Endogenous estrogens and estrogen metabolites (EM) in human peritoneal fluid may play an important role in health and disease, yet little is known regarding their types and levels present in human peritoneal fluid, primarily due to the lack of an analytical method that is capable of directly quantifying their absolute abundances. In this report, we describe the application of a capillary LC-MS/MS method for identifying and quantifying biologically active and total endogenous EM in human peritoneal fluid. The method requires only 50 μL of peritoneal fluid, yet can quantify 13 distinct EM. Calibration curves for each EM were linear over a 103-fold concentration range and the lower LOQ was 50 fg on-column. For a charcoal stripped human peritoneal fluid sample containing 10 pg/mL of each EM, accuracy ranged from 83 to 118%, and intrabatch precision ranged from 0.2 to 4.4% RSD and interbatch precision ranged from 5.5 to 15.5% RSD. The analyses of human female perito-neal fluid shows that at least 10 biologically active and 11 total endogenous EM can be positively identified and quantitatively measured. Many of the biologically active forms are present in high abundance and possess distinct biological activities which warrant further study. Although micellar EKC gave baseline separation of a standard mixture of 10 EM, the LOQs using UV detection were not suitable for the assay of the low level estrogens in biological samples.
Capillary liquid chromatography; Electrospray ionization-tandem mass spectrometry; Endogenous estrogens and estrogen metabolites; Human peritoneal fluid; Selected reaction monitoring
The rapid rise and application of proteomic technologies has resulted in an exponential increase in the number of proteins that have been discovered and presented as ‘potential’ biomarkers for specific diseases. Unfortunately, the number of biomarkers approved for use by the Food and Drug Administration has not risen in likewise manner. While there are a number of reasons for this discrepancy, this glut of ‘potential’ biomarkers also indicates the need for validation methods to confirm or refute their utility in clinical diagnostics. For this reason, the emphasis on developing methods to target and measure the absolute quantity of specific proteins and peptides in complex proteomic samples has grown.
mass spectrometry; biomarker validation; targeted proteomics; multiple-reaction monitoring; AQUA; SISCAPA
Stem cells are controlled, in part, by genetic pathways frequently dysregulated during human tumorigenesis. Either stimulation of Wnt/β-catenin signalling or overexpression of telomerase is sufficient to activate quiescent epidermal stem cells in vivo, although the mechanisms by which telomerase exerts these effects are not understood. Here we show that telomerase directly modulates Wnt/β-catenin signalling by serving as a cofactor in a β-catenin transcriptional complex. The telomerase protein component TERT (telomerase reverse transcriptase) interacts with BRG1 (also called SMARCA4), a SWI/SNF-related chromatin remodelling protein, and activates Wnt-dependent reporters in cultured cells and in vivo. TERT serves an essential role in formation of the anterior–posterior axis in Xenopus laevis embryos, and this defect in Wnt signalling manifests as homeotic transformations in the vertebrae of Tert−/− mice. Chromatin immunoprecipitation of the endogenous TERT protein from mouse gastrointestinal tract shows that TERT physically occupies gene promoters of Wnt-dependent genes. These data reveal an unanticipated role for telomerase as a transcriptional modulator of the Wnt/ β-catenin signalling pathway.
The inflammatory microenvironment promotes skin tumorigenesis. However, the mechanisms of how cells protect themselves from inflammatory signals have yet to be revealed. Downregulation of IKKα promotes skin tumor progression from papillomas to squamous cell carcinomas, which is frequently accompanied by genomic instability, including aneuploid chromosomes and extra centrosomes. In this study, we found that IKKα promoted oligomerization of nucleophosmin (NPM), a negative centrosome duplication regulator, which further enhanced NPM and centrosome association, inhibited centrosome amplification, and maintained genome integrity. Levels of NPM hexamers and IKKα were conversely associated with skin tumor progression. Importantly, pro-inflammatory cytokine-induced IKKα activation promoted the formation of NPM oligomers and reduced centrosome numbers in mouse and human cells, whereas kinase-dead IKKα blocked this connection. Therefore, our findings suggest a previously unknown mechanism in which an IKKα-NPM axis may use the inflammatory signal to suppress centrosome amplification, promote genomic integrity, and prevent tumor progression.
