N-Propargylglycine: a unique suicide inhibitor of proline dehydrogenase with anticancer activity and brain-enhancing mitohormesis properties.

Proline dehydrogenase (PRODH) is a mitochondrial inner membrane flavoprotein critical for cancer cell survival under stress conditions and newly recognized as a potential target for cancer drug development. Reversible (competitive) and irreversible (suicide) inhibitors of PRODH have been shown in vivo to inhibit cancer cell growth with excellent host tolerance. Surprisingly, the PRODH suicide inhibitor N-propargylglycine (N-PPG) also induces rapid decay of PRODH with concordant upregulation of mitochondrial chaperones (HSP-60, GRP-75) and the inner membrane protease YME1L1, signifying activation of the mitochondrial unfolded protein response (UPRmt) independent of anticancer activity. The present study was undertaken to address two aims: (i) use PRODH overexpressing human cancer cells (ZR-75-1) to confirm the UPRmt inducing properties of N-PPG relative to another equipotent irreversible PRODH inhibitor, thiazolidine-2-carboxylate (T2C); and (ii) employ biochemical and transcriptomic approaches to determine if orally administered N-PPG can penetrate the blood-brain barrier, essential for its future use as a brain cancer therapeutic, and also potentially protect normal brain tissue by inducing mitohormesis. Oral daily treatments of N-PPG produced a dose-dependent decline in brain mitochondrial PRODH protein without detectable impairment in mouse health; furthermore, mice repeatedly dosed with 50 mg/kg N-PPG showed increased brain expression of the mitohormesis associated protease, YME1L1. Whole brain transcriptome (RNAseq) analyses of these mice revealed significant gene set enrichment in N-PPG stimulated neural processes (FDR p < 0.05). Given this in vivo evidence of brain bioavailability and neural mitohormesis induction, N-PPG appears to be unique among anticancer agents and should be evaluated for repurposing as a pharmaceutical capable of mitigating the proteotoxic mechanisms driving neurodegenerative disorders.

Brain transcriptomic, metabolic and mitohormesis properties associated with N-propargylglycine treatment: A prevention strategy against neurodegeneration.

INTRODUCTION: There is an urgent need for new or repurposed therapeutics that protect against or significantly delay the clinical progression of neurodegenerative diseases, such as Huntington's disease (HD), Parkinson's disease and Alzheimer's disease. In particular, preclinical studies are needed for well tolerated and brain-penetrating small molecules capable of mitigating the proteotoxic mitochondrial processes that are hallmarks of these diseases. We identified a unique suicide inhibitor of mitochondrial proline dehydrogenase (Prodh), N-propargylglycine (N-PPG), which has anticancer and brain-enhancing mitohormesis properties, and we hypothesize that induction of mitohormesis by N-PPG protects against neurodegenerative diseases. We carried out a series of mouse studies designed to: i) compare brain and metabolic responses while on oral N-PPG treatment (50 mg/kg, 9-14 days) of B6CBA wildtype (WT) and short-lived transgenic R6/2 (HD) mice; and ii) evaluate potential brain and systemwide stress rebound responses in WT mice 2 months after cessation of extended mitohormesis induction by well-tolerated higher doses of N-PPG (100-200 mg/kg x 60 days). WT and HD mice showed comparable global evidence of N-PPG induced brain mitohormesis characterized by Prodh protein decay and increased mitochondrial expression of chaperone and Yme1l1 protease proteins. Interestingly, transcriptional analysis (RNAseq) showed partial normalization of HD whole brain transcriptomes toward those of WT mice. Comprehensive metabolomic profiles performed on control and N-PPG treated blood, brain, and kidney samples revealed expected N-PPG-induced tissue increases in proline levels in both WT and HD mice, accompanied by surprising parallel increases in hydroxyproline and sarcosine. Two months after cessation of the higher dose N-PPG stress treatments, WT mouse brains showed robust rebound increases in Prodh protein levels and mitochondrial transcriptome responses, as well as altered profiles of blood amino acid-related metabolites. Our HD and WT mouse preclinical findings point to the brain penetrating and mitohormesis-inducing potential of the drug candidate, N-PPG, and provide new rationale and application insights supporting its further preclinical testing in various models of neurodegenerative diseases characterized by loss of mitochondrial proteostasis.

Therapeutic targeting of HYPDH/PRODH2 with N-propargylglycine offers a Hyperoxaluria treatment opportunity.

