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A major challenge to broadening oncology applications for inhibitors of the ubiquitin-proteasome system (UPS) is the identification of UPS-dependent cancer pathways predictive of tumors responsive to peptidomimetic inhibitors of its 20S core protease activity. To inform clinical studies evaluating UPS inhibitors as breast cancer therapeutics, seven phenotypically diverse human breast cancer cell line models were characterized for their cellular and molecular responses to the clinically approved 20S inhibitor bortezomib (PS341; Velcade), focusing on those overexpressing estrogen receptor (ER) or ERBB2/HER2, because these oncogenic receptor pathways are constitutively activated in approximately 80% of all breast cancers. All models demonstrated dose-dependent bortezomib reduction in intracellular 20S activity correlating with cell growth inhibition, and bortezomib IC(50) values (concentrations producing 50% growth inhibition) varied directly with pretreatment 20S activities (r = 0.74; *, p < 0.05), suggesting that basal 20S activity may serve as a clinical predictor of tumor responsiveness to UPS inhibition. Reduction in 20S activity (> 60%) was associated with early (24 h) intracellular relocalization of ER (nucleus to cytoplasm) and ERBB2 (plasma membrane to perinuclear lysosomes), buildup of ubiquitinated and Hsp70-associated receptor, degradation and loss of ER and ERBB2 function, and induction of cellular apoptosis. These models were also used to screen a pharmacologic panel of pathway-targeted anticancer agents [4-hydroxy-3-methoxy-5-(benzothiazolylthiomethyl)benzylidenecyanoacetamide (AG825), 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide (AZD6244/ARRY142886), 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY294002), 17-N-allylamino-17-demethoxy geldanamycin (17AAG), and (2E)-N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2-propenamide (LAQ824)] for those capable of sensitizing to bortezomib. In keeping with the observation that 20S reduction has little effect on mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling in either ER-positive or ERBB2-positive models, only the MEK-1/2 inhibitor AZD6244 consistently improved the antitumor activity of bortezomib.

Activation of nuclear factor-kappaB (NFkappaB) has been linked to the development of hormone-independent, estrogen receptor (ER)-negative human breast cancers. To explore the possibility that activated NFkappaB marks a subset of clinically more aggressive ER-positive breast cancers, NFkappaB DNA-binding was measured in ER-positive breast cancer cell lines and primary breast cancer extracts by electrophoretic mobility shift assay and ELISA-based quantification of specific NFkappaB p50 and p65 DNA-binding subunits. Oxidant (menadione 100 microMx30 min) activation of NFkappaB was prevented by pretreatment with various NFkappaB inhibitors, including the specific IkappaB kinase (IKK) inhibitor, parthenolide (PA), which was found to sensitize MCF-7/HER2 and BT474 but not MCF-7 cells to the antiestrogen tamoxifen. Early stage primary breast cancers selected a priori for lower ER content (21-87 fmol/mg; n=59) and known clinical outcome showed two- to four-fold increased p50 and p65 NFkappaB DNA-binding over a second set of primary breast cancers with higher ER content (>100 fmol/mg; n=22). Breast cancers destined to relapse (13/59) showed significantly higher NFkappaB p50 (but not p65) DNA-binding over those not destined to relapse (46/59; p=0.04). NFkappaB p50 DNA-binding correlated positively with several prognostic biomarkers; however, only NFkappaB p50 DNA-binding (p=0.04), Activator Protein-1 DNA-binding (AP-1; p

Oxidant-induced structural modifications within the cysteine-rich DNA-binding domain (DBD) of the overexpressed estrogen receptor (ER) likely contribute to its loss of DNA-binding function and altered transcriptional activity during human breast cancer development. Using recombinant ER protein as a model, procedures to detect such endogenously produced structural changes in the two Cys(4)-type zinc fingers within the DBD of ER extracted from breast cancer cells are being developed. Unfortunately, ex vivo oxidation of these ER-DBD cysteine residues can occur during routine ER purification and preparation procedures. Also, cysteine residues readily undergo thiol-disulfide exchange reactions that can result in artificial oxidation and incorrect disulfide bond assignments. These problems can be circumvented by an initial irreversible alkylation of all free thiols followed by reduction of any disulfides and treatment with a second alkylating agent, prior to proteolysis and high-performance liquid chromatography mass spectrometry analysis of peptides in the doubly alkylated ER digest, to differentiate between the originally free and the disulfide-bonded cysteine residues. Although the use of chemically identical but isotopically different alkylating agents was more effective than the use of chemically different alkylating agents, subsequent problems were encountered with incomplete alkylation of particular Cys residues in the native ER protein. To overcome this limitation, the initial alkylation was accompanied by denaturation and the second alkylation was carried out during the proteolytic digestion. These improved analytical strategies should facilitate the monitoring of structurally altered endogenous ER produced within oxidant-stressed human breast cancer cells.

