of suspected estrogen mimics for rhERα were calculated over a range of [(3) H]estradiol-17ß concentrations using Lundon OneSite® and Compete® software. CONCLUSION: ß-zearalanol, a mycoestrogen similar to zearalenone used as an ICCVAM validation substance for the in vitro estrogen receptor binding assays (ICCAM report), was employed as a model estrogen mimic to illustrate the approach, methods and calculations using these techniques.

Physical structure of constitutional isomers influences antiproliferation activity of thiosemicarbazone-alkylthiocarbamate copper complexes.

A series of hybrid thiosemicarbazone-alkylthiocarbamate copper complexes with similar electronic environments but distinct physical structures have been prepared, characterized, and evaluated for antiproliferation activity. The complexes include the constitutional isomers (1-phenylpropane-1-imine-(O-ethylthiocarbamato)-2-one-(N-methylthiosemicarbazonato))copper(II) (CuL1) and (1-phenylpropane-1-one-(N-methylthiosemicarbazonato)-2-imine-(O-ethylthiocarbamato))copper(II) (CuL2) along with (1-propane-1-imine-(O-ethylthiocarbamato)-2-one-(N-methylthiosemicarbazonato))copper(II) (CuL3). Complexes CuL1 and CuL2 differ in the positions of the pendent thiosemicarbazone (TSC) and alkylthiocarbamate (ATC) moieties on the 1-phenylpropane backbone. Complex CuL3 employs a propane backbone with the TSC in the 2-position as in CuL1. The isomer pair CuL1 and CuL2 have equivalent electronic environments with indistinguishable CuII/I potentials (E1/2 = -0.86 V vs. ferrocenium/ferrocene) and electron paramagnetic resonance (EPR) spectra (g∥ = 2.26, g⊥ = 2.08). The electronic structure of CuL3 has a similar E1/2 of -0.84 V and identical EPR parameters to CuL1, 2. Single crystal X-ray diffraction studies confirm a consistent donor environment with no substantial variation in the CuN or CuS bond distances and angles between the complexes. The antiproliferation activities of the CuL1-3 were evaluated against the lung adenocarcinoma cell line (A549) and nonmalignant lung fibroblast cell line (IMR-90) using the MTT assay. CuL1 had the highest A549 activity (A549EC50 = 0.065 µM) and selectivity (IMR-90EC50/A549EC50 = 20). The constitutional isomer CuL2 displayed decreased A549 activity (0.18 µM) and selectivity (10.6). The complex CuL3 displayed activity (0.009 µM) similar to CuL1 but with a lack of selectivity (1.0). Cellular copper loading determined by ICP-MS was consistent with the activity and selectivity trends. The complexes CuL1-3 did not induce reactive oxygen species (ROS) generation.

Expression of urokinase-type plasminogen activator (uPA), its receptor (uPAR), and inhibitor (PAI-1) in human breast carcinomas and their clinical relevance.

Serine proteases convert plasminogen to plasmin which is involved in tissue remodeling under physiologic and pathophysiologic conditions, including breast carcinoma invasion and progression. Both urokinase-type plasminogen activator (uPA) and pro-uPA associate with uPA receptor (uPAR) on target cells, where plasminogen activator inhibitors (e.g., PAI-1) may modulate their activities. Expression levels of these factors were compared in breast carcinomas relative to patient characteristics, carcinoma features, and clinical outcome. uPA, uPAR, and PAI-1 were quantified by enzyme-linked immunosorbent assay (ELISA) in extracts of 226 biopsies while estrogen receptor (ER) and progestin receptor (PR) were determined by enzyme immunoassay (EIA) or radio-ligand binding. Each set of assays contained a novel reference specimen with known quantities of each of these five analytes. Levels in ng/mg protein of these biomarkers exhibited ranges: uPA (0-12.3); uPAR (0-19.5); PAI-1 (0-91.2). When considered independently, expression of uPA, uPAR, or PAI-1 was unrelated to patient age or menopausal status. Although no correlation was observed between each analyte with stage, grade, or ER/PR status, levels appeared to differ with pathology and nodal status. A dendrogram from hierarchical clustering of uPA, uPAR, and PAI-1 levels in 106 specimens revealed three clusters of breast cancer patients. Kaplan-Meier analyses of uPA, uPAR, and PAI-1 indicated a correlation with overall survival (OS), suggesting collective examination of these biomarkers is useful in predicting clinical outcome of breast cancer.

Molecular signatures of estrogen receptor-associated genes in breast cancer predict clinical outcome.