N-propargylglycine prevents 4-hydroxyproline catabolism in mouse liver and kidney. N-propargylglycine is a novel suicide inhibitor of PRODH2 and induces mitochondrial degradation of PRODH2. PRODH2 is selectively expressed in liver and kidney and contributes to primary hyperoxaluria (PH). Preclinical evaluation of N-propargylglycine efficacy as a new PH therapeutic is warranted.

FOXP3-positive regulatory T lymphocytes and epithelial FOXP3 expression in synchronous normal, ductal carcinoma in situ, and invasive cancer of the breast.

FOXP3-expressing T regulatory lymphocytes (Tregs) have been described as putative mediators of immune tolerance, and thus facilitators of tumor growth. When found in association with various malignancies, Tregs are generally markers of poor clinical outcome. However, it is unknown whether they are also associated with cancer progression. We evaluated quantitative FOXP3 expression in lymphocytes as well as in epithelial cells in a set of thirty-two breast tumors with synchronous normal epithelium, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) components. Tumors were stained for FOXP3 and CD3 expression and Tregs quantified by determining the ratio of colocalized FOXP3 and CD3 relative to 1) total CD3-expressing lymphocytes and 2) to FOXP3-expressing epithelial cells. The median proportion of FOXP3-expressing CD3 cells significantly increased with malignant progression from normal to DCIS to IDC components (0.005, 0.019 and 0.030, respectively; p ≤ 0.0001 for normal vs. IDC and p = 0.004 for DCIS vs. IDC). The median intensity of epithelial FOXP3 expression was also increased with invasive progression and most markedly augmented between normal and DCIS components (0.130 vs. 0.175, p ≤ 0.0001). Both Treg infiltration and epithelial FOXP3 expression were higher in grade 3 vs. grade 1 tumors (p = 0.014 for Tregs, p = 0.038 for epithelial FOXP3), but did not vary significantly with hormone receptor status, size of invasive tumor, lymph node status, or disease stage. Notably, Treg infiltration significantly correlated with epithelial up-regulation of FOXP3 expression (p = 0.013 for normal, p = 0.001 for IDC). These findings implicate both Treg infiltration and up-regulated epithelial FOXP3 expression in breast cancer progression.

Cell surface-bound IL-1alpha is an upstream regulator of the senescence-associated IL-6/IL-8 cytokine network.

Inflammation underlies most age-related diseases, including cancer, but the etiology is poorly understood. One proposed factor is the presence of senescent cells, which increase with age. The senescence response arrests the proliferation of potentially oncogenic cells, and most senescent cells secrete high levels of proinflammatory cytokines and other proteins. The complex senescence-associated secretory phenotype is likely regulated at multiple levels, most of which are unknown. We show that cell surface-bound IL-1alpha is essential for signaling the senescence-associated secretion of IL-6 and IL-8, 2 proinflammatory cytokines that also reinforce the senescence growth arrest. Senescent human fibroblasts expressed high levels of IL-1alpha mRNA, intracellular protein, and cell surface-associated protein, but secreted very little protein. An IL-1 receptor (IL1R) antagonist, neutralizing IL-1alpha antibodies, and IL-1alpha depletion by RNA interference all markedly reduced senescence-associated IL-6/IL-8 secretion. Depletion of the key IL-1R signaling component IRAK1 also suppressed this secretion, and IL-1alpha neutralizing antibodies prevented IRAK1 degradation, indicating engagement of the IL-1R signaling pathway. Furthermore, IL-1alpha depletion reduced the DNA binding activity of NF-kappaB and C/EBPbeta, which stimulate IL-6/IL-8 transcription. IL-1alpha was a general regulator of senescence-associated IL-6/IL-8 secretion because IL-1alpha blockade reduced IL-6/IL-8 secretion whether cells senesced owing to DNA damage, replicative exhaustion, oncogenic RAS, or chromatin relaxation. Furthermore, conditioned medium from IL-1alpha-depleted senescent cells markedly reduced the IL-6/IL-8-dependent invasiveness of metastatic cancer cells, indicating that IL-1alpha regulates the biological effects of these cytokines. Thus, cell surface IL-1alpha is an essential cell-autonomous regulator of the senescence-associated IL-6/IL-8 cytokine network.

Systematic mapping of posttranslational modifications in human estrogen receptor-alpha with emphasis on novel phosphorylation sites.