Telomeres play important functional roles in cell proliferation, cell cycle regulation, and genetic stability, in which telomere length is critical. In this study, quantitative proteome comparisons for the human breast epithelial cells with short and long telomeres (184-hTERTL vs. 184-hTERTS and 90P-hTERTL vs. 90P-hTERTS), resulted from transfection of the human telomerase reverse transcriptase (hTERT) gene, were performed using cleavable isotope-coded affinity tags. More than 2000 proteins were quantified in each comparative experiment, with approximately 77% of the proteins identified in both analyses. In the cells with long telomeres, significant and consistent alterations were observed in metabolism (amino acid, nucleotide, and lipid metabolism), genetic information transmission (transcription and translation regulation, spliceosome and ribosome complexes) and cell signaling. Interestingly, the DNA excision repair pathway is enhanced, while integrin and its ligands are downregulated in the cells with long telomeres. These results may provide valuable information related to telomere functions.
Breast epithelial cells; ICAT; Mass spectrometry; Quantitative proteomics; Telomere length
Metabolomic profiling has identified, sarcosine, a derivative of the amino acid glycine, as an important metabolite involved in the etiology or natural history of prostate cancer. We examined the association between serum sarcosine levels and risk of prostate cancer in 1122 cases (813 non-aggressive and 309 aggressive) and 1112 controls in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. Sarcosine was quantified using high-throughput liquid chromatography–mass spectrometry. A significantly increased risk of prostate cancer was observed with increasing levels of sarcosine (odds ratio [OR] for the highest quartile of exposure [Q4] versus the lowest quartile [Q1] = 1.30, 95% confidence interval [CI]: 1.02, 1.65; P-trend 0.03). When stratified by disease aggressiveness, we observed a stronger association for non-aggressive cases (OR for Q4 versus Q1 = 1.44, 95% CI: 1.11, 1.88; P-trend 0.006) but no association for aggressive prostate cancer (OR for Q4 versus Q1 = 1.03, 95% CI: 0.73, 1.47; P-trend 0.89). Although not statistically significant, temporal analyses showed a stronger association between sarcosine and prostate cancer for serum collected closer to diagnosis, suggesting that sarcosine may be an early biomarker of disease. Interestingly, the association between sarcosine and prostate cancer risk was stronger among men with diabetes (OR = 2.66, 95% CI: 1.04, 6.84) compared with those without reported diabetes (OR = 1.23, 95% CI: 0.95–1.59, P-interaction = 0.01). This study found that elevated levels of serum sarcosine are associated with an increased prostate cancer risk and evidence to suggest that sarcosine may be an early biomarker for this disease.
Recent studies have suggested that changes in serum phosphate levels influence pathological states associated with aging such as cancer, bone metabolism, and cardiovascular function, even in individuals with normal renal function. The causes are only beginning to be elucidated but are likely a combination of endocrine, paracrine, autocrine, and cell autonomous effects. We have used an integrated quantitative biology approach, combining transcriptomics and proteomics to define a multi-phase, extracellular phosphate-induced, signaling network in pre-osteoblasts as well as primary human and mouse mesenchymal stromal cells. We identified a rapid mitogenic response stimulated by elevated phosphate that results in the induction of immediate early genes including c-fos. The mechanism of activation requires FGF receptor signaling followed by stimulation of N-ras and activation of AP-1 and serum response elements. A distinct long-term response also requires FGF receptor signaling and results in N-ras activation and expression of genes and secretion of proteins involved in matrix regulation, calcification, and angiogenesis. The late response is synergistically enhanced by addition of FGF23 peptide. The intermediate phase results in increased oxidative phosphorylation and ATP production and is necessary for the late response providing a functional link between the phases. Collectively, the results define elevated phosphate, as a mitogen and define specific mechanisms by which phosphate stimulates proliferation and matrix regulation. Our approach provides a comprehensive understanding of the cellular response to elevated extracellular phosphate, functionally connecting temporally coordinated signaling, transcriptional, and metabolic events with changes in long-term cell behavior.
