Proteomic analysis identifies highly expressed plasma membrane proteins for detection and therapeutic targeting of specific breast cancer subtypes.

In recent years, there has been an emphasis on personalizing breast cancer treatment in order to avoid the debilitating side effects caused by broad-spectrum chemotherapeutic drug treatment. Development of personalized medicine requires the identification of proteins that are expressed by individual tumors. Herein, we reveal the identity of plasma membrane proteins that are overexpressed in estrogen receptor α-positive, HER2-positive, and triple negative breast cancer cells.

Integration of Breast Cancer Secretomes with Clinical Data Elucidates Potential Serum Markers for Disease Detection, Diagnosis, and Prognosis.

Cancer cells secrete factors that influence adjacent cell behavior and can lead to enhanced proliferation and metastasis. To better understand the role of these factors in oncogenesis and disease progression, estrogen and progesterone receptor positive MCF-7 cells, triple negative breast cancer MDA-MB-231, DT22, and DT28 cells, and MCF-10A non-transformed mammary epithelial cells were grown in 3D cultures. A special emphasis was placed on triple negative breast cancer since these tumors are highly aggressive and no targeted treatments are currently available.

17β-Estradiol alters oxidative damage and oxidative stress response protein expression in the mouse mammary gland.

Although substantial evidence has demonstrated that parity and 17β-estradiol (E2) reduce mammary carcinogenesis, it is not clear how this protection is conferred. Thus, we examined the effects of parity and E2 treatment in the mammary glands of ovariectomized 15 week-old virgin mice, 15 week-old primiparous mice, and 9 month-old retired breeders. E2 treatment significantly increased lipid peroxidation, protein carbonylation, and protein nitrosylation in the virgin mice, but not in the age-matched primiparous mice or retired breeders.

Expression of estrogen receptor α in the mouse cerebral cortex.

Although estrogen receptor alpha (ERα) and 17β-estradiol play critical roles in protecting the cerebral cortex from ischemia-induced damage, there has been some controversy about the expression of ERα in this region of the brain. We have examined ERα mRNA and protein levels in the cerebral cortices of female mice at postnatal days 5 and 17 and at 4, 13, and 18 months of age. We found that although ERα transcript levels declined from postnatal day 5 through 18 months of age, ERα protein levels remained stable.

Nanopore-based assay for detection of methylation in double-stranded DNA fragments.

DNA methylation is an epigenetic modification of DNA in which methyl groups are added at the 5-carbon position of cytosine. Aberrant DNA methylation, which has been associated with carcinogenesis, can be assessed in various biological fluids and potentially can be used as markers for detection of cancer. Analytically sensitive and specific assays for methylation targeting low-abundance and fragmented DNA are needed for optimal clinical diagnosis and prognosis.

17β-estradiol modulates gene expression in the female mouse cerebral cortex.

17β-estradiol (E2) plays critical roles in a number of target tissues including the mammary gland, reproductive tract, bone, and brain. Although it is clear that E2 reduces inflammation and ischemia-induced damage in the cerebral cortex, the molecular mechanisms mediating the effects of E2 in this brain region are lacking. Thus, we examined the cortical transcriptome using a mouse model system.

Plasma membrane proteomics of human breast cancer cell lines identifies potential targets for breast cancer diagnosis and treatment.

The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies.

17β-Estradiol alters oxidative stress response protein expression and oxidative damage in the uterus.

The steroid hormone 17β-estradiol (E2) has profound effects on the uterus. However, with the E2-induced increase in uterine cell proliferation and metabolism comes increased production of reactive oxygen species (ROS). We examined the expression of an interactive network of oxidative stress response proteins including thioredoxin (Trx), Cu/Zn superoxide dismutase (SOD1), apurinic endonuclease (Ape1), and protein disulfide isomerase (PDI).

17β-estradiol increases expression of the oxidative stress response and DNA repair protein apurinic endonuclease (Ape1) in the cerebral cortex of female mice following hypoxia.

While it is well established that 17β-estradiol (E2) protects the rodent brain from ischemia-induced damage, it has been unclear how this neuroprotective effect is mediated. Interestingly, convincing evidence has also demonstrated that maintaining or increasing the expression of the oxidative stress response and DNA repair protein apurinic endonuclease 1 (Ape1) is instrumental in reducing ischemia-induced damage in the brain. Since E2 increases expression of the oxidative stress response proteins Cu/Zn superoxide dismutase and thioredoxin in the brain, we hypothesized that E2 may also increase Ape1 expression and that this E2-induced expression of Ape1 may help to mediate the neuroprotective effects of E2 in the brain.