A systematic study of posttranslational modifications of the estrogen receptor isolated from the MCF-7 human breast cancer cell line is reported. Proteolysis with multiple enzymes, mass spectrometry, and tandem mass spectrometry achieved very high sequence coverage for the full-length 66-kDa endogenous protein from estradiol-treated cell cultures. Nine phosphorylated serine residues were identified, three of which were previously unreported and none of which were previously observed by mass spectrometry by any other laboratory. Two additional modified serine residues were identified in recombinant protein, one previously reported but not observed here in endogenous protein and the other previously unknown. Although major emphasis was placed on identifying new phosphorylation sites, N-terminal loss of methionine accompanied by amino acetylation and a lysine side chain acetylation (or possibly trimethylation) were also detected. The use of both HPLC-ESI and MALDI interfaced to different mass analyzers gave higher sequence coverage and identified more sites than could be achieved by either method alone. The estrogen receptor is critical in the development and progression of breast cancer. One previously unreported phosphorylation site identified here was shown to be strongly dependent on estradiol, confirming its potential significance to breast cancer. Greater knowledge of this array of posttranslational modifications of estrogen receptor, particularly phosphorylation, will increase our understanding of the processes that lead to estradiol-induced activation of this protein and may aid the development of therapeutic strategies for management of hormone-dependent breast cancer.

Protein acetylation and histone deacetylase expression associated with malignant breast cancer progression.

PURPOSE: Excess histone deacetylase (HDAC) activity can induce hypoacetylation of histone and nonhistone protein substrates, altering gene expression patterns and cell behavior potentially associated with malignant transformation. However, HDAC expression and protein acetylation have not been studied in the context of breast cancer progression. EXPERIMENTAL DESIGN: We assessed expression levels of acetylated histone H4 (ac-H4), ac-H4K12, ac-tubulin, HDAC1, HDAC2, and HDAC6 in 22 reduction mammoplasties and in 58 specimens with synchronous normal epithelium, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) components. Differences among groups were tested for significance using nonparametric tests. RESULTS: From normal epithelium to DCIS, there was a marked reduction in histone acetylation (P < 0.0001). Most cases showed similar levels of acetylation in DCIS and IDC, although some showed further reduction of ac-H4 and ac-H4K12 from DCIS to IDC. Expression of HDAC1, HDAC2, and HDAC6 was also significantly reduced but by a smaller magnitude. Greater reductions of H4 acetylation and HDAC1 levels were observed from normal to DCIS in estrogen receptor-negative compared with estrogen receptor-positive, and in high-grade compared with non-high-grade tumors. CONCLUSION: Overall, there was a global pattern of hypoacetylation associated with progression from normal to DCIS to IDC. These findings suggest that the reversal of this hypoacetylation in DCIS and IDC could be an early measure of HDAC inhibitor activity.

Destabilization of ERBB2 transcripts by targeting 3' untranslated region messenger RNA associated HuR and histone deacetylase-6.

In addition to repressing ERBB2 promoter function, histone deacetylase (HDAC) inhibitors induce the accelerated decay of mature ERBB2 transcripts; the mechanism mediating this transcript destabilization is unknown but depends on the 3' untranslated region (UTR) of ERBB2 mRNA. Using ERBB2-overexpressing human breast cancer cells (SKBR3), the mRNA stability factor HuR was shown to support ERBB2 transcript integrity, bind and endogenously associate with a conserved U-rich element within the ERBB2 transcript 3' UTR, coimmunoprecipitate with RNA-associated HDAC activity, and colocalize with HDAC6. HDAC6 also coimmunoprecipitates with HuR in an RNA-dependent manner and within 6 hours of exposure to a pan-HDAC inhibitor dose, that does not significantly alter cytosolic HuR levels or HuR binding to ERBB2 mRNA. Cellular ERBB2 transcript levels decline while remaining physically associated with HDAC6. Knockdown of HDAC6 protein by small interfering RNA partially suppressed the ERBB2 transcript decay induced by either pan-HDAC or HDAC6-selective enzymatic inhibitors. Three novel hydroxamates, ST71, ST17, and ST80 were synthesized and shown to inhibit HDAC6 with 14-fold to 31-fold greater selectivity over their binding and inhibition of HDAC1. Unlike more potent pan-HDAC inhibitors, these HDAC6-selective inhibitors produced dose-dependent growth arrest of ERBB2-overexpressing breast cancer cells by accelerating the decay of mature ERBB2 mRNA without repressing ERBB2 promoter function. In sum, these findings point to the therapeutic potential of HuR and HDAC6-selective inhibitors, contrasting ERBB2 stability effects induced by HDAC6 enzymatic inhibition and HDAC6 protein knockdown, and show that ERBB2 transcript stability mechanisms include exploitable targets for the development of novel anticancer therapies.

