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. The proteins we identified are involved in maintaining protein structure, intracellular homeostasis, and cellular architecture; enhancing cell proliferation and invasion; and influencing cell migration. These proteins may be useful for breast cancer detection and/or treatment.

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 (E13.5). Thereafter, expression persists in the male and is substantially lower in the female gonad until birth. We hypothesize that the sexually dimorphic expression of Sf1 during gonadogenesis is mediated by sex-specific regulation of its promoter. To investigate dimorphic regulation within the fetal gonad, we developed an experimental strategy using transient transfection of E13.5 gonad explant cultures and evaluated various Sf1 promoter constructs for sexually dimorphic DNA elements. The proximal Sf1 promoter correctly targeted reporter activity to SF1-expressing cells in both XY and XX gonads. Stepwise deletion of sequences from the Sf1 promoter revealed two regions that affected regulation within female gonads. Mutation of both sequences together did not cause further disruption of reporter activity, suggesting the two sites might work in concert to promote activity in female somatic cells. Results from gel mobility shift assays and fetal gonad-chromatin immunoprecipitation showed that TCFAP2 binds to one of the two female-specific sites within the proximal promoter of Sf1. Together, we show that transient transfection experiments performed within developing testes and ovaries are a powerful tool to uncover elements within the Sf1 promoter that contribute to sex-specific expression.

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. Because increased ROS accumulation and/or unrepaired DNA damage can lead to initiation and progression of cancer and we had identified a number of oxidative stress and DNA repair proteins that influence estrogen responsiveness of MCF-7 breast cancer cells, it seemed possible that these proteins might be differentially expressed in normal mammary tissue, benign hyperplasia (BH), ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC). METHODS: Immunohistochemistry was used to examine the expression of a number of oxidative stress proteins, DNA repair proteins, and damage markers in 60 human mammary tissues which were classified as BH, DCIS or IBC. The relative mean intensity was determined for each tissue section and ANOVA was used to detect statistical differences in the relative expression of BH, DCIS and IBC compared to normal mammary tissue. RESULTS: We found that a number of these proteins were overexpressed and that the cellular localization was altered in human breast cancer tissue. CONCLUSIONS: Our studies suggest that oxidative stress and DNA repair proteins not only protect normal cells from the damaging effects of ROS, but may also promote survival of mammary tumor cells.

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.

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. Surprisingly, the antioxidant enzyme Cu/Zn superoxide dismutase (SOD1), which is known primarily as a scavenger of superoxide, was associated with the DNA-bound receptor. We have now demonstrated that SOD1 interacts with ERalpha from MCF-7 cell nuclear extracts and with purified ERalpha and that SOD1 enhances binding of ERalpha to estrogen response element-containing DNA. Although SOD1 decreases transcription of an estrogen-responsive reporter plasmid in transiently transfected U2 osteosarcoma cells, RNA interference assays demonstrate that SOD1 is required for effective estrogen responsiveness of the endogenous pS2, progesterone receptor, cyclin D1, and Cathepsin D genes in MCF-7 breast cancer cells. Furthermore, ERalpha and SOD1 are associated with regions of the pS2 and progesterone receptor genes involved in conferring estrogen-responsive gene expression. Interestingly, when MCF-7 cells are exposed to 17beta-estradiol and superoxide generated by addition of potassium superoxide (KO2) to the cell medium, SOD1 levels are increased and tyrosine nitration, which is an indicator of oxidative stress-induced protein damage, is significantly diminished. Our studies have identified a new role for SOD1 in regulating estrogen-responsive gene expression and suggest that the 17beta-estradiol- and KO2-induced increase in SOD1 may play a role in the survival of breast cancer cells and the progression of mammary tumors.

Interaction of estrogen receptor alpha with proliferating cell nuclear antigen.

The ability of estrogen receptor alpha (ERalpha) to modulate gene expression is influenced by the recruitment of a host of co-regulatory proteins to target genes. To further understand how estrogen-responsive genes are regulated, we have isolated and identified proteins associated with ERalpha when it is bound to DNA containing the consensus estrogen response element (ERE). One of the proteins identified in this complex, proliferating cell nuclear antigen (PCNA), is required for DNA replication and repair. We show that PCNA interacts with ERalpha in the absence and in the presence of DNA, enhances the interaction of ERalpha with ERE-containing DNA, and associates with endogenous estrogen-responsive genes. Interestingly, rather than altering hormone responsiveness of endogenous, estrogen-responsive genes, PCNA increases the basal expression of these genes. Our studies suggest that in addition to serving as a platform for the recruitment of DNA replication and repair proteins, PCNA may serve as a platform for transcription factors involved in regulating gene expression.