Inorganic phosphate; FGF receptor signaling; immediate early genes; angiogenesis; AP-1
Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for ‘high Met’ expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue.
After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH).
Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (<150 amol/µg to 4669.5 amol/µg. High correlation was observed between SRM Met expression and both MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898). IHC did not correlate well with SRM (n = 44; R2 = 0.537) nor FISH GCN (n = 31; R2 = 0.509). A Met SRM level of ≥1500 amol/µg was 100% sensitive (95% CI 0.69–1) and 100% specific (95% CI 0.92–1) for MET amplification.
The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel clinical tool for efficient tumor expression profiling, potentially leading to better informed therapeutic decisions for patients with GEC.
The ability to interrogate thousands of proteins found in complex biological samples using proteomic technologies has brought the hope of discovering novel disease-specific biomarkers. While most proteomic technologies used to discover diagnostic biomarkers are quite sophisticated, "proteomic pattern analysis" has emerged as a simple, yet potentially revolutionary, method for the early diagnosis of diseases. Utilizing this technology, hundreds of clinical samples can be analyzed per day and several preliminary studies suggest proteomic pattern analysis has the potential to be a novel, highly sensitive diagnostic tool for the early detection of cancer.
proteomic patterns; cancer detection; serum; mass spectrometry
Endometrial cancer risk is strongly influenced by obesity, but the mechanisms of action remain unclear. Leptin and adiponectin, secreted from adipose tissue, reportedly play a role in such carcinogenic processes as cell proliferation, angiogenesis, and insulin regulation. In this case–control study, nested within the Breast and Bone Follow-up of the Fracture Intervention Trial (n = 15 595), we assessed pre-diagnostic serum leptin, total adiponectin, and high-molecular-weight (HMW) adiponectin in relation to endometrial cancer among postmenopausal women. During the 10-year follow-up, 62 incident endometrial cases were identified and matched to 124 controls on age, geographical site, time of fasting blood draw at baseline (1992–1993), and trial participation status. Adipokines and C-peptide were measured by ELISA. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated via conditional logistic regression, with exposures categorized in tertiles (T). Multivariable models considered C-peptide, BMI (kg/m2), and estradiol (E2) as potential confounders. Endometrial cancer risk was significantly associated with higher leptin levels, adjusted for E2 and C-peptide (ORT3 vs T1 = 2.96; 95% CI, 1.21–7.25; P trend <0.01). After further adjustment for BMI, the estimates were attenuated and the positive trend was no longer statistically significant (ORT3 vs T1 = 2.11; 95% CI, 0.69–6.44; P trend = 0.18). No significant associations were observed with adiponectin or HMW adiponectin and endometrial cancer. Our findings with leptin suggest that the leptin–BMI axis might increase endometrial cancer risk through mechanisms other than estrogen-driven proliferation. Continued exploration of these pathways in larger prospective studies may help elucidate mechanisms underlying observed obesity–endometrial cancer associations.
endometrial cancer; adiponectin; leptin; obesity
Infants are exposed to the endocrine disruptor bisphenol A (BPA) through breast milk and baby formula. Detoxication by conjugation of BPA may be limited in infants. We demonstrate BPA exposure in 11 neonates and 1 young infant, but find no evidence of a low capacity for BPA conjugation.