Targeting Mitochondrial Proline Dehydrogenase with a Suicide Inhibitor to Exploit Synthetic Lethal Interactions with p53 Upregulation and Glutaminase Inhibition.

Proline dehydrogenase (PRODH) is a p53-inducible inner mitochondrial membrane flavoprotein linked to electron transport for anaplerotic glutamate and ATP production, most critical for cancer cell survival under microenvironmental stress conditions. Proposing that PRODH is a unique mitochondrial cancer target, we structurally model and compare its cancer cell activity and consequences upon exposure to either a reversible (S-5-oxo: S-5-oxo-2-tetrahydrofurancarboxylic acid) or irreversible (N-PPG: N-propargylglycine) PRODH inhibitor. Unlike 5-oxo, the suicide inhibitor N-PPG induces early and selective decay of PRODH protein without triggering mitochondrial destruction, consistent with N-PPG activation of the mitochondrial unfolded protein response. Fly and breast tumor (MCF7)-xenografted mouse studies indicate that N-PPG doses sufficient to phenocopy PRODH knockout and induce its decay can be safely and effectively administered in vivo Among breast cancer cell lines and tumor samples, PRODH mRNA expression is subtype dependent and inversely correlated with glutaminase (GLS1) expression; combining inhibitors of PRODH (S-5-oxo and N-PPG) and GLS1 (CB-839) produces additive if not synergistic loss of cancer cell (ZR-75-1, MCF7, DU4475, and BT474) growth and viability. Although PRODH knockdown alone can induce cancer cell apoptosis, the anticancer potential of either reversible or irreversible PRODH inhibitors is strongly enhanced when p53 is simultaneously upregulated by an MDM2 antagonist (MI-63 and nutlin-3). However, maximum anticancer synergy is observed in vitro when the PRODH suicide inhibitor, N-PPG, is combined with both GLS1-inhibiting and a p53-upregulating MDM2 antagonist. These findings provide preclinical rationale for the development of N-PPG-like PRODH inhibitors as cancer therapeutics to exploit synthetic lethal interactions with p53 upregulation and GLS1 inhibition.

A novel serine phosphorylation site detected in the N-terminal domain of estrogen receptor isolated from human breast cancer cells.

Activated estrogen receptor (ERalpha) plays a critical role in breast cancer development and is a major target for drug treatment. Serine phosphorylation within the N-terminal domain (NTD) contributes to ERalpha activation and may also cause drug resistance. Previous biochemical identification of phosphorylated ERalpha residues was limited to protein artificially overexpressed in transfected cell lines. We report mass spectrometric methods that have allowed the identification of a new site within the NTD of ERalpha isolated from cultured human breast cancer cells. Immunoprecipitation, trypsin digestion, and analysis by nano-LC-ESI-MS/MS (Q-STAR, MDS Sciex) and vMALDI-MS(n) (Finnigan LTQ, Thermo-Electron) identified peptides containing 8 of 14 serine residues within the NTD, one being partially phosphorylated Ser-167, known but not previously reported by MS. Chymotrypsin digestion revealed other known sites at Ser-102/104/106 and 118. Tandem methods developed for the peptide containing Ser-118 and the use of hypothesis-driven experiments--i.e., the assumption that an intact phosphopeptide showing no molecular ion might yield fragment ions including loss of phosphoric acid in vMALDI-MS/MS--allowed the identification of a novel site at Ser-154. Quantitation by selected reaction monitoring demonstrated 6-fold and 2.5-fold increases in Ser-154 phosphorylation in estradiol- and EGF-treated cells, respectively, compared to controls, confirmed by immunoblotting with a novel rabbit polyclonal antibody. Thus, the protein isolation and MS strategies described here can facilitate discovery of novel phosphorylation sites within low abundance, clinically important cancer targets like ERalpha, and may thereby contribute to our understanding of the role of phosphorylation in the development of breast cancer.

Rapid alteration of microRNA levels by histone deacetylase inhibition.