Detection and quantification of methylation in DNA using solid-state nanopores.

Epigenetic modifications in eukaryotic genomes occur primarily in the form of 5-methylcytosine (5 mC). These modifications are heavily involved in transcriptional repression, gene regulation, development and the progression of diseases including cancer. We report a new single-molecule assay for the detection of DNA methylation using solid-state nanopores.

17β-Estradiol-mediated increase in Cu/Zn superoxide dismutase expression in the brain: a mechanism to protect neurons from ischemia.

A number of studies have demonstrated that 17β-estradiol (E(2)) protects the brain from ischemia and yet the mechanism by which this hormone brings about its protective effect is unclear. Interestingly, like E(2), overexpression of the oxidative stress response protein Cu/Zn superoxide dismutase (SOD1), which plays a critical role in regulating reactive oxygen species, also protects the brain from ischemia. Because we previously showed that E(2) treatment of cultured mammary cells increases SOD1 expression, we hypothesized that E(2) might increase SOD1 expression in the brain and that this E(2)-mediated increase in SOD1 expression might help to protect the brain from ischemia.

ERα-associated protein networks.

Estrogen receptor α (ERα) is a ligand-activated transcription factor that, upon binding hormone, interacts with specific recognition sequences in DNA. An extensive body of literature has documented the association of individual regulatory proteins with ERα. It has recently become apparent that, instead of simply recruiting individual proteins, ERα recruits interconnected networks of proteins with discrete activities that play crucial roles in maintaining the structure and function of the receptor, stabilizing the receptor-DNA interaction, influencing estrogen-responsive gene expression, and repairing misfolded proteins and damaged DNA.

Two regions within the proximal steroidogenic factor 1 promoter drive somatic cell-specific activity in developing gonads of the female mouse.

Targets of steroidogenic factor 1 (SF1; also known as NR5A1 and AD4BP) have been identified within cells at every level of the hypothalamic-pituitary-gonadal and -adrenal axes, revealing SF1 to be a master regulator of major endocrine systems. Mouse embryos express SF1 in the genital ridge until Embryonic Day 13.5 (E13.

Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II.

Histone H1 phosphorylation affects chromatin condensation and function, but little is known about how specific phosphorylations impact the function of H1 variants in higher eukaryotes. In this study, we show that specific sites in H1.2 and H1.

Nuclear hormone receptor architecture - form and dynamics: The 2009 FASEB Summer Conference on Dynamic Structure of the Nuclear Hormone Receptors.

Nuclear hormone receptors (NHRs) represent a large and diverse family of ligand-activated transcription factors involved in regulating development, metabolic homeostasis, salt balance and reproductive health. The ligands for these receptors are typically small hydrophobic molecules such as steroid hormones, thyroid hormone, vitamin D3 and fatty acid derivatives. The first NHR structural information appeared approximately 20 years ago with the solution and crystal structures of the DNA binding domains and was followed by the structure of the agonist and antagonist bound ligand binding domains of different NHR members.

Immunohistochemical analysis of oxidative stress and DNA repair proteins in normal mammary and breast cancer tissues.

Background: During the course of normal cellular metabolism, oxygen is consumed and reactive oxygen species (ROS) are produced. If not effectively dissipated, ROS can accumulate and damage resident proteins, lipids, and DNA. Enzymes involved in redox regulation and DNA repair dissipate ROS and repair the resulting damage in order to preserve a functional cellular environment.

Long-range transcriptional control of progesterone receptor gene expression.

Estrogen receptor alpha (ERalpha) binds to specific target DNA sequences, estrogen response elements (EREs), to regulate estrogen-responsive gene expression. The progesterone receptor (PR) gene has been used extensively as a marker of estrogen responsiveness. Although we previously identified cis elements within 1 kb of the PR-B transcription start site that are associated with ERalpha and help to confer estrogen responsiveness, the identification of ERalpha binding sites far removed from the transcription start site suggested that long-range regulation of this gene may occur.

Isolation of proteins associated with the DNA-bound estrogen receptor alpha.