Neonate; Biomarker; Endocrine Disruptor; Environment; Exposure
Although estrogen and the enzymes responsible for its metabolism have been detected within the lung, the ability of this tissue to metabolize estrogen has not been demonstrated previously. The goal of this study was to characterize the profile of estrogen metabolites within the murine lung and to determine the effect of tobacco smoke exposure on metabolite levels. Use of liquid chromatography–tandem mass spectrometry led to the detection of three estrogens (E1, E2 and E3) and five estrogen metabolites (2-OHE1, 4-OHE1, 4-OHE2, 2-OMeE1 and 2-OMeE2) within the perfused lung, with 4-OHE1 being the most abundant species. Levels of 4-OHEs, carcinogenic derivatives produced primarily by cytochrome P450 1B1 (Cyp1b1), were 2-fold higher in females than males. Deletion of Cyp1b1 in females led to a dramatic reduction (21-fold) in 4-OHEs, whereas levels of 2-OHE1 and the putative protective estrogen metabolite 2-OMeE2 were increased (2.4- and 5.0-fold, respectively) (P = 0.01). Similar quantitative differences in estrogen metabolite levels were observed between Cyp1b1 null and wild-type males. Exposure of female mice to tobacco smoke for 8 weeks (2h per day, 5 days per week) increased the levels of 4-OHE1 (4-fold) and 2-OHE2 (2-fold) within the lung while reducing the total concentration of 2-OMeEs to 70% of those of unexposed controls. These data suggest that tobacco smoke accelerates the production of 4-OHEs within the lung; carcinogenic metabolites that could potentially contribute to lung tumor development. Thus, inhibition of CYP1B1 may represent a promising strategy for the prevention and treatment of lung cancer.
NANOG is a stem cell transcription factor that is essential for embryonic development, reprogramming normal adult cells and malignant transformation and progression. The nearly identical retrogene NANOGP8 is expressed in multiple cancers, but generally not in normal tissues and its function is not well defined. Our postulate is that NANOGP8 directly modulates the stemness of individual human colorectal carcinoma (CRC) cells. Stemness was measured in vitro as the spherogenicity of single CRC cells in serum free medium and the size of the side population and in vivo as tumorigenicity and experimental metastatic potential in NOD/SCID mice. We found that 80% of clinical liver metastases express a NANOG with 75% of the positive metastases containing NANOGP8 transcripts. 3 to 62% of single cells within 6 CRC lines form spheroids in serum free medium in suspension. NANOGP8 is translated into protein. The relative expression of a NANOG gene increased 8–122 fold during spheroid formation, more than the increase in OCT4 or SOX2 transcripts with NANOGP8 the more prevalent family member. shRNA to NANOG not only inhibits spherogenicity but also reduces expression of OCT4 and SOX2, the size of the side population and tumor growth in vivo. Inhibition of NANOG gene expression is associated with inhibition of proliferation and decreased phosphorylation of G2-related cell cycle proteins. Overexpression of NANOGP8 rescues single cell spherogenicity when NANOG gene expression is inhibited and increases the side population in CRC. Thus, NANOGP8 can substitute for NANOG in directly promoting stemness in CRC.
NANOG; NANOGP8; stemness; colorectal cancer; cancer stem cell
Metabolic profiling of cancer cells has recently been established as a promising tool for the development of therapies and identification of cancer biomarkers. Here we characterized the metabolomic profile of human breast tumors and uncovered intrinsic metabolite signatures in these tumors using an untargeted discovery approach and validation of key metabolites. The oncometabolite 2-hydroxyglutarate (2HG) accumulated at high levels in a subset of tumors and human breast cancer cell lines. We discovered an association between increased 2HG levels and MYC pathway activation in breast cancer, and further corroborated this relationship using MYC overexpression and knockdown in human mammary epithelial and breast cancer cells. Further analyses revealed globally increased DNA methylation in 2HG-high tumors and identified a tumor subtype with high tissue 2HG and a distinct DNA methylation pattern that was associated with poor prognosis and occurred with higher frequency in African-American patients. Tumors of this subtype had a stem cell–like transcriptional signature and tended to overexpress glutaminase, suggestive of a functional relationship between glutamine and 2HG metabolism in breast cancer. Accordingly, 13C-labeled glutamine was incorporated into 2HG in cells with aberrant 2HG accumulation, whereas pharmacologic and siRNA-mediated glutaminase inhibition reduced 2HG levels. Our findings implicate 2HG as a candidate breast cancer oncometabolite associated with MYC activation and poor prognosis.