Our goal is to identify new molecular targets for drug design and improve understanding of the molecular basis of clinical behavior and therapeutic response of breast cancer (BC). Pure populations of BC cells were procured by laser capture microdissection (LCM) from deidentified tissue specimens. RNA from either LCM-procured cells or whole tissue sections was extracted, purified, and quantified by RT-qPCR using beta-actin for relative quantification. RNA was amplified, Cy5-labeled, and hybridized for microarray. Spectrophotometric and BioAnalyzer analyses evaluated aRNA yield, purity, and transcript length for gene microarray. Unsupervised and supervised methods selected 7,000 genes with significant variation. Expression profiles of BC cells were dominated by genes associated with estrogen receptor-alpha (ERalpha) status; over 3,000 genes were identified as differentially expressed between ERalpha+ and ERalpha(-) BC cells. Other prominent gene expression patterns divided ERalpha+ BCs into subgroups, which were associated with significantly different clinical outcomes (p < 0.01). While exploiting larger gene sets derived from LCM-cells and reports using whole tissues, a preliminary 14 gene subset was selected by UniGene Cluster analysis. Additionally, ERE-binding proteins (ERE-BP) were detected by EMSA, which were not recognized by ERa antibodies. Kaplan-Meier analysis indicated that patients with ERE-BP positive BCs had lower over-all survival than those with ERE-BP negative cancers. Collectively, these results will establish molecular signatures for assessing clinical features of BC and aid in the selection of molecular targets for drug development.

Biosensors for detecting estrogen-like molecules and protein biomarkers.

A novel evanescent-based biosensor (Endotect, ThreeFold Sensors, Inc.) was developed with laser-based fiber optics using fluorescent dye-labeled recombinant human estrogen receptor-alpha (rhERalpha) and hERbeta as probes. A three-tiered approach evaluating various steps in the formation of the estrogen-receptor complex and its subsequent activity was developed for instrument calibration to detect estrogen mimics in biological samples, water and soil. Using this approach, binding affinities and activities of certain known estrogen mimics were determined for their use as calibrator molecules. Results indicated rhERalpha and rhERbeta may be employed as probes to distinguish estrogen mimics with a broad range of affinities. In addition, application of the biosensor for detecting DNA-binding proteins in human tissue extracts was demonstrated. The later studies suggest the biosensor may be used as a clinical laboratory tool for assessing tumor marker proteins.

A three-tiered approach for calibration of a biosensor to detect estrogen mimics.

A three-tiered approach was developed to determine the influence of a chemically-diverse group of compounds exhibiting estrogen mimicry using recombinant human estrogen receptor (rhER) activity to calibrate a receptor protein-based biosensor. In the initial tier, a ligand competition array was developed to evaluate compounds inhibiting [3H]estradiol-17beta binding to rhER. Each of six different concentrations of [3H]estradiol-17beta was mixed with increasing concentrations of an unlabeled putative mimic. Each of these mixtures was incubated with a constant amount of rhERalpha and then receptor-bound [[3H]estradiol-17beta was measured. This array protocol analyzes ligand binding affinities of hERalpha with a potential inhibitor over the entire range of receptor protein saturation. When either hERalpha or hERbeta binds to an estrogenic ligand, the receptor monomer forms both homo- and hetero-dimers. Then the ligand-receptor dimer complex activates transcription by associating with an estrogen response element (ERE), which is a specific DNA sequence located upstream of estrogen-responsive genes. The second tier for ligand evaluation utilized an electrophoretic mobility shift assay (EMSA), which was performed with an ERE sequence labeled with [alpha[32]P]dATP and incubated with rhER in the presence or absence of unlabeled ligand. ERE-hER complexes were separated by electrophoresis and analyzed using phosphor imaging technology. To assess biological effects of an estrogen mimic on expression of an ER-target gene, a yeast cell-based bioassay was constructed with recombinant DNA technology using Saccharomyces cerevisiae. Each of these engineered yeast cells contained a rhERalpha expression plasmid (YEpE12) and a separate reporter plasmid (YRG2) containing an ERE sequence upstream of a beta-galactosidase reporter gene. Incubation of these yeast cells with an estrogenic compound allows formation of ligand-hERalpha complexes, which recognize the ERE sequence regulating beta-galactosidase expression. Estrogenic compounds, which were evaluated as calibrators for ligand-based and ERE-based biosensors, elicit varying responses in each of the three tiers of the protocol.

Effects of body protection vests and experience levels in prevention of equestrian injuries.