Regulating gene expression is a complex process requiring the interaction of multiple transcription factors with their cognate recognition sequences. While these DNA-bound transcription factors are the primary drivers of gene expression, the capacity of a transcription factor to alter gene expression is tempered by its association with a host of coregulatory proteins that are recruited to the DNA-bound transcription factor. We have developed a novel approach to isolate large complexes of proteins associated with the DNA-bound estrogen receptor alpha (ERalpha) using an agarose-based electrophoretic mobility shift assay (EMSA).

Thioredoxin and thioredoxin reductase influence estrogen receptor alpha-mediated gene expression in human breast cancer cells.

Accumulation of reactive oxygen species (ROS) in cells damages resident proteins, lipids, and DNA. In order to overcome the oxidative stress that occurs with ROS accumulation, cells must balance free radical production with an increase in the level of antioxidant enzymes that convert free radicals to less harmful species. We identified two antioxidant enzymes, thioredoxin (Trx) and Trx reductase (TrxR), in a complex associated with the DNA-bound estrogen receptor alpha (ERalpha).

Apurinic/apyrimidinic endonuclease 1 alters estrogen receptor activity and estrogen-responsive gene expression.

Apurinic/apyrimidinic endonuclease 1 or redox factor-1 (Ape1/Ref-1) is a pleiotropic cellular protein involved in DNA repair and, through its redox activity, enhances the binding of a select group of transcription factors to their cognate recognition sequences in DNA. Thus, we were intrigued when we identified Ape1/Ref-1 and a number of DNA repair and oxidative stress proteins in a complex associated with the DNA-bound estrogen receptor alpha (ERalpha). Because Ape1/Ref-1 interacts with a number of transcription factors and influences their activity, we determined whether it might also influence ERalpha activity.

Using RNA interference to study protein function.

RNA interference can be extremely useful in determining the function of an endogenously-expressed protein in its normal cellular environment. In this chapter, we describe a method that uses small interfering RNA (siRNA) to knock down mRNA and protein expression in cultured cells so that the effect of a putative regulatory protein on gene expression can be delineated. Methods of assessing the effectiveness of the siRNA procedure using real time quantitative PCR and Western analysis are also included.

Isolation of novel coregulatory protein networks associated with DNA-bound estrogen receptor alpha.

Background: DNA-bound transcription factors recruit an array of coregulatory proteins that influence gene expression. We previously demonstrated that DNA functions as an allosteric modulator of estrogen receptor alpha (ERalpha) conformation, alters the recruitment of regulatory proteins, and influences estrogen-responsive gene expression and reasoned that it would be useful to develop a method of isolating proteins associated with the DNA-bound ERalpha using full-length receptor and endogenously-expressed nuclear proteins.

Results: We have developed a novel approach to isolate large complexes of proteins associated with the DNA-bound ERalpha.

The role of retinoblastoma-associated proteins 46 and 48 in estrogen receptor alpha mediated gene expression.

The differential recruitment of coregulatory proteins to the DNA-bound estrogen receptor alpha (ERalpha) plays a critical role in mediating estrogen-responsive gene expression. We previously isolated and identified retinoblastoma-associated proteins 46 (RbAp46) and 48 (RbAp48), which are associated with chromatin remodeling, histone deacetylation, and transcription repression, as proteins associated with the DNA-bound ERalpha. We now demonstrate that RbAp46 and RbAp48 interact with ERalphain vitro and in vivo, associate with ERalpha at endogenous, estrogen-responsive genes, and alter expression of endogenous, ERalpha-activated and -repressed genes in MCF-7 breast cancer cells.

A new small molecule inhibitor of estrogen receptor alpha binding to estrogen response elements blocks estrogen-dependent growth of cancer cells.

Estrogen receptor alpha (ERalpha) plays an important role in several human cancers. Most current ERalpha antagonists bind in the receptor ligand binding pocket and compete for binding with estrogenic ligands. Instead of the traditional approach of targeting estrogen binding to ER, we describe a strategy using a high throughput fluorescence anisotropy microplate assay to identify small molecule inhibitors of ERalpha binding to consensus estrogen response element (cERE) DNA.

Effects of Cu/Zn superoxide dismutase on estrogen responsiveness and oxidative stress in human breast cancer cells.

The effects of estrogen on gene expression in mammary cells are mediated by interaction of the estrogen receptor (ER) with estrogen response elements in target DNA. Whereas the ER is the primary initiator of transcription, the recruitment of coregulatory proteins to the DNA-bound receptor influences estrogen responsiveness. To better understand how estrogen alters gene expression, we identified proteins associated with the DNA-bound ERalpha.