Improved understanding of the molecular mechanisms by which small-molecule inhibitors of histone deacetylases (HDAC) induce programs, such as cellular differentiation and apoptosis, would undoubtedly assist their clinical development as anticancer agents. As modulators of gene transcript levels, HDAC inhibitors (HDACi) typically affect only 5% to 10% of actively transcribed genes with approximately as many mRNA transcripts being up-regulated as down-regulated. Using microRNA (miRNA) array analysis, we report rapid alteration of miRNA levels in response to the potent hydroxamic acid HDACi LAQ824 in the breast cancer cell line SKBr3. Within 5 hours of exposure to a proapoptotic dose of LAQ824, significant changes were measured in 40% of the >60 different miRNA species expressed in SKBr3 cells with 22 miRNA species down-regulated and 5 miRNAs up-regulated. To explore a potential functional link between HDACi induced mRNA up-regulation and miRNA down-regulation, antisense experiments were done against miR-27a and miR-27b, both abundantly expressed and down-regulated in SKBr3 cells by LAQ824. Correlating a set of genes previously determined by cDNA array analysis to be rapidly up-regulated by LAQ824 in SKBr3 with a database of potential 3' untranslated region miRNA binding elements, two genes containing putative miR-27 anchor elements were identified as transcriptionally up-regulated following miR-27 antisense transfection, ZBTB10/RINZF, a Sp1 repressor, and RYBP/DEDAF, an apoptotic facilitator. These findings emphasize the importance of post-transcriptional mRNA regulation by HDACi in addition to their established effects on promoter-driven gene expression.

ERpS294 is a biomarker of ligand or mutational ERα activation and a breast cancer target for CDK2 inhibition.

ERα phosphorylation at hinge site S294 (pS294) was recently shown to be essential for ER-dependent gene transcription and mediated by an unknown cyclin-dependent kinase (CDK). This study was undertaken to identify the exact CDK pathway mediating pS294 formation, and to determine if this phosphorylation event occurs with, and can be targeted to treat, the ligand-independent growth of breast cancers expressing endocrine-refractory ESR1 mutations. Using a newly developed anti-pS294 monoclonal antibody, a combination of CDK specific siRNA knockdown studies and a broad panel of CDK selective inhibitors against ligand (E2)-stimulated MCF7 cells, we first identified CDK2 as the primary mediator of pS294 formation and showed that CDK2-selective inhibitors like Dinaciclib, but not CDK4/6 inhibitors like Palbociclib, can selectively prevent pS294 formation and repress ER-dependent gene expression. We then expressed the ER-activating mutations ERmut(Y537S) and ERmut(D538G) in MCF7 cells, and demonstrated their ability to induce ligand-independent and tamoxifen-resistant growth, associated with constitutive and CDK2-dependent pS294 expression. Following robust growth of E2-independent and TAM-resistant MCF7mutER(Y537S) tumors in vivo, nude mice were also treated with either Dinaciclib or Palbociclib at doses and injection schedules unable to retard tumor growth as single agents; the TAM plus Palbociclib combination arrested further tumor growth without affecting pS294 formation, while the TAM plus Dinaciclib combination produced tumor regression associated with loss of pS294 expression. These findings, and our proposed mechanistic model, provide new rationale for the clinical evaluation of CDK2 inhibitors given in combination with endocrine agents as a new treatment strategy against ESR1 mutation expressing breast cancers.

Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies.

BACKGROUND: Recent studies indicate that microRNAs (miRNAs) are mechanistically involved in the development of various human malignancies, suggesting that they represent a promising new class of cancer biomarkers. However, previously reported methods for measuring miRNA expression consume large amounts of tissue, prohibiting high-throughput miRNA profiling from typically small clinical samples such as excision or core needle biopsies of breast or prostate cancer. Here we describe a novel combination of linear amplification and labeling of miRNA for highly sensitive expression microarray profiling requiring only picogram quantities of purified microRNA. RESULTS: Comparison of microarray and qRT-PCR measured miRNA levels from two different prostate cancer cell lines showed concordance between the two platforms (Pearson correlation R2 = 0.81); and extension of the amplification, labeling and microarray platform was successfully demonstrated using clinical core and excision biopsy samples from breast and prostate cancer patients. Unsupervised clustering analysis of the prostate biopsy microarrays separated advanced and metastatic prostate cancers from pooled normal prostatic samples and from a non-malignant precursor lesion. Unsupervised clustering of the breast cancer microarrays significantly distinguished ErbB2-positive/ER-negative, ErbB2-positive/ER-positive, and ErbB2-negative/ER-positive breast cancer phenotypes (Fisher exact test, p = 0.03); as well, supervised analysis of these microarray profiles identified distinct miRNA subsets distinguishing ErbB2-positive from ErbB2-negative and ER-positive from ER-negative breast cancers, independent of other clinically important parameters (patient age; tumor size, node status and proliferation index). CONCLUSION: In sum, these findings demonstrate that optimized high-throughput microRNA expression profiling offers novel biomarker identification from typically small clinical samples such as breast and prostate cancer biopsies.