OBJECTIVES: To investigate the risk reduction and benefit of wearing body protection/safety vests in equestrian sports. METHODS: A comparison of equestrians wearing body protective vests and those not wearing vests was performed using incident report data of 718 participants in the United States Pony Clubs during 2011-2017. Data obtained included age, gender, certification level of member, type of activity, description of incident, description of injuries, what protective equipment was worn and other possible contributing factors. RESULTS: While wearing body protective vests when riding on the flat or for show jumping was not correlated with a decrease in injuries, wearing vests for cross country was correlated with decrease in reported injuries (p=0.036) and showed a trend towards a lower incident severity level (p=0.062). Wearing body protection during cross country reduced the relative risk of injury by 56%. While the volume of incidents varied with a rider's experience level, the number of serious injuries did not appear to correlate with lesser equestrian experience. CONCLUSIONS: While equestrian sports are considered to have a certain degree of risk associated with them, there are ways to make them safer. Wearing safety equipment, such as helmets and body protection, and obtaining education and experience can lessen the chance of incurring serious injuries.

A systems biology approach identifies a regulatory network in parotid acinar cell terminal differentiation.

OBJECTIVE: The transcription factor networks that drive parotid salivary gland progenitor cells to terminally differentiate, remain largely unknown and are vital to understanding the regeneration process. METHODOLOGY: A systems biology approach was taken to measure mRNA and microRNA expression in vivo across acinar cell terminal differentiation in the rat parotid salivary gland. Laser capture microdissection (LCM) was used to specifically isolate acinar cell RNA at times spanning the month-long period of parotid differentiation. RESULTS: Clustering of microarray measurements suggests that expression occurs in four stages. mRNA expression patterns suggest a novel role for Pparg which is transiently increased during mid postnatal differentiation in concert with several target gene mRNAs. 79 microRNAs are significantly differentially expressed across time. Profiles of statistically significant changes of mRNA expression, combined with reciprocal correlations of microRNAs and their target mRNAs, suggest a putative network involving Klf4, a differentiation inhibiting transcription factor, which decreases as several targeting microRNAs increase late in differentiation. The network suggests a molecular switch (involving Prdm1, Sox11, Pax5, miR-200a, and miR-30a) progressively decreases repression of Xbp1 gene transcription, in concert with decreased translational repression by miR-214. The transcription factor Xbp1 mRNA is initially low, increases progressively, and may be maintained by a positive feedback loop with Atf6. Transfection studies show that Xbp1 activates the Mist1 promoter [corrected]. In addition, Xbp1 and Mist1 each activate the parotid secretory protein (Psp) gene, which encodes an abundant salivary protein, and is a marker of terminal differentiation. CONCLUSION: This study identifies novel expression patterns of Pparg, Klf4, and Sox11 during parotid acinar cell differentiation, as well as numerous differentially expressed microRNAs. Network analysis identifies a novel stemness arm, a genetic switch involving transcription factors and microRNAs, and transition to an Xbp1 driven differentiation network. This proposed network suggests key regulatory interactions in parotid gland terminal differentiation.

Gender-associated expression of tumor markers and a small gene set in breast carcinoma.

Breast carcinomas in both genders share pathological features, although differences in incidence, prognosis and survival are reported. Expression of 33 genes was investigated in male and female breast carcinomas in association with ER, PR, HER-2/neu and EGF-receptor. Among 98 male breast cancers, 82 were ER+ and 78 were PR+. ER and PR protein levels were greater in males compared to females, although no differences were observed in ESR1 and PGR expression. A difference was observed in binding affinities of PR but not ER between genders. No differences were observed in HER-2/neu, EGFR protein, or patient age. Expression of NAT1, TBC1D9, IL6ST, RABEP1, PLK1 and LRBA was elevated in carcinomas of males compared to those of females, in which ER status appeared to be related to expression. Over-expression of protein products of these genes represents novel molecular targets for development of gender-specific therapeutics and companion diagnostics.

Protein tyrosine phosphatase 4A2 expression predicts overall and disease-free survival of human breast cancer and is associated with estrogen and progestin receptor status.