Prospective studies have consistently found that postmenopausal breast cancer risk increases with circulating estrogens; however, findings from studies of estrogens and mammographic density (MD), an intermediate marker of breast cancer risk, have been inconsistent. We investigated the cross-sectional associations of urinary estrogens, and their 2-, 4-, and 16-hydroxylated metabolites with MD.
Postmenopausal women without breast cancer (n=194), ages 48-82 years, and reporting no current menopausal hormone therapy use were enrolled at a clinic in Western NY in 2005. Urinary estrogens and estrogen metabolites were measured using mass spectrometry. Percent MD and dense area (cm2) were measured using computer-assisted analyses of digitized films. Linear regression models were used to estimate associations of log-transformed estrogen measures with MD while adjusting for age, body mass index (BMI), parity, and past hormone therapy use.
Urinary concentrations of most individual estrogens and metabolites were not associated with MD; however, across the interdecile range of the ratio of parent estrogens (estrone and estradiol) to their metabolites, MD increased by 6.8 percentage points (p=0.02) and dense area increased by 10.3 cm2 (p=0.03). Across the interdecile ranges of the ratios of 2-, 4-, and 16-hydroxylation pathways to the parent estrogens, MD declined by 6.2 (p=0.03), 6.4 (p=0.04), and 5.7 (p=0.05) percentage points, respectively. All associations remained apparent in models without adjustment for BMI.
In this study of postmenopausal women, less extensive hydroxylation of parent estrogens was associated with higher MD.
Hydroxylation of estrogens may modulate postmenopausal breast cancer risk through a pathway involving MD.
Estrogens; metabolism; mammography; breast neoplasms; risk factors; human; female; middle-aged
Cse4 is posttranslationally modified in Saccharomyces cerevisiae. Ipl1 contributes to Cse4 phosphorylation in vivo and in vitro. Phosphorylation of Cse4 at centromeres is enhanced in response to nocodazole or reduced cohesion. The results suggest that phosphorylation of Cse4 ensures faithful chromosome segregation.
The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein–labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.
Mitochondria play central roles in integrating pro- and anti-apoptotic stimuli and JNK is well-known to have roles in activating apoptotic pathways. We establish a critical link between stress-induced JNK activation, mitofusin 2, which is an essential component of the mitochondrial outer membrane fusion apparatus, and the ubiquitin-proteasome system (UPS). JNK phosphorylation of mitofusin 2 in response to cellular stress leads to recruitment of the ubiquitin ligase (E3) Huwe1/Mule/ARF-BP1/HectH9/E3Histone/Lasu1 to mitofusin 2, with the BH3 domain of Huwe1 implicated in this interaction. This results in ubiquitin-mediated proteasomal degradation of mitofusin 2, leading to mitochondrial fragmentation and enhanced apoptotic cell death. The stability of a non-phosphorylatable mitofusin 2 mutant is unaffected by stress and protective against apoptosis. Conversely, a mitofusin 2 phosphomimic is more rapidly degraded without cellular stress. These findings demonstrate how proximal signaling events can influence both mitochondrial dynamics and apoptosis through phosphorylation-stimulated degradation of the mitochondrial fusion machinery.