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. Purified ERalpha and HeLa nuclear extracts were combined with oligos containing ERalpha binding sites and fractionated on agarose gels. The protein-DNA complexes were isolated and mass spectrometry analysis was used to identify proteins associated with the DNA-bound receptor. Rather than simply identifying individual proteins that interact with ERalpha, we identified interconnected networks of proteins with a variety of enzymatic and catalytic activities that interact not only with ERalpha, but also with each other. Characterization of a number of these proteins has demonstrated that, in addition to their previously identified functions, they also influence ERalpha activity and expression of estrogen-responsive genes. CONCLUSION: The agarose gel fractionation method we have developed would be useful in identifying proteins that interact with DNA-bound transcription factors and should be easily adapted for use with a variety of cultured cell lines, DNA sequences, and transcription factors.

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. Our findings reveal that RbAp48 limits expression of estrogen-responsive genes and that RbAp46 modulates estrogen responsiveness in a gene-specific manner. The ability of RbAp46 and RbAp48 to interact with ERalpha and influence its activity reveals yet another role for these multifunctional proteins in regulating gene expression.

The deoxyribonucleic acid repair protein flap endonuclease-1 modulates estrogen-responsive gene expression.

The ligand-occupied estrogen receptor alpha (ERalpha) initiates changes in gene expression through its interaction with target DNA. The capacity of ERalpha to modulate gene expression is influenced by the association of the receptor with a variety of coregulatory proteins. To further understand the role of these coregulatory proteins in ERalpha-mediated transcription, we have isolated and identified proteins associated with ERalpha when it is bound to the consensus estrogen response element. One of the proteins identified in this complex, flap endonuclease-1 (FEN-1), is required for DNA replication and repair. We show that FEN-1 interacts directly with ERalpha and enhances the interaction of ERalpha with estrogen response element-containing DNA. More importantly, chromatin immunoprecipitation and RNA interference assays demonstrate that endogenously expressed FEN-1 associates with the native pS2 gene in MCF-7 cells and influences estrogen-responsive gene expression. Interestingly, estrogen differentially regulates expression of FEN-1 in mouse uterine epithelial, stromal, and myometrial cells. Together, our studies help to elucidate the functional consequence of the ERalpha-FEN-1 interaction and increase our understanding of the elaborate regulatory mechanisms that drive estrogen-responsive gene expression and DNA repair.

Estrogen receptor alpha regulates expression of the breast cancer 1 associated ring domain 1 (BARD1) gene through intronic DNA sequence.

We have used a chromatin immunoprecipitation (ChIP)-based cloning strategy to isolate and identify genes associated with estrogen receptor alpha (ERalpha) in MCF-7 human breast cancer cells. One of the gene regions isolated was a 288bp fragment from the ninth intron of the breast cancer 1 associated ring domain (BARD1) gene. We demonstrated that ERalpha associated with this region of the endogenous BARD 1 gene in MCF-7 cells, that ERalpha bound to three of five ERE half sites located in the 288bp BARD1 region, and that this 288bp BARD1 region conferred estrogen responsiveness to a heterologous promoter. Importantly, treatment of MCF-7 cells with estrogen increased BARD1 mRNA and protein levels. These findings demonstrate that ChIP cloning strategies can be utilized to successfully isolate regulatory regions that are far removed from the transcription start site and assist in identifying cis elements involved in conferring estrogen responsiveness.

Protein disulfide isomerase serves as a molecular chaperone to maintain estrogen receptor alpha structure and function.

The effects of the steroid hormone 17beta-estradiol are mediated through its interaction with the nuclear estrogen receptor (ER). Upon binding 17beta-estradiol, the ER initiates changes in gene expression through its interaction with specific DNA sequences, estrogen response elements (EREs), and recruits coregulatory proteins that influence gene expression. To better understand how estrogen-responsive genes are regulated, we have isolated and identified proteins associated with ERalpha when it is bound to the consensus ERE. One of these proteins, protein disulfide isomerase (PDI), has two distinct functions: acting as a molecular chaperone to maintain properly folded proteins and regulating the redox state of proteins by catalyzing the thiol-disulfide exchange reaction through two thioredoxin-like domains. Using a battery of biochemical and molecular techniques, we have demonstrated that PDI colocalizes with ERalpha in MCF-7 nuclei, alters ERalpha conformation, enhances the ERalpha-ERE interaction in the absence and presence of an oxidizing agent, influences the ability of ERalpha to mediate changes in gene expression, and associates with promoter regions of two endogenous estrogen-responsive genes. Our studies suggest that PDI plays a critical role in estrogen responsiveness by functioning as a molecular chaperone and assisting the receptor in differentially regulating target gene expression.