Expression of protein tyrosine phosphatase PTP4A2 (also known as PRL2) has been examined in a variety of human carcinomas, although its role in breast cancer remains inconclusive. Since the majority of previous breast cancer studies utilized tissue biopsies composed of heterogeneous cell populations, we hypothesized that an examination of PTP4A2 expression in carcinoma cells isolated by laser capture microdissection (LCM) would provide a more accurate means of assessing its predictive value. From investigations of 247 human breast cancer biopsies collected under standardized, stringent conditions, total RNA was extracted from LCM-procured carcinoma cells to perform microarray analyses to identify gene signatures associated with breast cancer behavior. Expression of PTP4A2 was corroborated by real-time quantitative polymerase chain reaction (qPCR) and referenced to estrogen and progesterone receptor levels. Patient outcomes for overall and disease-free survival were more favorable (p = 0.004 and p = 0.001, respectively) when the expression of PTP4A2 in breast carcinomas was increased compared to patients with biopsies with decreased PTP4A2 levels. PTP4A2 expression determined either by microarray or qPCR was elevated in either estrogen receptor (ER)-positive or progestin receptor (PR)-positive breast cancer biopsies compared to ER-negative or PR-negative biopsies. However, PTP4A2 expression was only correlated with overall survival in PR-positive breast carcinomas. These data suggest that PTP4A2 mRNA expression alone may serve as a biomarker for prediction of a breast cancer patient's risk of recurrence and overall survival.

Co-expression of genes with estrogen receptor-α and progesterone receptor in human breast carcinoma tissue.

UNLABELLED: Abstract Background: To detect genes associated with the expression of ESR1 and PGR - as well as of their protein products, estrogen receptor (ER) and progesterone receptor (PR) - 221 de-identified invasive ductal carcinomas of the breast were investigated. Our long-term goal is to decipher relationships between the expression of ER- and PR-associated genes and breast cancer behavior to improve diagnostics and identify new molecular targets for drug design. MATERIALS AND METHODS: Frozen tissue sections were evaluated for structural integrity and pathology after hematoxylin and eosin staining. ER and PR protein levels were quantified by either enzyme immunoassay or radio-ligand binding assay. Total RNA preparations were reverse transcribed for qPCR measurements of ESR1, PGR and 31 gene candidates. RESULTS: Both ESR1 and PGR expression levels were correlated with their cognate receptor protein expression (Pearson correlations of 0.82 and 0.68, p<0.001, respectively), to assess molecular relationships between clinically relevant biomarkers in tissue specimens. Coordinate expression of EVL, NAT1, TBC1D9, SCUBE2, RABEP1, SLC39A6, TCEAL1, FUT8, XBP1, PTP4A2 or GATA3 with either ESR1 or PGR was detected. CONCLUSIONS: Examination of relationships between ESR1 and PGR gene expression and that of other genes of interest indicated: a high degree of correlation between ESR1 levels and expression of NAT1, SCUBE2, XBP1 and GATA3; and a high degree of correlation between PGR expression and that of NAT1, ESR1, SCUBE2 and RABEP1. These results suggest that direct relationships of these genes exist with estrogen and progestin receptor mediated pathways. Pathway analysis software provided additional evidence of gene interactions.

Relationships of ESR1 and XBP1 expression in human breast carcinoma and stromal cells isolated by laser capture microdissection compared to intact breast cancer tissue.

Results from investigations of human genomics which utilize intact tissue biopsy specimens maybe compromised due to a host of uncontrolled variables including cellular heterogeneity of a sample collected under diverse conditions, then processed and stored using different protocols. To determine the cellular origin and assess relationships of mRNA expression of two genes reported to be co-expressed in human breast carcinoma (estrogen receptor-α, ESR1 and X-box binding protein 1, XBP1), gene expression analyses were performed with intact tissue sections and compared with those of laser capture microdissection (LCM)-procured carcinoma and stromal cells from serial sections of the same tissue. Frozen sections of human breast carcinomas were first evaluated for structural integrity and pathology after hematoxylin and eosin (H&E) staining. Total RNA preparations from intact tissue sections and LCM-procured carcinoma and stromal cells were reverse transcribed for measurements of ESR1 and XBP1 expression by quantitative PCR (qPCR). These results were compared with those obtained from microarray analyses of LCM-procured carcinoma cells. Levels of ESR1 and XBP1 were detected in the intact breast cancer tissue sections suggesting coordinate gene expression. Although coordinate expression of these genes was observed in the LCM-procured carcinoma cells, it was not discerned in LCM-procured stromal cells. The origin of coordinate expression of ESR1 and XBP1 observed in whole tissue sections of human breast cancer biopsies is due principally to their co-expression in carcinoma cells and not in the surrounding stromal cells as substantiated using LCM-procured cells. Collectively, a microgenomic process was established from human tissue preparation to RNA characterization and analysis to identify molecular signatures of specific cell types predicting clinical behavior.