Breast cancer incidence increases with age but this relationship has not been fully explored with regard to expression of estrogen receptor (ER) and ER-inducible genes (PR, pS2, Bcl2, cathepsin D), or the age-dependence of oxidant stress markers that also affect ER-inducible gene expression. In this three-part study, we first correlated age at diagnosis with expression of breast cancer markers ER, PR, pS2, Bcl2, and cathepsin D, quantitated by enzyme immunoassays from a European collective of approximately 3000 cryobanked primary breast cancers and approximately 300 adjacent non-malignant breast tissues. Results were then compared with ER and PR data reported to the SEER registry for 83,541 US cancers diagnosed during 1992-1997. Lastly, a homogeneous subset of 70 ER-positive tumors preselected from the European collective was blindly analyzed for age-specific changes in the DNA-binding content of redox-sensitive transcriprtion factors, AP1 and Sp1, and the oxidant stress-activated protein kinase, phosphorylated(P)-Erk5. Increases in breast tumor ER from patients aged <30 to >80 years mirrored 10-fold lower increases in non-malignant breast tissue ER content up to age 60, rising faster thereafter and reaching a near 25-fold differential between malignant and non-malignant breast tissue by age 80. ER-inducible markers PR, pS2, Bcl2, and cathepsin D were overexpressed in tumors relative to non-malignant breast tissue but, unlike ER, did not increase with patient age. While SEER data demonstrated that the increase in US breast cancer incidence rates after age 50 is confined to ER-positive tumors in patients of all ethnic subsets, these patients also showed a striking increase in the proportion of higher-risk ER-positive/PR-negative breast cancers arising after age 50. Mechanistically essential for ER-inducible PR expression, Sp1 DNA-binding function (but not Sp1 content) was lost with age in ER-positive tumors; and this functional defect correlated with increased tumor content of the oxidant stress marker, P-Erk5. Altogether these findings support two hypotheses: (i) dysregulated ER expression underlies the age-specific increase in breast cancer incidence after age 50; and (ii) oxidative stress and loss of Sp1 DNA-binding may contribute to an increasing incidence in higher-risk ER-positive/PR-negative breast cancers with aging.

Full-length (67 kDa) immunoreactive estrogen receptor (ER) extracted from a third of untreated ER-positive primary breast tumors appears unable to bind to its cognate estrogen response element (ERE). We have observed partial reversibility of this ER DNA-binding defect upon treatment of these tumor extracts with excess thiol reducing agent (DTT), suggesting that ER DNA-binding is subject to redox modulation as is reported for other zinc-finger proteins and transcriptional activators. Treatment of recombinant ER DNA-binding domain (ER-DBD) or ER-enriched extracts from CHO(ER) and MCF-7 cells with thiol-reacting oxidants (diamide, iodosobenzoate, H2O2) or alkylator (iodoacetamide) produces a dose-dependent loss in ER DNA-binding capacity. Thiol-specific oxidative loss in ER DNA-binding is fully reversible by DTT reduction, unlike the defect caused by thiol-specific alkylation. Circular dichroism spectrometry shows that both forms of treatment substantially modify ER secondary structure, inducing loss of alpha-helical content within the ER-DBD that is reversible after thiol oxidation but not after thiol alkylation. Oxidant (H2O2, menadione) exposure of cultured CHO(ER) or MCF-7 cells impairs the ability of endogenous ER to bind DNA and transactivate an ER-responsive reporter gene (ERE-tk-CAT), demonstrating that extracellular redox stress can modulate intracellular ER function. Since these thiol-specific oxidant and alkylator treatments have no significant effect on either recombinant ER ligand-binding or intracellular immunoreactive ER content, our findings suggest that DNA-binding and transactivation are the most sensitive intracellular ER functions impaired by oxidant stress in some ER-positive human breast tumors.

Polyamines are known to inhibit sequence specific DNA-binding activity of several zinc-finger transcription factors, including estrogen receptor (ER) binding to its cognate estrogen response element (ERE). The mechanism accounting for this disruption of protein-DNA interaction is unknown, although polyamine induction of DNA conformational changes has been suggested. To determine if polyamines can directly impair ER action, we compared the effects of putrescine (Putr), spermidine (Spd), and spermine (Spm) on ER DNA-binding (ER-ERE complex formation), ER ligand-binding (estradiol), ER structure (circular dichroism and sucrose gradient sedimentation), and the capacity of ER to transactivate an ERE-tk-CAT reporter in transient transfection assays. Polyamine concentrations causing 50% inhibition of ER-ERE formation (IC50 values) were found to be 1 mM for Putr, 4 mM for Spd, and 3 mM for Spm. This loss of ER DNA-binding was associated with a direct and irreversible effect on the ER DNA-binding domain (ER-DBD). Additionally, polyamines were observed to inhibit ER ligand-binding with IC50 values of 10 mM for Putr, 2 mM for Spd, and < 0.1 mM for Spm; and this correlated with a measureable change in higher-order ER structure (5S to 3.5S sedimentation) and inhibition of intracellular ER transactivation. These findings suggest that in ER-positive human breast tumors with increased polyamine (especially Spm) content, ER structure and function may be directly altered by tight-ion polyamine complexing that results in loss of ER-mediated gene regulation.

