Oxidative Stress and Estrogen Receptor Structural Changes

Institution: Buck Institute for Age Research
Investigator(s): Bradford Gibson, Ph.D. - Christopher Benz, M.D. -
Award Cycle: 2004 (Cycle 10) Grant #: 10YB-0125 Award: $1,122,520
Award Type: SPRC Full
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2004)
Oxidative stress induces aging and age-related diseases like breast cancer. There are >30 years of accumulated scientific evidence demonstrating the critical importance of an "activated" estrogen receptor (ER, alpha isoform) mechanism driving both the initiation and promotion of most human breast cancers, in addition to serving as the molecular target for lifesaving endocrine therapy. Despite unequivocal biological and clinical evidence for profound oxidant stress-induced changes in breast cancer ER structure and function, lack of sufficiently powerful analytical tools have impeded research directed at an understanding of how various oxidants actually deform ER structure and enhance ER breast tumorigenic mechanisms, and how these effects might be reversed or prevented. Quinone oxidants, common in the human diet and endogenously produced by estrogen catabolism, generate both reactive oxygen species and "arylated" proteins. The model quinone, menadione/vitamin K3, induces differential phosphorylation, irreversible arylation, and loss of DNA-binding in full-length and variant forms of the endogenous ER expressed in human breast cancer cells . Using this quinone as a model and a powerful "mass spectrometry" approach, we plan to detect arylated and phosphorylated amino acids at the molecular level in full-length and variant forms of ER extracted from human breast cancer cells.

For this CBCRP collaborative grant, we assembled a cross-disciplinary team of translational breast cancer researchers and protein chemists who previously proved the feasibility of applying novel mass spectrometry approaches to monitor oxidant-induced structural changes in ER. Our aims are to use chemical and proteomic strategies detect quinone-induced arylation of all cysteines (Cys) and phosphorylation of all serines (Ser), threonines (Thr), and tyrosines (Tyr) within all ER forms that can be purified from control/treated ER-overexpressing breast cancer cells. This includes optimization of each step required for the MS analysis of these modified ER amino acids, and the final goal is to measure oxidant-altered ER from treated breast cancer cells. For this research we plan to use recombinant and purified ER from MCF-7 breast cancer cells.

Newer and more powerful protein chemistry procedures are needed to achieve a detailed understanding of the dysfunctional ER found in breast epithelial cells exposed to various oxidants. If we are successful in optimization and validation of the mass spectrometry approach in this project, then the ER from primary human breast biopsy specimens can then be analyzed for the type and location of modified Cys, Ser, Thr, and Tyr residues. The final goal is to relate the pattern of ER structural changes correlated with known oxidant stresses. This will be a first step to relate environmental, dietary, and lifestyle effects for new model of breast cancer based on structural modifications of the ER.

Final Report (2007)
Lack of sufficiently powerful analytical tools have impeded our understanding of how common chemical exposures can alter estrogen receptor (ER, alpha isoform) structure and function, and thereby potentially affect ER-driven breast tumorigenic mechanisms. For this CBCRP-SPRC project we assembled a cross-disciplinary team of translational breast cancer researchers and protein chemists to apply novel mass spectrometry (MS) approaches to monitor post-translational chemical changes in intracellular ER structure.

Our previous studies indicated that various redox-based chemical stresses can cause different types of reversible or irreversible ER structural changes even within the same amino acid residue. This projectís three specific aims included:

Aim 1 required over two years to fully complete, but this did not impede our initiation and progress on Aim 2, which required a similarly long duration. Remarkably, Aim 3 was definitively initiated at the end of year 2 and was fully completed by June 2007. A significant impediment to successful accomplishment of Aim 1 was encountered with receipt of variable quality commercially prepared immuno-capture and ER enrichment reagents needed to purify ER from human breast cancer cell extracts. While we were ultimately successful using an excellent batch of ER antibody-conjugated agarose beads, two other poor quality reagent batches were received that caused major problems and delays; we continue to work with the commercial source to try and improve their manufacturing processes and assure adequate quality control checks so that other investigators do not encounter these same difficulties. Completion of Aim 2 required and exhaustive evaluation of all MS procedural steps ranging from choice of protease digestion to type of denaturing condition in order to optimize the analytical MS coverage of full-length (67 kDa) ER protein, particularly with respect to the specific amino acids considered most susceptible to the post-translational modifications of interest: e.g. Cys residues reactive to oxidants; and Serine, Threonine and Tyrosine residues susceptible to phosphorylation. Ultimately, we also turned to two new MS instrument platforms, vMALDI-LTQ and 4000Qtrap mass spectrometers (that only became operational in 2006), to take advantage of their complementary performance strengths. Finally our perseverance and diligence paid off as our optimized approaches (Aims 1 and 2) were fully reduced to practice on ER extracted from human breast cancer cells exposed to various stress and growth conditions. Along with confirmation of structural changes in intracellular ER similar to those we had observed using recombinant ER protein and that others had observed in other experimental models, we also identified several novel structural changes in ER that had not previously been detected even though some were suspected. We also performed critical validation of the cross-disciplinary clinical relevance of our basic research by showing that two of our newly detected MS defined structural changes in ER could be translated into novel immunodiagnostic assays for application to very small clinical samples of ER-positive breast cancers.