Proliferating cells consume more glucose to cope with the bioenergetics and biosynthetic demands of rapidly dividing cells as well as to counter a shift in cellular redox environment. This study investigates the hypothesis that manganese superoxide dismutase (MnSOD) regulates cellular redox flux and glucose consumption during the cell cycle. A direct correlation was observed between glucose consumption and percentage of S-phase cells in MnSOD wild type fibroblasts, which was absent in MnSOD homozygous knockout fibroblasts. Results from electron paramagnetic resonance spectroscopy and flow cytometry assays showed a significant increase in cellular superoxide levels in S-phase cells, which was associated with an increase in glucose and oxygen consumption, and a decrease in MnSOD activity. Mass spectrometry results showed a complex pattern of MnSOD-methylation at both lysine (68, 89, 122, and 202) and arginine (197 and 216) residues. MnSOD protein carrying a K89A mutation had significantly lower activity compared to wild type MnSOD. Computational-based simulations indicate that lysine and arginine methylation of MnSOD during quiescence would allow greater accessibility to the enzyme active site as well as increase the positive electrostatic potential around and within the active site. Methylation-dependent changes in the MnSOD conformation and subsequent changes in the electrostatic potential around the active site during quiescence vs. proliferation could increase the accessibility of superoxide, a negatively charged substrate. These results support the hypothesis that MnSOD regulates a “metabolic switch” during progression from quiescent through the proliferative cycle. We propose MnSOD as a new molecular player contributing to the “Warburg effect.”
MnSOD; methylation; glucose; cell cycle; Warburg effect
Cytosolic foreign DNA is detected by pattern recognition receptors and mainly induces Type-I IFN production. We found that transfection of different types of DNA into various untreated cells induces Type-III IFN (IFN-lambda1) rather than Type-I IFN, indicating the presence of uncharacterized DNA sensor(s). A pull-down assay using cytosolic proteins identified that Ku70 and Ku80 are the DNA binding proteins. The knockdown studies and the reporter assay revealed that Ku70 is a novel DNA sensor inducing the IFN-lambda1 activation. The functional analysis of IFNL1 promoter revealed that PRDI and ISRE sites are predominantly involved in the DNA-mediated IFNL1 activation. A pull-down assay using nuclear proteins demonstrated that the IFN-lambda1 induction is associated with the activation of IRF-1 and IRF-7. Thus we show for the first time that Ku70 mediates type III IFN induction by DNA.
Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. While affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes pre-fractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Pre-fractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data shows that pre-fractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.
strong cation exchange; immuno-precipitation; protein complex isolation; mass spectrometry; FLAG
MicroRNA 34a (miR-34a) is a potential tumor suppressor gene and has been identified as a miRNA component of the p53 network. To better understand the biological pathways involved in miR-34a action, a parallel global protein and mRNA expression profiling on miR-34a treated neuroblastoma cells (IMR32) was performed using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray respectively. Global profiling showed that miR-34a causes much smaller mRNA expression changes compared to changes at the protein level. A total of 1495 proteins represented by 2 or more peptides were identified from the quantitative ICAT analysis, of which 143 and 192 proteins are significantly up- or down-regulated by miR-34a, respectively. Pathway analysis of these differentially expressed proteins showed the enrichment of apoptosis and cell death processes in up-regulated proteins but DNA replication and cell cycle processes in the down-regulated proteins. Ribosomal proteins are the most significant set down-regulated by miR-34a. Additionally, biological network analysis to identify direct interactions among the differentially expressed proteins demonstrated that the expression of the ubiquitous transcription factor YY1, as well as its downstream proteins, is significantly reduced by miR-34a. We further demonstrated that miR-34a directly targets YY1 through a miR-34a-binding site within the 3’ UTR of YY1 using a luciferase reporter system. YY1 is a negative regulator of p53 and it plays an essential role in cancer biology. Therefore, YY1 is another important direct target of miR-34a which closely regulates TP53 activities.
miR-34a; YY1; ICAT; proteomics; neuroblastoma
Elevated levels of circulating estrogens are linked to breast cancer risk among postmenopausal women but little is known about the importance of estrogen metabolism. A recently developed liquid chromatography tandem mass spectrometry-based method (LC-MS/MS) measuring a panel of 15 estrogen metabolites (EM) has been evaluated in one study, linking high levels of 2-pathway metabolites relative to the parent estrogens to reduced breast cancer risk. We analyzed this panel of EM in a nested case-control study of postmenopausal breast cancer.