Preliminary studies have suggested that measuring the ability of immunoreactive 67-kDa estrogen receptor (ER) to bind DNA and form in vitro complexes with its cognate estrogen response element (ERE) might serve to identify breast tumors most likely to respond to antiestrogens like tamoxifen. Data from two different surveys of untreated primary breast tumors confirmed that only 67% (74 of 111) of ER-positive tumors express a receptor capable of forming ER-ERE complexes by gel-shift assay, with tumors of lower ER content having significantly reduced ER DNA-binding frequency (56%) relative to those of higher ER content (82%; P = 0.007). In contrast to these untreated tumors, a panel of 41 receptor-positive breast tumors excised after acquiring clinical resistance to tamoxifen during either primary (n = 26) or adjuvant therapy (n = 15) showed a significantly greater ER DNA-binding frequency, with nearly 90% capable of forming ER-ERE complexes (P < 0.02). To assess experimentally whether ER DNA-binding function is altered during the development of antiestrogen resistance, nude mouse MCF-7 tumor xenografts were analyzed before and after the acquisition of in vivo resistance to either tamoxifen or a pure steroidal antiestrogen, ICI 182,780. Tamoxifen-resistant MCF-7 tumors retained full expression of 67-kDa DNA-binding ER, and despite a markedly reduced ER content in the ICI 182,780-treated tumors, the expressed ER in these antiestrogen-resistant tumors exhibited full ability to form ER-ERE complexes. These findings indicate that breast tumors with acquired antiestrogen resistance continue to express ER of normal size and DNA-binding ability and suggest that the failure of antiestrogens to arrest tumor growth during emergence of clinical resistance results from an altered gene-regulatory mechanism(s) other than ER-ERE complex formation.

Evidence to date indicates that structurally abnormal estrogen receptor (variant ER) can be detected in some human breast tumors. Based on in vitro ability to bind DNA sequences containing the cognate estrogen response element (ERE), these variant receptors may be categorized into DNA-binding ER (Type-1 variants) and non-DNA-binding ER (Type-2 variants). To look for Type-2 variants of normal size (67 kDa ER) that lack the ability to form immunoreactive ER-ERE complexes, a panel of 40 cryopreserved primary breast tumors were extracted and analyzed by enzyme immunoassay (ER-EIA), gel-shift, and Western blot techniques. For the 33 tumor extracts containing > or = 10 fmol/mg ER (by ER-EIA), the amount of 67 kDa ER detectable by D75 anti-ER monoclonal antibody under fully denatured and reduced assay conditions (Western blotting) did not correlate well with the presence or intensity of D75 immunoreactive ER-ERE bands seen under native conditions by gel-shift assay. Overall, 30% (10 of 33) of these extracts containing 67 kDa ER failed to produce immunoreactive ER-ERE complexes, with this frequency varying from over 40% in tumor samples with lower ER content (10-49 fmol/mg) to 11% in tumor samples with the highest ER content (> 100 fmol/mg). These results indicate that Type-2 variant receptors characterized as non-DNA-binding 67 kDa ER may be present in a significant fraction of ER-positive primary breast tumors; preliminary evidence suggests that further study of abnormalities in ER tertiary or quaternary structure, such as those produced by intracellular oxidation of ER thiol groups, is warranted.

The likelihood a breast cancer will respond to antiestrogen therapy depends on the tumor content of immunoreactive or ligand-binding estrogen receptor (ER). To investigate the failure of many ER-positive breast cancers to respond to antiestrogen therapy, we examined by gel-shift assay the ability of tumor ER to bind its cognate estrogen response element (ERE). Analysis of 38 primary breast cancers showed that some tumors containing abundant immunoreactive ER failed to demonstrate DNA binding ER. In many other ER-positive tumors, the fraction of DNA binding ER was low and consisted primarily of truncated receptor forms, which on Western analysis were revealed to be 50 kD homodimers and 67-50 kD ER heterodimers. The use of protease inhibitors during tumor extraction and the demonstration of nuclear-localizing ER and ERE-binding COUP (chicken ovalbumin upstream promoter) protein in these tumors indicated that the truncated forms of ER were likely present in vivo. The presence of intact DNA binding ER correlated with higher tumor content of immunoreactive sex steroid receptors (ER and/or PR), standard predictors of tumor responsiveness to antiestrogen, suggesting that loss or truncation of DNA binding ER may be an important prognostic parameter accounting for some forms of clinical resistance to antiestrogen therapy.