Thus, we have suitable methodology to monitor the effects of oxidative stress on the key protein that is critical to the biology of ER-positive breast cancer at the molecular level. It is now expected that future studies will shed new light on breast tumorigenic pathways and reveal environment exposures likely contributing to the development and progression breast cancer.

Symposium Abstract (2005)
Estrogen receptor (ER, alpha isoform) is a 67 kDa zinc finger transcription factor that plays a fundamental role in both normal reproductive gland development and breast carcinogenesis, and also represents a critical molecular target for breast cancer therapy. Our previous studies indicate that different chemical oxidants (e.g. diamide, peroxide and menadione/K3) cause alternative types of ER structural changes, which in a few clinical tumors appear to be reversible, but in the majority the ER changes are apparently irreversible. In this study, we are investigating the structural consequences of chemical exposures thought to modify essential zinc finger cysteine residues in human ER and our objectives are as follows: i) Employ selected chemical oxidants for in vitro chemical derivatization of recombinant ER followed by digestion strategies that will allow for cataloguing chemical modifications of thiol (cysteine) residues in ER alpha, especially within the critical DNA-binding domain (DBD). ii) Optimize protocols for ER enrichment and purification from cultured human breast cancer cells (MCF-7); treatment of these cells with selected chemical oxidants/quinones followed by mass spectrometric analysis of the purified ER to pinpoint chemically modified ER cysteine residues.

The current study employs mass spectrometry and tandem mass spectrometry to probe ER zinc finger structural changes induced by a redox-reactive vitamin K3 analog menadione/K3, a commonly used cysteine alkylator, iodoacetic acid/IAA, and a bulkier thiol alkylating fluorophore, bromobimane/BrB. Double enzyme digestion of ecombinant ER, using endoproteinase Lys-C and Asp-N, gives rise to four diagnostic peptides derived from the two zinc fingers in ER-DBD, each containing two cysteine residues. By comparing peptide mass fingerprints generated from the chemically treated recombinant protein samples with those from controls, cysteine residues within the DBD that exhibit heightened sensitivity to modification by K3, BrB and IAA have been characterized. Tandem mass spectrometry of a synthetic peptide encompassing the C-terminal half of the structurally more labile second zinc finger of ER (ZnF2B) demonstrates that the two nucleophilic thiols in ZnF2B (Cys-237, Cys-240) are not chemically equivalent in their reactivity to BrB or K3; consistent with their relative positioning near basic amino acids in the sequence which alters their pKa and reactivity to thiol reagents. These findings demonstrate representative ER zinc finger chemical modifications and the differential susceptibility of cysteine residues in ER zinc fingers to such modifications. We have also developed an affinity purification protocol for extraction and mass spectrometric analysis of endogenous ER from MCF-7 cells. Treatment of growing MCF-7 cells with each of the above mentioned chemical oxidants/quinones is expected to produce intracellular thiol modifications within endogenous ER which can be confirmed by our current mass spectrometry protocols. These optimized analytical procedures will ultimately reveal the spectrum of naturally occurring post-translational changes found in breast tumor ER, and hopefully uncover links between environmental chemical exposures, ER structural changes, and different mechanisms of breast cancer development.