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. The breast cancer cells secreted factors of variable potency that stimulated proliferation of the relatively quiescent MCF-10A cells. The conditioned medium from each cell line was subjected to mass spectrometry analysis and a variety of secreted proteins were identified including glycolytic enzymes, proteases, protease inhibitors, extracellular matrix proteins, and insulin-like growth factor binding proteins. An investigation of the secretome from each cell line yielded clues about strategies used for breast cancer proliferation and metastasis. Some of the proteins we identified may be useful in the development of a serum-based test for breast cancer detection, diagnosis, prognosis, and monitoring.

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

Although substantial evidence has demonstrated that parity and 17ß-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. Mammary gland expression of the oxidative stress response protein Cu/Zn superoxide dismutase was consistently reduced in all of the E2-treated mice regardless of parity. Expression of the oxidative stress and DNA repair protein apurinic endonuclease (Ape1) was significantly increased only in the mammary glands of the E2-treated retired breeders. These findings suggest that E2 and parity help to reduce mammary oncogenesis by maintaining the structure and function of proteins, lipids, and DNA.

A novel estrogen receptor alpha-associated protein, template-activating factor Ibeta, inhibits acetylation and transactivation.

Estrogen receptor-alpha (ERalpha) functions as a ligand-activated transcription factor that alters expression of estrogen-responsive genes in target cells. Numerous regulatory proteins interact with ERalpha to influence estrogen-mediated transactivation. We have identified a novel coregulatory protein, template-activating factor-Ibeta (TAF-Ibeta), which binds to ERalpha in vitro when the receptor is not complexed with an estrogen response element. The central region of TAF-Ibeta interacts with both the DNA-binding domain and the carboxy-terminal region of ERalpha. Coimmunoprecipitation experiments demonstrate that TAF-Ibeta is associated with the unoccupied, but not the estrogen-occupied, ERalpha in MCF-7 breast cancer cells. Overexpression of TAF-Ibeta inhibits ERalpha-mediated transcription in a dose- dependent manner. TAF-Ibeta represses p300-mediated acetylation of histones and ERalpha in vitro and decreases ERalpha acetylation in vivo. TAF-Ibeta also binds to other nuclear receptor superfamily members and represses thyroid hormone receptor beta- induced transcription in transient transfection assays. Taken together, these data provide evidence that TAF-Ibeta regulates transcription of estrogen- responsive genes by modulating acetylation of histones and ERalpha and that the effects of TAF-Ibeta extend to other nuclear receptor superfamily members as well.

Fos and Jun inhibit estrogen-induced transcription of the human progesterone receptor gene through an activator protein-1 site.

The progesterone receptor (PR) gene is activated by estrogen in normal reproductive tissues and in MCF-7 human breast cancer cells. Although it is typically thought that estrogen responsiveness is mediated through estrogen response elements (EREs), the human PR gene lacks a palindromic ERE sequence. We have identified an activating protein-1 (AP-1) site at +745 in the human PR gene that bound purified Fos and Jun and formed a complex with Fos/Jun heterodimers present in MCF-7 nuclear extracts. Surprisingly, mutating the +745 AP-1 site in the context of a 1.5-kb region of the PR gene significantly enhanced estrogen receptor (ER) alpha-mediated transactivation, suggesting that the wild-type +745 AP-1 site plays a role in inhibiting PR gene expression in the presence of hormone. In support of this idea, transient transfection assays demonstrated that increasing levels of Fos and Jun repressed transcription of a reporter plasmid containing the +745 AP-1 site. Fos levels were transiently increased, ERalpha levels were decreased, and Jun was dephosphorylated after MCF-7 cells were treated with estrogen. Chromatin immunoprecipitation assays demonstrated that Jun was associated with the +745 AP-1 site in the endogenous PR gene in the presence and in the absence of estrogen, but that ERalpha and Fos were only associated with the +745 AP-1 site after estrogen treatment of MCF-7 cells. Our studies suggest that the human PR gene is regulated by multiple transcription factors and that the differential binding of these dynamically regulated trans-acting factors influences gene expression.

A novel estrogen receptor alpha-associated protein alters receptor-deoxyribonucleic acid interactions and represses receptor-mediated transcription.