Validated high-throughput screening of drug-like small molecules for inhibitors of ErbB2 transcription.

A whole cell high-throughput screening assay was developed and tested against > 2,000 structurally and functionally diverse drug-like small molecules to identify lead compounds capable of cell permeability and selective silencing of ErbB2 transcription. Screening employed reporter sublines clonally selected from ErbB2-negative MCF7 breast cancer cells after stable genomic integration of the ErbB2 proximal promoter driving a luciferase reporter; anti-ErbB2 activities (50% inhibitory concentration values) were compared to inhibition of control MCF7 sublines bearing integrated reporters driven by either a mutated ErbB2 promoter or the cyclin D1 promoter. Of the seven resulting lead compounds, four emerged from the National Cancer Institute (NCI)/ Developmental Therapeutics Program (DTP) Structural Diversity Set (NSC-131547, NSC-176328, NSC-259968, and NSC-321237); three others emerged from a panel of anticancer compounds with known mechanistic actions and included a minor groove DNA-binding antibiotic (NSC-58514, chromomycin A3), a hydroxamic acid inhibitor of histone deacetylases (NSC-709238, trichostatin A), and a tripeptide aldehyde proteasome inhibitor (MG-132). For optimization, 58 scaffold analogs of the four NCI/DTP structural leads and nine functional analogs of the mechanistic leads were secondarily screened to identify seven compounds with comparable or superior activity relative to the leads, including an approved anticancer drug, PS-341 (bortezomib). PS-341 activity was validated against cultured ErbB2-positive breast cancer cell lines (SKBr3 and BT474) and a trastuzumab-resistant ErbB2-positive breast cancer xenograft model (B585), in which PS-341 antitumor activity correlated with selective down-regulation of ErbB2 mRNA and protein levels, confirming the ErbB2- silencing potential of proteasome inhibitors.

Activation of nuclear factor-kappaB (NFkappaB) identifies a high-risk subset of hormone-dependent breast cancers.

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

Reactivity of zinc finger cysteines: chemical modifications within labile zinc fingers in estrogen receptor.

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. We are investigating the structural consequences of chemical exposures thought to modify essential zinc finger cysteine residues in human ER. The current study employs mass spectrometry to probe ER zinc finger structural changes induced by a redox-reactive vitamin K3 analog, menadione; a commonly used cysteine alkylator, iodoacetic acid; and a thiol alkylating fluorophore, monobromobimane. Although they are slower to react, the sterically bulkier reagents, monobromobimane and menadione, effectively alkylate the most susceptible ER zinc finger cysteine sulfhydryl groups. Menadione arylation results first in Michael addition of the hydroquinone followed by rapid oxidation to the corresponding quinone, evidenced by a 2 Da mass loss per cysteine residue. Mass spectrometric analysis performed under MALDI conditions reveals both hydroquinone and quinone forms of arylated menadione, whereas only the quinone product is detectable under ESI conditions. 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 bromobimane or menadione, consistent with their unequal positioning near basic amino acids that affect thiol pKa, thereby rendering Cys-240 more reactive than Cys-237. These findings demonstrate important differential susceptibility of ER zinc finger cysteine residues to thiol reactions.

Transcriptional repression of ErbB2 by histone deacetylase inhibitors detected by a genomically integrated ErbB2 promoter-reporting cell screen.