Between 1977 and 1987, 6,915 women provided blood samples to the Columbia Missouri Serum Bank and were followed for incident breast cancer through December 2002. We studied 215 postmenopausal breast cancer cases and 215 matched controls who were postmenopausal and not using exogenous hormones at the time of blood draw. EM were examined individually, grouped by pathway (hydroxylation at the C-2, C-4 or C-16 positions of the steroid ring) and by ratios of the groupings. Logistic regression models controlling for matching and breast cancer risk factors were used to calculate quartile-specific odds ratios (ORs) and 95% CIs.
Significant elevated risks were not observed for individual EM, except for quartiles of 16-epiestriol (P trend = 0.07). The OR for total EM, the parent estrogens estrone and estradiol, and 2-pathway catechol EM (2-hydroxyestrone and 2-hydroxyestradiol) were elevated but the trends were not statistically significant. Among 2-pathway metabolites, risks for the highest levels of 2-hydroxyestrone-3-methyl ether and 2-methoxyestradiol were reduced; ORs for women in the highest versus lowest quartiles were 0.57 (95% CI = 0.33 to 0.99) and 0.53 (95% CI = 0.30 to 0.96), respectively. Overall, women with higher levels of 2-pathway EM had a reduced risk of breast cancer, which remained after accounting for levels of parent EM, 4-pathway EM and 16-pathway EM (all trends, P <0.11).
Women with more extensive hydroxylation along the 2-pathway may have a reduced risk of postmenopausal breast cancer. Further studies are needed to clarify the risks for specific EM and complex patterns of estrogen metabolism. This will require aggregation of EM results from several studies.
Estrogens are recognized causal factors in breast cancer. Interindividual variation in estrogen metabolism may also influence the risk of breast cancer and could provide clues to mechanisms of breast carcinogenesis. Long-standing hypotheses about how estrogen metabolism might influence breast cancer have not been adequately evaluated in epidemiological studies because of the lack of accurate, reproducible, and high-throughput assays for estrogen metabolites.
We conducted a prospective case–control study nested within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). Participants included 277 women who developed invasive breast cancer (case subjects) and 423 matched control subjects; at PLCO baseline, all subjects were aged 55–74 years, postmenopausal and not using hormone therapy, and provided a blood sample. Liquid chromatography–tandem mass spectrometry was used to measure serum concentrations of 15 estrogens and estrogen metabolites, in unconjugated and conjugated forms, including the parent estrogens, estrone and estradiol, and estrogen metabolites in pathways defined by irreversible hydroxylation at the C-2, C-4, or C-16 positions of the steroid ring. We calculated hazard ratios (HRs) approximating risk in highest vs lowest deciles of individual estrogens and estrogen metabolites, estrogens and estrogen metabolites grouped by metabolic pathways, and metabolic pathway ratios using multivariable Cox proportional hazards models. All statistical tests were two-sided.
Nearly all estrogens, estrogen metabolites, and metabolic pathway groups were associated with an increased risk of breast cancer; the serum concentration of unconjugated estradiol was strongly associated with the risk of breast cancer (HR = 2.07, 95% confidence interval [CI] = 1.19 to 3.62). No estrogen, estrogen metabolite, or metabolic pathway group remained statistically significantly associated with the risk of breast cancer after adjusting for unconjugated estradiol. The ratio of the 2-hydroxylation pathway to parent estrogens (HR = 0.66, 95% CI = 0.51 to 0.87) and the ratio of 4-hydroxylation pathway catechols to 4-hydroxylation pathway methylated catechols (HR = 1.34, 95% CI = 1.04 to 1.72) were statistically significantly associated with the risk of breast cancer and remained so after adjustment for unconjugated estradiol.
More extensive 2-hydroxylation of parent estrogens is associated with lower risk, and less extensive methylation of potentially genotoxic 4-hydroxylation pathway catechols is associated with higher risk of postmenopausal breast cancer.