Estrogen receptor alpha (ER alpha) serves as a ligand-activated transcription factor, turning on transcription of estrogen-responsive genes in target cells. Numerous regulatory proteins interact with the receptor to influence ER alpha-mediated transactivation. In this study, we have identified pp32, which interacts with the DNA binding domain of ER alpha when the receptor is free, but not when it is bound to an estrogen response element. Coimmunoprecipitation experiments demonstrate that endogenously expressed pp32 and ER alpha from MCF-7 breast cancer cells interact. Although pp32 substantially enhances the association of the receptor with estrogen response element-containing DNA, overexpression of pp32 in MCF-7 cells decreases transcription of an estrogen-responsive reporter plasmid. pp32 Represses p300-mediated acetylation of ER alpha and histones in vitro and inhibits acetylation of ER alpha in vivo. pp32 Also binds to other nuclear receptors and inhibits thyroid hormone receptor beta-mediated transcription. Taken together, our studies provide evidence that pp32 plays a role in regulating transcription of estrogen-responsive genes by modulating acetylation of histones and ER alpha and also influences transcription of other hormone-responsive genes as well.

Expression of estrogen receptor α in the mouse cerebral cortex.

Although estrogen receptor alpha (ERα) and 17ß-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. Importantly, expression of the E2-regulated progesterone receptor gene was sustained in younger and in older females suggesting that age-related changes in estrogen responsiveness in the cerebral cortex are not due to the absence of ERα protein.

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. We present a nanopore-based direct methylation detection assay that circumvents bisulfite conversion and polymerase chain reaction amplification. Building on our prior work, we used methyl-binding proteins (MBPs), which selectively label the methylated DNA. The nanopore-based assay selectively detects methylated DNA/MBP complexes through a 19 nm nanopore with significantly deeper and prolonged nanopore ionic current blocking, while unmethylated DNA molecules were not detectable due to their smaller diameter. Discrimination of hypermethylated and unmethylated DNA on 90, 60, and 30 bp DNA fragments was demonstrated using sub-10 nm nanopores. Hypermethylated DNA fragments fully bound with MBPs are differentiated from unmethylated DNA at 2.1- to 6.5-fold current blockades and 4.5- to 23.3-fold transport durations. Furthermore, these nanopore assays can detect the CpG dyad in DNA fragments and could someday profile the position of methylated CpG sites on DNA fragments.

Estrogen receptor alpha and activating protein-1 mediate estrogen responsiveness of the progesterone receptor gene in MCF-7 breast cancer cells.

The progesterone receptor (PR) gene is activated by estrogen in MCF-7 human breast cancer cells. Although the human PR gene does not contain an estrogen response element (ERE), we have identified a putative activating protein-1 (AP-1) site at +90 in the PR gene that was hypersensitive to deoxyribonuclease I cleavage in genomic Southern analysis, bound purified Fos and Jun, formed a complex with Fos/Jun heterodimers present in MCF-7 nuclear extracts in gel mobility shift assays, and functioned as an estrogen-responsive enhancer in transient cotransfection assays. When the +90 AP-1 site was mutated in the context of the PR gene, estrogen responsiveness was significantly decreased. Purified estrogen receptor (ER) enhanced binding of Fos and Jun to the +90 AP-1 site and bound to an adjacent imperfect ERE half-site. Mutating this ERE half-site diminished the binding of ER, Fos, and Jun and decreased transcription. Chromatin immunoprecipitation assays demonstrated that the ER, Fos, and Jun were present at the +90 AP-1 site in the endogenous PR gene only after treatment of MCF-7 cells with estrogen. These studies suggest that the cooperative interaction of the ER with Fos and Jun proteins helps confer estrogen responsiveness to the endogenous PR gene.

Estrogen receptor alpha and Sp1 regulate progesterone receptor gene expression.

The progesterone receptor (PR) gene is induced by estrogen in reproductive and mammary tissues and in MCF-7 human breast cancer cells even though the human PR gene lacks an estrogen response element. We have identified a region from -80 to -34 in the PR gene that contains two Sp1 sites and confers estrogen responsiveness to a heterologous promoter in an estrogen and estrogen receptoralpha (ERalpha)-dependent manner. Sp1 present in MCF-7 nuclear extracts and purified Sp1 bind to and protect both Sp1 sites from DNase I cleavage, but the proximal Sp1 site is preferentially protected. Mutation of either Sp1 site decreases Sp1-DNA complex formation and ERalpha-mediated transactivation. ERalpha enhances Sp1 binding, but does not interact directly with the -80/-34 region. Our studies suggest that ERalpha confers estrogen responsiveness to the PR gene by enhancing Sp1 interaction with the Sp1 site in the -80/-34 region of the human PR gene.