The antitumor activity of histone deacetylase (HDAC) inhibitors has been linked to gene expression induced by acetylation of histone and nonhistone proteins; but the molecular basis for their antitumor selectivity remains largely unknown. With development of a genomically integrated, ErbB2 promoter-reporting breast cancer cell screen, ErbB2 promoter inhibiting activity was observed by the HDAC inhibitors trichostatin A (TSA) and sodium butyrate. Paradoxically, these agents stimulated the episomal form of this ErbB2 promoter-reporter introduced by transient transfection. Transcriptional run-off assays in ErbB2 amplified and overexpressing breast cancer cells confirmed that within 5 h, TSA exposure profoundly inhibits ErbB2 transcript synthesis from the amplified oncogene yet preserves transcription from single copy genes such as the epithelial-specific Ets family member, ESX. Northern analyses of ErbB2-overexpressing breast cancer lines (SKBR3, BT-474, and MDA-453) showed that within 24 h of submicromolar treatment by TSA, ESX transcript levels increase while ErbB2 transcript levels rapidly decline, with no TSA effect apparent on the open chromatin configuration of either gene as monitored by DNase I hypersensitivity. Actinomycin D studies confirmed that in addition to inhibiting ErbB2 transcript synthesis, TSA selectively destabilizes mature ErbB2 transcripts enhancing their decay. Whereas TSA markedly reduced ErbB2 protein levels in these overexpressing cell lines, TSA treatment of MCF/HER2-18 cells engineered to overexpress the ErbB2 receptor under control of a heterologous promoter increased their expression of ErbB2 protein. These findings suggest that further studies are warranted to determine whether ErbB2-positive human cancers represent unusually sensitive clinical targets for HDAC inhibitor therapy.

Sources of superoxide/H2O2 during mitochondrial proline oxidation.

p53 Inducible gene 6 (PIG6) encodes mitochondrial proline dehydrogenase (PRODH) and is up-regulated several fold upon p53 activation. Proline dehydrogenase is proposed to generate radicals that contribute to cancer cell apoptosis. However, there are at least 10 mitochondrial sites that can produce superoxide and/or H2O2, and it is unclear whether proline dehydrogenase generates these species directly, or instead drives production by other sites. Amongst six cancer cell lines, ZR75-30 human breast cancer cells had the highest basal proline dehydrogenase levels, and mitochondria isolated from ZR75-30 cells consumed oxygen and produced H2O2 with proline as sole substrate. Insects use proline oxidation to fuel flight, and mitochondria isolated from Drosophila melanogaster were even more active with proline as sole substrate than ZR75-30 mitochondria. Using mitochondria from these two models we identified the sites involved in formation of superoxide/H2O2 during proline oxidation. In mitochondria from Drosophila the main sites were respiratory complexes I and II. In mitochondria from ZR75-30 breast cancer cells the main sites were complex I and the oxoglutarate dehydrogenase complex. Even with combinations of substrates and respiratory chain inhibitors designed to minimize the contributions of other sites and maximize any superoxide/H2O2 production from proline dehydrogenase itself, there was no significant direct contribution of proline dehydrogenase to the observed H2O2 production. Thus proline oxidation by proline dehydrogenase drives superoxide/H2O2 production, but it does so mainly or exclusively by providing anaplerotic carbon for other mitochondrial dehydrogenases and not by producing superoxide/H2O2 directly.

RPL24: a potential therapeutic target whose depletion or acetylation inhibits polysome assembly and cancer cell growth.

Partial loss of large ribosomal subunit protein 24 (RPL24) function is known to protect mice against Akt or Myc-driven cancers, in part via translational inhibition of a subset of cap(eIF4E)-dependently translated mRNAs. The role of RPL24 in human malignancies is unknown. By analyzing a public dataset of matched human breast cancers and normal mammary tissue, we found that breast cancers express significantly more RPL24 than matched normal breast samples. Depletion of RPL24 in breast cancer cells by >70% reduced cell viability by 80% and decreased protein expression of the eIF4E-dependently translated proteins cyclin D1 (75%), survivin (46%) and NBS1 (30%) without altering GAPDH or beta-tubulin levels. RPL24 knockdown also reduced 80S subunit levels relative to 40S and 60S levels. These effects on expression of eIF4E-dependent proteins and ribosome assembly were mimicked by 2-24 h treatment with the pan-HDACi, trichostatin A (TSA), which induced acetylation of 15 different polysome-associated proteins including RPL24. Furthermore, HDAC6-selective inhibition or HDAC6 knockdown induced ribosomal protein acetylation. Via mass spectrometry, we found that 60S-associated, but not, polysome-associated, RPL24 undergoes HDACi-induced acetylation on K27. Thus, RPL24 K27 acetylation may play a role in ribosome assembly. These findings point toward a novel acetylation-dependent polysome assembly mechanism regulating tumorigenesis.