Symposium Abstract (2007)
There is more than 30 years of accumulated evidence supporting the critical role of activated estrogen receptor alpha (ERa) in driving development of most human breast cancers and serving as the prime molecular target for lifesaving endocrine therapeutics such as anti-estrogens and aromatase inhibitors. While crystallographic and nuclear magnetic resonance structure analyses have provided critical understanding of how estrogenic and anti-estrogenic ligands modify the secondary structure of the C-terminal ligand-binding domain of ERa, lack of structural information about the N-terminal region of ERa and limited availability of sufficiently powerful analytical tools to catalog ERa post-translational modifications (PTMs) have hampered our understanding of ligand-dependent and ligand-independent ERa activation. Nearly 40 residues across the 595 amino acids (67 kDa) of human ERa are suspected of undergoing PTMs in vivo, including 8 putative phosphorylation sites which are thought to regulate many ERa functions, particularly gene transactivation. The highly disordered N-terminal 184 amino acids of ERa encompass its critical ligand-independent activation function domain, AF1 (aa 38 to 149), and 14 serine residues of which only five are known to be phosphorylated (S102, S104, S106, S118, S167) in response to activators of receptor tyrosine kinases (e.g. EGF/IGF) and other growth factors that directly activate such intracellular serine phosphorylating kinases as Erk1/2, Akt, PKA and PKC. Modern biochemical and immunochemical methods capable of detecting phospho-amino acids have severe limitations, including presupposed knowledge of the modified amino acid of interest and availability/quality of site- and modification-specific antibodies. To address these limitations we have extended modern mass spectrometry (MS) and proteomic approaches, including use of ESI-MS/MS (Q-STAR/4000 Q-TRAP) and vMALDI-MSn (vMALDI-LTQ), to interrogate potential phosphorylation sites within the N-terminal domain of ERα extracted and purified from human breast cancer cells (MCF7) and to compare the effects of ligand-dependent (estradiol, E2) and ligand-independent growth factor (EGF) stimulation. These mass spectrometry approaches allowed us to achieve >90% coverage of full-length ERα and analysis (MS and MSn) of all N-terminal serines. Immunoblotting and MS analyses allowed semi-quantitative detection of S104/S106, S118 and S167 phosphorylation changes in ERα after MCF7 exposure to E2 or EGF. Remarkably, we identified by MS and MSn a novel serine site (S154) showing approximately 5 fold increase in phosphorylation in response to E2 as well as an Erk-activating dose of the vitamin K quinone, menadione (K3); we also confirmed the absence of phosphorylation on other candidate N-terminal serine sites. An antibody specific to phospho-S154, now under development, will facilitate clinical evaluation of this PTM as found in ER-positive human breast cancers. In sum, this novel finding illustrates the analytical power and potential clinical impact of applying comprehensive proteomic and MS strategies to interrogate ERα PTM from human breast cancer cells; hopefully these findings will shed light on the types of environmental and endogenous stimuli involved in human breast cancer development.

Reactivity of Zinc Finger Cysteines: Chemical Modifications Within Labile Zinc Fingers in Estrogen Receptor
Periodical:Journal of the American Society for Mass Spectrometry
Index Medicus: J Am Soc Mass Spectrom
Authors: Christian Atsriku, Gary K.Scott, Christopher Benz, Michael Baldwin
Yr: 2005 Vol: 16 Nbr: 12 Abs: Pg: 2017-2026

Vitamin K3 (Menadione)-Induced Oncosis Associated with Keratin 8 Phosphorylation and Histone H3 Arylation
Periodical:Molecular Pharmacology
Index Medicus: Mol Pharmacol
Authors: G. Scott, C Atsriku, P Kaminker, J Held, B Gibson, M Baldwin C Benz
Yr: 2005 Vol: 6 Nbr: 3 Abs: Pg:606-615

Ligand-independent MAPK-dependent activation and serine phosphorylation of wildtype and delta7 isoform of estrogen receptor alpha by redox active quinone, vitamin K3/menadione
Periodical:American Association for Cancer Research
Index Medicus: AACR
Authors: Scott GK, Zhou Y, Yau C, Benz CC
Yr: 2005 Vol: 45 Nbr: Abs: Pg:a5601

Novel pathways associated with quinone-induced stress in breast cancer cells.
Periodical:Drug Metabolism Reviews
Index Medicus: Drug Metab. Rev.
Authors: Benz CC, Atsriku C, Yau C, Britton D, Schilling B, Gibson BW, Baldwin MA, Scott GK.
Yr: 2006 Vol: 38 Nbr: 4 Abs: Pg:601-13

Quantification of cysteine oxidation in human estrogen receptor by mass spectrometry.
Periodical:Analytical Chemistry
Index Medicus: Anal Chem
Authors: Atsriku C, Benz CC, Scott GK, Gibson BW, Baldwin MA.
Yr: 2007 Vol: 79 Nbr: 8 Abs: Pg:3083-90